Saudi Pharmaceutical Journal (2011) 19, 185–191King Saud University Saudi Pharmaceutical Journal www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE A validated HPTLC method for determination of terbutaline sulfate in biological samples: Application to pharmacokinetic study Md. Faiyazuddin a,b,*, Abdul Rauf a, Niyaz Ahmad a, Sayeed Ahmad c, Zeenat Iqbal a, Sushma Talegaonkar a, Aseem Bhatnagar d, Roop K. Khar a, Farhan J. Ahmad a a Formulation Research Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110 062, India b Faculty of Pharmacy, Integral University, Dasauli, Kursi Road, Lucknow 222 026, Uttar Pradesh, India c Natural Bioactive Research Laboratory, Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110 062, India d Department of Nuclear Medicine, Institute of Nuclear Medicine & Allied Sciences (INMAS), Brig. Mazumdar Road, Delhi 110 054, India Received 13 November 2010; accepted 11 March 2011 Available online 1 April 2011 KEYWORDS Terbutaline sulfate; High-performance thin-layer chromatography; Biological ﬂuids; Ex vivo stability; Pharmacokinetic study Abstract Terbutaline sulfate (TBS) was assayed in biological samples by validated HPTLC method. Densitometric analysis of TBS was carried out at 366 nm on precoated TLC aluminum plates with silica gel 60F254 as a stationary phase and chloroform–methanol (9.0:1.0, v/v) as a mobile phase. TBS was well resolved at RF 0.34 ± 0.02. In all matrices, the calibration curve appeared linear (r2 P 0.9943) in the tested range of 100–1000 ng spot 1 with a limit of quantiﬁcation of 18.35 ng spot 1. Drug recovery from biological ﬂuids averaged P95.92%. In both matrices, rapid degradation of drug favored and the T0.5 of drug ranged from 9.92 to 12.41 h at 4 C and * Corresponding author at: Formulation Research Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110 062, India. Tel.: +91 7668963486. E-mail address: [email protected]
(Md. Faiyazuddin). 1319-0164 ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.jsps.2011.03.004 Production and hosting by Elsevier this drug was stable for at least 2 months (without losses >10%). New Delhi) and processed. 1.1. Herring and Johnson. 2000). Frozen at 20 C. 2001). Daraghmeh et al. Materials and methods 2. only about 10–20% of inhaled dose reaches the lungs but after nanosizing the drug candidate >50% can be targeted deeper to alveolar region (Bhavna et al. TBS is a selective b2 adrenoceptor agonist. 2002).. 2000. the validated method could be used to carry out pharmacokinetic studies of the TBS from novel drug delivery systems. 1996)...2 lg mL 1 for every mg of an oral dose. For validation of method. Fesser et al. voltammetery (Beltagi et al.. Several analytical techniques like. All rights reserved.. Germany) as a stationary phase for TBS (Faiyazuddin et al. Sagar et al...2. The equipment parameters include constant application rate of 160 nL s 1. India. Faiyazuddin et al. Peak plasma levels are 1. Mumbai. Jacobson and Peterson. Merck (Mumbai. The method was validated with respect to accuracy. reached within 2–3 h.2. 2. All chemicals and reagents used were of analytical grade and were purchased from E.0). 2005). robustness. chronic bronchitis and emphysema and other chronic obstructive pulmonary diseases (COPD) with reversible bronchial hyper-reactive conditions (Borgstro¨m et al. TBS is a short-acting bronchodilator which can be administered orally. chemiluminescence (Li et al. Zhang and Zhang.. Shahin et al. b-[(tert-butylamino) methyl]-3. slit dimension of 5 mm · 0. Orally administered terbutaline is absorbed incompletely. parenterally or by suitable inhalation systems (DPI or nebulization).. Although the above methods were found promising for the determination of TBS in bio-ﬂuids but high expertise need limits routine analysis.186 Md. Methodology 2.. 1992). 2009). we adopted our previously validated stability indicating high performance thin layer chromatographic method (Faiyazuddin et al. 2010). 2004). from 6.0.03 ± 114 ng mL 1. 1998.1. 2001).. high performance liquid chromatography (Chiap et al. 1993).0:1. capillary electrophoresis (Boer and Ensing. Reverchon and Porta. Production and hosting by Elsevier B. solid phase extraction or both (Black and Hansson. ª 2011 King Saud University. E. precision. biological samples were collected from volunteers (Hamdard University. Owing to its potency. Bredberg et al. TBS undergoes high ﬁrst-pass metabolism in the gut-wall and liver and limits bioavailability up to 15% (Daraghmeh et al.. 1984. Following inhalation.69 ± 96 ng mL 1).V. ultraviolet spectroscopy (Selek et al. and electrospray high-ﬁeld asymmetric waveform ion mobility spectrometry–mass spectrometry (ESI-FAIMS– Figure 1 Chemical structure of terbutaline sulfate. Materials Terbutaline sulfate (Assigned purity >98.45 mm and scanning speed of 20 mm s 1. selectivity and limit of quantiﬁcation and limit of detection according to FDA guidelines (FDA. Densitometric scanning was performed on a CAMAG TLC scanner III in ﬂorescent mode at 366 nm and operated by win CATS software (Version 1. Isolation of unchanged compounds and metabolites from biological samples by GC–MS necessitates elimination of matrix interferences by liquid–liquid extraction (Henze et al. 1989.. 2003. 200 lm thickness. 1999. the most useful bioanalytical method reported is GC–MS (Borgstro¨m et al. 2003.. 2009). Switzerland) sample applicator. 1994... 1) is a synthetic b2-adrenoceptor (b2AR) agonist that is widely used as a bronchodilator in acute and long-term treatment of bronchial asthma.. 2008) have been reported in the literature for quantiﬁcation of TBS. The mobile phase consisted of chloroform–methanol (9.. 2007).. which was capable of evaluating TBS concentrations in saliva and plasma with a single step extraction with methanol.31 to 9. MS) (Mie et al. 2000). . Fig. Spisso et al.2. Kim et al. Plasma was received from Yash Pharma Laboratories.. Merck. 2010). LC–tandem MS (Dickson et al. In order to circumvent above mentioned problems. with 610% of the adult population suffering from asthma or related conditions (Bhavna et al. India). Therefore. v/v). Asthma has a diurnal rhythm and in most of the patients. Introduction Asthma is one of the most frequent diseases inﬂuencing the human pursuit. 2000. 1501.. 2005. but most of them either employ large sample volumes or have long extraction processes. Terbutaline sulfate. 2002).5-dihydroxy-benzyl alcohol (C12H19NO3) (TBS. 2002. Hyderabad. 1989. which is signiﬁcantly higher than the maximum saliva concentration (Csmax. GC–MS (Spisso et al.. The maximum plasma concentration (Cpmax) was found to be 5875. 2009). Chromatography The HPTLC analysis was performed on precoated silica gel aluminum plate 60F254 (20 cm · 10 cm. 2002). 2.. pulmonary function gets reduced from midnight sustained up to 8 h. For pharmacokinetic studies..54%) was received as gift sample from Netco Limited. India. Thus a perfect therapeutic agent should have effective measures in preventing bronchospasm for the period of 6– 8 h during which most individuals sleep. TBS is employed in very mere quantities and its determination in biological ﬂuids requires the detection of nanogram or subnanogram/mL levels (Damasceno et al.13 h at 20 C. LC-electrochemical detection (Edholm et al. 2000). The samples were spotted as bands of width 6 mm wide and 10 mm apart by means of CAMAG syringe using a Linomat V (CAMAG. Couper and Drummer. Degradation rate constant (Kobs). QC samples were prepared at concentrations of 20. 4 and 8 h. The time from spotting to chromatography and from chromatography to scanning was varied from 0. Limit of detection (LOD) and limit of quantiﬁcation (LOQ).2. 2001).2.638 ± 0. 2.5. One microliter of each sample was spotted on the TLC plate to obtain the ﬁnal calibration range of 100–1000 ng spot 1. with the following equations: Y = (4. 4.4.0 ± 0.5) and shelf life (T0. sensitivity. 9 aliquots of each QC sample (in each matrix) were stored at 20 C for 24 h. whereas accuracy was expressed as percent recovery.13) in plasma and saliva. The cycle was repeated three times.2.0. 8.A validated HPTLC method for determination of terbutaline sulfate in biological samples 2. 2.033)X + (1255. biological samples were brought to room temperature and extraction was performed. the samples were centrifuged (5 min. 2. Pharmacokinetic study In order to authenticate the applicability of proposed method. Then supernatant was transferred to glass microvials and the solvent was evaporated (<50 C). 60 and 80 lg mL 1. Results and discussion 3. Ten microliters of each working solution of TBS were used to spike plasma and saliva samples (1 mL) in order to obtain calibration standards ranging from 10 to 100 lg mL 1.2.2. For the purpose of stability concern. 10 and 20 min.0 h after dosing. 600 and 800 ng spot 1 in all matrices. The stability of the drug in frozen samples ( 20 C) was determined by periodic analysis over a period of 2 months.0.2. Precision and accuracy were evaluated by performing replicate analyses of QC samples at three levels (200.2. After spiking in plasma and saliva. The correlation coefﬁcients (r2) for calibration curves were equal to or better than . 2. concentration ﬁtted well to a straight line.0 ± 0.4. All blood samples were collected in heparinized tubes and plasma was procured after centrifugation (1000g. In order to determine LOD and LOQ each of blank 187 plasma and saliva samples were spotted six times on TLC plate and the standard deviations (r) of the magnitude of analytical response were determined.2.0.0. QC samples at ﬁnal concentration of 200. respectively. plasma and saliva samples were thawed at room temperature (RTP) for about 10 min and 1 mL of plasma and saliva calibration standards and QC samples were transferred into small eppendorf tubes and mixed with 1 mL of methanol.5. 2. 2. The plasma and saliva samples were reconstituted with 100 lL of methanol. Each concentration was spotted six times on the TLC plate.2.3.23 ± 14. Long term stability. recovery and robustness according to the USFDA guidelines for validation of bioanalytical methods (FDA.3.1.2. 600 and 800 ng spot 1) in plasma and saliva. The peak area vs.019)X + (978. Run time stability at room temperature of processed samples after extraction was determined for QC samples. The LOD was expressed as 3. Ex vivo stability 2. 3. 1.5.>2000g). 2.5:1. 20 min).4. Recovery studies.2.5. samples were analyzed immediately after preparation (control) and after a stipulated time period.2. aliquots were stored at 4 and 20 C. v/v) (9. 2. The stability was assessed after 0.3r/ slope. The precision was expressed in terms of percentage coefﬁcient of variation (CV. Chloroform–methanol (9. 2.5.5. 2. 2. also in six replicates for each kind of sample. Inter-day repeatability was studied by comparing the results of assays performed on different days on the same spiked samples. The amounts of mobile phase and durations of saturation were varied at 20 ± 2 mL and 30 ± 10 min.9) of TBS in plasma and saliva were also obtained at 4 and 20 C.722 ± 0.2. %). To test the stability. 12.0:1. 600 and 800 ng spot 1 were obtained after spotting.0 and 24. Working solutions ranging from 1 to 10 mg mL 1 were prepared by properly diluting the stock solution with methanol. TBS was well resolved at RF 0. accuracy. After vigorous vortexing for 1 min. All the samples were cleaned up as described in Section 2. Extraction efﬁciency or recovery was determined for TBS in triplicate at 200.1. The blood and saliva samples were collected at 1. The speciﬁcity was investigated by screening three different batches of blank plasma and saliva samples. Bioanalytical method validation The method was validated for speciﬁcity. precision. Previous to analysis.4. Short term stability.34.3. Robustness. The effects on the results were expressed as standard deviations. Calibration standards (CS) and quality control (QC) samples of plasma and saliva A stock solution was prepared by dissolving 100 mg of TBS in 10 mL of methanol.2. Sample preparation Previous to analysis. The RF values of endogenous compounds in the matrices were compared with those of TBS. The peak areas obtained after extraction were compared with peak areas resulting from standard solutions at the same concentrations. Robustness studies were done in triplicate at a concentration level of 600 ng spot 1.65 ± 10.5) were tried to assess robustness of the method. half-life (T0. respectively. whereas LOQ was expressed as 10r/slope of the calibration curve of the TBS. Calibration curves The linear regression data for the calibration curves in plasma and saliva are shown in Table 1. Three aliquots of each QC sample were analyzed after extraction and rest of the aliquots were returned to 20 C for another 24 h. The reproducibility of the method was checked by determining precision on a different instrument.4. Speciﬁcity. Precision and accuracy. All the samples were frozen at 20 C until analysis. the pharmacokinetic study with marketed TBS tablet preparation was performed in three healthy volunteers after a single oral dose (5 mg).2. 2.6.1. three different concentrations of QC samples were used. Stability of TBS in methanol (stock solution) was assessed at 4 and 20 C. 2.95) and Y = (3. Freeze thaw stability.4. Intra-day repeatability was determined by treating spiked samples in six replicates on the same day.3.2.4. then they were left to completely thaw at room temperature. In favor of freeze thaw stability studies.2.0. SE and % RSD of the peak areas for each parameter at a concentration level of 600 ng spot 1 in plasma and saliva samples. respectively.1.34).58% and 58.0. 2(b) and 3(b) respectively. As exposed in both ﬁgures (Figs. LOD and LOQ The LOD and LOQ of TBS were found to be 7.019 3.13 966. 3.86% and 4.103 978.746 0.3.37% and 5. Extraction recovery In plasma. Each experiment was performed in triplicate and the mean concentration of TBS was calculated. The percent recovery averaged 94.75% of the initial concentrations in plasma and saliva. v/v).41 h at 4 C.0015 100–1000 0. Plasma and saliva samples spiked at concentration of 600 ng spot–1 are shown in Figs.8 ± 7. Stock Figure 2 Chromatograms of (a) drug-free plasma and (b) plasma spiked at a concentration of 600 ng spot 1. Ex vivo stability 0.02.11. The low LOD and LOQ values indicate the adequate sensitivity of the proposed method.2. indicating that the drug was least stable in plasma (Fig.19%) was obtained after storage at 4 C for 8 h.19% in plasma and saliva.033 4.11 4. two freeze thaw cycles can be tolerated without losses higher than 10%.5.5) and shelf life (T0.9943. The degradation rate constant (Kobs) of TBS in plasma and saliva were 0.9) of drug in plasma. and saliva were obtained from the slope of the straight lines at both temperatures. ranged 96.682 0. 3. Chromatographic conditions: mobile phase.9943 ± 0. The values for interday precision were 64. Faiyazuddin et al. No signiﬁcant difference was observed in the slopes of standard curves (ANOVA. in all biomatrices studied. 3. chloroform–methanol (9.013 3.52) and % RSD (61.722 ± 0. The long term freezer stability ( 20 C) indicated that TBS was stable in the studied matrices during 2 months. The intra and inter-day accuracy calculated as percent recovery. In contrast.638 ± 0.65 ± 10.56–990.2. RF: 0.18 6.698–3. 4).12) obtained after introducing small deliberate changes in the developed method indicated the robustness of the HPTLC method.076 h 1.41% for TBS. however in saliva (n = 6) the recovery was 98.72%.35 ± 2. the mean recovery (n = 6) averaged 95.05).35 ng spot 1. Bioanalytical method validation 3.9982 ± 0.0:1. a Linear regression data for the calibration curves 95% conﬁdence limit. . Intercept Mean ± SD Conﬁdence limita Standard error 1255. A monoexponential decline in drug (TBS) concentration was observed at both 4 and 20 C.23 ± 14. After storage at 20 C for 8 h. Parameter Value Plasma Saliva Linearity range (ng spot 1) Correlation coeﬃcient (r2 ± SD) 100–1000 0. with no signiﬁcant differences between values for intra and inter-day precision. half-life (T0. respectively at 20 C.19%).2. 0.92 to 12. The low values of CV (<5.92 ± 4.35%.2.25–1272.95 1236. The freeze thaw data indicate that.135 Table 1 (n = 6).12%) and saliva (68. Moreover. 3. P > 0. Robustness Table 3 summarizes the average values of SD.15% for the matrices studied. Speciﬁcity Chromatograms of TBS (drug) free plasma and saliva samples are represented in Figs. respectively.3. the drug degradation was substantial and the percentage recovery of TBS decreases to 40.13 h at 20 C and from 9. respectively. higher recovery of TBS in plasma (55.5 ± 4.2 ± 3. Slope Mean ± SD Conﬁdence limita Standard error 4.34 ± 0.188 Md.4. The extraction efﬁciency is not statistically different over the range of concentration studied. TBS eluted was free of interferences in all of the drug free plasma and saliva samples.41 and 18.31 to 9. The stability of TBS was investigated in plasma and saliva samples. respectively.2. TBS peak RF 0. The corresponding half-life values ranged from 6. as a minimum. The low values of SE (62. Precision and accuracy The intra and inter-day precision and accuracy of the assay are described in Table 2. indicate the method’s reproducibility.77–99.37% in plasma and saliva. 2(a) and 3(a) respectively. The intra-day precision was 62.2 ± 2.597–4.2. 2 and 3.2. UV detection at 366 nm.008 3.0018 3. which is signiﬁcantly higher than the maximum saliva concentration (Csmax.68 2.86 1.72 5. After 12 h no signiﬁcant losses occurred. 1501.58 97.93 1. The maximum plasma concentration (Cpmax) was found to be 5875.25 ± 8. 3. 20 and 22 mL) Duration of saturation (20.66 1. %) Accuracyb (%) Plasmac 200 600 800 194.53 4. v/v) Mobile phase volume (18.41 1.11 592.12 ± 10. The result indicates that higher plasma levels were obtained after oral administration of TBS.27 ± 11.57 2. Parameter SDb (peak area) SEb % RSD Mobile phase composition: chloroform–methanol (9.42 788. Actual concentration (ng spot 1) Intra-day reproducibility Inter-day reproducibility Concentration found (ng spot 1) (mean ± SD) Precision (CV.76 3.02.83 Salivac 200 600 800 195.48 1. Accuracy = mean concentration/nominal concentration · 100.54 ± 8.69 ± 96 ng mL 1).4. Chromatographic conditions: mobile phase.24 594.54 98.61 193.0:1. %) = SD/mean concentration · 100. Fig. 30 and 40 min) Time from spotting to chromatography (0. Stability at room temperature of processed samples after extraction was determined.98 1.45 791.64 ± 13.71 98. v/v).25 ± 13. 4 shows the mean concentration–time proﬁles for TBS.57 593. TBS peak RF 0.37 1. Table 3 Robustness of the HPTLC method (na = 3.A validated HPTLC method for determination of terbutaline sulfate in biological samples 189 Figure 3 Chromatograms of (a) drug-free saliva and (b) saliva spiked at a concentration of 600 ng spot 1.91 197.40 99.11 0.52 1.72 790.09 1.5:1.16 ± 9.03 2. chloroform–methanol (9. Table 2 Precision and accuracy of HPTLC method for TBS. .0.84 a b Mean of three determinations (n = 3).32 ± 5.46 591.89 ± 14. Mean of six determinations (n = 6).0 ± 0. %) Accuracy (%) Concentration found (ng spot 1) (mean ± SD) Precisiona (CV.72 97. Average of results obtained from plasma and saliva.87 98.64 793.15 a b c a b Precision as coefﬁcient of variation (CV.5.55 4.16 98.06 1.71 96.72 ± 12.86 97.53 4. 10 and 20 min) Time from chromatography to scanning (0. 600 ng spot 1).35 ± 12. Experimental Cpmax values after oral administration were comparable with reported data indicating that this method is suitable for pharmacokinetic studies.12 1.58 98.03 ± 114 ng mL 1. Pharmacokinetic study The method was used for analysis of plasma and saliva samples obtained after oral administration of single TBS (5 mg).96 1.12 0.12 99.59 2.62 2. solutions of TBS were stable for 2 days at 20 C and for 6 days at 4 C without measurable degradation.0 ± 0.34 ± 0. 10 and 20 min) 3.38 2.28 ± 11. UV detection at 366 nm.77 98.19 2. Dickson. Khar. Sara. Anal. Badwan.31 to 9. 2002. Mac Neil. L..D. Pharmacokinetic evaluation in man of terbutaline given as separate enantiomers and as the racemate. The present method is also used to study the stability of TBS in plasma and saliva. Bull. The methanol used to elute the plasma samples enables rapid extraction with few impurities and no interfering substances. 1984. F. Lee. Mittal. J. J.pdf>. Al-Omari.. J. 27. Drummer. 2005. Chromatogr. Lubda. Pharm. Jaber. Hansson. Jo¨nsson. 1018–1023. Guidance for Industry Bioanalytical Method Validation.. 685. Lee.H. R.. N.. P. Couper. respectively. B: Biomed.B. Black. Schanzer. Appl. G. 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