Pharmaceutics 356CChapter 13 Solutions Dr. Bill Williams
[email protected] PHR 4.214 471-4681 1 Recalls and Safety Alerts • • • Recalls are actions taken by a firm to remove a product from the market. Recalls may be conducted on a firm's own initiative, by FDA request, or by FDA order under statutory authority. Class I Recall – A Class I recall is a situation in which there is a reasonable probability that the use of or exposure to a violative product will cause serious adverse health consequences or death. • Class II Recall – A Class II recall is a situation in which use of or exposure to a violative product may cause temporary or medically reversible adverse health consequences or where the probability of serious adverse health consequences is remote. • Class III Recall – A Class III recall is a situation in which use of or exposure to a violative product is not likely to cause adverse health consequences. 2 Recalls and Safety Alerts • A market withdrawal occurs when a product has a minor violation that would not be subject to FDA legal action. The firm removes the product from the market or corrects the violation. For example, a product removed from the market due to tampering, without evidence of manufacturing or distribution problems, would be a market withdrawal. • A medical device safety alert is issued in situations where a medical device may present an unreasonable risk of substantial harm. In some case, these situations also are considered recalls. 3 Recent Recalls • • RECALLS AND FIELD CORRECTIONS: DRUGS -- CLASS III _____________________________ PRODUCT a) Children's Tylenol (Acetaminophen) Oral Suspension, 160 mg, 4 Fl oz (120mL) bottles, Bubblegum Flavor, NDC #50580-407-04. Recall # D-096-3. b) Children's Tylenol (Acetaminophen) Oral Suspension, 160mg, 4 Fl oz (120mL), Grape Flavor, NDC #50580-296-04. Recall # Recall D-097-3. CODE a) Lot #EFM041 exp 7/04; b) Lot #EFM040 exp 7/04. RECALLING FIRM/MANUFACTURER McNeil Consumer & Specialty Pharmaceuticals, Fort Washington, PA, by letter on November 20, 2002. Firm initiated recall is ongoing. REASON Defective container; product packaged with incorrect dosing cups marked with metric measurements rather than with U.S. standard measurements VOLUME OF PRODUCT IN COMMERCE 116172 bottles. DISTRIBUTION Nationwide. 4 Recent Recalls • • RECALLS AND FIELD CORRECTIONS: DRUGS -- CLASS III _____________________________ PRODUCT a) R-Tanna S Pediatric Suspension (Chlorpheniramine Tannate 4.5mg and Phenylephrine tannate 5 mg) 4 fl oz (118mL) bottles, Rx only. Recall # D055-3; b) CP-Tannic Suspension (Chlorpheniramine tannate 4.5mg and Phenylephrine tannate 75mg) 4fl oz (118mL) and 16fl oz (473mL) bottles, Rx only. Recall # D-056-3. CODE a) Lot# GB024; b) Lot# GA984, GA986, GB005, GB033 and GB036. RECALLING FIRM/MANUFACTURER Recalling Firm: Kiel Laboratories, Inc., Gainesville, GA, by fax on/about September 12, 2002. Firm initiated recall is ongoing. REASON SUBPOTENT; R-Tanna S Pediatric Suspension for phenylephrine tannate (3 month stability); CP-Tannic Suspension for chlorpheniramine tannate ( 1 month stability). VOLUME OF PRODUCT IN COMMERCE a) 5463 bottles; b) 12817 bottles. DISTRIBUTION OH and MS. 5 Recent Recalls • • RECALLS AND FIELD CORRECTIONS: DRUGS -- CLASS II _____________________________ PRODUCT Robinul Forte Tablets (glycopyrrolate tablets USP) 2mg, physician sample cartons of 8 tablets (2 cards @4 tablets per card), Rx only. Recall # D-0583. CODE Lot Number H020572. RECALLING FIRM/MANUFACTURER Recalling Firm: First Horizon Pharmaceutical Corp., Alpharetta, GA, by letters dated September 6, 2002. Manufacturer: Mikart, Inc., Pharmaceutical Manufacturers, Atlanta, GA. Firm initiated recall is ongoing. REASON Product contains glass particles. VOLUME OF PRODUCT IN COMMERCE 386 cartons (8 tablets each/2 blister cards of 4 tablets per card). DISTRIBUTION Nationwide. 6 PHR 356C • Moving to lecture notes 7 Solubility (Chapter 13) 8 Introduction • You, as the Pharmacist, must be aware of the solubility characteristics of the solutes and the features of the common pharmaceutical solvents • Each chemical agent has its own solubility in a given solvent • For many medicinal agents, their solubilities are stated in the USP, Merck Index, other reference books 9 Solubility • Solubility: Concentration of a solute in a saturated solution at a given temperature • Saturated Solution: When a solvent at a certain temperature has dissolved all of the solute that it can • Supersaturated Solution--Crystallization • The solubility of a pure chemical substance at a given temperature and pressure is constant (USP, Merck)--However, its RATE of Solution depends on the particle size of the substance and the extent of agitation • Solubility given usually as “1 gram in X ml” of water, alcohol, etc. 10 General Rules of Solubility • Inorganic (examples) 1. If both the cation and anion of an ionic compound are monovalent, the solute-solute attractive forces are usually easily overcome, and therefore these compounds are generally water soluble (NaCl, LiBr, KI) 2. If both the cation and anion are multivalent, the solute-solute interaction may be too great to be overcome by the solute-solvent interaction and the compound may have poor water solubility (CaSO4, BaSO4) 3. Salts of alkali metals (Na, K, Li) – usually water soluble 11 Solubility 12 Dissolution Profile 13 General Rules of Solubility 4. Ammonium and quaternary ammonium salts – water soluble 5. Nitrates, nitrites, acetates, chlorates, lactates – generally water soluble 6. Sulfates, sulfites, thiosulfates – generally water soluble 7. Acid salts of corresponding insoluble salt – tend to be more water soluble than original salt 14 Solubility • Inorganic (examples) 8. Hydroxides and oxides – generally water insoluble 9. Sulfides – generally water insoluble 10. Phosphates, carbonates, silicates, borates, hypochlorites – generally water insoluble 15 Solubility • Organic (examples) 1. Molecules with one polar functional group--Usually soluble to total chain lengths of five carbons 2. Branched chains--More soluble than straight chained 3. Decrease in water solubility with an increase in molecular weight 4. Increased structural similarity between solute and solvent is accompanied by an increased solubility 16 Important Terms • Definitions: – Solution – Solute – Solvent • Examples: – – – – – Alcohol USP Diluted Alcohol NF Glycerin USP Propylene Glycol USP Purified Water USP 17 Expression of Solubility • Percent solubilities: --%w/v g/100 ml solution --%v/v ml/100 ml solution --%w/w g/100 g solution • Ratio strength --volume-in-volume (ml solute/ml solution) --weight-in-volume (g solute/ml solution) --weight-in-weight (g solute/g solution) 18 Polarity • Solubility due in large part to polarity of solvent • Three categories: 1. Polar (water) --High dielectric constant --Break covalent bonds of strong electrolytes by acid/base rxns. --Dipole interaction forces (hydrogen bonding) 2. Semi-Polar (Ketones, alcohols) --Induces polarity in nonpolar solvents --Co-Solvents 19 Co-Solvents • Used to increase aqueous solubility of weak electrolytes and non-polar molecules • Solute more soluble in mixture of solvents (alcohol/water mixture) • Examples: --Propylene glycol --Polyethylene glycol --Glycerin --Alcohol 20 Co-Solvent Example • Valium (Diazepam) Injection 5mg/mL – – – – – 40% Propylene Glycol 10% Alcohol 5% Na Benzoate/Benzoic Acid 1.5% Benzyl Alcohol QS Purified Water • Diazepam – crystalline, insoluble in water • Inject NGT 1mL/min to avoid venous thrombosis, phlebitis, irritation • Do not mix with other solution or drug in IV or infusion 21 Polarity • 3. Non-Polar Solvents (Carbon tetrachloride, Benzene, Mineral Oil) --Low dielectric constant --Cannot reduce attraction, break covalent bonds, nor ionize weak electrolytes • Dielectric Constant: --Measure of polarizability of a molecule --Polarizability (Increase DC--Increase Polarizability) --”like dissolves like” 22 Solubility and Rate of Dissolution • Solid drug must pass into solution to be absorbed • Rate of dissolution may influence therapeutic response and blood levels • Decreasing particle size and increasing surface area will increase rate of dissolution at a given temperature – (Sw = 6/ρd) 23 In Vitro Dissolution Curve Amount Dissolved Time 24 25 Dissolution Process • Influenced by: – Free surface energy (surfactant) – Shape of particles (surface area) – Temperature – Agitation (type and degree; diffusion layer) – Amount already in solution (near saturation) – Viscosity, volume of solvent 26 Effect of PSD of Phenobarbital on Dissolution M50 = 150 micron M50 = 710 micron (with 0.2% Tween 80 in media) Amount Dissolved M50 = 710 micron Time 27 Noyes-Whitney Equation • dC/dt = KS (Cs – Ct) – where • K=D/h • • • • • • • dC/dt – dissolution rate K – dissolution rate constant S – surface area of exposed solid Cs – concentration of saturated solution Ct – concentration at time t D – diffusion coefficient of solute in solution h – thickness of diffusion layer • Sink conditions – if Ct << Cs, then omit Ct • So dC/dt = KSCs 28 Stagnant Diffusion Layer 29 Concentration Gradient 30 Isomers • Definition: Fumaric Acid Sublimes at 200oC 0.72g/100mL More Stable Maleic Acid 130oC 78g/100mL 31 Polymorphs • Polymorphs – Definition – Properties • Amorphous Solids – Definition – Examples (cefuroxime axetil) 32 pH and Solubility • Many drugs are weak acids or weak bases • Many insoluble in water • Water soluble salts formed by reaction with strong acid or strong base • Examples – Phenobarbital (1:1000) + NaOH – Codeine (1:120) + Sulfuric Acid Phenobarbital Na (1:1) + HOH Codeine Sulfate (1:30) + HOH • Solubility depends on pH – What if adjust pH in wrong direction?? 33 pH and Solubility • To enhance stability, it may be necessary to form poorly water soluble salts: – Examples • • • • Erythromycin stearate Erythromycin estolate Chloramphenicol palmitate Hydroxyzine pamoate 34 USP Terms of Solubility Descriptive Term Parts Solvent for 1 Part Solute Very soluble Freely soluble Soluble Sparingly soluble Slightly soluble Very slightly soluble Practically insoluble or Insoluble Less than 1 From 1 to 10 From 10 to 30 From 30 to 100 From 100 to 1000 From 1000 to 10,000 More than 10,000 35 Pharmaceutical Liquids 36 Advantages and Disadvantages of Liquids • Advantages 1. Inability to swallow capsules and tablets 2. Only suitable dosage form--Often the case with deliquescent or hygroscopic powders 3. Irritation--Some drugs may be less of an irritant when dissolved in a soothing liquid 4. Dosing Flexibility--Can have a greater variation of drug combinations and dosages in liquid formulations • Disadvantages 1. Leakage 2. Costly to ship 3. Patient compliance 4. Taste 5. Preservative(s)--Bacteriostatic Agents (WATER*) 37 Waters • • Six USP Monographs: Purified Water, USP --Less than 0.001% total solids— --Intended for oral and topical dosage forms --Prepared by distillation or reverse osmosis 1. 2. 3. Distillation Ion-exchange Reverse Osmosis 38 Pressure water saline water saline or water saline Osmosis Reverse Osmosis 39 Waters • Water for Injection, USP --Prepared by distillation or RO --Total Solids not more than 1mg/100 ml --Use within 24 hours --Free from pyrogens (bacterial byproduct) --Intended for injections which are to be sterilized after manufacture 40 Waters • Sterile Waters—Four grades 1. Sterile Water for Injection, USP --Package less than or equal to 1 liter --Single use only 2. Sterile Water for Irrigation, USP --Package in larger containers --Single use --Type I—Highly resistant borosilicate glass --Type II—Treated soda-lime glass 41 Sterility Tests • Fluid Thioglycate Medium – Range of microbes – 30 – 35C for 7 days • Soybean-Casein Digest Medium – Molds and fungi – 20-25C for 7 days • Test Results: NO GROWTH 42 Waters • Sterile Waters—Four grades, cont. 3. Bacteriostatic Water for Injection, USP --Sterile --Contains antimicrobial agents which must be stated on label—toxicity issues --Preserve in single-dose or multi-dose vials NGT 30 ml --Not for seniors or newborns 43 Bacteriostatic Agents • • • • • • • • Methyl paraben Propyl paraben Benzyl alcohol Benzalkonium chloride Phenol Thimerosal Chlorobutanol Phenylmercuric nitrate 0.1% 0.01% 0.9-2.0% 0.01% 0.25-0.5% 0.01% 0.25-0.5% 0.01% 44 Waters 4. Sterile Water for Inhalation, USP --Distillation or RO --No antimicrobial agent --Labeling 45 Waters Schematic 46 Other Solvents • • • • • Alcohol, USP Glycerin, USP Propylene Glycol, USP Polyethylene glycols, USP Oils --Vegetable Oils --Synthetic fatty acid esters (ethyl oleate; propylene glycol dicaprylate caprate) --Liquid paraffin (MO) • Note: All EXCEPT the OILS may be used in internal, external and parenteral products either alone or in conjunction with water 47 Oils • Used as solvents in External products --i.e. Liniments and ear-drops --Nose drops—danger of lipid pneumonia • Used alone or as co-solvent • Most substances are more soluble in vegetable oils or synthetic fatty acid esters than in liquid paraffin • Heavy liquid petroleum or MO is used as a laxative • Always use the Oil Soluble or Free Base of the drug to form an oil solution 48 Aromatic Waters • Clear aqueous solutions saturated with volatile oils or other aromatic or volatile substances • Peppermint Water, USP—Flavoring Agent • Strong Rose Water, USP—Fragrance • Hamamelis Water—”Witch Hazel”—An Astringent • Stability Issues --Store in tight, light resistant glass containers --Prone to oxidation 49 Syrups 50 Syrups • Concentrated aqueous solutions of a sugar or sugar substitute with or without flavoring agent • Vehicle--Better taste, increased palatability • Components of Syrups --Sugar or sugar substitute --Preservative(s) --Flavors --Color(s) • Some contain medicament and are a complete preparation in themselves--Medicated syrups Example: Chloral Hydrate Syrup 51 Syrups • Syrup, NF (Simple Syrup) --Contains 85g sucrose in 100 ml syrup --High concentration of sucrose 1. Give desired sweetness 2. Prevent microbial growth --Specific gravity: 1.313 --May crystallize in warmer weather – “cap lock” --Diluted--need preservatives--subject to mold and fungi growth (i.e. Parabens)—WHY? 52 Non-Sucrose Syrups • Alternative components of syrups --Dextrose --Glycerin --Sorbitol (64%w/w—60% sweetness of sucrose—produces gas and GI motility) --Also Sorbitol used to prevent crystallization around cap of bottles --Xylitol (Expensive) --Aspartame (Expensive—Some problems) 53 Non-Sucrose Syrups • Syrups for Diabetics --Methyl cellulose—Non-caloric, good suspending agent also, masks bitter tasting drugs—Increases viscosity --Other celluloses— Hydroxyethylcellulose, etc. 54 Preparation of Syrups • Preparation of Syrups: 1. Solution with the aid of heat 2. Solution by admixture in the cold 3. Dissolution of sucrose in a medicated or flavored liquid 4. By percolation 55 Preparation of Syrups • Solution with Heat: --Quick --Can hydrolyze sucrose (a di-saccharide)—Dextrose and Fructose (Inversion and result is Invert Sugar—Darker color—Too much heat and is said to be caramelized) --Caramelized sugars are more subject to fermentation and microbial contamination --Example using heat: --Acacia Syrup --Benzoic Acid 0.1%--WHY? 56 Preparation of Syrups • Preparation by Admixture: --Prepared without heat to avoid inversion or damage to actives --Mix with vigorous agitation --Example: Ferrous Sulfate Syrup— Turns green 57 Ferrous Sulfate Syrup USP • Composition – function of each? – – – – – Ferrous sulfate Citric Acid (weak chelating agent) Peppermint Spirit Sucrose (reducing agent) Purified Water • • • • Ferrous Sulfate Ferric Hydroxide Ferric Ion Ferrous Ion Fe+2SO4-2 7 H2O (soluble) Fe+3(OH)3-1 (insoluble) Fe+3 Fe+2 (oxidizes to Fe+3 ) 58 Oral Solutions 59 Oral Solutions • Two categories: 1. Colored and flavored aqueous solutions of medicaments 2. Dry powder mixtures for reconstitution --Usually antibiotics which are unstable in aqueous solution --Usually refrigerate—Use within 7-14 days --Dry granules—Contain flavors and other additives 60 Preparation of Oral Solutions • By Simple Admixture --Method depends on the nature of the solute and solvent • By Chemical Interaction --For water insoluble salt in an aqueous vehicle --Options: 1. Change the solvent (alcohol, etc.) 2. Use a soluble form of the drug --Example: Strong Iodine Solution (Lugol’s Solution) I2 + KI KI3 --Water insoluble Iodine in a salt solution of Potassium Iodide (soluble salt complex) 61 Preparation of Oral Solutions • Isotonic Solutions: --Same osmotic pressure as biological fluids with which they are being compared (Tears, blood, etc.) --Body fluids have an osmotic pressure corresponding to that of a 0.9% solution of Sodium Chloride --Primarily Ophthalmic solutions • Hypotonic Solutions: --Osmotic Pressure < 0.9% NaCl solution --Hemolysis--Why? • Hypertonic Solutions: --Osmotic Pressure > 0.9% NaCl solution --Crenation or cell shrinking—Why? 62 Preparation of Oral Solutions • Sodium Chloride Equivalents: --Dextrose, etc. Example: For Atropine Sulfate SCE = 0.12, so 1 g Atropine Sulfate equals the tonic effect of 0.12 g NaCl See text 63 Osmolality (mOsmol/Kg) • Classifications: > 350 329 – 350 270 – 328 250 – 269 0 – 249 Hypertonic Slightly Hypertonic Isotonic Slightly Hypotonic Hypotonic 64 Example – FDA aware of 4 deaths due to hemolysis – (since 1994) • Albumin (Human) 25% (plasma exchange) Dilute 1 part to 4 parts SWFI Albumin (Human) 5% Trissel’s Handbook on Injectable Drugs (1994) – states “…prepare a 5% From a 25% with 1 part of the 25% and 4 parts of SWFI, NS, or D5W. Hospital pharmacists relied on this information! Plasmapheresis – replacement mixture accounts for significant fraction of the patient’s calculated blood volume Revised in 1996 – NO SWFI Hypotonic albumin solutions prepared with SWFI; large volumes with plasmapheresis accounting for significant fraction of patient’s blood 65 Examples of Oral Solutions • Powders for Reconstitution to Oral Solutions 1. Cloxacillin sodium for Oral Solution, USP (antibiotic) 2. Nafcillin sodium for Oral Solution, USP (antibiotic) 3. Oxacillin sodium for Oral Solution, USP (antibiotic) 4. Penicillin V Potassium for Oral Solution, USP (antibiotic) 5. Potassium Chloride for Oral Solution, USP (Potassium Supplement) 66 Examples of Oral Solutions • Oral Solutions 1. Furosemide Oral Solution (Lasix)—for edema and hypertension 2. Cimetidine HCl Liquid (Tagamet)—Peptic ulcer disease & hypersecretory disorders 3. Ergocalciferol Solution—Vitamin D2 in propylene glycol 67 Oral Liquid Vehicles • Used for their solvent, suspending and/or emulsifying properties • Used to dilute drugs to suitable volume for dosing • Used to enhance flavor and palatability • Used to provide stable and elegant preparations 68 Oral Liquid Vehicles (Official) • • • • • • • • Aromatic Elixir NF (Simple Elixir) Compound Benzaldehyde Elixir NF Peppermint Water NF Sorbitol Solution USP Suspension Structured Vehicle NF Sugar-Free Suspension Structured Vehicle NF Syrup NF (Simple Syrup) Xanthan Gum Solution NF 69 Oral Liquid Vehicles (Nonofficial) • • • • • • • • • • • Acacia Syrup Cherry Syrup Citric Acid Syrup (Syrup of Lemon) Cocoa Syrup (Cacao Syrup, Chocolate Syrup) Raspberry Syrup Tolu Syrup (Tolu Balsum Syrup, Syrup of Tolu) Wild Cherry Syrup Coca-Cola Syrup Ora-Sweet Syrup Vehicle Ora-Sweet SF Sugar-Free Syrup Vehicle Syrpalta 70 • See reference: L. Allen, Int. J. Pharm. Compounding, 5(1)2001,65-67. Non-Aqueous Solutions 71 Non-Aqueous Solutions • Hydrocarbons --Aliphatic hydrocarbons (Mineral Oil) --Chlorinated hydrocarbons (Trichloroethane) • Oils --Used for liniments, sunscreens, lotions (i.e. Topicals) – reduces irritation 1. Fixed Oil—Almond, Peanut, Sesame 2. Volatile Oil—Wintergreen Oil, Turpentine Oil, Peppermint 72 Alcohols • Ethanol for Internal Preparations --Glycerin is a high molecular weight alcohol • Rubbing Alcohol, USP --70% v/v ethanol with water, denaturants, perfume oils, and color additives --must contain: 355mg sucrose octaacetate or 1.40mg denatonium benzoate --Regulated by ATF -- Denaturants: 73 Alcohols • Isopropyl Rubbing Alcohol, USP --70% v/v IPA with water, stabilizers, perfume oils and color additives --Used as a Disinfectant (skin, equipment) 74 Collodions • Formed from reaction of: Cotton HNO3 H2SO4 Pyroxylin (cellulose tetranitrate) --Forms protective layer on skin • Three USP Collodion products 1. Collodion USP—To protect cuts, lacerations 2. Flexible Collodion, USP—Camphor and Castor oil 3. Salicylic Acid Collodion, USP—Keratolytic agent (warts) 75 Spirits and Elixirs 76 Spirits • Spirits --Alcoholic or hydro-alcoholic solutions of volatile or aromatic substances --Alcohol > 60% --”Salting Out”—Too much water added, aromatic substance will salt out --Methods of Preparation: 1. Simple solution 2. Maceration 3. Distillation 4. Chemical reaction Official USP Spirits: 1. Aromatic Ammonia Spirit, USP (For fainting, syncope) 2. Camphor Spirit, USP 3. Peppermint Spirit, USP 77 Elixirs • Elixirs: --Clear, sweetened hydro-alcoholic solution intended for oral use --Palatability --Range of 4 to 40% alcohol --Often contain co-solvents --Alcohol as preservative 78 Elixirs --Classes of Elixirs: 1. Non-medicated --Vehicle and Diluent 2. Medicated (pg. 321 in text for other examples) --Examples: 1. Antihistamine (Benadryl— Diphenhydramine HCl Elixir 2. Sedative and Hypnotic Elixirs— Phenobarbital Elixir 3. Expectorants—Terpin Hydrate Elixir 4. Cardiotonic Elixirs--Digoxin 79 Tinctures and Fluid Extracts (Liniments & Liquefied Phenol) 80 Tinctures and Fluid Extracts • • • • • Prepared by extraction Protect from air, light, heat—tightly sealed bottles Inactive plant compounds extracted from actives Pectin, celluloses, extraneous material removed Methods of extraction: 1. Maceration (“soak”) Example: Cascara Sagrada Fluid Extract 2. Percolation Uses column containing drug 81 Tinctures • Alcoholic or hydro-alcoholic solutions from vegetable substances or chemical substances • Contain extracts or chemical agents • Alcohol Content: 15-80% • Stability and Storage • Preparation of tinctures: 1. Maceration—Compound Benzoin Tincture 2. Percolation—Opium Tincture, Belladonna Tincture 3. Solution—Iodine Tincture (I2 + KI KI3) 82 Tinctures • Official Tinctures: --Belladonna Tincture --Iodine Tincture --Strong Iodine Tincture --Compound Benzoin Tincture 83 Fluid Extracts • Liquid preparations of vegetable drugs (plant extracts) containing alcohol • Adjusted (standardized) so 1 ml tincture contains 1g of drug • Preparation—Percolation • Official Fluid Extracts—Laxatives --Aromatic Cascara Fluidextract --Cascara Sagrada Fluidextract --Senna Fluidextract 84 Liniments • Oily or alcoholic liquids or semi-solids for External Use (Ben-Gay, Theragesic) • Counter-irritant • Oils less irritating to skin than alcohols in base • Actives ingredients: --Methyl salicylate --Camphor --Menthol --Thymol --Various Oils • There is no Official Liniment 85 Pyrogens and Pyrogen Testing • Pyrogens: Fever-producing organic substances arising from microbial contamination and are responsible for many of the febrile reactions which occur in patients following injection • Material is thought to be a lipopolysaccharide (endotoxin) from the outer cell wall of the bacteria; and endotoxins are soluble • Is thermostable and may remain in water even after sterilization by autoclaving or by bacterial filtration • Are oxidized (Potassium permanganate—oxidizing agent) • USP Pyrogen Test: --Uses healthy rabbits— inject, check rabbit temperature for norms --Replaced now by Limulus amebocyte lysate (LAL test) (test solution + cell lysate from amebocytes of horseshoe crab) Protein fraction of amebocyte coagulates if endotoxin present --USP Bacterial Endotoxins Test utilizes LAL 86 Pyrogens and Pyrogen Testing • Gram (–) bacilli are most potent • Pyrogens come from: – Water used as solvent – Container used in preparation – Chemicals used in preparation • Remove pyrogens by: – Dry heat (250C for 30 min; 180 C for 3-4 hours) – Washing with pyrogen free Water For Injection USP to remove water soluble pyrogens – Too small to filter out; SO PREVENT • Pyrogens cause chills, fever, headache, pain in legs and limbs 87