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Chemical Reagents and Derivatization Procedures in Drug AnalysisNeil D. Danielson, Patricia A. Gallagher, and James J. Bao in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 7042–7076 © John Wiley & Sons Ltd, Chichester, 2000 CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 1 Chemical Reagents and Derivatization Procedures in Drug Analysis Neil D. Danielson and Patricia A. Gallagher Miami University, Oxford, USA James J. Bao Procter and Gamble Pharmaceuticals, Mason, USA 1 Introduction 2 Gas Chromatography 2.1 Alkylation 2.2 Acylation 2.3 Silylation 2.4 Sample Handling 3 High-performance Liquid Chromatography 3.1 Alkaloids 3.2 Amines 3.3 Antibiotics 3.4 Barbiturates 3.5 Carbonyl Compounds and Carboxylic Acids 3.6 Catecholamines 3.7 Hydroxy Compounds 3.8 Steroids 3.9 Sulfur Compounds 4 Capillary Electrophoresis 4.1 Derivatization for Ultraviolet Detection 4.2 Derivatization for Fluorescence Detection 4.3 Special Applications 4.4 Derivatization Modes 5 Conclusion Abbreviations and Acronyms Related Articles References 1 2 2 3 5 7 8 8 10 13 14 14 16 16 17 18 19 19 20 21 22 23 23 23 24 to enhance the volatility, temperature stability, and/or detectability. The GC section considers derivatization of drugs by reagent class: alkylation, acylation, and silylation. The GC sample handling section describes an approach which combines the extraction and derivatization steps together. Both pre- and postcolumn derivatization are common approaches for high-performance liquid chromatography (HPLC) and many of these methods have been adapted for CE. Derivatization for HPLC is often directed toward aliphatic amines, carboxylic acids, or alcohols that are difficult to detect at low levels by absorbance, luminescence, or electrochemical means. In addition, small hydrophilic molecules upon prederivatization are often converted into larger more hydrophobic compounds, making reversed-phase HPLC easier or even feasible. The HPLC section considers derivatization of drugs based on type: alkaloids, amines, antibiotics, barbiturates, carbonyl compounds and carboxylic acids, catecholamines, hydroxy compounds, steroids, and sulfur compounds. For the GC and HPLC sections, tables are included giving the structures of the more important derivatizing agents, the analytes, and the corresponding reaction products. A brief rationale for derivatization chemistry with CE concludes this article. For CE, derivatization is often done to improve detectability since the path length for absorbance detection is very short and fluorescence can be effective using laser-based systems. 1 INTRODUCTION Derivatization in conjunction with chromatography or CE can be done either in an off-line mode, often prior to the separation step (prederivatization), or in an online mode (postcolumn derivatization). Generally, only prederivatization is used in GC, principally to enhance the volatility, temperature stability, and/or detectability. Both pre- and postcolumn derivatization are common approaches for HPLC and many of these methods have been adapted for CE. Derivatization for HPLC is often directed toward aliphatic amines, carboxylic acids, or alcohols that are difficult to detect at low levels by absorbance, luminescence, or electrochemical means. In addition, small hydrophilic molecules upon prederivatization are often converted into larger more hydrophobic compounds, making reversed-phase HPLC easier or feasible. For CE, derivatization is often done to improve detectability since the path length for absorbance detection is very short and fluorescence can be effective using-laser based systems. The desirable conditions to be met for precolumn derivatization are (1) the reaction stoichiometry and product structure should be known, (2) the reaction This article describes both pre- and postcolumn derivatization chemistry used in conjunction with either chromatography or capillary electrophoresis (CE) to facilitate the determination of drugs. Generally, only prederivatization is used in gas chromatography (GC), principally Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) Copyright © John Wiley & Sons Ltd 4 DMFDA is a rapid methylating agent which provides quantitative yields upon mixing in a nonaqueous solvent with the sample of interest (Table 1. The alcohol is evaporated. and t-butyl acetals may be used analogously. n-butyl. OH. amines. and sulfur compounds. Ethyl. A specific example and an overview for each of these reaction classes will be described below. Several good review sources describing derivatization reagents. reducing volatility. causing peak broadening. Sample handling before analysis is minimal. GC. Compounds with these functional groups tend to form intermolecular hydrogen bonds. Because postcolumn derivatization involves mixing the column effluent with a reagent or passing it through a reactor to form the derivative before it is detected. and N-alkylamines. and silylation reaction chemistries are the most common approaches to derivatization for GC and will be the primary focus in this article. The conditions desirable to be met for postcolumn derivatization are (1) although the reaction must be rapid. Alkylation reactions can also be used to prepare ethers from alcohols such as phenol. or carboxyl group. Generally. catecholamines. NH. Generally the esters are extracted into n-heptane followed by evaporation of the solvent. One principal chromatographic use is the conversion of carboxylic acids into esters that are stable and volatile with good chromatographic characteristics. and sulfonamides from amines. and (3) the reactor volume must be small to minimize dilution and band broadening. At least one commercial company provides a good overview of the commercial reagents and the apparatus available. the automation step is built in. acylation. 1 Two other references are fairly comprehensive in describing many of the published methods for GC derivatization either by reaction class 2 or type of organic compound. which is important for labile unsaturated fatty acids.a. and thiols into halogenated derivatives that can 2 GAS CHROMATOGRAPHY Prior to analysis by GC. The GC section considers derivatization of drugs by reagent class: alkylation. and silylation. and dilantin. Esterification of carboxylic acids has traditionally been done through reaction with an alcohol in the presence of an acid catalyst. found in an organic acid or amine. a 1 – 2-mg sample of the acid is heated with 100 µL of the alcohol (methanol or ethanol) containing 3 M HCl for 30 min at 70 ° C. reactions. thioethers from sulfur compounds. leaving the ester as a residue. hydroxy compounds. carbonyl compounds and carboxylic acids.g. into the hot injection port (250 – 300 ° C). phenols. steroids. The HPLC section considers the derivatization of drugs based on type: alkaloids. benzyl) group. They are also thermally unstable and can interact with either fused silica or the stationary phase. and be reproducible. barbiturates.N-dimethylaminoethylene derivatives and amino acids are simultaneously modified as this derivative at the amino group and as the methyl ester at the carboxyl group. thiols to thioethers. amides. sulfonamides. reagent 4). One advantage of the prederivatization approach is that simple equipment is commercially available to allow for the reaction chemistry to be done in the batch mode. hydroxyl. the reaction products do not have to be very stable. PHARMACEUTICALS AND DRUGS Alkylation. compounds containing functional groups with active hydrogens such as COOH. antibiotics. the use of a robot to carry out the entire prederivatization chemistry and an HPLC instrument equipped with an autosampler means that the sample throughput will usually be limited by the chromatographic step. and phenols to methyl ethers. and applications for GC are available. phenolic alkaloids. and (3) the derivative should be stable and readily separable and distinguishable from the starting material. 3 . HPLC. Alternatively. The derivative is immediately swept onto the analytical column for separation and detection. (2) the reagent itself must have no or a very low detection response. Primary amines are converted into their N. The remainder of the article is divided into three major sections. reagent 2). PFB-Br can convert carboxylic acids. and SH need to be protected. 2. and CE. In addition. Carboxylic acids are converted to methyl esters. TMAH is the most common reagent used but trimethyl (a. a short discussion of sample handling is included.a-trifluoro-m-tolyl)ammonium hydroxide can be used at a lower injection temperature. within about 2 min. by an aliphatic or aliphatic – aromatic (e. propyl. Aliphatic hydroxyl groups are not methylated. 5 Upon injection of the QUAT mixed with the drug having a reactive amino. reagent 1).1 Alkylation Alkylation represents the replacement of an active hydrogen. A faster reaction in only about 2 min using a boiling water-bath is possible with a BF3 – methanol solution (Table 1. formation of the appropriate methyl derivative will occur (Table 1. reducing the chance of error from sample loss. sedatives. Subsequent sample analysis using an autoinjector and a standard unmodified chromatograph is straightforward. This method is particularly good for barbiturates. acylation. Pyrolysis of a quaternary ammonium salt (QUAT) in the presence of the organic analyte can be a convenient methylation procedure.2 should be reasonably fast and proceed quantitatively (or at least reproducibly). A brief rationale for derivatization chemistry with CE concludes this article. xanthine bases. be easily detected by electron capture (EC) (Table 1. pentafluorobenzyl bromide. thioesters. reagent 3). Sulfonamides R SH 4. A typical chromatogram taken on a packed OV-225 glass column is shown in Figure 1.5 µg of the CBD (2). and amides. A detection limit of 50 ng mL 1 of plasma was possible. SH. Interestingly. R COOH R NH2 R OH R SH R COOH R OH R COOCH3 R NH (CH2)2 N(CH3)2 R O CH3 R S CH3 O R C O (PFB) R O (PFB) (PFB) HN R S (PFB) R O CH3 R NH CH3 R COOCH3 SO2NHR 3. 0 4 8 12 16 24 28 Time (min) Figure 1 Typical chromatogram from a plasma sample containing 2. NH) into esters. CH3 N+ −OH H3C CH3 (TMAH) R OH R NH2 R COOH a DMFDA. trimethylanilinium hydroxide. it can handle larger amounts of sample.N-dimethylformamide dimethylacetal. TMAH.2 Acylation Acylation is the use of a carboxylic acid or carboxylic acid derivative to convert compounds that have active hydrogens (OH. respectively. 6 The analysis of cannabidiol (CBD) and the internal standard tetrahydrocannabidiol in blood plasma involved derivatization with PFB-Br followed by purification on a Florisil™ column.0 µg of the internal standard tetrahydrocannabidiol (1) and calculated to contain 1. Reagenta F H F B: O CH3 F (BF3−methanol) O CH3 (CH3)2N C H O CH3 (DMFDA) F F F F CH2Br F (PFB-Br) Compounds R COOH Derivatives R COOCH3 2.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 3 Table 1 Common GC derivatives – alkylation No. Although a packed column has limited separation capability. [Reproduced with permission from Jones et al. N. the reaction is run in an organic solvent in the presence of a base such as a tertiary amine for 5 min at 40 ° C. Acetone washings of this solution were evaporated to dryness and the residue was dissolved in hexane. For carboxylic acids. 1. 7 ] . 2. 7 The derivatization procedure involved refluxing a hexane extract with PFB-Br overnight with stirring. A portion of this hexane solution was injected into a gas chromatograph equipped with an EC detector. Acyl derivatives tend to direct the mass 2 1 inj. tertiary amines have been derivatized with pentafluorobenzyl chloroformate prior to GC with EC detection. PFB-Br. di-. such as TFAA.50 10.00 6.4 spectrometry (MS) fragmentation patterns of compounds providing useful structural information. [Both figures reproduced with permission from Jones and Mell. HFAA. react readily with alcohols. trifluoroacetic anhydride. Metoclopromide.00 2 10. Generally the reaction times are 1 – 3 h at 75 ° C. A GC/MS example of derivatization of amphetamine and methamphetamine by HFAA 10 is shown in Figure 2(a) and (b). Perfluoro acid anhydrides [R(CDO) O (CDO)R]. Figure 2 (a) Total ion current chromatogram of HFAA-derivatized amphetamine (1) and methamphetamine (2). 8 These reagents are often used in bifunctional derivatization schemes involving silylation chemistry. trifluoroacetylates primary and secondary amines and carbohydrates under mild nonacidic conditions. shown in Table 2 as reagent 3. and tetrasaccharides was obtained. C2F5 (PFAA). phenolic amines can be silylated with N-trimethylsilylimidazole (TMSI) to protect the hydroxyl group and acylated to protect the amine group.00 7.50 7. Table 2 Common GC derivatives – acylation No. C2F5 (PFAI).00 8. or heptafluorobutylimidazole (HFBI) can react with hydroxyl groups and primary or secondary amines to generate the halogenated derivative and the relatively inert by-product imidazole (Table 2.00 6.00 9. being amenable to GC. 9 For carbohydrates.50 8.50 9.50 10. The splitting of the peaks 5. MBTFA. Indoleamines and indole alcohols. Perfluoroacylimidazoles such as trifluoroacetylimidazole (TFAI). For example. pentafluoropropionylimidazole (PFAI).50 6. O O R′ C O C R′ R′=CF3 (TFAA). 1. The baseline level in this chromatogram is only about 10% of that in (a). has been derivatized with HFBI for GC with EC detection.00 8. an antispasmodic agent related to procainamide. The overloaded peak at about 5. which are acid-sensitive compounds.50 3. tri-. which are particularly useful for EC detection (Table 2. a single derivative for mono-. 10 ] 10. Drugs of abuse are often derivatized in this way before gas chromatography/mass spectrometry (GC/MS) confirmation.and p-chlorobenzylimidazole have been used because the detectability of the EC detector is enhanced. Triethylamine is added as part of the reaction mixture to neutralize the acidic by-products and drive the reaction to completion.00 7.50 Compounds Derivatives 400 000 350 000 300 000 250 000 200 000 150 000 100 000 50 000 0 Abundance 2 1 .6 min is due to excess HFAA. phenols.00 R OH R2 R1 NH R NH2 O R O C R′ R1 R2 O N C R′ (a) 550 000 500 000 450 000 400 000 350 000 300 000 250 000 200 000 150 000 100 000 50 000 0 5.00 9. The N-methyltrifluoroacetamide byproduct is stable and volatile. pentafluoropropionic anhydride. Reagenta N O N C R′ PHARMACEUTICALS AND DRUGS MBTFA.50 6.50 Time (min) R′=CF3 (TFAI). reagent 1). However. or C3H7 (HFBI) Abundance O R NH C R′ O R O C R′ O O C R′ R2 O R1 N C R′ O R NH C CF3 R2 O R1 N C CF3 O R O C CF3 O R S C CF3 2. PFAA and HFAA. the volatility and stability of the derivatives under GC conditions tend to be lower compared with the fluorinated types. O O F3C C N C CF3 CH3 (MBTFA) R NH2 R2 R1 NH R OH R SH (b) Time (min) a TFAA.50 8. N-methylbistrifluoroacetamide.50 9. and urine for the preparation of perfluoroacyl derivatives. or C3H7 (HFAA) 1 R OH OH R2 R1 NH 7. (b) Total ion current chromatogram of HFAA derivatized amphetamine (1) and methamphetamine (2) after extraction of the excess derivatizing agent. heptafluorobutyric anhydride. reagent 2). have been modified by this procedure. PFAA. The splitting of the drug peaks in both chromatograms is due to the presence of the deuterated internal standards (amphetamine-d5 and methamphetamine-d8 ). Other halogenated imidazoles such as p-bromobenzyl. 2 mm ID capillary column. reconstituted with acetonitrile. MSTFA has similar donor strength to BSA and BSTFA but generates an even more volatile by-product. Separations were effected on a relatively short 12 m ð 0. BSTFA (Table 3. TMCS is often used in conjunction with HMDS (Table 3. It does not react with amines or amides and is effective for most steroids. and urine samples after isolation by SPE and subsequent derivatization with BSTFA with TMCS before analysis by GC/MS. After cooling. N-methyltrifluoroacetamide (Table 3. Silylacetamides are represented in the most popular group of silylation agents. was removed by the procedure outlined below. Representative chromatograms are shown in Figure 3(a – c). The drugs of interest were extracted from 2 mL of urine by solid-phase extraction (SPE) columns with a 10% 2-propanol in chloroform elution solvent. and amide groups can all be protected. GC of silyl derivatives is generally straightforward. often to derivatize carboxylic acids (Table 3.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 5 is due to the presence of deuterated internal standards. reagent 2) to improve the silylation of sugars and related compounds. The limit of quantification and limit of detection were both of the order of 50 ng mL 1 and the linearity was observed up to at least 4000 ng mL 1 . for amines. For urine. reagent 1). and then the aqueous phase was discarded. thiol. column contamination and eventual degradation of the stationary phase. BSA is a highly reactive TMS donor (Table 3. Often an excess of the silylating agent is used in the derivatization reaction to minimize the problem of moisture or other acidic components in the sample. saliva. For alcohols.1 ng mg 1 . secondary amines. plasma. reagent 5). primary > secondary. it is recommended that the excess silylation reagent be evaporated from the sample using a stream of nitrogen before injection into the GC column. and allowed to react with a BSTFA – TMCS mixture at 60 ° C for 30 min before analysis by GC/MS. 11 After spiking with internal standards. the limit of detection was about 1 ng mL 1 and for hair 0. This permits derivatization of lower molecular weight analytes without potential overlap of the trifluoroacetamide by-product. Both excess MSTFA and the by-product often elute with the solvent peak. The addition of TMCS to BSTFA will promote the derivatization of amides. and metabolites in hair. The eluate was evaporated to dryness. which causes a high background level. Sodium periodate oxidation of interfering drugs was carried out if necessary. usually with trimethylsilyl (TMS) groups. It also reacts quickly and smoothly with carboxyl groups. the presence of excess silylating agent.1 M trimethylamine. saliva. If possible. Steric hindrance is also a factor within a class of organic compounds. 12 The derivatized compounds were separated by a GC instrument with a methylphenylsilicone column and nitrogen – phosphorus detection. HFAA was added and the mixture was heated in a sealed tube for 1 h at 70 ° C. A 4% ammonia solution was shaken with the remaining hexane volume and then this organic layer was removed for injection into the GC/MS system. heroin. eliminating the presence of extra peaks in the chromatogram. 13 Reaction conditions involve heating at 70 ° C for 1 h before removal of the excess reagent by N2 and reconstitution . with only one major concern. Cocaine and its metabolites benzoylecgonine and ecgonine methyl ester were sequentially derivatized with thyliodide to obtain the ester derivative and then MSTFA to form the o-TMS derivatives. including those with unhindered and highly hindered OH groups. the tube was shaken. The ease of silylation of these functional groups follows the order alcohol > phenol > carboxylic acid > amine > amide. The extract was acidified with 10% HCl in methanol and the solvent evaporated before reconstitution of the residue in hexane with 0. alcohol. Methyltestosterone was derivatized using either the dimethylethylsilyl (DMES)imidazole or dimethylisopropylsilyl (DMIPS)-imidazole to the corresponding silyl ether derivative. reagent 3). and plasma. Excess HFAA. This avoids several problems such as large reagent blank peaks in the chromatogram and fouling of the flame ionization detector by SiO2 deposits. A polar stationary phase such as poly(ethylene glycol) (Carbowax™ 20 M) and free fatty acid phase (FFAP) will be derivatized by excess silylation reagent and cannot be used. and. A comprehensive article on drug detection methodology describes the determination of cocaine. plasma. amine. 2. Again TMCS can be added to MSTFA to promote the derivatization of amides and hindered amines and hydroxyl groups.3 Silylation Silylation is probably the most widely used derivatization scheme for GC. reagent 4) has the advantage of giving a more fluorinated by-product than BSA. and hindered hydroxyl groups. owing to their ability to react quickly and quantitatively under mild conditions. saliva. reagent 6). Silyl compounds other than those that derivatize with a TMS group have also been used in conjunction with GC. and hair extracts were passed through SPE columns and the drugs eluted with a methylene chloride – 2 propanol – ammonia solvent. In general. The strongest reagent for silylation of hydroxyl groups is TMSI (Table 3. Trimethylchlorosilane (TMCS) is the simplest reagent that can be used. the reactivity order is primary > secondary > tertiary. Active hydrogens from acid. deionized water was added to the tube. silylation reagents are unstable and must be protected from moisture. R OH R O Si(CH3)3 O R C O Si(CH3)3 3.1 – 1. thiol. N. CH3 CH3 H3C Si O C N Si CH3 CH3 CF3 CH3 (BSTFA) Same as BSA. primary and secondary amines by adding t-butyldimethylsilyl (TBDMS) groups.O-bis (trimethylsilyl)trifluoroacetamide. N. MSTFA. Because the desired application was the assay of methyltestosterone in bulk powder and tablets. Flophemesyl derivatives also have favorable .6 Table 3 Common GC derivatives – silylation No. separation on a packed column was adequate in the concentration range 0. N-methyl-N-trimethylsilyltrifluoroacetamide. carboxyl.5 mg mL 1 (Figure 4a – c). These advantages have been demonstrated in the determination of short. CH3 CH3 H3C Si O C N Si CH3 CH3 CH3 CH3 (BSA) R OH R NH2 R1 NH R2 O R C NH2 R COOH R O Si(CH3)3 R N Si(CH3)3 Si(CH3)3 R1 N Si(CH3)3 R2 O Si(CH3)3 R C N Si(CH3)3 O R C O Si(CH3)3 4. a strong M 57 fragment is noted which can be used to determine the original compound’s molecular weight. BSTFA. N CH3 N Si CH3 CH3 (TMSI) No reaction with amines a HMDS. 1. leaving group O H F3C C N Si (CH3)3 more volatile than that of BSA Same as BSA. This is a selective reaction with only primary and secondary hydroxyl groups in steroids reacting in the presence of unprotected ketone groups. These TBDMS derivatives are more stable to hydrolysis than TMS compounds and. leaving group O H F3C C N CH3 5. N-methylN-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) will derivatize hydroxyl.O-bis(trimethylsilyl)acetamide. 14 Pentafluorophenyldimethylsilyl (flophemesyl) derivatives are often generated from steroids by reaction with flophemesylamine at room temperature for 15 min. hexamethyldisilazane. BSA. CH3 O F3C C N Si CH3 H3C CH3 (MSTFA) is very volatile R OH (steroids) R COOH R O Si(CH3)3 O R C O Si(CH3)3 6. when analyzed by GC/MS. Alternatively.and long-chain carboxylic acids. GC with EC detection provides detection limits in the nanograms – picograms range. Reagenta CH3 H3C Si CH3 Cl (TMCS) CH3 CH3 H3C Si N Si CH3 H CH3 CH3 (HMDS) PHARMACEUTICALS AND DRUGS Compounds R COOH Derivatives 2. of the residue in cyclohexane. drugs are found in aqueous matrices such as urine and plasma. Direct derivatization of drugs in untreated biological samples for GC analysis has the primary advantages of improved extractability of . DMCS (b) and (c) DMIPS derivatives. cocaine (6).0 7. normorphine (22). benzoylnorecgonine (13). ecgonine ethyl ester (4). morphine (17). 11 ] Figure 4 Typical gas chromatograms of (1) methyltestosterone and (2) norethandrolone as (a) TMS.0 8. benzoylecgonine (10). and (c) a hair sample collected from a heroin user. Analytes are identified as follows: anhydroecgonine methyl ester (1). [2 H-3]cocaine (5).0 11. [2 H-3]morphine (16).0 10. [Reproduced with permission from Zakhari et al. [2 H-9]heroin (23). producing diagnostic ions which carry more of the current than TMS derivatives. cocaethylene (8). 6-acetylmorphine (21). norcodeine (18).0 6.0 9. 15 2. 13 ] required not only to isolate the drug in a nonaqueous solvent which is compatible with most derivatization reagents but also to remove inorganic salts which are not compatible with GC. (b) drug-free control hair. extraction with an organic solvent is 3 4 1 2 7 5 13 6 8 11 12 14 17 16 18 9 10 15 20 19 21 23 22 2770 2840 1 2 (1) CH2 CH3 (2) H C 2H 5 (a) 24 2880 (a) 2 5 7 9 1 2960 2 14 16 19 20 (b) 23 2980 (b) ×75 2 1 ×5 ×1 ×5 ×75 10 14 6 9 7 11 8 12 3 4 5 15 16 ×15 1 3060 2 17 20 19 21 24 23 2 (c) 4 6 Time (min) 4. [2 H-6]-6-acetylmorphine (19). heroin (24). [1 H-3]benzoylecgonine (9). ecgonine methyl ester (3). An alternative approach called direct derivatization combines the extraction and derivatization steps together. [2 H-3]codeine (14). norcoceathylene (12). norcocaine (11).CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 7 R2 R1 N OCH 3 R1 R2 advantages for GC/MS. [Reproduced with permission from Wang et al. [2 H-3]-6-acetylmorphine (20). Note the bulkier derivatives are shifted to a cleaner area in the GC trace.0 Time (min) Figure 3 Single ion monitoring recordings of extracts from (a) standard cocaine/opiate hair. [2 H-3]ecgonine methyl ester (2). codeine (15). [2 H-3]cocaethylene (7).4 Sample Handling Generally.0 (c) 5. 40 Morphine has been detected fluorimetrically after postcolumn oxidation with alkaline potassium hexacyanoferrate(II) to form the dimer pseudomorphine. scopolamine. Conversely. which is the basis for this part of this article. codeine. 37 The organic mobile phase was monitored fluorimetrically with detection limits of 40 – 100 ng. A precolumn ion-pair derivatization method for atropine.46 Using this UV photochemical reaction. Acylation involving perfluorinated anhydrides and chloroformates has also been used for the determination of drugs such as metanephrine and normetanephrine. Reserpine. and their metabolites were separated by reversed-phase HPLC using 9. 41 Morphine can be oxidized to the fluorescent dimer pseudomorphine using alkaline hexacyanoferrate(III). The presence of 4% of the nonionic surfactant Triton X100 at the micellar level in the derivatization reagent solution increased the signal about twofold. but also a twophase reaction where the derivatization takes place in the organic phase while extraction of the analyte is continuing from the aqueous phase. 16 Extractive alkylation has been applied to valproic acid and ketoprofen. phencyclidine. The derivatizing agent was 50 mg of K3 Fe(CN)6 in 250 mL of 4 M ammonia solution delivered at 0. an acetylcholinesterase inhibitor. 42 A detection limit of 0.1 Alkaloids Separation of atropine and ergotamine by normal-phase liquid chromatography (LC) followed by postcolumn ion-pair extraction with an aqueous solution of 9. and others. in which the tetrabutylammonium ion acted as the anion-pairing agent to pull the compound into the organic phase. Again. which appears to have been the most active time for research in this field. 43 The heroin metabolite 6-acetylmorphine has been determined in urine by reversed-phase HPLC with fluorescence detection after automated precolumn oxidation with hexacyanoferrate(III). Most of the books reviewing fundamental reactions for pre. such as normorphine. Morphine could be determined at the 2 – 30 µg mL 1 level in biological samples. and a detection limit of 0. permitting the identification of these compounds in complex chromatograms. Danielson et al. was separated from its degradation products and reacted with coulometrically generated bromine. were also reactive. 24 – 31 Some specific review articles on derivatization chemistry of pharmaceutical compounds with HPLC have also been published.2 pmol was possible for morphine after precolumn dansylation. 47 Electrochemical detection of unreacted bromine was inversely proportional to the amount of drug.10-dimethoxyanthracene-2-sulfonic acid in the mobile phase. hyoscyamine. was detected fluorimetrically after postcolumn reaction with nitrous acid and UV irradiation. Physostigmine. naborphine.or postcolumn derivatization in conjunction with HPLC is large in part because either aqueous or organic solvent-compatible reactions are possible. respectively. Direct derivatization of drugs in untreated biological samples can mean not only derivatization in the sample matrix followed by extraction. 38 The resultant ion pairs (Table 4. 32 – 36 Although the other articles do not try to be comprehensive. 44 A detection limit of 1 ppb was reported.10dimethoxyanthracene-2-sulfonate has been reported. The mobile phase. reagent 3) were extracted with chloroform postcolumn on-line and detected fluorimetrically with detection limits of 1 – 6 ng mL 1 in plasma. an antihypertensive agent. methadone. 39. An on-line photochemical reaction detector caused decreased fluorescence of ergot alkaloids. PHARMACEUTICALS AND DRUGS 3 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY The variety of chemistries that can be adopted for either pre. Other opiates. a 20-fold increase in signal over the native fluorescence of reserpine was found. where alkylation to generate the methyl or phenacyl derivative was possible. The excitation and emission wavelengths were 323 and 432 nm. A comparison of the UV and fluorescence response was made using the instrumentation shown in Figure 5. The reason for the improvement was credited to an increase in the actual dimer formation yield.5 ng could be attained. 36 published another fairly complete review covering the period 1979 – 87. a specific example and an overview for each major class of pharmaceutical compounds will be given. 17 – 23 Many review articles have focused on postcolumn derivatization with HPLC. was propelled through a C18 reversed-phase column. provided ultraviolet (UV) detection limits of about 200 ng. 45.1 M KBr solution. involving picric acid and normal-phase chromatography. 3.and postcolumn derivatization chemistry in conjunction with HPLC were published in the 1980s. Ahuja 35 gave a virtually complete review of the topic up to 1979. Chloroformates can de-alkylate tertiary amines to form stable carbamates. 48 A similar system has been shown to convert cannabinol into a fluorescent . and ergotamine. The selectivity advantage of the fluorescence detector for the determination of morphine in a urine extract was shown and urine and serum samples were analyzed with detection limits of about 10 ng.8 a derivatized polar compound and avoidance of stability problems. a 12.5 methanol – 0. Fluorescence detection of morphine and related opiates was improved after postcolumn derivatization with alkaline hexacyanoferrate(III) and micelle formation with Triton X.5 : 87.4 mL min 1 . Phenolic compounds such as elioquinol have been determined in an analogous fashion. norcodeine. RSH (F) H3C BrH2C 5. R3NH (F) OCH3 O O N N CH3 CH3 H3C RSH2C O RCHN OCH3 O N N O CH3 + SO3− SO3− +NHR3 4. RNH2 (F) CHO .CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 9 Table 4 Common fluorescent (F) and electrochemical (E) derivatives for HPLC No. 41 ] . CN− CHO OCH3 OCH3 N R 3. Steroid (E) O H2NHN NO2 NHN NO2 HO OH Mobile phase reservoir Pump I Injector Column UV detector 3-Way valve Waste Tee Dual-pen recorder Reaction coil Derivatization reagent reservoir Pump II Gauge Waste Fluorescence detector Figure 5 HPLC system for morphine using postcolumn derivatization and fluorescence detection. R′′SH Derivative S R′′ N R′ 1. RCOOH (E) H2N OH OH 6. [Reproduced with permission from Nelson et al. Compounds Reagent(s) CHO CHO . R′ NH2 (F) CN 2. Tranexamic acid has been detected using OPA.10 derivative providing detection limits of less than 1 ng in urine. 87 Secondary amines. A more recent modification of this method is to use naphthalene-2. such as piperazines. 52 baclofen.R)-sulfoximine in plasma 67 and mexiletine. Both primary and secondary amines can react (Table 5. tryptamine. 72 The determination of debrisoquine and its hydroxy metabolites was possible by reaction through the guanidine moiety to form fluorescent compounds. The o-phthalaldehyde (OPA). 81 Some UV methods have been developed for aliphatic amines.92 ephedrine.and postcolumn modes. Precolumn reaction of chlorpheniramine with .2 Amines Primarily fluorescent methods have been developed for amino acids and peptides. b-phenethylamine was determined with a detection limit of 0. Riboflavin. reagent 1A) and other acid chlorides for subsequent UV detection. 61 A dinitrophthalic anhydride reaction of peptides permitted either electrochemical or absorbance detection. and thiamine. 17 – 20 Other amino acids such as s-carboxymethyl-L-cysteine. An ion-pair extraction detector using dimethoxyanthracene sulfonate (Table 4. 64 Biogenic amines such as tyramine. 85 The cardiotonic drug heptaminol has been derivatized with aminoazobenzene-4-isothiocyanate for UV detection at 420 nm 86 or OPA for electrochemical detection. 50 Pilocarpine was quaternized with p-nitrobenzyl bromide before reversed-phase HPLC separation and detection at 254 nm. 53. 75 Thiamine (vitamin B1 ) was oxidized with hexacyanoferrate(III) in base to give the fluorescent thiochrome in either the precolumn 76 or postcolumn 77 mode. Specific articles describing common amino acids are cited in general references. 89 The determination of isophenindamine in the presence of phenindamine tartrate has been achieved by forming charge transfer complexes using AgNO3 and reversedphase HPLC. 59 The peptide leupeptin was reacted through the guanidino moiety with benzoin to form a fluorescent derivative. milk. reagent 1B) and the aromatic tag provides good retention for the smaller amino acids.5-dimethoxybenzene fluorobenzene. a nonapeptide. 74 Indolethylamines were condensed with an aldehyde or a-keto acid to form fluorescent carboline derivatives.4-diaminopteridine-6-carboxyaldehyde. such as isoniazid. was derivatized with fluorescamine.54 and melphalan 55 have also been derivatized with OPA. mercaptoethanol (or other thiol) reaction (Table 4. 73 PHARMACEUTICALS AND DRUGS A postcolumn photochemical reactor caused the cleavage of methotrexate to form the highly fluorescent 2. reagent 2).3 ng. 82 Both primary and secondary amines were derivatized with 9-fluorenylmethyl chloroformate (FMOC) before separation by reversed-phase HPLC with UV detection at 254 nm. 51 3. 68 Fluorescent derivatization with fluorescamine has been applied to tocainide 69 and in the postcolumn mode for sulfapyridine.1 ng mL 1 . and beans. 56 Detection limits down to 200 fmol with improved stability of the derivatives were stated advantages. 88 Enantiomers of metoprolol have been separated after reaction with the chiral reagent (S)-( )-phenylethyl isocyanate.65 or precolumn 66 mode. already fluorescent. 80 Fluorescein isothiocyanate (FITC) modified a bronchodilator before HPLC – fluorescence analysis. g-Aminobutyric acid was modified with dansyl chloride before reversed-phase HPLC and fluorescent detection. 90 Antihistamines and other pharmaceuticals with a tertiary amine moiety have been derivatized to provide for luminescence detection. 78 Hydrazine compounds.4 µg. 93 and hyoscyamine. the fluorescamine reaction provided detection limits of 0. The OPA precolumn fluorescent reaction has also been applied to L-buthionine-(S. were reacted with m-fluorobenzyl chloride before reversed-phase separation and detection at 220 nm. and serotonin were reacted with OPA in a post. reagent 3) has permitted the fluorescent determination of tertiary amines such as bromopheniramine and chlorpheniramine.95 Detection limits of 200 – 500 pg were possible. 79 A precolumn coumarin reaction permitted fluorescent detection of 5-fluoro-20 -deoxyuridine. 70 Enantiomers of mexiletine have also been resolved. After SPE of a urine sample. 57 Detection at 360 nm was possible for N-acetylcysteine after reaction with 2. 60 Felypressin. Phenyl isothiocyanate has been used as a precolumn derivatizing agent for amino acids in conjunction with reversed-phase HPLC. 71 After conversion of the drug panthenol to aminopropanol. 62 An ion-pair detection technique has been applied to hydrophobic amino acids and peptides. 58 was used to form a fluorescent derivative of phydroxybestatin. 83 Amphetamine and methamphetamine have been reacted with 4-methoxybenzoyl chloride (Table 5. 49 Post-column photochemical derivatization has the advantage of no sample handling and without the problems of pumping a reagent or detection background from a reagent.4-dinitro-11. 91. after precolumn oxidation to thiochrome using hexacyanoferrate(III).3-dicarboxyaldehyde with cyanide ion (Table 4. providing detection limits of 0. reagent 1) has been well studied in both pre.2-Diamino-4. On-line reaction of cannabinoids with Fast Blue Salt B produced colored derivatives for detection at 490 nm. were determined by HPLC in a variety of food powders such as flour. 94. were converted into UV derivatives with m-toluoylacyl chloride. 63 A variety of other fluorescent methods have also been reported for specific drugs. 84 A comparison of methods for amphetamines has been made. Compounds RNH2 (A) Cl Reagent(s) O C OCH3 (B) NCS (B) (A) R O C Derivative N H OCH3 NHCSNHR 2. 103 Tertiary tetrahydroisoquinolines.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 11 Table 5 Common UV derivatives for HPLC No. 99 Tamoxifen and its metabolites in human serum can be UV photochemically activated to form fluorescent phenanthrenes.1 ng mL 1 detection limits. 100 Postcolumn UV irradiation of tamoxifen and its derivatives caused rearrangement to a substituted phenanthrene. Penicillin R C O H N N O S CH3 CH3 COOH NaOH. which could then be reacted with (R)-(C)-1-phenylethyl isocyanate (Table 5. EDTA R C O H N N H S CH3 CH3 COO− −OOC benzyl chloroformate gives a fluorescent derivative with a detection limit of 0. and other metabolites can be detected at 530 nm in urine after HPLC. 102 by using diethylthiobarbituric acid as a color-forming agent. and these compounds were separated by reversed-phase HPLC. such as diclofensine. permitting fluorescent detection with 0. cotinine. R3N (A) O H C C Cl O CH2 N H (B) C C N C O H 2 H2 O C O C O C NHR2 4. 1.1 ng mL 1 . 98 Antihistamines in urine have also been separated by HPLC and detected with good selectivity using this ruthenium metal complex (Figure 6a – c). HgCl2. RSH O Cl C H2C C C2H5 Cl OCH2COOH Cl O C RS H2C CH C2H5 Cl OCH2COOH 6. 97 Aliphatic tertiary amines can be determined at the picomole level by postcolumn chemiluminescent detection. R2NH SO2Cl SO2NR2 3. such as diphenhydramine. Racemic phenothiazines were N-demethylated with vinyl chloroformate to form their secondary amines. RCOOH CH2Br Br R O C C H2 O Br 5. were converted through the tertiary amine group into fluorescent derivatives using 2-naphthyl chloroformate. 101 Derivatives of nicotine. were oxidized before photochemical conversion to fluorescent . 96 Antihistamines. reagent 3). using tris(bipyridyl)ruthenium(III). 29 µg mL 1 diphenhydramine taken with (a) UV (214 nm). 99 ] 0 5 Absorbance (290 nm) A Absorbance (300 nm) 0 (b) 5 10 isoquinolinium derivatives. Figure 8(a) and (b) shows the improvement in response over conventional UV detection using the derivatization 104 (a) Time (min) Time (min) Figure 8 Comparison of UV detection (300 nm) after HPLC separation (a) and reactor response (290 nm) to derivatized platinum (b) for a mixture of cis-dichloroplatinum complexes: (1) cis-dichloro(1. [Reproduced with permission from Holeman et al. Using knitted open-tubular reactors.2-diaminocyclohexane)platinum(II).26 µg mL browith (A) 0. 106 ] scheme. (2) cis-dichloro(ethylenediamine)platinum(II). which then combines rapidly with bisulfate to give a UVabsorbing complex.7 min for the bisulfate reaction were found to be optimal. [Reproduced with permission from Marsh et al. (B) 0. (b) UV (254 nm). 105 Postcolumn detection of platinum(II) antineoplastic agents such as cisplatin is possible. 106 ] 1 0. The cisplatin-derived species are first reacted with a oxidant such as dichromate to form an activated species. Detection limits of 5 – 10 µg mL 1 were possible.05 au 2 3 B C 0 (c) 6 12 18 24 Time (min) 1 1 1 2 3 Figure 6 Chromatograms of an undiluted urine sample spiked pheniramine. [Reproduced with permission from Marsh et al. as shown in Figure 7.1 mL min –1) A Mobile phase (0.12 Pump 1 PHARMACEUTICALS AND DRUGS Analytical column Pump 2 K 2Cr2O 7 (0.15 µg mL mopheniramine. peroxylate chemiluminescence detection was carried out. Cisplatin was also determined in a plasma ultrafiltrate sample at the 5-ng level. delay times of 26 s for the dichromate reaction and 4. and (3) cis-dichlorodiammineplatinum(II). After postcolumn ion-pair extraction of secoverine.0025 au 0. 106 . and (c) Ru(bpy)3 3C chemiluminescence detection. and (C) 0.7 mL min –1) ∆t1 B C A Detector ∆t2 B λ = 290 nm Pump 3 0 (a) 6 12 18 24 (b) 0 6 12 18 24 NaHSO 3 (0.1 mL min –1) Figure 7 Schematic representation of the Pt HSO3 reaction detector for cisplatin-type compounds. 122 Monensin. detection limits are about 1 µg mL 1 .and postcolumn derivatization. and a reagent flow rate of 0. For example. have all been reported.139 and amikacin 140.3 Antibiotics This class of pharmaceuticals has received major attention for both pre. 111 and fludalanine 112 were assayed by using an OPA postcolumn reactor. and electrochemical methods have been reported for tetracycline-type antibiotics and related compounds. ampicillin. the derivatives were eluted with ethanol before separation by reversed-phase HPLC. such as amoxicillin. can be separated by reversed-phase HPLC with alkaline degradation in the presence of mercuric chloride 124 (Table 5. cephalosporins. has been commonly performed (Table 4. 113 Reversedphase separation was employed for all these separations because the OPA reaction is carried out in an alkaline buffer solution. 109 cephalosporin C. 120 Numerous other postcolumn UV. milk. Fluorescent derivatization of the primary amine group of many antibiotics with OPA and a sulfhydryl compound. 131 The excess bromine was detected at 0. Methanol promotes this reaction. penicillin N. 119 For all these OPA fluorescence methods. and meat samples have been detected at 450 nm after derivatization with pdimethylaminobenzaldehyde. 123 Penicillins.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 13 3. reagent 2) before normal-phase HPLC and UV detection at 254 nm. 128 Photothermal derivatization of ciprofloxacin and its metabolites permits fluorescent detection throughout a linear range of about 2 – 1000 ng mL 1 . a temperature of 90 ° C. 130 Electrochemically generated bromine was used as an oxidizing agent for cephalosporins and their decomposition products. 142 Aminoglyosides have also been reacted with 2. 136 Nitrobenzene derivatives of neomycin B and C can be separated by normal-phase HPLC and detected at 350 nm. cephalosporin C. 144 The secondary amine group of spectinomycin was derivatized with 2naphthalenesulfonyl chloride (Table 5. narasin. Formation of a mercuric mercaptide of penicillins permits UV detection at 310 nm with detection limits of 10 ng. 114 sisomicin.8 mL min 1 .5 and 1. and their precursors in biological samples. A comparison of this method with UV detection and postcolumn fluorescamine derivatization has recently been summarized. Neomycin and other aminoglycosides have been published. Spectinomycin after postcolumn oxidation with hypochlorite could also be derivatized with OPA in a second reaction coil. 125 Application of this latter method to penicillins in biological samples has been made. often mercaptoethanol. Excitation and emission . 108 penicillin N. and salinomycin in animal feeds have been reacted with vanillin to give products detectable at 520 nm. Phenacyl esters of some natural penicillins were prepared using dibromoacetophenone before reversedphase HPLC with UV detection. 126. 143 Ion-pair formation of methscopolamine bromide with an aromatic anion during reversedphase HPLC permitted UV detection. 110 cycloserine.4-dinitrobenzene to form 2. reagent 6). 115 phosphinothricin and its alanine analog 116. Precolumn reaction with OPA for gentamicin.4. Neomycin and other aminoglycosides have been converted to benzoyl derivatives before UV detection at 230 nm. 134 Chemiluminescence detection of clindamycin phosphate using tris(bipyridyl)ruthenium(III) 135 gave detection limits of 8 ppb compared with 970 ppb using UV detection at 214 nm.and postcolumn OPA reaction conditions for gentamycin has been made. 121 Sulfonamides in egg. 137 Similar methods using 1-fluoro-2.108 penicillin V after enzymatic conversion to 6-aminopenicillanic acid.7 before injection of the derivatives on to a C18 reversed-phase column.117 before reversed-phase separation.141 have also been published. b-lactams found in microbial fermentation broths were separated on a C18 HPLC column and detected fluorimetrically with excitation at 350 nm and emission at 450 nm after reaction with OPA and mercaptoethanol. Detection limits of less than 0. which absorbs at 274 nm. reagent 1).4 V using a glassy carbon electrode. a reaction coil of 12 m.4-dinitrophenyl derivatives of neomycin B and C 138. forming the ring-cleaved product. and 6-aminopenicillanic acid were separated in about 12 min at a flow rate of 1. 129 Photolysis with electrochemical detection of penicillins and cefoperazone has provided detection limits of about 6 ng. 132 Acidification of a serum sample sometimes improved recovery of cephalosporins when micellar chromatography was used with sodium dodecyl sulfate (SDS) in the mobile phase. Gentamicin. It was discovered later that sodium hypochlorite could replace the mercuric chloride reagent while maintaining a 1-min hydrolysis time. fluorimetric. A variety of reagents are also available for precolumn derivatization of antibiotics and UV or fluorimetric detection.127 Fluorescent detection of streptomycin reacted through the guanidino groups with naphthoquinone-4-sulfonate in alkaline solution has been reported in serum samples. respectively. Amikacin isomers were first adsorbed on a silica gel column before reaction with OPA. 118 A comparison of pre. 133 Fluorescamine has been used in an automated system for amoxycillin in biological fluids. and others. Cephamycin. 107. 146 b-Lactams were prederivatized with FMOC for 5 min at 20 ° C in borate buffer at pH 7.5 mL min 1 with postcolumn conditions involving the OPA reagent at pH 12. giving a detection limit of 50 ng mL 1 . permitting UV detection at 350 nm.0 µg mL 1 were achieved for penicillin N and cephalosporin C.6-trinitrobenzenesulfonic acid. 145 The reagent FMOC formed fluorescent derivatives of natural penicillins. 150 – 152 Two precolumn derivatization studies with UV detection were checked with real samples.25 mm ID Teflon knitted open tubular reactor mounted around a mercury lamp. It was determined by HPLC that de-alkylation at the 5-position to give ethylbarbituric acid was the mechanism of the photochemical reaction. 161 However. which provided an irradiation time of about 190 s. 163 Danthron (1.159 A wavelength shift for maximum absorbance from 220 to 240 nm provides for detection limits of about 6 µg for butabarbital.5 Carbonyl Compounds and Carboxylic Acids Most of these methods refer to carboxylic acids. Dansylhydrazine formed fluorescent derivatives of tetraphenylacetone isomers before separation. 154. 156 An online solid-phase anion-exchange extraction provided enhanced chromatographic selectivity of barbiturates in urine. particularly in biological samples such as serum. 110 A similar method using 1. 164 A comparison of UV and fluorescence chromatograms for danthron spiked in a urine sample showed the selectivity advantage of fluorescence (Figure 11a and b).2. amoxicillin.4 Barbiturates Precolumn methods primarily include reaction with various alkylating reagents. Vitamin K1 in human plasma was electrochemically reduced at 0. 154 Detection limits of 5 ng for phenobarbital have been reported. 149 A comparison of this method with the postcolumn OPA fluorescence method has been made.8 mM sulfuric acid.4-triazole and Hg(II) for ampicillin. and other antibiotics has been studied.05 µg mL 1 for 6-aminopenicillanic acid and isopenicillin N. 153 Acetonitrile (%) .14 B D 100 PHARMACEUTICALS AND DRUGS 3.155 2-Naphthacyl bromide forms strongly absorbing derivatives of barbiturates at 254 nm with detection in plasma or serum below the therapeutic range. short-chain carboxylic acids were modified using pbromophenacyl bromide (Table 5. 158.4 V and the fluorescent derivative detected at levels as low as 25 – 50 pg. (D) ampicillin. 146 ] wavelengths for the fluorescent detection were 260 and 313 nm. Application of the method to fermentation broths was made over a range of 0. 162 Comparison of this method with UV and electrochemical oxidation detection systems has been made. reagent 4) to form esters that could be easily detected by UV absorbance. Using a 25 m ð 0. Detection limits were 0. (C) cephalosporin C. 165 5-Bromomethylfluorescein was studied for the prederivatization of carboxylic acids for detection by either Relative fluorescence C A 0 0 10 20 30 40 50 Retention time (min) Figure 9 Reversed phase HPLC of FMOC derivatives of cephalosporins (5 µg mL 1 ) in a fermentation broth using a borate buffer – acetonitrile gradient mobile phase. (B) Deacetoxycephalosporin C. respectively. 157 Postcolumn UV detection of barbiturates can be conveniently enhanced by mixing with a pH 10 borate buffer.05 – 100 µg mL 1 .01 and 0. A typical chromatogram is given in Figure 9 showing the modest acetonitrile gradient until after elution of ampicillin in which the acetonitrile is taken to 100%. first a short summary of the methods involving the reduction of quinone compounds to form fluorescent hydroxy derivatives will be given. Separation of the derivatives on a C18 column with detection at 365 nm was carried out and the stability of tobramycin in ophthalmic solutions was determined. The UV detection of barbiturates can be significantly enhanced through postcolumn photochemical derivatization. (A) Deacetylcephalosporin C. The highly reactive chlorine of N-chloromethylphthalimides permits derivatization of OH and NH functional groups to form UV-absorbing compounds.4-dinitrofluorobenzene for 20 min at 70 ° C in 0. [Reproduced with permission from Shah and Adlard. 148 An imidazole – mercuric chloride reagent can convert penicillins into mercury-stabilized penicillinic acids after reaction at 50 ° C for 50 min prior to UV detection at 325 nm. Barbiturates have no significant absorbance above 230 nm and detection in the 200 – 220-nm range can be complicated by the presence of interfering peaks. respectively. 160 3. Tobramycin plus impurities neamine and kanamycin and also the degradation product nebramine were derivatized with 2. excellent signal enhancement at 270 nm was possible for barbiturates (Figure 10a and b). 147 A maleimide reagent gave a fluorescent derivative for penicillamine.8-dihydroxyanthraquinone) has been reduced with dithionite and determined by either flow injection analysis in Modane tablets or HPLC in urine. [Reproduced with permission from Wolf and Schmid. 175 Ascorbic acid from 5 to 800 mg L 1 was detected by chemiluminescence after postcolumn reaction with lucigenin. Model analytes included prostaglandins (unsaturated carboxylic acids) and the drug cefuroxime. (a) without and (b) with on-line photochemical reaction. 106 ] Figure 11 Separation of danthron spiked in urine (2 ng mL 1 ). 170. [Reproduced with permission from Miller and Danielson. (a) UV detection (254 nm). (4) mephobarbital. and also standard aliphatic and aromatic carboxylic acids. Dicarboxylic acids did not react with bromomethylfluorescein. 167 A UV-absorbing naphthacyl ester of the prostaglandin carboprost has been formed before normal-phase HPLC. 60 : 40 methanol – water at 1. 178 UV detection of bis(dinitrophenyl)hydrazine derivatives of ascorbic and dehydroascorbic acid was . mobile phase. (2) butethal.15% Relative fluorescence Danthron Lamp off 0 (a) 10 0 (b) Lamp on 10 Time (min) Time (min) 0 2 4 6 8 (a) 0 (b) 4 8 Time (min) Time (min) Figure 10 Chromatogram of a standard sample of barbiturates detected at 270 nm.04 AUFS UV 0. and the dehydroascorbic acid was reduced to ascorbic acid with dithiothreitol for UV detection at 267 nm. 168 Fluorescent derivatives of prostaglandins using 9-anthryldiazomethane provided detection limits of 100 pg after reversed-phase HPLC.2-phenylenediamine both ascorbic acid and dehydroascorbic acid were separated by reversed-phase ion-pair HPLC.174 Prostaglandins have been labeled with the fluorescent reagent 9-anthoyldiazomethane through the carboxyl group. A solution of 90 mM dithionite in 2% borate buffer was pumped into the effluent at 0. Five prostaglandin derivatives were separated by reversedphase HPLC with fluorescent detection with excitation at 365 nm and emission at 418 nm at the 8-ng level. (b) Fluorescence detection (388 nm excitation. 172 Oxidation of prostaglandins to the corresponding 15-oxo derivatives using pyridinium dichromate permitted UV detection at 228 nm and picomole detection limits. 510 nm emission).6 mm C18 . reagent 5).CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 15 3 1 2 5 4 0. (5) secobarbital. possibly owing to solubility problems in the organic reaction medium. 164 ] UV absorbance or fluorescence (standard and laser induced).4 mL min 1 . 169 The prostaglandin arbaprotstil was derivatized with panacyl bromide before column switching using fluorescent derivatization. Column. (3) pentobarbital. 166 Fatty acids and prostaglandins were converted into p-hydroxanilides using p-aminophenol (Table 4. These hydroxy derivatives were then oxidized through electrochemical detection after reversed-phase HPLC.2 mL min 1 . (1) Aprobarbital. 250 ð 4. 173. 176 After derivatization with 1.171 Prostaglandins have also been derivatized with p-(9-anthroyloxyl)phenacyl bromide. This esterification reaction can be carried out under mild conditions such as 40 ° C for 30 min. 177 The same two compounds were separated by reversed-phase HPLC. 179 Four forms of ascorbic acid. Fluorescent catecholamine derivatives have been generated using 1. A detection limit of 3 pg of derivatized ibuprofen was found. 182 Postcolumn alkaline hydrolysis has also been applied to aspirin in plasma in order to form the fluorescent salicylic acid. This alkylation reaction takes place in dry acetonitrile for 20 min at 50 ° C and separation of the derivative is possible on a C18 column with an acetonitrile – water – tetrahydrofuran mobile phase with the ion-pairing agent tetrabutylammonium ion. RR. 217 Derivatization can be an effective method to facilitate the chiral separation of pharmaceuticals. can be converted into a UV-absorbing compound using a sodium hydroxide solution before HPLC separation. were detected postcolumn using benzamidine and fluorescence. SR.p-nitrobenzyl-N. Qinghaosu. betaine. The limit of detection . 206 Indirect photometric detection using n-heptyl p-aminobenzoate in the mobile phase and a C18 column has been applied to menthol. Norepinephrine. 207 Enantiomers of propranolol and 4-hydroxypropranolol were formed upon derivatization with (C)-1-phenylethyl isocyanate 208 or (C)tetraacetyl b-D-glucopyranosyl isothiocyanate 209. 181 Finally. 205 Oncolumn periodate oxidation of ephedrine sulfate. 190 3. 185 Flunoxaprofen enantiomers have been separated after reaction with (S)( )-1-phenylethylamine. Enantiomers of derivatized or underivatized propranolol have been separated. 197 This method was compared with the PHARMACEUTICALS AND DRUGS OPA – thiol reaction in the postcolumn mode. 214 A derivative of qinghaosu has been esterified with diacetyldihydrofluorescein before HPLC and UV detection. 186 Artesunic acid was determined after derivatization with o. to form benzyl alcohol has been carried out in a seven-laboratory study. 202. 215 Cholic acid and its derivatives have been labeled with 1-anthroylnitrile before HPLC and fluorescent detection at the femtomole level. and SS diastereomers was straightforward on a C18 column using a 60 : 40 water – acetonitrile mobile phase (Figure 12a – c). dopamine. Indomethacin formed a fluorescent derivative after postcolumn alkaline hydrolysis.2-diphenylethylenediamine. an antimalaria component in a Chinese herb.16 possible at 497 nm. the previous two plus isoascorbic acid and its dehydro form. 183 The enantiomeric composition of ibuprofen in human plasma has been resolved after prederivatization with (S)-( )-1-(naphthenyl)ethylamine 184 or ethyl chloroformate – leucinamide. 198. and normetanephrine have been measured at the low picogram level after reversed-phase HPLC. reagent 1). providing detection limits in the femtomole range. 211 Diastereomeric derivatization of 1-methyl3-pyrolidinol 212 and proxyphylline 213 for UV detection has been accomplished. 216 Trospium has been converted into the corresponding spiro alcohol before fluorescent derivatization with benoxaprofen chloride and reversed-phase HPLC. a variety of methods directed toward pain relievers and other compounds have been published. 204 3. Isotachysterol derivatives of vitamin D improved detection at 290 nm after normal-phase HPLC. a nasal decongestant. Chemiluminescence detection of the acridinium label is possible by postcolumn addition of potassium hydroxide solution.203 Catecholamines catalyze the reaction between formaldehyde and o-dintrobenzene and permit their detection at 560 nm.6 Catecholamines Precolumn fluorescent derivatization of catecholamines with OPA and a thiol has been well established (Table 4.192 An electrochemical cell placed between the injector and column was used for oxidation of adrenaline and levodopa to the corresponding quinones for detection in the visible region. giving a detection limit of 5 pg. Nadolol diastereomers were derivatized with (R)-( )-1-(1naphthyl)ethyl isothiocyanate to chiral urea derivatives through the secondary amine group by reaction for 5 min at 45 ° C. 187 Choline. Fluorescence detection with excitation at 285 nm and emission at 340 nm provided selectivity for the determination of nadolol in plasma samples. 200 Dopamine isomers in serum and urine have been separated before hydrolysis and reaction with p-aminobenzoic acid to form fluorescent products.N 0 -diisopropylisourea by HPLC with UV detection.7 Hydroxy Compounds Most precolumn methods focus on enhancing UV detection. 193 Resolution of norephedrine enantiomers after derivatization with either acetylglycosyl isothiocyanates 194 or 4-methyl-5-phenyl-2-oxazolidone 195 has been reported. 196 Postcolumn fluorescent reaction of norepinephrine and epinephrine with trihydroxyindole provided 1-pg detection limits. A new acridinium sulfonylamide label permits the chemiluminescent detection of carboxylic acids such as the test compound ibuprofan. 191. Separation of the RS. and related compounds have been reacted to form either 40 -bromophenacyl esters 188 or p-nitrobenzyl oxines 189 to permit UV detection at 254 nm.199 The same two catecholamines can be converted into fluorescent products by heating in an alkaline borate buffer after ion-exchange chromatography. 201 Glycylglycine as an alternative postcolumn reagent to glycinamide increased the rate of formation of fluorescent derivatives after ion-exchange HPLC. 180 Total ascorbic acid was determined fluorimetrically after reaction with diaminodimethoxybenzene.210 and separated by reversed-phase HPLC. 229 Further work using this method with reversed-phase HPLC and optimized reaction conditions permitted detection limits as low as 7 – 10 ng. epiandosterone. (b) plasma spiked with 50 ng mL 1 of each diasteromer and (c) plasma obtained 2 h after oral administration of 1 mg kg 1 of racemic nadolol. 235 The reactive site is an a-hydroxycarbonyl group. 227 Ketosteroids. 233 Hydroxysteroids were derivatized with anthroylnitrile. showing the feasibility of the reaction for fluorescent detection after HPLC. and HCl. such as corticosterone. dehydroepiandrosterone. a widely used cardiac glycoside. 224 3.5 ng mL 1 . peroxide. were derivatized with p-nitrophenylhydrazine (Table 4. reaction 6) and detected electrochemically at levels as low as 200 pg. 232 A similar method for this class of compounds using 3-chloroformyl-7-methoxycoumarin and reversedphase HPLC has been published.220 Anabolic stilbenes. 221 The antihypertensive agent fenoldopam was formed using a postcolumn enzyme reactor from the corresponding glucuronide. 228 Isonicotinoyl hydrazine was used to tag steroids. 237. to permit fluorescent detection after normal-phase HPLC. equilin. 3 D RR -nadolol. such as digoxigenin. 218 A few of the post-column approaches are outlined below. such as DES. was improved upon reduction to the 17-a-hydroxy compounds using sodium borohydride. has been 1 2 3 4 1 2 3 4 30 35 40 45 Time (min) Figure 12 Typical chromatograms of (a) blank control dog plasma. and estrone. and detected electrochemically. A similar method using 4-nitrobenzoyl chloride has been reported. representing 50 pg injected. has been detected fluorimetrically by postcolumn reaction with a solution of ascorbic acid. 240 Dihydrodigoxin. were derivatized with 1-naphthoyl chloride before separation and fluorescent detection 239 and 5-ng amounts could be determined in urine or feces. such as equilin. and separation could be achieved by reversed-phase HPLC. Digoxin and its metabolites. 4 D S.8 Steroids Esterified estrogens were converted into their free phenolic forms by acid hydrolysis. 2 D RS -nadolol. Using a photochemical reactor. Peaks: 1 D SR -nadolol. 236 Digoxin. 225 The chromatographic behavior of estrogen carbonyls.238 A detection limit less than 1 ng was attained. 222 .6dibromoquinone-4-chlorimide to form a colored product with a maximum absorbance at 650 nm.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 17 200 180 160 140 120 100 80 60 40 20 (a) 0 200 180 160 140 120 100 80 60 40 20 (b) 0 200 180 160 140 120 100 80 60 40 20 0 0 (c) 5 10 15 20 25 Cyclodextrins in biological fluids were assayed by negative colorimetric detection after postcolumn complexation with phenolphthalein. diethylstilbestrol (DES) has been converted into a fluorescent derivative and determined at the low parts per billion level in biological matrixes. and etiocholanolone. and detection limits of 2 pg were comparable to those with precolumn fluorescent methods. [Reproduced with permission from Hoshino et al. 218 ] was 2. such as androsterone. This chiral derivatization method has been used for the determination of enantiomers of other b-blocker drugs. have been measured in urine by HPLC and an offline chemiluminescence immunochemical assay. 223 Vitamin B6 (pyridoxine) has been postcolumn derivatized with 2. 226 Prederivatization of conjugated estrogens with dansyl chloride permitted fluorescent detection after separation by normal-phase HPLC (Table 4).231 17-Oxosteroids were labelled with dansylhydrazine and then chromatographed on a silica column with subsequent fluorescent detection from 60 to 100 pg. 234 Six corticosteroids were separated within 25 min and reacted with a lucigenin – KOH solution for chemiluminescence detection. Corticosteroids were also postcolumn derivatized with glycinamide in the presence of hexacyanoferrateIII) before fluorimetric detection at the 5-ng level. a major metabolite of digoxin. 219. S -nadolol. 230. The total time for the derivatization and separation was 33 min.18 separated from digoxin with dual UV and postcolumn fluorescent derivatization detection. AS D autosampler. such as ampicillin and ranitidine. 250 An analogous method has been reported for phenothiazines. Dansylaziridine 243 was used to determine cysteine and other thiols. and MP C D mobile phases A. [Reproduced with permission from Funakoshi et al.3-dimercaptopropane-1-sulfonic acid. and P(C) D pumps. 244 This method was also adopted for the determination of dithiols. and 3.9 Sulfur Compounds Most methods employ a precolumn reaction to form fluorescent or UV derivatives. however. 259 . the residue reconstituted in water was injected on to C1. P(B). and C3 D columns 1. Then the busulfan – DCC derivative from C1 was backflushed on to C2. and saccharides. such as 2. 255 A detection limit of 10 pg was found. aldehydes. was accomplished using on-line generated bromine and electrochemical detection of the excess bromine. 258 On-line precolumn derivatization to improve reproducibility has been demonstrated for the determination of busulfan in human serum. The solid and dotted lines in the six-port valves indicate valve positions 0 and 1. such as glutathione and ergothioneine. respectively. 246 Resolution of the optical isomers of diltiazem was accomplished using UV detection after derivatization with optically pure 2-naphthylsulfonyl-2-pyrollidinecarbonyl chloride.10-dihydroxyanthracene-2. where it was derivatized with diethyldithiocarbamate (DCC) in mobile phase A for 5 min. C2. 253. Cl. 252 Applications of photochemical reactors to a variety of pharmaceuticals. 259 ] detection at 435 nm with detection limits in the low parts per billion range. and methimazole.5 µg mL 1 . 251 Photochemical activation of phenothiazines and demoxepam in 2 min and subsequent fluorescence detection provided detection limits a factor of 10 better than UV detection. valve 2 was used to take a heart cut of this peak of interest and inject it on to C3. The reagent pyrenemaleimide provided derivatization of N-acetylcysteine with a detection limit of 10 pmol. C0 D cleanup column. reagent 5) forms UV-detectable derivatives of thiols. where it was separated using mobile phase B. After extraction of busulfan from serum. such as thioridazine. the resultant products from the oxidation reaction with bromine are detected fluorimetrically. Detection was at 278 nm and the lower limit of quantitation in serum was 10 ng mL 1 . DET(A) and DET B D UV detectors A and B. 257 Quenched peroxalate chemiluminescence has been employed using immobilized reagents in a postcolumn reactor for thioridazin. 249 Postcolumn derivatization of thioethers. and C3 were C18 types with different lengths and/or diameters. 248 Ethacrynic acid (Table 5. 247 Three N-substituted maleimides were compared for precolumn derivatization of thiols such as penicillamine before electrochemical detection at the picogram level. sulforidazine. have been determined in the range 50 – 500 ng. N-acetyl-L-cysteine. Biological thiols. and C. MP(A). 241 The photoreduction of anthroquinone-2. and mercaptopropionylglycine with detection limits down to 0. in urine at levels down to 10 pmol. cardiac glycosides. 245 Fluorescent derivatization of 2-mercaptopropionylglycine using N-(7dimethylamino-4-methyl-3-coumarinyl)maleimide could probably be extended to other thiol compounds. 242 3. All three columns C1. such as phenothiazines. The resulting separation using mobile phase C is shown in Figure 14(a) and (b). 2.6-disulfonate to the fluorescent 9. reagent 4) before ion-exchange chromatography. VAL-1 and VAL-2 D six-port valves 1 and 2. were reacted with monobromobimane (Table 4. Because the background interference from mostly excess DCC overlapped completely the busulfan– DCC derivative peak. which is electrochemically active. such as alcohols. C2. 256 Postcolumn complexation of disulfiram and two of its metabolites using Cu2C allowed colorimetric Waste PHARMACEUTICALS AND DRUGS AS CO P(A) MP (A) VAL 1 MP (B) P(B) C1 C2 DET (A) VAL 2 MP (C) DET (B) C3 Waste P(C) Waste Figure 13 Schematic diagram of the HPLC system. Using a knitted Teflon reactor. ethers. and diginatin. such as captopril. P(A). have been summarized. digoxigenin.254 A precolumn derivatization method for phenothiazine involves desulfurization with Raney nickel to produce diphenylamine. MP(B). amines. A schematic diagram of the HPLC system is shown in Figure 13. B.6-disulfonate occurs only in the presence of hydrogen atom-donating substrates. such as digoxin. the anticancer drug prospidin is a piperazinium derivative with chloroxypropyl groups but no UV/VIS chromophore. 266 Most of the naturally occurring amino acids do not have proper UV/VIS chromophores for CE analysis. 263 and dyes used to derivatize molecules for CE with laserinduced fluorescence (LIF) detection in drug analysis. and C3. The indirect detection method used 5 mM 4-methylbenzylamine in 1 : 4 methanol – water as absorbing background electrolyte for detection at 210 nm. 24 4.1 and 2 mg L 1 for indirect detection and derivatization methods. Since UV/VIS and LIF detection are the most common detection methods used in CE for pharmaceutical analysis. Derivatization is a necessity for the determination of amino acids at reasonable concentrations.1 Derivatization for Ultraviolet Detection Although most organic compounds have UV/VIS chromophores. Derivatization in conjuction with GC and HPLC is a well-developed field. 261 Instead of listing the derivatization reagents suited for labeling amino.0006 a. the solution was further diluted to about 1. i. for UV/VIS detection in CE. C2. Dansyl chloride dissolved in acetonitrile (3. For example. The arrow indicates the retention time of the busulfan derivative. derivatization is still important. Because rimantadine is almost transparent in the UV/VIS range. Both are antiviral agents used for prophylaxis and treatment of influenza A. The major advantages of CE over chromatographic separation techniques are its simplicity and efficiency. enhancing . the enantiomeric forms of novel depsipeptide antitumor antibiotic BMY-45012. derivatization chemistry previously developed for HPLC is often applicable to CE. hydroxyl.7 mg mL 1 with water. 0 (a) 8 16 24 Inj. carboxyl. 260 At least one comprehensive review specifically focused on the derivatization in CE has appeared. The detection limits were 0.8 mg) was dissolved in water – acetonitrile solvent. One way to improve the sensitivity of CE detection is to derivatize the analytes with more favorable detection characteristics by adding either an ultraviolet/visible (UV/VIS) chromophore or a fluorophore. keto. 262 diastereomer derivatization. With the advent of CE.150 the detectability. 259 ] 4 CAPILLARY ELECTROPHORESIS CE has developed into a versatile separation technique well suited for the determination of pharmaceutical and biomedical samples. 264 The pros and cons of the various modes of derivatization and a detailed comparison of this topic between LC and CE can be found in the literature. generates a product that absorbs light at 254 nm. CE suffers from poor sensitivity in detection owing to the extremely small sample volume involved. The derivatization method used rimantadine to react with 1. 267 The compounds were subjected to total hydrolysis in a vacuum hydrolysis tube with 6 M HCl at 110 ° C for 24 h.2-naphthoquinone-4-sulfonic acid in alkaline medium. with loss of HCl.u. The methods were used to determine rimantadine in pharmaceutical products and for dissolution testing of Flumadin tablets. [Reproduced with permission from Funakoshi et al. we intend to focus on the purpose of derivatization in CE from a practical point of view. UV/VIS detection of the chromophores does not give a satisfactory response owing to the very short light pathlength (50 – 100 µm) in CE. respectively. were determined by CE with UV/VIS detection. either indirect detection or derivatization has to be used to detect this compound. aldehyde. and sulfhydryl groups. and its analogs. 265 Rimantadine is a synthetic analog of amantadine. For subsequent derivatization.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 19 Busul fan/16. By using dansyl chloride to derivatize the amino acids. we shall discuss the most recent work related to this area. A 50-µL sample solution was mixed with 50 µL of dansyl chloride solution and an aliquot of Inj. 261 Several other review articles dealt with more specific topics. However. 0 (b) 8 16 Time (min) Time (min) Figure 14 Typical chromatograms of (a) drug-free serum and (b) serum spiked with busulfan (100 ng mL 1 ) obtained with columns Cl.0 mg mL 1 ) was utilized for the derivatization of standard native amino acids and those present in the hydrolyzate.e. 0. Separation of the desired product from excess derivatizing agent is possible in 10 min by CE with a detection limit of 1 ng L 1 .2. Derivatization with DCC at 37 ° C for 90 min in a basic solution. Very often. The hydrolyzed residue (about 7. and sometimes necessary. CE determination of the derivative at 280 nm was performed in an uncoated capillary (44 cm ð 75 µm ID) using a 40 mM tetraborate buffer at pH 9. such as postcolumn derivatization. LIF offers a certain selectivity because. unlike UV absorbance. 3. have been reviewed previously. (5) 3-mercaptopropionic acid. On the other hand. Amines can easily be derivatized with FITC isomer I and analyzed by CE using alkaline buffers with or H3C PHARMACEUTICALS AND DRUGS C CH2 + H3C C CH3 X O I N (CH2 )4 N CH3 SO3− CY5. and most organic compounds do not fluoresce. The mixture was held at room temperature for 2 h and used directly for injection. Captopril. Ramseier et al. That value is relevant for toxicology drug screening and confirmation. electrophoresis in microstructures is shown to provide faster separation and higher efficiencies without loss of accuracy and precision. The principles of fluorescence and the prerequisites for a good fluorophore. [Reproduced with permission from Couderc et al. 270 This wavelength was compatible with a semiconductor laser at 670 nm.3a. there was only a twofold gain in sensitivity on switching from UV to fluorescence detection of the amikacin derivative. methamphetamine. Since the available excitation wavelengths of various lasers are limited. (4) L-cysteine. under optimized conditions. and more and more industrial scientists are using LIF detection to detect trace amounts of analytes in complex matrices. If laser excitation can be used (as discussed in the next section). emission at 482 nm). Relative peak height . with direct labeling of 10 µL of urine that had been alkalinized and diluted for derivatization. CE also separated other labeled thiols such as cysteine and reduced glutathione from derivatized captopril (Figure 15). First. The detection limit of 2. (2) captopril. In practice. derivatization is often required when using CE with LIF detection. 4. 269 This is critical to pharmaceutical applications because sensitivity is often the most important parameter to consider when analyzing biological samples. Using a methanol – borate running electrolyte. Using 5 mL of urine with a ‘spiked amine’ to FITC ratio of 1 : 250.4-methylenedioxymethamphetamine and b-phenylethylamine in human urine by CE using chip-based and fused-silica capillary instrumentation with LIF detection. (7) reduced glutathione. LIF selectively detects compounds that are fluorescent at certain wavelengths. 268 However. it offers excellent sensitivity.IA: X = −NH − H HS • CH3 N • O C HO O Captopril 1 2 7 5 6 4 3 40 30 20 10 0 Migration time (min) Figure 15 Electropherogram of a number of thiols labeled with CY5. reported the determination of FITC-derivatized amphetamine. 271 The results obtained via direct labeling of fortified urine were compared with those generated after FITC labeling of urinary extracts that were prepared by SPE. The following examples demonstrate the applicability of derivatization in CE with LIF detection for pharmaceutical analysis. a value that is too high for practical purposes. almost any detection mode can be combined with a derivatization procedure.IA: (1) unreacted label. was derivatized through the thiol group with a dicarbocyamine label to give a fluorescent derivative with a wavelength maximum at 675 nm. the limit of identification was 10 µg mL 1 . the gain in sensitivity should be significantly higher. 264 ] without dodecyl sulfate micelles. 261 LIF detection is now commercially available for CE.2 Derivatization for Fluorescence Detection In principle. fluorescence monitoring is favored in most cases. (6) 2-mercaptoacetic acid. which was used for LIF detection in conjunction with CE. LIF detection has several advantages. a level of 10 10 M is feasible. Compared with fused-silica capillaries. (3) DL-homocysteine. an antihypertensive agent.08.20 borate buffer at pH 9. The aminocychitol antibiotic amikacin can be derivatized with 1-methoxycarbonylindolizine-3.5-dicarbaldehyde at room temperature for 15 min before determination in plasma by micellar CE with UV detection at 280 nm and standard fluorescence detection (excitation at 414 nm.5 ð 10 8 M for captopril is limited by dilution due to the derivatization reaction. the fact that most organic compounds do not fluoresce is also a major limitation of LIF detection. the SPE extract had a sensitivity of 200 ng mL 1 urine. including the potential of using diode lasers in combination with a labeling procedure. Second. In contrast.3a. the fluorogenic reagent FITC was dissolved in acetone (0.5 mg of rMAb were buffer exchanged into 800 µL of 0. 190 µL of the antibody – dye conjugate was loaded on to a second NAP-5 column and collected in 700 µL of 0.g. The reaction tubes were vortex mixed and then incubated overnight at 40 ° C. 4.0 ð 10 4 M) under basic conditions (borate buffer. pH 9.3.3) using a NAP-5 column. 272 Before derivatization.2 Oligosaccharide Determination The derivatization of the pseudo-oligosaccharide acarbose (2) and its main metabolite (3) with 7-aminonaphthalene-1. 0.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 21 4.9 M NaCNBH3 solution in dimethyl sulfoxide (DMSO) were added to each sample residue.1 Chiral Confirmation With proper derivatization.08 M ANDS solution in acetic acid – water (3 : 17 v/v) and a 50-µL volume of 0.01 M) as a stock solution. such as D-serine and L-b-hydroxyl-Nmethylvaline. Liu et al.4 mg mL 1 ) dissolved in DMSO was then added to 190 µL of rMAb solution and the resultant mixture incubated for 2 h at 30 ° C. A 100-µL volume of 0. 267 ]. 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRSE) for CE/LIF analysis. after incubating with CH3O N N O O O H3C HO H N O O N N O H N O N O CH OR1 3 O N N CH3 CH3 OH N CH3 O O N H O N N OCH3 N CH3 CH3 R2O CH3 O N O O N H O (1) Compound BMY45012 Analog-G439B Analog-G451 Analog-G435 Analog-G439A R1 CH(CH3)2 CH2CH3 C(CH3)3 C(CH3)3 CH(CH3)2 R2 CH(CH3)2 CH2CH3 C(CH3)3 CH(CH3)2 CH3 . The labeled sample. The efficient separation of these derivatives by CE using 100 mM triethylammonium phosphate buffer at pH 1. the proposed structure of which is shown as (1) [reproduced with permission from Liu et al. 273 Samples containing 2.5mL samples of urine or spiked urine were evaporated to dryness.08).3. These methodologies provide a quick and sensitive approach for the determination of amino acid racemization in pharmaceutical natural products and have proven to be useful for structural elucidation refinement. For fluorescence detection. CE/LIF with proper derivatization has been shown to be promising as a general method.3. Amino acids were analyzed by complete hydrolysis and the hydrolyzate was derivatized with either dansyl chloride for UV absorbance detection or FITC for LIF detection in CE.5 allowed the quantitation of acarbose and (3) in human urine after application of 300 mg of acarbose.3 Special Applications 4.3). The reaction was allowed to proceed for 2 – 4 h in the dark at room temperature and then stored at 20 ° C prior to use.1 M sodium hydrogencarbonate (pH 8. a generic analytical approach for the analysis of size-based rMAb variants is desired. and its analogs. Amino acids were derivatized with FITC-derivatizing solution (5. Both a metal chelate chiral CE method and a cyclodextrin-mediated host – guest interaction approach in micellar electrokinetic chromatography (MEKC) with LIF detection confirmed the presence of several chiral amino acids. CE has been used for the chiral separation of enantiomeric forms of derivatized amino acids. e. The rMAb can be derivatized with a neutral fluorophore. and the nonchiral amino acid sarcosine in the proposed structure. A 10-µL volume of 5-TAMRSE (1.3 Derivatization of Protein Samples With the increased interest in the therapeutic use of the recombinant monoclonal antibody (rMAb) technique.3-disulfonic acid (ANDS) (Scheme 1) in human urine allowed the on-column LIF detection of the pseudo-oligosaccharides in human urine in the nanomolar range. After incubation.1 M sodium hydrogencarbonate (pH 8. The reagent solutions were freshly prepared before derivatization. reported the determination of enantiomeric forms of amino acids derived from the novel depsipeptide antitumor antibiotic BMY-45012. 4. which in turn was reacted with IFN-a. and free-solution or end-column systems. including coaxial capillary reactors. 262 Various systems to merge the reagent solution with the separation medium have been developed. derivatization in CE can be accomplished by either pre. can be separated by CE using a hydrophilic polymer as a sieving matrix. The precolumn derivatization is similar to methods used for derivatization in HPLC. 274 ] Scheme 1 Reaction of (2) and (3) for derivatization with ANDS by reductive amination. O R COOH + HO N DCC O HO CH2OH OH C N HO H Schiff base SO3H O R O N + DCU SO3H O NaCNCH3 O Active ester (isolated) + NH2 IFN α pH 7.4 Derivatization Modes (3) CH2OH O CH2OH OH O HO C O HO H SO3H H2N SO3H O HO OH HO Carbohydrate In general. the geometry of the system. the interference from the derivatization by-products may be less of a problem than in LC. CE was used for the study of palmitoyl derivatization of interferon-a2b (p-IFN-a). Since CE has a much superior separation power. and on-line detection. Using these precolumn labeling conditions. CE is very sensitive to the ionic strength of the sample. 272 ] SDS. the detection of rMAb at a low-nanomolar concentration (9 ng mL 1 ) is obtained with no apparent decrease in resolution or changes to the distribution of rMAb analyte species in comparison with an unlabeled sample. 261 The details about the instrumental developments and applications of post-column derivatization in CE can also be found in the literature. For all reactor types. . gap reactors. However. R COOH palmitic acid. ease of use. speed.2 CH2OH OH O CH2 NH HO HO SO3H O R C N IFN α H SO3H Scheme 2 Synthetic process for the fatty acrylation of IFN-a. The first step involved the preparation of an intermediate (NHSP). Additional sample preparation may be needed if the mixture in the derivatization reaction has a high ionic strength. CE was able to study the effect of reaction time and reagent/protein ratio. [Reproduced with permission from Foldvari et al.22 CH2OH HO HO CH3 HO O HN CH2OH HO HO O O CH2OH HO HO O O OH HO HO PHARMACEUTICALS AND DRUGS (2) CH2OH HO HO CH3 HO O HN CH2OH HO HO O O OH HO HO of the bulk manufacture of a protein pharmaceutical and in providing a size-based separation of product-related variants and also nonproduct impurities. The CE/LIF with derivatization demonstrated comparable resolution and sensitivity to silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) but offered the advantages of enhanced precision and robustness. 4.or postcolumn derivatization. [Reproduced with permission from Rethfeld and Blaschke. 274 The derivative was prepared by covalent attachment of the fatty acid to lysine residues in the protein through a reaction with Nhydroxysuccinimide palmitate ester (Scheme 2). An overview of the advantages and limitations of postcolumn derivatization for CE can be found in a recent paper. This assay can be used in monitoring consistency Postcolumn detection strategies have also been developed for CE. To minimize peak broadening. and (4) sheath flow.O-Bis(trimethylsilyl)acetamide N. Column Selection for. careful design in terms of connecting a reagent capillary to form a tee is necessary. (2) gap. With proper design. As the cost of laser technology comes down. in Drug Analysis ž Planar Chromatography in Pharmaceutical Analysis ž Solid-phase Extraction and Clean-up Procedures in Pharmaceutical Analysis . and CE have diminished the importance of chemical derivatization for these techniques. which often contain multiple polar substituents. mainly OPA and its naphthalene analog have been used. Because of the small (50 – 75 µm) ID of the capillaries used. HPLC. (3) free-solution. MS detection for HPLC and CE is still expensive and requires considerable operator expertise. However. The strict requirements on the reaction rate in postcolumn derivatization in CE limit the number of different reagents that have been used.N-Dimethylformamide Dimethylacetal Dimethylisopropylsilyl Dimethyl Sulfoxide Electron Capture Free Fatty Acid Phase Fluorescein Isothiocyanate 9-Fluorenylmethyl Chloroformate Gas Chromatography Gas Chromatography/Mass Spectrometry Heptafluorobutyric Anhydride Heptafluorobutylimidazole Hexamethyldisilazane High-performance Liquid Chromatography Liquid Chromatography Laser-induced Fluorescence N-Methylbistrifluoroacetamide Micellar Electrokinetic Chromatography Mass Spectrometry N-Methyl-N-trimethylsilyltrifluoroacetamide N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide o-Phthalaldehyde Pentafluoropropionic Anhydride Pentafluoropropionylimidazole Pentafluorobenzyl Bromide Quaternary Ammonium Salt Recombinant Monoclonal Antibody Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Solid-phase Extraction t-Butyldimethylsilyl Trifluoroacetic Anhydride Trifluoroacetylimidazole Trimethylanilinium Hydroxide Trimethylchlorosilane Trimethylsilyl N-Trimethylsilylimidazole Ultraviolet Ultraviolet/Visible 5-Carboxytetramethylrhodamine Succinimidyl Ester RELATED ARTICLES Pharmaceuticals and Drugs (Volume 8) Eluent Additives and the Optimization of Highperformance Liquid Chromatography Procedures ž Gas and Liquid Chromatography. the detection limits of postcolumn derivatization can be comparable to those of precolumn derivatization. chemical derivatization of drugs is critical for GC because these samples. Chemical derivatization with HPLC to permit fluorescence detection is certainly one of the more desirable methods for routine use to solve selectivity and detectability problems for drug samples not amenable to GC. are simply not volatile or thermally stable. plate numbers of over 100 000 could be realized. For LIF detection. With careful instrument design. FFAP FITC FMOC GC GC/MS HFAA HFBI HMDS HPLC LC LIF MBTFA MEKC MS MSTFA 5 CONCLUSION It can be argued that the advances made in MS detection for GC. normal mixing through a diffusion process is sufficient. the same statement will eventually be true for CE also. The most frequently applied reactors are (1) co-axial.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 23 and the method used to propel the reaction mixture (by pressure or by voltage) appear to be critical to preserve the separation efficiency.3-disulfonic Acid N.O-Bis(trimethylsilyl)trifluoroacetamide Cannabidiol Capillary Electrophoresis Diethyldithiocarbamate Diethylstilbestrol Dimethylethylsilyl N. MTBSTFA OPA PFAA PFAI PFB-Br QUAT rMAb SDS SDS/PAGE SPE TBDMS TFAA TFAI TMAH TMCS TMS TMSI UV UV/VIS 5-TAMRSE ABBREVIATIONS AND ACRONYMS ANDS BSA BSTFA CBD CE DCC DES DMES DMFDA DMIPS DMSO EC 7-Aminonaphthalene-1. Anal. Krossa. Pharm. Jansen. Walsh. Bedford. J. Elsohly. Biomed. 15. Vessman. D. 279 – 284 (1985). High Performance Liquid Chromatography in Biochemistry. A. C. H.D. 7.. Pharm. Frei. Jones. Mell.D. 1981. A.Th. J. ‘Reaction Detectors in Modern Liquid Chromatography’. Van Den Berg. R. Brinkman. 15. 1529A – 1539A (1985). Poole.J. Separation and Continuous Flow Techniques. Chromatographia. Hahn. 2. P. Marcel Dekker. Vol.. Arfwidsson. Frei.J. Derivatization Chemistry for Gas Chromatography. R. J.A.D. M.S. U. 1 and 2. . 175 – 184 (1983). Jones. Toubar. 1983. Halket. 2. 27.A. Chromatogr. Chapter 4.. 123 – 142 (1980). Lett. Plasma. S. IL. Frei.H.J.I. 33. K. Pharm. Vols. Ross-Lee. Biomed. Reaction Detection in Liquid Chromatography. Darwin. J.W.). Engelhardt. 1986. A.M. J. 1985.J. Beeren.R. W. 911 – 914 (1990). ‘Simultaneous Detection in Urine of Cocaine and Its Main Metabolites’.W. Anal.. Ramachandran. ‘A Simple Wash Procedure for Improving Chromatography of HFAA Derivatized Amphetamine Extracts for GC/MS Analysis’.F. J. 226. 4. J. Handbook of Analytical Derivatization Reactions. 660.M. 1986. J. M.B. Organic Trace Analysis by Liquid Chromatography. Kim. C. Chromatographia. 29. Hooper. Biological/Biomedical Applications of Liquid Chromatography. 19. L. ‘Determination of Cannabidiol in Plasma by Electron Capture Gas Chromatography’. J.F. 26. 57. New York. 20.R. J. Frei. W. M. New York. Heroin.W.. Wiley. 161 – 166 (1982). J.F.. O. 3. R.. Scholten. 177 – 187 (1979). M. J. A.Th. Cole. 17. 15.. Karlsson. ‘Pyrolytic Methylation/Gas Chromatography: A Short Review’. R. 669 – 682 (1978). ‘Preparation and Gas Chromatography of Highly Volatile Trifluoracetylated Carbohydrates Using N-Methylbis(trifluoracetamide)’. 21. PHARMACEUTICALS AND DRUGS 14. 24. 17. ‘Simultaneous Assay of Cocaine. A. 1 and 2. CRC Press. J. 705 – 709 (1991). Chromatogr. Krull (ed. Horning. Sci. Gyllenhaal. N. Sci.W. Chromatogr. Plenum Press. Brinkman. Chromatogr.T. C.24 Gas Chromatography (Volume 12) Gas Chromatography: Introduction ž Column Technology in Gas Chromatography ž Sample Preparation for Gas Chromatography Liquid Chromatography (Volume 13) Liquid Chromatography: Introduction ž Capillary Electrophoresis ž Column Theory and Resolution in Liquid Chromatography ž Normal-phase Liquid Chromatography ž Reversed Phase Liquid Chromatography 13. R. U. Frei. Pierce Chemical Co. ‘Post-column Reactor Systems in Liquid Chromatography’. Metcalf. ‘Post-column Reaction and Extraction Detectors for Narrow-bore High Performance Liquid Chromatography’. Vol. Chem. R. J.. Brooks. Boca Raton. 190 – 200 (1987). U. 199. Webber.-F. 18. Hsu. New York. S. Henschen (ed.G. Chapter 5. Jansen. Lawrence (eds.A. A. 149. Chromatogr. Kroll. J. G. VCH. F. Rizk. W. Segura.A. Deelder.K. Chromatogr. A. 23. 5. ‘Reaction Detector with Three Dimensional Coiled Open Tubes in HPLC’. B. 25. New York.L. Sullivan. 99 – 105 (1981). Cone. F.-L. I. Drugs. ‘Reaction Detectors in HPLC’. L. Chromatogr. L. 28. Handbook of Derivatives for Chromatography. 30. ‘A Review of Reaction Detection in HPLC’. Delatorre. K. Knapp.W.. 22.J. 1999. New York. 1981. Vessman. Zlatkis.M. 1979. Sci. Lawrence (eds.F. 23. Gupta. Neue. 15. Deerfield Beach. I. Singhawangcha. Biomed.E. 6. Schmitz. 24.C. J. J. Hawk.M. 1993. FL. J. 11. Academic Press. Brinkman. Chromatographia. J. S. J. ‘Determination of Tertiary Amines as Pentafluorobenzyl Carbamates by Electron Capture Gas Chromatography’. 4. 9. Plenum Press. ‘New Electron Capturing Pentafluorophenyldialkylchlorosilanes as Versatile Derivatizing Reagents for Gas Chromatography’. Wiley. Middleditch. M. Turner.B. Vessman. Sunshine.S. and Urine by Gas Chromatography – Mass Spectrometry’. 17.D. 10. 7. 514 – 515 (1966). L.. B. U.R. J. 196 – 197 (1977). A... Lawrence. M. 8. J... R. 447 (1993). 1. J. R.). and Metabolites in Hair. Wang. ‘Simultaneous Gas Chromatography of Volatile and Nonvolatile Carboxylic Acids as t-Butyldimethylsilyl Derivatives’.J. REFERENCES 16.S.. New York. Zlatkis. 289 – 301 (1989). W. J.A.S.W. Blau. 1.B. K. 152 – 160 (1979). 1981.). Marcel Dekker. Sci. Chem. Rockford.E.W. Saliva. J.H. ‘Direct Derivatization of Drugs in Untreated Biological Samples for Gas Chromatographic Analysis’. ‘Rapid Preparation of Fatty Acid Esters from Lipids for Gas Chromatographic Analysis’. Eadie.Th. 828 – 834 (1986).. E. Anal.S. Toxicol. New York. W.A. 183. J. Frei. Chromatogr. Chapter 7. Hartvig. ‘Electron-capture Gas Chromatographic Assay for Metoclopramide in Plasma’. Tyrer.H.C. 223 (1974). 2nd edition. Schewe. Cami. J. Kabi. H. 279 – 290 (1994).E. 468. Chromatogr. FL.D. ‘Post-column Reaction Detectors for HPLC’. Zakhari. A. 9. Vols. 8. W. 17. 403 – 408 (1982). H. J. F. R. Anal. A. Chemical Derivatization in Analytical Chemistry. J. E.). Anal. Ortuno. 12.A. CRC Handbook of Chromatography. ‘Gas Chromatographic Assay of Methyltestosterone in Tablets’. Sye. Anal. Bochner. Chromatogr. MacGee. Anal. E. H. 415. J. J. P. Greig. 336. 39. Derks.R. Honigberg. ‘The Application of Chemical Derivatization to Clinical Drug Analysis’.C. 40. Brinkman. Stobaugh.E. Sci. T. J. 43.T. 53. 683 – 691 (1978).. Danielson.S. 417.M. 144 – 148 (1983). Ellis. S.J. J. A. 41. Chromatogr.P. P. K. The Effect of Micelle Formation’. Frei.L. J.T.M. Sternson. C. S. J. N. L.L. Chromatogr.L. Xenobiotica. K.E. R. 173 – 178 (1986).C. I. Brinkman. Chromatogr. Honigberg.Th. E.D. 330. Nelson. ‘Automated Determination of Drugs in Blood Samples After Enzymatic Hydrolysis Using Precolumn Switching and Post-column Reaction Detection’. Rendi. 407 – 414 (1982). 411.W. Chromatogr. T. Tsuji. 205. Van Der Kleyn. J. 337 – 344 (1984). 12. Wuis. 337. E. and Metabolites by Postcolumn Ion-pair Extraction and On-line Fluorescent Detection of the Counterion with Applications’. Chromatogr. 208 – 215 (1987). J. 166.A. R. Karler. H.. Bedford. O. Biomed. Pharm. Werkhovern-Goewie. Badarani. R.. Sternson. R. B.. R. 35.J.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 25 31. J.. H.J. ‘Post-column Air-segmentation Photochemical Reactor for the Fluorometric Detection of Reserpine by Liquid Chromatography’. Durruc. 252. J. . M. 349 – 357 (1979). II. D. G. J. PereboomDe Fauw. 370. 133 – 140 (1978). C. 38. G. 52. 234. Frei. Stewart. N. M. Chromatogr. 59 – 65 (1984). ‘Automation of Pre-column Derivatization in High-performance Liquid Chromatography. 55.. GLC and HPLC Determination of Therapeutic Agents. Chromatogr.C. ‘High-performance Liquid Chromatographic Analysis of Baclofen in Plasma and Urine of Man After Precolumn Extraction and Derivatization with o-Phthaldialdehyde’. 176. a Rationally Designed Fluorogenic Reagent for Primary Amines’.. ‘New Derivatization Reactions in Pharmaceutical Analysis’. ‘Post-column Air-segmentation Reactor for the Fluorometric Detection of Reserpine by Liquid Chromatography’. C. 56. ‘High-performance Liquid Chromatographic Assay for Pilocarpine in Aqueous Humor’. Thevenin. Marigo.C. 229 – 236 (1987).G.C. De Jong.. Chromatogr. Chromatogr. 49. 341 – 350 (1985).W. Stewart. J. 368 – 370 (1979). J. M.and Post-column Derivatization Chemistry in Conjunction with HPLC for Pharmaceutical Analysis’.M. Ahuja. J. Sci. Burgers. ‘Liquid Chromatography with Pre-column Dansyl Derivatisation and Fluorimetric Detection Applied to the Assay of Morphine in Biological Samples’. 50. U. 51. 72. J. R. 42. Sci. 37. ‘Post-column Derivatization Procedure for the Colorimetric Analysis of Tissue Cannabinoids Separated by High-performance Liquid Chromatography’. J. A.M.. S. J. Gal. Renyei. 26. J. Loeber. 630 – 635 (1983). F. Derendorf. J. J.B. Gfeller. Hermann.F. 497 – 506 (1983). M. Higuchi. Dorizzi. Beneken Kolmer. Chromatogr. Garrett.P. Little.M. A. Pharm. Chromatogr. J.W. J.W. Vree. 36. 473 – 481 (1979). E. P.K. J. ‘High-performance Liquid Chromatography Detection of Morphine by Fluorescence After Post-column Derivatisation. Frigerio. 1096 – 1101 (1987). S.A. Dirks.P. Lang. M. J. Van Tuillert. Van Beijsterveldt.J. Twitchett.. 79 – 90 (1983). Van Der Kleyn. U. Haddouche. ‘Naphthalene-2. Zomer. Anal.R. 17.L. Lang. T. Chromatogr. L. Lubli. J. Morozowich (eds. R. 255. ‘Automated Precolumn Derivatisation HPLC’. Rapoport. Ph. W. 47.A. ‘High-performance Liquid Chromatography Detection of Morphine by Fluorescence After Post-column Derivatisation’. Marcel Dekker. R. J. A. Scholten. I. ‘Determination of the Heroin Metabolite 6-Acetylmorphine by Highperformance Liquid Chromatography Using Automated 45.R. L. 303 – 323 (1981). 434 – 439 (1985). J. 264.A. Wuis..M.A. Pre-column Derivatization and Fluorescence Detection’. 323 – 331 (1985). ‘Enantioselective High-performance Liquid Chromatographic Determination of Baclofen After Derivatization with a Chiral Adduct of o-Phthaldialdehyde’. Moffat. Chromatogr.B. G. Nolan.J. ‘Identification of Ergot Alkaloids with a Photochemical Reaction Detector in Liquid Chromatography’. Gfeller. P.). ‘Determination of S-CarboxymethylL-cysteine in Plasma by High-performance Liquid Chromatography with Column Switching Following Precolumn Derivatization’. Tagliaro.. Lawrence. J. 32.T. R.. P. J.F.A. Huen. R. E. J.G. 46. 33. Chem.W.E. 339. de Montigny. ‘Pre. 419 – 422 (1987).W. Carlson. J. A. ‘High-performance Liquid Chromatographic Assay of Methadone. Srinivasachar. ‘Continuous Post-column Ion-pair Extraction Detection of Some Basic Organic Compounds in Normal-phase Chromatography’.H.J.I. K. Gorog. Lauko. 54.E.R. J. J.. Williams. Chromatogr. 149. Targove. 1978. 185. Thenot. Hegedus.H. Phencyclidine.. 168 – 172 (1979).Th. 288 – 292 (1982). 44. Frei. Huen. ‘High-performance Liquid Chromatographic Analysis of Melphalan in Plasma. 298. Sweeney..K. Chromatogr. D.G. C. ‘Evaluation of a Chemical Reaction Detector with Electrochemical Production of Bromine for the Liquid Chromatographic Determination of Physostigmine’. J. 48. Anal. Chromatogr. Borys. P. Nelson.. Chromatogr.. Miller... J. J. B. Brain and Peripheral Tissue by oPhthalaldehyde Derivatization and Fluorescence Detection’. Thevenin. Isaksson. E. Chromatographia. ‘Chemical Derivatization for the Liquid Chromatography of Compounds of Pharmaceutical Interest’.P. Vree. Application to Ion-pair Partition Chromatography’.3-dicarboxaldehyde/Cyanide Ion. 1. Chromatogr. New York. Givens. 34. E. 362 – 371 (1988). ‘Photochemical Detection in High-performance Liquid Chromatography and Its Application to Cannabinoid Analysis’.. J. 17.P...J.H. 385 – 396 (1987). Stahel. Chromatogr. De Ruiter. 59. K. Chromatogr. J. E. ‘Determination of pHydroxybestatin in Human Serum by High-performance Liquid Chromatography Using Fluorescence Detection’. A. J. ‘Poly(styrene – divinylbenzene) as Reversed-phase Adsorbent for the High-performance Liquid Chromatographic Analysis of Thiochrome Derivatives of Thiamine and Phosphorylated Esters’.. Okada. 315 – 326 (1982). Venema. T. J. F. Smrz. Chromatogr.A. H.M. J. PHARMACEUTICALS AND DRUGS 58. Ishida. J. Pachla.. Tscherne. Maddock. Lewis.A. 60. 172 – 177 (1985). Sedman. J. 549 – 555 (1983). W. H. ‘Determination of 5-fluoro-20 -deoxyuridine in Human Plasma by High-performance Liquid Chromatography with Precolumn Fluorimetric Derivatization’. S. ‘High-performance Liquid Chromatographic Method for Monitoring Leupeptin in Mouse Serum and Muscle by Pre-column Fluorescence Derivatization with Benzoin’.. 69.B. P. Turgeon. 341. Leever.and (R)-( )Hydroxy Metabolites in Urine’.F. J. 275 – 282 (1987). J. ‘Liquid Chromatographic Determination of Mexiletine and Tocainide in Human Plasma with Fluorescence Detection After Reaction with a Modified o-Phthalaldehyde Reagent’. J. 81. Musch. Leonard. 267 – 274 (1985). 106. Cole. Rennert. 464 – 468 (1983). Schoffeniels. Y. Thalheimer. 371 – 376 (1983). 232. D. Chromatogr. Umagat. Salamounova. Michaelis. J. D. Meese. J. Toomey. ‘Determination of L-Buthionine(S. ‘High-performance Liquid Chromatographic Assay for Mexiletine Enantiomers in Human Plasma’. 113 – 122 (1985). 1368 – 1370 (1980). 344. G. 287 – 293 (1985). A. J.. Chromatogr. M. R. M.. Y. Reynolds.. J. ‘Pre-column Derivatization with Fluorescamine and High-performance Liquid Chromatographic Analysis of Drugs. J.D.4-tetrahydro-5H(1)-benzopyrano [3. Chromatogr. J. W.R.4-c]pyridin-5-one in Plasma by Liquid Chromatographic Column Switching After Derivatizing the . Biomed. ‘Derivatizing Reagents for High-performance Liquid Chromatography Detection of Peptides at the Picomole Level’.J. ‘Determination of g-Aminobutyric Acid by Reversed-phase Highperformance Liquid Chromatography and Pre-column Labeling for Fluorescence Detection’. M. Chromatogr. 343. K. Grech-Belanger. Y. ‘High-performance Liquid Chromatographic Method for Determination of Sulfapyridine in Human Saliva Using Post-column. 327. Fellman. O. 70.. J. 74. Chromatogr. 344 – 350 (1987). ‘Ion-pairing Detection Technique in Reversed-phase High-performance Liquid Chromatography of Drugs and Related Compounds’.M. Bakker. Ishida.S.E. Dandrifosse. 345. 59. Elworthy. Chromatogr. J. G. 80. Tassinari. P.. J. ‘Determination of Plasma Tranexamic Acid Using Cation-exchange High-performance Liquid Chromatography with Fluorescence Detection’. 66. 73. Chromatogr. Miura.L. 62. J. ‘Derivatization in Aqueous Solution. 486 – 491 (1984). J. M. Chan. M.R. 385. 121 – 124 (1982). 67. 75. E. Chromatogr. W. 176 – 182 (1987). 213 – 223 (1987). ‘Liquid Chromatographic Determination of Felypressin After Precolumn Derivatization with Fluorescamine’.. Greismann. 295. Gupta. M. Isolation and Separation of Tetrahydro-bcarbolines and Their Precursors by Liquid Chromatography’. and Multivitamin Preparations’. H. R. Chromatogr. ‘Simultaneous Determination of Thiamine and Riboflavin in Selected Foods by HPLC’. 63. Korf. Westhead. Acetylhydrazine and Diacetylhydrazine in Biological Fluids by High-performance Liquid Chromatography’. Sista.O. 79. ‘Determination of Isoniazid.3. ‘Column Liquid Chromatography of Methotrexate and Its Metabolites Using a Post-column Photochemical Reactor and Fluorescence Detection’.. Duff. C. J. P. Lombet..J. G. J. Kai. 266. Murrill. Baba. C.. Tsementzis. J. ‘High-performance Liquid Chromatographic Determination of Four Biogenic Amines in Chocolate’. 72. 337. In-line Derivatization with Fluorescamine’. J. Bosin. C. 344. K. J. Anal. J. 273. Gal.C. Chromatogr. Widahl-Nasman. C. Chromatogr.A. ‘Analysis of 3-(2-(Ethylamino)propyl)-1. 260. Augustin. Chromatogr.A. T.. Bontemps. Bettendorf. Chromatogr. Woodward. ‘High-performance Liquid Chromatographic Analysis of Thiamine in Rice Flour with Fluorimetric Post-column Derivatization’.M. 230.... Ohta. 401 – 408 (1983).. W. Chromatogr. J. Chem.. ‘Improved Method for the Determination of N-Acetylcysteine in Human Plasma by High-performance Liquid Chromatography’. M.. J. R. 64. Hitchcock. 52. Food Sci. Artz. E. ‘High Performance Liquid Chromatographic Determination of Panthenol in Bulk. Jarvis.N. O.R)-sulfoximine in Plasma by High-performance Liquid Chromatography with o-Phthalaldehyde Derivatization and Fluorometric Detection’. Lew. Chromatogr.2.E.O. Chromatogr. 416. 291. 259 – 265 (1985). Salamoun. P. Ohkura. Foth. 71. W.K. Kai. Crommen. Haayer. J. H. J. 423. E. Eichelbaum. D. Pharm. Kahl.26 57. A. 419. 394 – 402 (1981).Y. Castro-Parra. J. J. 2048 – 2050 (1982). Anal. Acetylisoniazid. 76. 61.J.. 65. Chromatogr. 47. Chromatogr. G. Kiss. M. 221 – 230 (1985). E. L. H. L. Von Sassen. J.L. Grandfils.L. Analyst. 77.. P. 261 – 267 (1985). ‘Automated Precolumn Derivatization Device to Determine Neurotransmitter and Other Amino Acids by Reversed-phase High-performance Liquid Chromatography’. Premix. 281 – 284 (1984). Meek. T. Groningsson. 1. 109 – 117 (1985). J. 338. J. Suzuki. Hurst. ‘High-performance Liquid Chromatographic Method for the Analysis of Debrisoquine and Its (S)-(C). J.. Dye. Application to Tocainide’. Chromatogr. 185 – 194 (1984).. Eichelbaum. Ohkura. 68. 284. 78. J. 20 -bipyridine)ruthenium(III)’. J. Cohen. 421. 154. 342 – 344 (1980). 219 – 225 (1978). Huen. 679. 1..A. 172. 341. J. 69.F. Tarvin. Hartinger.. 94. 85.. Acta. M. High Resolut. Lawrence. 98. J. Nguyen. Herraez-Hernandez. Chromatogr. Stone. Brinkman.W. High Resolut. 91. R. ‘High-performance Liquid Chromatographic Separation and Quantitation of Polyfunctional Amines as Their m-Toluoyl Derivatives’. R. ‘Determination of Isophenindamine in Phenindamine Tartrate Using an Argentated High-performance Liquid Chromatographic Mobile Phase’. Jefferies. 111 – 128 (1983).20 bipyridine)ruthenium(III) Chemiluminescence Detection’.M. ‘Determination of Metoprolol Enantiomers in Plasma and Urine Using (S)-( )-Phenylethyl Isocyanate as a Chiral Reagent’. ‘Fluorescence Derivatization of Tertiary Amines with 2Naphthyl Chloroformate’. Following Pre-column o-Phthalaldehyde Derivatization and Reversed-phase High-performance Liquid Chromatography’. 332 – 339 (1978). ‘Extraction Detector for High-performance Liquid Chromatography Using Solvent Segmentation of the Column Effluent’. Secondary Amine with Fluorescein-6-isothiocyanate’. Nieder. T.P. 272. Chromatogr. C. L. J. Clark. P. Chem. 63 – 72 (1987). Sci.A. 336. R. Mailhe.A. 102.F.A.D. 292. 351 – 358 (1983).A. 105. Pharm. Anal. Jaeger. ‘Reversed-phase Liquid Chromatography of Basic Drugs and Pesticides with a 95. ‘Dosage de l’Heptaminol dans des Preparations Pharmaceutiques par Chromatographie Liquide Apres Derivation par le 4-N. Pflugmann. A.. 561 – 569 (1983). G. Nicolas.A. Danielson.B. Chromatogr. Chromatogr.L. Strojny. P. J. Sci. B.F.P.. R. 141 – 151 (1979). M. Thompson. 101. Leroy. 24. 83.A.C.J. T. and Its Application to Biopharmaceutic Investigations’. an Antidepressant Agent. ‘Rapid Analysis of Amino Acids Using Pre-column Derivatization’. Wintersteiger. 100. Frey. Frei.. Chromatogr. Chromatogr. Umagat. Gubitz.C.M. J. 218. I. ‘Precolumn Derivatization of Amines for Enhanced Detectability in Liquid Chromatography’.. M. Frey... 468 – 472 (1984). Wellons.A. Gfeller. N.F. N. J. Frei.Th.M..M.M. Chromatogr. van den Berg. J. ‘Liquid Chromatography of Aliphatic Trialkylamines with Post-column Chemiluminescent Detection Using Tris(2. J. 96. Chromatogr. R. R. J. U. ‘An HPLC Fluorescence Detection System for Amines’.J. 52. 87. ‘A Post-column Extraction System for the Determination of Tertiary Amines by Liquid Chromatography with Chemiluminescence Detection’. Brown. J. J.. 90. Commun. Chim. 419. Thevenin.R.. Bidlingmeyer.F. Y.W. 161 – 164 (1987). Honigberg.. 269 – 278 (1988). Kwakman. S.C. Sci. G. F. 99. R.B.. 16. Photochemical Reaction and Fluorescence Detection. G. J. 207 – 217 (1987). Spruit. ‘Highly Sensitive Determination of Chlorpheniramine as Fluorescence Derivative of Highperformance Liquid Chromatography’. Lawrence. 313 – 331 (1985). R.D. Lawrence.M. 344. J. Frei. ‘Indirect High-performance Liquid Chromatographic Resolution of Racemic Tertiary Amines as Their Diastereomeric Urea Derivatives After N-Dealkylation’. J. 88. Chromatogr. Holeman.NDimethylaminoazobenzene-4-isothiocyanate’. Brinkman. Fluorigenic Ion-pair Extraction Detector’. 520 – 524 (1987). SevillanoCabeza. 103. J. Commun. J. J. A. J. G. 700 – 704 (1980). N. 1091 – 1094 (1975). Campins-Falco. 420. J.G. H. Mutschler. Brinkman.H. J. 2. ‘Determination of Amines Using the Fluorimetric Ionpair Technique as a Post-column Reactor for Highperformance Liquid Chromatography’. J. P. Miyamoto. 272. ‘Determination of Tamoxifen and Metabolites in Human Serum by Highperformance Liquid Chromatography with Post-column Fluorescence Activation’. J.A.M. Trumpler. ‘Postcolumn Derivatization Using the Fluorometric Ion-pair Technique’. Farrell..Th. C.. Carey. J. J. J.A. E. V. 213 – 214 (1978). ‘An Investigation of Highperformance Liquid Chromatographic Methods for the Analysis of Amphetamines’. G. J. 97. P. N. R.D. Anal. A. L. 73 – 80 (1979). U.Th.J. J. . de Jong.. 277 – 284 (1994).L. Honigberg..L. Chromatographia. Noffsinger.. Thevenin. J. 375 – 380 (1987). U. Maibaum. A. J. 171. Bauer. ‘Simultaneous Determination of Nicotine. 84. H. Chromatogr.J.. J. Chromatogr. I. J. Moreau.Th. 282. 93 – 104 (1984). H. Chromatogr. Cotinine and Five Additional Nicotine Metabolites in the Urine of Smokers Using Pre-column Derivatisation and High-performance Liquid Chromatography’. Tscherne. Chromatogr.W. J. Spahn.W. Frei. 125 – 136 (1997). B. ‘Quantification of Tamoxifen and N-Desmethyltamoxifen in Human Plasma by Highperformance Liquid Chromatography. Chromatogr. J. and Its Major Metabolites in Human Plasma by High-performance Liquid Chromatography with Fluorometric Detection’. 92. 395 (1987). J. Chromatogr.. Barlow. Huen. Brinkman. ‘Determination of Diclofensine. R. Pharm. I. 387. Wells. 74. Jordan. Chromatogr. 413.M. 104. 11 – 14 (1979). Gfeller. ‘Liquid Chromatography of Antihistamines Using Post-column Tris(2. 86. Chromatogr. 436.R. Danielson. 89. A. M. Chromatogr. ‘Derivatization of Amines in Solidphase Extraction Support with 4-Fluorenylmethyl Chloroformate for Liquid Chromatography’. ‘Electrochemical Detection of Sympatomimetic Drugs. S.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 27 82. Bain. Chromatogr. de Silva.. Chromatogr. R. 51 – 56 (1981). U. 93. van Buuren. J. Lammers. 489 – 493 (1978). Chromatographia. F. ‘Automated Oncolumn Derivatization of the Antibiotics Phosphinothricin and Phosphinothricyl-alanyl-alanine with PHARMACEUTICALS AND DRUGS 107.J. Haginaka. Wakai. 525 – 527 (1983). P. 56. G. J. Rice.. 2. 281. 101 – 111 (1987). Beek. Kubo. S. Anhalt.J. ‘Gentamicin C: Separation of C1 . R. Tawa. 58. A. 298. Kobayashi. 112. A.J.M.. Chromatogr. ‘Gentamicin Determination by High-performance Liquid Chromatography’.. R. G. K. Westerlund. Rindler. 445 – 457 (1984). Hamilton. Plaga. 16. 229 – 241 (1987). Anal. Vanderhaeghe. J.E. Schuster. 121 – 129 (1987). W. M. 153. J. 315. Kinoshita. Maitra. Guze.M. Plaga.. 357 – 367 (1985). Blanchflower. T. Wakai. M. R. J. 25. H. ‘Determination of the Antibiotic Fludalanine in Plasma and Urine by High-performance Liquid Chromatography Using a Packed-bed. ‘Determination of Amikacin Isomers by High Pressure Liquid Chromatography’. J. J. Narasin and Salinomycin in Feeds Using Post-column Derivatisation’. Chromatogr. Chromatogr..M. Yoshikawa.T. S. J. 338. Buchberger. Chromatogr. Carlqvist. Seidl. D. J. M. Meat and Milk Using Post-column Derivatization with Dimethylaminobenzaldehyde’. G. Chromatogr. Chem. 435. Chromatogr. Hwang. 111.. W. H. Weigand. Wakai. 129. 97 – 112 (1988). Saunders. J. Rogers.J. Using Fluorescence Detection Following Post-column Derivatization’. 109. J. Schotz.. ‘Separation of Amino Acids and Antibiotics by Narrow-bore and Normal-bore Highperformance Liquid Chromatography with Pre-column Derivatization’. Liq. ‘Determination of Spectinomycin by High-performance Liquid Chromatography with Fluorometric Detection’. 413. W. C1a . Chromatogr. Adlard. Anal. T. Chromatogr. Y. 116. 117. ‘Simultaneous High-performance Liquid Chromatographic Determination of Monensin. 103 – 108 (1979). J. 257.W. ‘Post-column Reaction Detector for Platinum(II) Antineoplastic Agents’. 123. ‘Gentamicin C-component Ratio Determination by High-pressure Liquid Chromatography’. 125. ‘Determination of the Component Ratio of Commercial Gentamicins by High-performance Liquid Chromatography Using Pre-column Derivatization’. D. ‘Liquid Chromatographic Determination of Amoxicillin and Its Metabolites in Human Urine by Postcolumn Degradation with Sodium Hypochlorite’. 120. ‘Monitoring of Veterinary Drug Residues by a Combination of Continuous Flow Techniques and Column-switching High-performance Liquid Chromatography.. H. Wakai.. Theodorsen. Scholl. L. 113. ‘Analysis of Penicillins in Biological Material by Reversed Phase Liquid Chromatography and Post-column Derivatization’. ‘Simultaneous Quantification of Cycloserine and Its Prodrug Acetylacetonylcycloserine in Plasma and Urine by High-performance Liquid Chromatography Using Ultraviolet Absorbance and Fluorescence After Post-column Derivatization’. Holt.. J. K. and C2b Components by HPLC Using Isocratic Ion-exchange Chromatography and Post-column Derivatisation’..V. ‘High-performance Liquid Chromatographic Determination of Ampicillin and Its Metabolites in Rat Plasma. J. 128. Bayne.S. Holt. S. 124.W. C2 .P. 57. 115.F. T. 121. McCorkle. Haginaka. G. Chromatogr. 91 – 100 (1983). 108. S. Acta. Weber. Fresenius’ Z. Anal. 91 – 100 (1983). D. F.M. C. Chromatogr. 122. Koshide.F.P. Claes. M. Bayne. 114... 1568 – 1571 (1985). H.J. 126. J. Post-column Reactor with o-Phthalaldehyde and 2-Mercaptoethanol’. 119. 118.G. J.N. H. Coombes. 414. Analyst. Schmidt. Maglietto. ‘Sensitive and Selective Determination of Picogram Amounts of Ciprofloxacin and Its Metabolites in Biological Samples Using . Musson. M. J.. 110. Busson. Rogers. Sulfonamides in Egg.P.A.. ‘Fluorescence Determination of Streptomycin in Serum by Reversedphase Ion-pairing Liquid Chromatography’. Chromatogr. S. Haginaka. R. Anal. W. Uno. 353.T. Marsh.A.P. Musson. Sternson. Using Fluorescence Detection Following Post-column Derivatization’.28 106.. Steyn. 176. Aerts. I. 400. Fiedler. ‘High Performance Liquid Chromatographic Determination of b-Lactam Antibiotics. Yasuda. ‘Trace Determination of Penicillin V with High-performance Liquid Chromatography’. Repta. 2653 – 2655 (1986).G. Meyers. Chromatogr.Th. Sancilio.C. H. Anal. J. M. R. 58. Chem. Chromatogr. Nachtmann. Pharm. Takahashi. J.A. ‘Liquid Chromatographic Determination of Penicillins by Post-column Alkaline Degradation’. 187 – 214 (1979). Gravellese. ‘Liquid Chromatographic Determination of Penicillins by Postcolumn Degradation with Sodium Hypochlorite’.. 110. J.. Bile and Urine by Post-column Degradation with Sodium Hypochlorite’. o-Phthalaldehyde and Microbore Column High-performance Liquid Chromatography for Quantitative Determination in Biological Cultures’. Chromatogr. K. 257. Winsauer. D.L. Hirose. Nerad.. J. Chem. J. J. 425. 1896 – 1898 (1986). J. J. J. H. K. 219 – 226 (1987). T.M. H. A. 1283 – 1287 (1985).C. 210 – 216 (1986).E. T. D.. A. Katagi. Saunders. J. Fiedler. W. Maglietto.. 491 – 497 (1984). Suec. K. B. 381 – 385 (1983).B. Adlard. 823 – 836 (1979). U.D. 127. 169 (1988). Chromatogr. C2a . ‘High-performance Liquid Chromatographic Determination of b-Lactam Antibiotics. 143 – 152 (1988). Haginaka. Chem. 386. ‘Pre-column Derivatization of Sisomicin with o-Phthalaldehyde-b-mercaptopropionic Acid and Its Application to Sensitive Highperformance Liquid Chromatographic Determination with Fluorimetric Detection’. Brinkman. J. L.K. Chem. G. 325 – 336 (1988). Chromatogr. Nagai. D. 149. 1277 – 1281 (1985).D. Yasuda. 150. Iwamori. Chromatogr.4-Triazole and Mercury (II) Chloride’. J. K. ‘Determination of Cephalosporins and Decomposition Products by Liquid Chromatography with Indirect Electrochemical Detection’. 231. Binns. J.. 142.W. J. 134. Krishnamoorthy. Targove. 175. off. Haginaka. 138. P. Chem. K. J. Analyst. Blauw.A. 60. 276. Fabre. Chem.. 341. T. Barends. ‘New Detection Modes for the Determination of Cephalosporins and Their Decomposition Products’. Chromatographia. ‘High Performance Liquid Chromatography of Clindamycin Antibiotics Using Tris(bipyridyl)ruthenium(III) Chemiluminescence Detection’.. 505 – 509 (1990). 136. . 131. M.W. Nomura.L.Th. Haginaka. Chromatogr. 385 – 391 (1984). 333.W. M. 285 – 296 (1985). Gionti. Amoxicillin and Ciclacillin in Serum and Urine Using a Pre-column Reaction with 1. K.. 625 (1987).. Gusciora. T. 148. ‘Derivatization Techniques for High-performance Liquid Chromatographic Analysis of b-Lactams’.B.D. 145. 147. J. A. Holt. 348. C. H. Holt. Haginaka.. W. A. Smits. R. Wakai. 140. W. ‘High-performance Liquid Chromatographic Assay of Carbenicillin. Kryger. Anal. 344. Chromatogr. T. 141. Mancini. R. 1099 – 1106 (1979). A.E. Shah.S. Chromatogr. Birkett. ‘High-performance Liquid Chromatographic Assay of Ampicillin. 4. ‘Highperformance Liquid Chromatographic Determination of Penicillins Following Derivatization to Mercurystabilized Penicillenic Acids’. 235. Jenkins..CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 29 130. Chem. 2165 – 2181 (1998). Helboe. 144. Nakagawa. J. 297. K. ‘Analysis of D-Penicillamine in Plasma by Fluorescence Derivatisation with N-[p-(2Benzoxazolyl)phenyl]maleimide and High-performance Liquid Chromatography’. J. J. E. Kok. G. Pharm. Gambardella. G. M. M. ‘Determination of Amikacin in Microlitre Quantities of Biological Fluids by High-performance Liquid Chromatography Using 1-Fluoro-2.A. M.M. ‘Automated Determination of Amoxycillin in Biological Fluids by Column Switching in Ion-pair Reversed-phase Liquid Chromatographic Systems with Post-column Derivatization’.J. D. D. P. R. ‘Analysis of Aminoglycoside Antibiotics as Benzoyl Derivatives 143. J. ‘Determination of Amikacin in Serum by Highperformance Liquid Chromatography with Ultraviolet Detection’.Th. H. Beaubien.2. Selavka. K. VanMeter.2. Westerlund. W. Chromatogr. Wakai. 1185 – 1188 (1985). Analyst. 139. Saunders. J. Liq. by High-performance Liquid Chromatography and Its Application to the Quantitation of Neomycin in the Perilymph’.. J. M.4-dinitrobenzene Derivatization’. Russ.. 21.W. J. Fabre. ‘High-performance Liquid Chromatographic Assay of Sulbactam Using Pre-column Reaction with 1. 69 – 72 (1984). Guadalupi. Adlard. J. M. Miller. Davis.. J. 62.H.. Anal. Katimy.O..T. J. J. 215 – 220 (1982). 24. R. Anal.M. Sci. 424. Miners. K. J. Yasuda. 110. Chromatographic Behavior and Recovery of Cephalosporins’. ‘Direct Serum Injection with Micellar Liquid Chromatography. Anal. ‘Reversed Phase Liquid Chromatography of Phenacyl Esters of Some Natural Penicillins’. J. Grushka.E. ‘Liquid Chromatographic Determination of Methscopolamine Bromide in Various Veterinary Formulations’. 229 – 240 (1985). J. J. M. 153. ‘Analysis for Penicillins and Cefoperazone by HPLC – Photolysis – Electrochemical Detection (HPLE – hn-EC)’. H. Adlard. Chromatogr. 73. Tsuji. G.J. Wong. Chromatogr. Tsuji. Chromatogr. Sci. M. 28. D. Liq. Pakuts. J. 33 – 41 (1978).. 337. Y. J. 187 – 193 (1985). 115 – 122 (1985). ‘Determination of b-Lactams and Their Biosynthetic Intermediates in Fermentation Media by Pre-column Derivatization Followed by Fluorescence Detection’. Neomycin B and Neomycin C in Neomycin Sulfate’. Harada. 1..4-dinitrobenzene’. 83 – 93 (1986). J.. Chromatogr. G. G. 145 – 154 (1982). ‘High-performance Liquid Chromatographic Analysis of Neomycin in Petrolatum-based Ointments and in Veterinary Formulations’. 132. 2019 – 2031 (1983). P. 110. 275. Wakai. 135.S. Chromatogr.R. S. Fearnley. J. ‘Improved High-performance Liquid Chromatographic Method for Simultaneous Determination of Neamine. Ticarcillin and Sulbenicillin in Serum and Urine Using Pre-column Reaction with 1. T. Blanchin. Lam.M. Chromatogr. Bratin.. C. J. J. K. 152. Rogers. M.4-Triazole and Mercury (II) Chloride’. 59. Carlqvist. Chromatogr. R.. P. L. Brooks. J. Liq. 2732 – 2734 (1987). J. 146. ‘Normal-phase High-performance Liquid Chromatographic Determination of Neomycin Sulfate Derivatized with 1-Fluoro-2. Chromatogr. High-performance Liquid Chromatography and Photothermal Post-column Derivatization’. J. 321 – 330 (1987). I. Assoc. Nakagawa. J. Montana. 137.M. Chromatogr. Mangia.J. A. Hulshoff. ‘Development and Validation of a Stability-indicating HPLC Method for the Determination of Tobramycin and Its Related Substances in an Ophthalmic Suspension’. 141 – 152 (1979). 385 – 394 (1983). M.4Triazole’. Pharm. 136 – 141 (1988). Adlard.. Chromatogr.2. J. Saunders. I. Uno. H. 6. Chromatogr. Wakai. 133. H.. Whitehouse.P.A. Y. S. 151. Punziano. ‘Quantitative Determination and Separation of Analogs of Aminoglycoside Antibiotics by High-performance Liquid Chromatography’.W. Tsuji. C. ‘Derivatization of Secondary Amines with 2-Naphthalenesulfonyl Chloride for Highperformance Liquid Chromatographic Analysis of Spectinomycin’.B. Hartman. Smith. Kok. Goetz. J. A. N. Haginaka. Biomed. 89 – 96 (1983)..F. McCleary. 416.. Danielson. J.. 365 – 380 (1985). Rogers. Krull. J. 591 – 595 (1987).. 367 – 372 (1980).F. 391.P.E. J.. J. R. ‘Selective On-line Sample Handling for the Determination of Barbiturates in Urine by Liquid Chromatography with Precolumn Technology’. Brown. Hiroshima. 419 – 426 (1987). 69. 164. 166. H. J. L. Angus. 170. J.W. Chromatogr. Roseboom. Kimata..T. Chromatogr. 635 – 637 (1978). J. Pharm. 156.H. ‘High-performance Liquid Chromatographic Separation and Determination of Prostaglandins. S. Tjaden.R. Bile Acids and Prosta-glandins by Derivatization to an Electrochemically Active Form’. 1089 – 1091 (1983). T. Doehl.S.-L. J. Clark.W.W. J. R. C. 175. 186. 72. Chromatogr. R. 271 – 275 (1982). Chromatogr. Greibrokk. T. Chim. Lindner. 271 – 275 (1982). Meier. Renema. J. Chromatogr. J. J. L. ‘Determination of (15R). A. Anal. 162.W. Langenberg.L. Hashida.. Cox. Santi. L. ‘High-performance Liquid-chromatographic Assay for Prostaglandins with the Use of p-(9-Anthroyloxy) phenacyl Bromide’. Seki.A.E. IV. 176. Lindner. Pullen. Veazy. ‘Determination of Tetraphenylacetone in Human Plasma by High-performance Liquid Chromatography with Fluorescence Derivatization Using Dansylhydrazine’. M. W.H. Tjaden. . Stein. M. T. J. Acta. W. Chem. J. 535 – 541 (1979).. 305. ‘Electrochemical Detector for High Performance Liquid Chromatography. 395.L.and (15S)-15-methylprostaglandin E2 in Human Plasma with Picogram per Milliliter Sensitivity by Columnswitching High-performance Liquid Chromatography’. S. W. Miller. a Synthetic Prostaglandin’. T. 158. ‘Application of Postcolumn Ionization in the High-performance Liquid Chromatographic Analysis of Butabarbital Sodium Elixir’. Hirosawa... Sci. 61 – 72 (1984).Th. 349. M. ‘Rapid and Sensitive Determination of Dehydroascorbic Acid in Addition to Ascorbic Acid by Reversed-phase High-performance Liquid Chromatography Using a Post-column Reduction System’. Scott. Hatsumi. J. J. Oxidized by Pyridinium Dichromate’. 172.. Bull. Pharm. 381. ‘Rapid Concentration and Analysis of Short Chain Carboxylic Acids: Variation on a Theme’. Keating. 249 – 255 (1982).. ‘Simultaneous Determination of Ascorbic Acid and Dehydroascorbic Acid by Reversed-phase Ion-pair High-performance Liquid Chromatography with Pre-column Derivatisation’. S. Chromatogr.R.A. 176. 173. Chromatogr. Chromatogr. 171. J. C.A. Anal. ‘Comparison of Two Highpressure Liquid Chromatographic Assays for Carboprost. Danielson. J.R.. Borrero. ‘High-performance Liquid Chromatographic Separation and Ultraviolet Detection of Prostaglandins. 192. 163. 431 – 437 (1986). 178. U. 1396 – 1399 (1980).R. R. J. Chromatogr. Hatsumi. K. ‘N-chloromethylphthalimides as Derivatization Reagents for High-performance Liquid Chromatography’. 282. O. B. Thomas.W. 168. 343. Wolf. 13. T. B. L. Patience. P.W.. J.D. Chromatogr. Brinkman. 435 – 442 (1983). J. 253.D. 271 – 283 (1985). 253. 2207 – 2216 (1990). 410 – 415 (1988). K. 2365 – 2369 (1985). ‘9-Anthryldiazomethane Derivatives of Prostaglandins for Highperformance Liquid Chromatographic Analysis’.. N. Langenberg. Karnes. Chromatogr. P. Sci. Frei. Kanazawa. J. Optimization and Applications’. J. Haddad.. Analyst. 1573 – 1579 (1996). 177. 431 – 438 (1985). J. Mukherjee. U.-I.. H. Kimata. ‘Improved Detectability of Barbiturates in High Performance Liquid Chromatography by Post-column Ionization’. O. Sticher. 121.W. Single and Dual Electrochemical Detection’. Chromatogr. Oxidized by Pyridinium Dichromate. 50. J. 161. Schmid. 200. J. E. Liq. Chem. Chromatogr. 377 – 385 (1984). Royer. ‘Fluorimetric Determination of Danthron in Pharmaceutical Tablets and in Urine’. 2941 – 2947 (1980). ‘Chemiluminescence Highperformance Liquid Chromatographic Detector Applied to Ascorbic Acid Determinations’.P. Chem. J.. Pullen. R.. Ziegler. 153 – 162 (1980)..H. J. ‘Improved Detectability of Barbiturates in High-performance Liquid Chromatography by Pre-column Labelling and Ultraviolet Detection’. 174.. 234. Pharm. Doehl.P.. ‘9-Anthryldiazomethane Derivatives of Prostaglandins for Highperformance Liquid Chromatographic Analysis’.E. T. Kawabe. 165. 28. R. Auguste. 245. PHARMACEUTICALS AND DRUGS 155. 55 – 64 (1979). 293 – 299 (1987). ‘Determination of (Endogenous) Vitamin K1 in Human Plasma by Reversedphase High-performance Liquid Chromatography Using Fluorometric Detection After Post-column Electrochemical Reduction. M. N. K. ‘Reaction of 5-Bromomethylfluorescein (5-BMF) with Cefuroxime and Other Carboxyl-containing Analytes to Form Derivatives Suitable for Laser-induced Fluorescence Detection’. Analysis of Fatty Acids. U. Ohmae. ‘Isolation of Plasma Components by Double Antibody Precipitation and Filtration: Application to the Chromatographic Determination of Arbaprostil [(15R)-15-methylprostaglandin E2 ]’. 424. J. 167. 289. 57. Wise. ‘Enhanced UV-detection of Barbiturates in HPLC Analysis by On-line Photochemical Reaction’. Nielen.30 154. B. DeJong.J. Cox. ‘N-Chloromethyl-4-nitro-phthalimid als ¨ Derivatiserungsreagens fur die Hochleistungs-Flussigkeits-Chromatographie’. J. Chromatogr.. ‘Improved Method for the Determination of Vitamin K1 Epoxide in Human Plasma with Electrofluorimetric Reaction Detection’. 157. Chromatogr. Greibrokk. Hirosawa. Chan. S. 225 – 230 (1982). Ikenoya.. R. J. Carpenter. 169. Comparison with Ultraviolet. 159. Hulshoff.. Chromatogr. E. M. Anal. Chromatogr. Nieman..J. 198. Chromatogr. 160. J. Nakamura. 13.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 31 179. M. Ishida. ‘Determination of Total Ascorbic Acid in Human Serum by High-performance Liquid Chromatography with Fluorescence Detection’. ‘Resolution of Enantiomers of Norepinephrine and Epinephrine by Reversed-phase High-performance Liquid Chromatography’. 327 – 330 (1981). Van Boekel. Gorham. 176 – 180 (1983).. Wada.. F. 261 – 267 (1985). J. K. Chromatogr. 344. 249 – 255 (1980). J. Xie.. N.. Nishimura. Chromatogr.J. 415.A. T. Kormos.A.W. H. Leinfelder. ‘Determination of Plasma Aspirin and Salicylic Acid Concentrations After Low Aspirin Doses by High-performance Liquid Chromatography with Post-column Hydrolysis and Fluorescence Detection’. 344. S. Brinkman. J. A. Ingalls. Johnson. J. 31 – 40 (1998). Y. Chromatogr. 182. Chromatogr. Hutt. 193. 410. Mitsui. 342. Chromatogr. ‘Determination of Dopamine-3. A. 185.E. Kinoshita. Kinoshita. Gerhardt. Nohta. ‘High-performance Liquid Chromatography of Plasma Catecholamines Using 1. 261. 195. Chromatogr. Yui. Y. L. 271 – 283 (1984). W. Urine.. Chromatogr. Wainer. Applied to Analysis of Ascorbic Acid in Beer’. 199.and -4-o-Sulfate in Human Plasma and Urine by Anion-exchange Highperformance Liquid Chromatography with Fluorimetric Detection’. Brater. 202. ‘Separation and Detection of Small Amounts of Catecholamines by Highperformance Liquid Chromatography’. J. 184. Lou. 183. Yanagihara. Iwata. H. 201. M. C. Yamaguchi. D. Gehrke. Kimura.. Dasler. J. J. Sacchi. 197. ‘Determination of Carnitine. Chromatogr. Hara. 181. E. A.A. 190. M. T. D. ‘Determination of Ascorbic Acid in Human Urine by High-performance Liquid Chromatography Coupled with Fluorimetry After Post-column Derivatization with Benzamidine’. A. Okamoto. 107 – 111 (1983).. 268.. Weir. Bochner. Zanoboni-Muciaccia. C. 385.M. Chromatogr. Nimura. J. M.H.E.. Yamaguchi. 221. Zou.L. ‘Highly Sensitive Measurement of Indomethacin Using a Highperformance Liquid Chromatographic Technique Combined with Post-column In-line Hydrolysis’.-Q. ‘Determination of the Enantiomeric Composition of Ibuprofen in Human Plasma by High-performance Liquid Chromatography’. and Tissue’. Hoppel. C. C. Chemilumin.W. 167. 188. A. Yamaguchi.. Steijger. 177. G. A. Y.D.C. J. 1.D. Chem..-D.S. 415. Y. Itokawa. T. K. 245 – 254 (1985). Doyle. J. Liq. 336. 192. Nimura.Th. Davis. Gehrke. Chromatogr. P. Seibert. Chromatogr. J. ‘Comparison of the Sensitivity of Various Post-column Methods for Catecholamine Analysis by High-performance Liquid Chromatography’. A. Y. 350. J. 339. Glycinebetaine Aldehyde and Related Compounds’. ‘Novel Post-column Derivatization Method for the Fluorimetric Determination of Norepinephrine and Epinephrine’.D. J. ‘An Acridinium Sulfonylamide as a New 191.M. U. 198. 61 – 70 (1985). and Betaine as 40 -Bromophenacyl Ester Derivatives by High-performance Liquid Chromatography’.O. ‘Fluorimetric Determination of Catecholamines Using Glycylglycine as the Reagent . 24. J. Butyrobetaine. 206. Jr. D.. Yui. Kuo. 1317 – 1324 (1978). Hamidzadeh. M.B. J. De Zeeuw. ‘High-performance Liquid Chromatographic Method for the Separation and Estimation of Choline. X. Chemiluminescent Label for the Determination of Carboxylic Acids in Liquid Chromatography’. K. D. Chromatogr. Y. I. Chromatogr. Noguchi. C. Ishida. H. Clin. S. 196.N 0 -diisopropylisourea by High-performance Liquid Chromatography and Ultraviolet Absorption’. Chromatogr. 376 – 379 (1979). Asai. 586 – 588 (1981). ‘Determination of the Enantiomers of Indoprofen in Blood Plasma by High-performance Liquid Chromatography After Rapid Derivatization by Means of Ethyl Chloroformate’. Williams. Chem. Avgerinos. O. A. 189.. C. J.. Schieffer. Chromatogr.C. Yamatodani. ‘Resolution of Norephedrine as its 2-Oxazolidone Derivative: Enantiomeric Separation on a Chiral Highperformance Liquid Chromatographic Stationary Phase and Preparative Regeneration of the Resolved Isomers’. 339 – 346 (1985). 157 – 162 (1986). Forgione. C.J. 214 – 220 (1987).. T. Minkler. M..M. M.P. ‘High-performance Liquid-chromatographic Separation and Fluorescence Measurement of Biogenic Amines in Plasma.A. Yamamoto. Biolumin. Gustafson. Pedrazzini. ‘Pre-column Electrochemical Cell for High-performance Liquid Chromatography’. Y. 351 – 355 (1985). H. 287 – 291 (1987). Kawai. Kasahara. ‘Determination of Flunoxaprofen Enantiomers in Biological Fluids by High-performance Liquid Chromatography’. 186. 217 – 221 (1987). ‘Determination of Artesunic Acid After Chemical Derivatization with o. 213.. Ohkura.. T. 420. Kawai. T.J. 187. Chromatogr. 51.W. Chromatogr. J.. C. 75 – 83 (1987). ‘Ultramicro Method for the Determination of Picogram Amounts of Norepinephrine and Epinephrine by High-performance Liquid Chromatography’.W. 205 – 217 (1978).pNitrobenzyl-N. McDonnell. Chromatogr. Y.. K. K.. Bjorkman. D. Chromatogr. S. Aldridge. Meeuwissen. T.L. ‘Highperformance Liquid Chromatography of the Bis(dinitrophenyl)hydrazone Derivative of Ascorbic Acid. T. Cunningham.D.S. Lingeman. Mell. 180. J. T. J. 380. 200. 1573 – 1575 (1979). 261 – 277 (1978). Chromatogr. 194. J.2-Diphenylethylenediamine as Precolumn Fluorescence Derivatization Reagent’.-I. Z. ‘Pre-column Fluorescent Derivatization for High Pressure Liquid Chromatography with o-Phthalaldehyde: Separation of Urinary Catecholamines’. J. N. K. J.T. Y.. Seki. S.. Kamminga. Seki. L.R. Anal. 425 – 431 (1987). M... S. Gal. T. Roos. V. 360. J.o-Dibenzoyltartaric Acid Anhydride’. 112 – 114 (1987). Chromatogr. Chromatogr. Chem. Yajima. Assoc. B.. ‘Studies in Steroids. Ruud-Christensen. M.J. ‘Estimation of Catecholamines by Ion-exchange Chromatography on Asahipak ES-502C. Salvesen. Determination of Serum Bile Acids by High-performance Liquid Chromatography with Fluorescence Labeling’. Furusawa. Chromatogr. C. Van Den Berg.-D. J. 329. Yamaguchi. J.T. Walle. R.W. Hoshino. 414 – 419 (1983). 58.M.K. 692 – 695 (1980).A. Pharm. 661.W. 214. 311 – 313 (1979). Lynn. ‘Identification of Anabolic Stilbene Derivatives in Bovine Urine by High-performance Liquid Chromatography and Off-line Chemiluminescent Immunochemical Detection’. ‘Rapid.. H. H. L. Specific Method for Diethylstilbestrol Analysis Using an In-line Photochemical Reactor with High-performance Liquid Chromatography and Fluorescence’. 189 – 194 (1988). Kiba. J. Chikai. Hubbard. Anal. B. I. J. 9 – 13 (1985). Stephany. Yanagihara. K. Gripshover. 217. Chromatogr. ‘Sensitive and Specific Postcolumn Fluorimetric Determination of Diethylstilboestrol Residues in Extracts of Urine and Animal Tissues at the 1-ppb Level’. ‘Determination of Catecholamines by High-performance Liquid Chromatography with Catalytic Photometric Detection’. Parkin. 433 – 435 (1984). J. ‘Determination of Cyclodextrins in Biological Fluids by Highperformance Liquid Chromatography with Negative Colorimetric Detection Using Post-column Complexation with Phenolphthalein’. 226. ‘High-performance Liquid Chromatographic Assay of Menthol Using Indirect Photometric Detection’.32 for Post-column Derivatization’. J. . M. Demian. Noguchi.. Seamark. ‘Application of Precolumn Reaction to High-performance Liquid Chromatography of Qinghaosu in Animal Plasma’. J. 436 – 439 (1984). T. Tscherne. X. 235. G. R. Chromatogr. D. G. Off. 267. R. Chromatogr. J.H. 213. M.A. 215. R.R)o. J. R. E. J. Visser. 99 – 110 (1985). 207. J. 345. Chromatogr. U. ‘Sub-nanogram Detection of Dihydroartemisinin After Chemical Derivatization with PHARMACEUTICALS AND DRUGS 203.J. M. 281 – 289 (1994). J.D. Okada. 461 – 470 (1982).. N. 225. Chromatographia. Chromatogr. J. T.J. 210. 147 – 152 (1985). D. 216. C. 287. ‘Use of a Post-column Immobilized b-Glucuronidase Enzyme Reactor for the Determination of Diastereomeric Glucuronides of Fenoldopam in Plasma and Urine by High-performance Liquid Chromatography with Electrochemical Detection’. 224. 219. Anal. J.. Diacetyldihydrofluorescein Followed by High-performance Liquid Chromatography and UV Absorption’.. J. K. 239 – 255 (1983). J.E. Chem. J. ‘Determination of Nadolol Diastereomers in Dog Plasma Using Chiral Derivatization and Reversed-phase Highperformance Liquid Chromatography with Fluorescence Detection’. T. 325 – 333 (1987). Y. Z. 276. 213 – 216 (1985). 218. Chromatogr.J. J. Sedman.H. K. 209. Chromatogr. Goto. R. 63. 222. A.K. ‘Use of Borohydride Reduction in the Separation of Estrogen Carbonyls’. R. Schladitz-Keil. Verbeke. Walle. Seki. Using Glycylglycine as the Post-derivatizing Agent’. 231 – 247 (1986). 449 – 451 (1986). Trafford. ‘Separation of Enantiomers of Derivatised or Underivatised Propranolol by Means of High-performance Liquid Chromatography’. ‘Separation and Quantitation of Esterified Estrogens in Bulk Mixtures and Combination Drug Preparations Using High-performance Liquid Chromatography’. Both-Miedema. A.. 407 – 412 (1984). E. Mei-Yi. B. J. 624 – 627 (1979). Chromatogr. Mizuno. Gupta.J. 387.. 424.W. 341. J.6-Dibromoquinone4-chlorimide’. Belloli. D. A. J. 208.. Vanhee.W. 265.. J. Okahira. 353. N. Ziemniak. 424 – 430 (1984). Fong. Christ. R. Medwick. T. ‘Separation of (R)and (S)-Proxyphylline as Diastereoisomeric Camphanates by Reversed-phase Liquid Chromatography’. 223.C. Shishan. M. 17. Midha. Capitano. T.. ‘Post-column Derivatization of Vitamin B6 Using 2. M. Chromatogr. ‘Separation of the Enantiomers of Intact Sulfate Conjugates of Adrenergic Drugs by High-performance Liquid Chromatography After Chiral Derivatization’. Boppana. 206. ‘Silica Gel High-performance Liquid Chromatography for the Simultaneous Determination of Propranolol and 4-Hydroxypropranolol Enantiomers After Chiral Derivatization’.. P.. T. Jansen. Saito.H.. Lou.C. 289 – 300 (1983).. Frijlink. Chromatogr. Drenth.... Wilson.A.B. Spahn. 303. T..F. Sci. 303. 212. 220. 211. ‘Fluorimetric Determination of the Quaternary Compound Trospium and Its Metabolite in Biological Material After Derivatization with Benoxaprofen Chloride’.-L. J. 271 – 273 (1980). ‘Resolution of the Enantiomers of Propranolol and Other Beta-adrenergic Antagonists by High-performance Liquid Chromatography’.H. Walle.R. Sci. 289 – 292 (1986). Chromatogr.L. 278. ‘High-performance Liquid Chromatographic Determination of Enantiomeric Purity of 1-Methyl-3-pyrrolidinol via Derivatization with (R. E. 23. Z. Chromatogr. Williams. Chromatogr. Chromatogr. Fujita. Chromatogr. 199 – 203 (1983). Goto. Chromatogr. J.K. 415. Y.F. G. J. Nambara. Y. T. K. 532 – 535 (1987). Clark.. 205. Suzuki. 204. J. 68. 310. 197. Kawamoto. Wilson. ‘High-performance Liquid Chromatography of Vitamin D: Enhanced Ultraviolet Absorbance by Prior Conversion to Isotachysterol Derivatives’. J. Zomer.. 332. Enkelaar-Willemsen. Mutschler. M. Chromatogr.K. 221. Chromatogr. CLXXXVII. Winfield. J. ‘On-column Periodate Reaction Method for Analysis of Ephedrine Sulfate in Solid Dosage Forms: Collaborative Study’. Xie. Komatsu. F. M. Hamada.H. J. ‘Analysis of Biological Thiols: Quantitative Determination of Thiols at the Picomole Level Based Upon Derivatization with Monobromobimanes and Separation by Cation-exchange Chromatography’. E. ‘Development of a Highperformance Liquid Chromatographic Assay for Digoxin Using Post-column Fluorogenic Derivatization’. E. 14. 245. G. A. 240. E. 244. ‘Chemiluminescence with Lucigenin as Post-column Reagent in High-performance Liquid Chromatography of Corticosteroids and pNitrophenacyl Esters’. ‘New Sensitive Derivatization of Hydroxysteroids for High-performance Liquid Chromatography with Fluorescence Detection’.S. ‘New Derivatization Reagent for the Resolution of Optical Isomers in Diltiazem Hydrochloride by High-performance Liquid Chromatography’. Cavrini. Maiorino. 374. 381. T. ‘Fluorimetric Determination of 2. ‘Derivatization of Ketosteroids for High-performance Liquid Chromatography with Electrochemical Detection’. Chromatogr. Tanaka. Reh. ‘Determination of Thiol Compounds by Selective Fluorescence Derivatization and HPLC’.M. 239. B. Mueller. Kwong. 18. E. Gatti. A. Robertson. 1779 – 1784 (1980). Suzaki. 233. N. Chromatogr.. Acta. 417.. T.. J. K. Maeda. 23. Iwasaki.. Ishii. Shimada. R.C. Y. J. McErlane. Nambara. Chromatogr. G. M. . 147. ‘Digoxin and Metabolites in Urine and Feces: A Fluorescence Derivatization – Highperformance Liquid Chromatographic Technique’. Anal. V. Chem. 297 – 310 (1986). Tsuji.. 282. Kuroda. Chromatogr. F. M. DiPietra.6-disulfonate’. 1129 – 1136 (1980). G. S. 303. 357 – 365 (1981). Chromatogr. 426 – 431 (1985). ‘High-performance Liquid Chromatography of 2-Mercaptoproprionylglycine and Its Metabolite 2Mercaptoproprionic Acid in Plasma and Urine After Treatment with Thiopronine’. Part 1. J.. Y. Anal. Anal. 680 (1987).W.Th. Y. Reuning. J. Auswahl Chemischer Reaktionen am Beispiel von Steroiden’. Chromatogr. J. Selection of Chemical Reactions’. 377. ‘Separation of Digoxin from Dihydrodigoxin and the Other Metabolites by High-performance Liquid Chromatography with Post-column Derivatization’. Chromatographia. J.W. R. Kwong. K. Chromatogr. Birks. Fresenius’ Z. 13. T. Shimoda. Chromatogr. 236. 1 – 12 (1981). ‘New Fluorimetric Detection Method of Corticosteroids After High-performance Liquid Chromatography Using Post-column Derivatization with Glycinamide’. Nishi..A. 397 – 400 (1983).. Tsuji.. McErlane. ‘Determination of Digoxin. 248. Kosower. 375 – 380 (1983). 242. 382.. Lett. Anal. Tsumagari. 147.A..M. G. Fahey.A. 247.H. Desta. Chromatogr.M. Chromatogr. Goto. ‘Application of Dansyl Derivatization to the High Pressure Liquid Chromatographic Identification of Equine Estrogens’. Lankmayr. J. 246. 119 – 125 (1986). P. H. S. ‘Sensitive Derivatization Reagents for Thiol Compounds in Highperformance Liquid Chromatography with Electrochemical Detection’..A. K. Carlsson. Nambara. Chromatographia. 357. ‘Analysis of Digoxin at Therapeutic Concentrations Using High-performance Liquid Chromatography with Post-column Derivatization’. A. M. 1131 – 1138 (1983).M. Chim. J. Medwick. Denneberg. 249. 352. ‘Determination of 17Oxosteroids in Serum and Urine by Fluorescence Highperformance Liquid Chromatography Using Dansyl Hydrazine as a Pre-labeling Reagent’. Goto. 193 – 209 (1983).M. T.. J. Chandrasekaran. Kakimoto. R. 235. 380. Chromatogr.W.3-Dimercaptopropane-1sulfonic Acid and Other Dithiols by Precolumn Derivatization with Bromobimane and Column Liquid Chromatography’. 421. 371 – 374 (1979).V. 626 – 630 (1980). Schwedt. Hoppe. M. R. Yamaguchi. Sci. Shamsa. Nambara. H. 13.. K. N. Reh. R. 213 – 220 (1986). 237. T. R. 243. T. T. G. R. T. Andersson. Dorian. 357 – 363 (1986). 89 – 98 (1986). C. R. Frei. 349 – 354 (1986). 232. 341. 234. 117 – 120 (1980). Anal. B. 261 – 267 (1987). Teil 1. ‘HPLC Determination of Thiol Drugs in Pharmaceutical Formulations Using Ethacrynic Acid as a Precolumn Ultraviolet Derivatization Reagent’. Waldorff.. Sams.L. K. E. ‘Liquid Chromatographic Detection of Cardiac Glycosides and Saccharides Based on the Photoreduction of Anthraquinone-2. 295. Jakobsen. J. K. K. Reh. ‘Fluorometric Reaction Detectors for the HPLC Analysis of Steroids’. Chem. M. A. Kagedal. Biochem. Gandelman. T. Schwedt. Nachtmann. M. Digoxigenin and Dihydrodigoxigenin in Urine by Extraction. L. A. 238.CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS 33 227. E.. Schwedt. ‘Fluorimetric Reaction Detectors for High-performance Liquid Chromatography. Ohkura. Liq. Chromatogr. 228. Chromatogr. E. M. Seki. Caldwell. J. 233 – 242 (1986). 226. Kawasaki. J. Chromatogr. Aposhian. Maeda. J. Chromatographia. Fujimoto. ‘3Chloroformyl-7-methoxycoumarin as a Fluorescent Derivatization Reagent for Alcoholic Compounds in Liquid Chromatography and Its Use for the Assay of 17Oxosteroids in Urine’. ‘Aufbau und Optimierung Fluorimetrischer Reaktionsdetektoren mit Luftsegmentierten Flussigkeitsstromen fur die Hockdruck-Flussigkeits-Chromatographie.W. Acta. Chim.. Anal. J. 229. 381 – 386 (1987). R. Shimizu. Weber. Hui. 230.. Brinkman. J. 231. R.P. M. 111.W. Newton. Derivatization and High-performance Liquid Chromatography’. 249 – 253 (1981). 6. N. J. J.L. Raggi.L. Donnerberg. Tanaka. 241. U. K. T. Roos. Fresenius’ Z. Saito. Shepard. Budna. Deyl. E. Chromatogr. 661.. W. R. 260. 255.. Y.. J.J. Couderc.S.T. Pharm. Chromatogr.G. U. M. H.H. J.W. W. Chem. Yashiki. P.H.34 250. 2777 – 2790 (1998).H. J.J. Brinkman. Underberg. Brinkman. 424. ‘Photochemical Reaction Detectors in Continuous-flow Systems – Applications to Pharmaceuticals’. ‘Analysis of the Antidiabetic Drug Acarbose by Capillary Electrophoresis’. ‘Drug Analysis by Capillary Eletrophoresis and Laser Induced Fluorescence’. 273. K. Nakajima. Irth.W.W. 1 – 17 (1994). 261. W. in Plasma by Column Liquid Chromatography and Post-column Derivatization’. W. Biomed. ‘Quenched Peroxyoxalate Chemiluminescence as a New Detection Principle in Flow Injection Analysis and Liquid Chromatography’. Lingeman. S.A.Th. J. 1203 – 1208 (1998). E. 463 – 471 (1981). R. Zhu. A. F. 2580 – 2583 (1985).L. 249 – 257 (1986). Chromatogr. W. F. 58. Velthorst. 433 – 439 (1998). 385.A. Chromatogr. B. PHARMACEUTICALS AND DRUGS 263.T.H. Column Switching and Ultraviolet Absorbance Detection’. A. Lingeman. J. Lingeman. 17. ‘Application of the Desulfurization of Phenothiazines for a Sensitive Detection Method by High Performance Liquid Chromatography’. Takashi. B.Th. Chem. De Jong. Miki. O. U... Pharm. Scholten.T.M. Ramseier. J. 249 – 253 (1997). A. Frei. Lee. 259. Babiuk. G. 295 – 311 (1998). Kerns. 266. Frei. 686. U.E. I. Welling. Kok. 354. J. Chromatogr. Thormann. U. 239 – 248 (1980). De Ruiter. Bayle.. Okun. M. W. Voogt. ‘Determination of Disulfiram and Two of Its Metabolites in Urine by Reversed-phase Liquid Chromatography and Spectrophotometric Detection After Post-column Complexation’. Liq. Hunt. ‘Capillary Electrophoresis Sodium Dodecyl Sulfate Non-gel Sieving Analysis of a Therapeutic Recombinant Monoclonal Antibody: A Biotechnology Perspective’.. 271.M. A. D. Frei. 200 – 204 (1994). Chromatogr. Krull. Chromatogr. 272. Klohr. Chromatographia. Westerlund. Yamashita. Scholten. ‘Palmitoyl Derivatives of Interferon a: Potential for Cutaneous Delivery’. G. ‘High-performance Liquid Chromatographic Determination of Busulfan in Human Serum with Online Derivatization. A. Electrophoresis. . Chromatogr. H. S.J. Takadate. 253.. Von Heeren. Brinkman. Matson.W. P. ‘Analysis of Amino Acid Enantiomers Derived from Antitumor Antibiotics Using Chiral Capillary Electrophoresis’. Anal. U. Gooijer. Frei. J. K. J. Anal. A. 258.L. Kok.1021/ac981209m S0003-2700(98)012098. M. B. Frei. Brinkman. 19. ‘Semi-conductor Laser Induced Fluorescence Detection in Capillary Electrophoresis Using Pre-column Thiol Derivatization’.J. Revilla. J. Chromatogr. 789 – 874 (1985). Z. 252. ‘Post-column Derivatization for Fluorescence and Chemiluminescence Detection in Capillary Electrophoresis’. R. H. C. Pharm. W. J. Lubal. Chromatogr. ISSN 0003-2700.W. Brinkman.. Havel. 265. Causse. A. C. A. J. Scholten.W. P. N. Anal. 17.H.Th. Chromatogr. Yamato. Chromatogr. 199. J. 57. Johansson. Frei. W. ‘Detection of Thioethers of Pharmaceutical Importance by Liquid Chromatography with Online Generated Bromine’. 274. Shimada. 205 – 210 (1996). Chromatogr. Chromatogr. R.Th. Liu. Wakabayashi. L. ‘Analysis of Fluorescein Isothiocyanate Derivatized Amphetamine and Analogs in Human Urine by Capillary Electrophoresis in Chip-based and Fused-silica Capillary Instrumentation’. Sci. ‘Determination of Amikacin in Human Plasma by High Performance Capillary Electrophoresis with Fluorescence Detection’. ‘On-line Electrochemical Reagent Production for Fluorescence Detection of Phenothiazines in Liquid Chromatography’.. S. Hulshof. Hu.W. U. ACS ASAP Article 10. Dabrah. High Resolut. Bardelmeijer.J. 717.A.A. T.M. 659. B. Kamminga.M. 1245 – 1248 (1986).S. J. B. C.M. J. Attah-Poku. 343 – 356 (1987). Welling. J. Oguri. H. ‘Fluorescence Detection in High Performance Liquid Chromatography’. Lingeman. J. Brinkman. V. Chim. Anal.Th.A. ‘Chiral Separation of Pharmaceuticals Possessing a Carboxy Moiety’. 16.M. 700. 267.Th. 269. J.H. 3 – 26 (1998). H. B. M. Electrophoresis.. ‘Use of PTFE in Post-column Photochemical Reactors for Liquid Chromatography – Application to Pharmaceuticals’. Hamacek.A. ‘Determination of NAcetylcysteine. G. Voogt. 660.L. 85 – 91 (1994). Nashabeh. Hughes.M. A. K.. 675. ‘General Strategies and Selection of Derivatization Reactions for Liquid Chromatography and Capillary Electrophoresis’. ‘Determination of Rimantadine in Pharmaceutical Preparations by Capillary Zone Electrophoresis with Indirect Detection or After Derivatization’. Foldvari. Halvax. 2967 – 2975 (1998). W. Q. Kok. C. Intact and Oxidized. Li. 137 – 146 (1980). Blaschke. A. van Zoonen. 797 – 798 (1994).T. Chem. Funakoshi. Frei. Anal. 207 – 214 (1997). 264. Acta. J. T. J. 8. 114. R. H. 270. 254. Aak. W.M. R. J.H. S. R. Kruger. Biomed. Anal. B. G. H.T. 313 – 319 (1996). Rethfeld.Th. K.. Kozlov. ‘Derivatization in Capillary Electrophoresis’. T. V. J.A. 262. R. Chao.A. Volk. 256.J. H. Yamaguchi. 95 – 102 (1988). 217.Y. ‘Liquid Chromatography of Demoxepam and Phenothiazines Using a Post-column Photochemical Reactor and Fluorescence Detection’. 87. S. 257. Chromatogr.Th.A. 19. Underberg. Mank. 807. I. M.A. Gooijer. Mino. J. P. 47. DeVries. J. 985 – 999 (1998).V. 268. ‘Determination of the Anticancer Drug Prospidin in Human Tissue by High Performance Capillary Electrophoresis Using Derivatization’. 251. D.