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Symmetry® Columns Applications Notebook

Symmetry® Columns Applications Notebook

Symmetry ® Columns Applications Notebook

Symmetry® SymmetryShield™ Symmetry300™ SymmetryPrep™ Intelligent Speed (IS™)

www.waters.com Symmetry® Columns

Setting the Standard for Reproducibility

As today’s chemists establish new Unmatched Year-to-year Reproducibility analytical methods for the latest pharmaceu-

® ® tical and biopharmaceutical products, the Column: Symmetry C18, Instrumentation: Waters Alliance HT 2795 selection of a reproducible HPLC column is 4.6 x 150 mm, 5 µm Waters 996 PDA detector Mobile Phase A: H2O Sampling Rate: 5 pts./sec essential. The selected column needs to Mobile Phase B: MeCN Mobile Phase C: pH 3.75; 100 mM provide the same chromatographic results NH4COOH in H2O Flow Rate: 1.4 mL/min over the life of the new drug product. Isocratic: 30% A; 60% B; 10% C RSD’s for Retention Times Injection Volume: 5.0 µL ® 1. Terbinafine HCl 0.7% Symmetry columns demonstrate superior Temperature: 30 °C reproducibility over many years. Batches Detection: UV @ 233 nm 2. Ibuprofen 0.8% 3. Lovastatin 0.6% randomly selected over the past 5 years 4. Simvastatin 0.7% demonstrate excellent reproducibility in the example shown. Reproducibility is our number one priority in supplying Symmetry® columns. This excellent reproducibility is a result of our commitment Batch 136 (1998) to maintaining the tightest specifications in the HPLC column industry. Symmetry® columns Batch 142 (1998) are engineered with high purity raw materials, tightly controlled manufacturing Batch 149 (1999) processes and column packing procedures that provide today’s scientists with the best, Batch 151 (2000) most reproducible HPLC column available. Batch 156 (2001) Our goal is to supply a family of HPLC columns that you can rely on for rugged and Minutes robust methods. Symmetry® columns let you increase your laboratorys' productivity, and allow easier method transfer between labs and around the globe. Comprehensive Certificate of Analysis A three-page Certificate of Analysis is included with every Symmetry® column. We report both our specifications, which are the tightest in the industry, and the results of the 22 critical tests each column must pass before it carries the Symmetry® brand.

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 1 Table of Contents

Introduction...... 1

Compound Index ...... 3-4

List of Applications ...... 5-8

Applications ...... 9-182

Ordering Information ...... 183-186

Notes ...... 187-192

2 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Compound Index Click in the box, Type desired page number, Then click OK

Acephate ...... 5, 9 Butyl ...... 79, 135, 150 Dichlorprop ...... 14 Acetaminophen ...... 5, 11-13 Butyrophenone ...... 147-148 Diclofenac Sodium ...... 6, 73 Acetanilide ...... 122-123, 147-148 Caffeine ...... 5, 45, 48, 140, 170 2,6-Diethylaniline ...... 10 Acetophenone ...... 147-148 Cafteric acid ...... 125 10,11-Dihydrocarbamezapine ...... 30 2-Acetylfuran ...... 147-148 Pantothenate ...... 180 Dihydroergotamine mesylate ...... 6, 74 Acifluorfen ...... 14 Captopril ...... 5, 46 Dilitiazem HCL ...... 75-76 Acyclovir ...... 5, 15-17 Captopril Disulfide ...... 46 Dinoseb ...... 14 Alachlor ...... 10 Carbadox...... 5, 47 HCL ...... 6, 77 Albuterol ...... 5, 18-19 ...... 48 Diquat ...... 7, 120 Allobarbital ...... 33 Carbamezapine ...... 30 Disopyramide ...... 6, 78 Alpha-Tocopherol (Vitamin E) ...... 179 Carbamezapine-10,11-epoxide ...... 30 Doxepin ...... 6, 29, 79, 173-175 6a-Methyl-17l-hydroxyprogesterone ...... 122-123 Carbaryl ...... 36, 81 Doxycycline ...... 167 2-Amino-7-chloro-5-oxo-5H-[1] Cefixime ...... 5, 49 EDDP (Methadone Metabolite) ...... 99 Benzopyrano[2,3-b]pyridine-3-carbonitrile . . . 122-123 Cefuroxime ...... 5, 50 Enalapril ...... 80 7-Amino Clonazepam ...... 86 Cephalexin ...... 5, 51 Ephedrine ...... 26-27 3-Aminofluoranthene ...... 122-123 Chloramben ...... 14 Epicatechin ...... 48 7-Amino Flunitrazepam ...... 86 Chloramphenicol ...... 5, 52 ...... 48 7-Amino Nitrazepam ...... 86 Chlorcyclizine HCL ...... 65 Estazolam ...... 99 Amitriptyline ...... 173-175, 29 Chlordiazepoxide ...... 6, 53-54 ...... 151-152 Amobarbital ...... 33-34 2-Chloro-2,6-diethylacetanilide ...... 10 ...... 151-152 Amoxapine...... 5, 21 Chlorogenic acid ...... 125 Ethosuximide ...... 153 Amoxicillin ...... 5, 22-24 Chlorpheniramine ...... 6-7, 55, 137, 160 Etodolac ...... 6, 83 Amphetamine ...... 5, 26-27 1-(3-Chlorophenyl)peperazine ...... 171 Fenoprofen ...... 131-132 Ampicillin ...... 5, 25 Cholecalciferol (Vitamin D3) ...... 179 Flunitrazepam ...... 6, 85-86 Angiotensin I ...... 115 Cichoric acid ...... 125 Flurbiprofen ...... 131-132 Angiotensin II ...... 115 ...... 6, 56-58 Folic Acid ...... 8, 181 Antipyrine ...... 5, 31 Clavulanate ...... 23 Furazolidone ...... 161 ...... 5, 32, 36, 81, 172 Clobetasol ...... 6, 59 Furosemide ...... 6, 88 AZT ...... 182 Clonazepam ...... 6, 60, 86 Guanosine hydrate ...... 17 β-hydroxyethyltheophylline (I.S.) ...... 45, 140 Cocaethylene ...... 62 Heptanophenone ...... 147-148 Bamethan (I.S.) ...... 19 Cocaine ...... 6, 61-62 Hexanophenone ...... 147-148 Barbital ...... 33 ...... 6, 11, 63-64, 139 Hydrastine ...... 20 Beclomethasone ...... 5, 35, 59 Codeine-6-Glucuronide (C-6-G) ...... 63-64 Hydrocodone Bitartrate ...... 5, 12 Benomyl ...... 5, 36, 81 Corticosterone ...... 151-152 Hydrocortisone ...... 6, 89, 122-123 Bentazon ...... 14 Cortisone ...... 151-152 Hydrocortisone-21-Acetate ...... 89 ...... 145-146 Cyanazine ...... 172 4-Hydroxyantipyrine ...... 31 Benzophenone...... 147-148 Cyclizine ...... 6, 65 Hydroxyatrazine ...... 32, 172 Benzoylecgonine ...... 62 Cytochrome C...... 6, 66-69 1-Hydroxybenzotriazole ...... 51 ...... 5, 37-38, 149-150 2,4-D ...... 14 Hydroxydesethylatrazine ...... 32 Betamethasone Valerate ...... 149-150 2,4-DB ...... 14 Hydroxydesisopropylatrazine ...... 32 Berberine...... 20 Demoxepam ...... 53-54 7-(2-Hydroxyethyl) Theophylline ...... 169 A ...... 5, 36, 81 Deoxidized (Insulin)...... 91 Ibuprofen ...... 6, 90, 131-132 2-Bromofluorene ...... 122-123 Desamino (Insulin) ...... 91 Imipramine ...... 173, 175 8-Bromoguanosine ...... 122-123 Desethylatrazine ...... 32, 172 Insulin(s) ...... 91-93 BSA ...... 39-42 Desethyldesisopropylatrazine ...... 32 Isorhamnetin ...... 84 Bupropion ...... 5, 43 Desisopropylatrazine ...... 32, 172 ...... 84 Busiperone ...... 5, 44 Dextromethorphan ...... 6, 70-71, 87 Ketamine ...... 124 Butabarbital ...... 33-34 Diazepam ...... 6, 72 ...... 94 Butalbarbital ...... 33 Dicamba...... 14 Ketoprofen ...... 131-132 Butalbital ...... 34 3,5-Dichlorobenzoic Acid...... 14 Lasix (furosemide) ...... 88

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Leu enkephalin ...... 82 Oxytocin ...... 7, 115 Sulfisoxazole ...... 156-157 Lidocaine HCL ...... 6, 95 Paclitaxel (Taxol) ...... 7, 116-117 2,4,5-T ...... 14 Maleate ...... 160 Papaverine HCL ...... 7, 118 citrate ...... 162-163 Maleic Acid ...... 80 Paracetamol ...... 7, 119 Taxol ...... 8, 116-117 Maprotiline HCL ...... 6, 97, 171 Paraquat ...... 7, 120 Terconazole ...... 94 MCPA ...... 14 Paraxanthine ...... 45, 140 Terfenadine ...... 8, 164-165 MCPP ...... 14 Peptides...... 7, 121 acetate ...... 166 Meclizine HCL ...... 98 Perylene ...... 122-123 ...... 166 Mephenytoin ...... 28 Phencyclidine (PCP) ...... 124 (I.S.) ...... 166 Mephobarbital ...... 33-34 Phenobarbital ...... 28, 30, 33-34 (I.S.) ...... 35, 166 Met enkephalin ...... 82 Phensuximide ...... 153 Tetracaine...... 6, 87 Methadone ...... 7, 99 2-Phenyl-2ethylmalonamide ...... 30 Tetracycline...... 167-168 Methamphetamine ...... 26-27 ...... 30 Theobromine ...... 8, 45, 140, 169-170 Methoxyverapamil ...... 29, 178 Phthalic Acid ...... 146 Theophylline ...... 8, 45, 140, 147-148, 169-170 Methsuximide ...... 153 p-Hydroxyphenobarbital ...... 30 Thiamine Hydrochloride ...... 180-181 Methylparaben ...... 95 Picloram ...... 14 Toluamide ...... 160 Methylphenidate HCL ...... 7, 100 ...... 149-152 2,4,5-TP ...... 14 Metolachlor...... 10 Prednisolone Acetate ...... 149 Trans-Retinol (Vitamin A) ...... 179 Metolachlor-metabolite ...... 10 ...... 7, 126 Trazodone ...... 5, 8, 21, 171 Metoprolol ...... 134 Primidone ...... 28, 30 Triamcinolone ...... 122-123 Metoprolol tartrate ...... 7, 101 Procainamide ...... 7, 127-129 Trimethoprim ...... 8, 158-159 Metronidazole ...... 7, 102-103 Prochlorperazine Edisylate ...... 130 Trimipramine ...... 173, 175 Minocycline ...... 167-168 Progesterone ...... 151-152 Tripelennamine ...... 8, 176 ...... 7, 104 Promethazine HCL ...... 133 Triprolidine HCL ...... 87, 138 Morphine-D3-glucuronide ...... 104 Propiophenone ...... 147-148 Uracil ...... 149, 179 Morphine-D6-glucuronide ...... 104 Propranolol ...... 7, 134-135, 160 Valerophenone ...... 147-148 N-Acetylprocainamide ...... 128-129 Propylparaben ...... 37-38 Verapamil ...... 8, 29, 177-178 N-Propionylprocainamide ...... 128-129 Protriptyline HCL ...... 173 Vitamins ...... 179-181 Nadolol ...... 7, 105 Pseudoephedrine HCL ...... 7, 136-139 Warfarin ...... 6, 87 Nalidixic acid...... 145, 161 Pyridoxal ...... 180 Zidovudine ...... 8, 182 Naloxone ...... 106-109 Pyridoxamine Dihydrochloride ...... 180 Naltrexone ...... 7, 106-109, 114 Pyrodoxine HCL ...... 180 Naphtho(2,3-a)pyrene ...... 122-123 ...... 84 Naproxen ...... 7, 90, 110-111 Quinidine sulfate ...... 8, 141 Niacinamide ...... 180 Ranitidine ...... 8, 63-64, 142-144 Nicergoline ...... 7, 112, 181 Riboflavin ...... 180-181 Nifedipine ...... 7, 113 Salbutamol ...... 5, 18-19 Nitrazepam ...... 86 Salicylic acid ...... 8, 145-146 4-Nitrophenol ...... 14 Secobarbital ...... 33-34 Norchlordiazepoxide ...... 53-54 Simazine ...... 172 Nordiazepam ...... 53-54 Succinimide ...... 8, 28, 153 Nordoxepin ...... 29, 174-175 Succinylsulfathiazole ...... 96, 154 Nortriptyline ...... 29, 173-175 Sulfadiazine ...... 97, 154-157 Norverapamil...... 8, 29, 178 Sulfadimethoxine ...... 156-157 Octanophenone ...... 147-148 Sulfadimidine ...... 156-157 O-dichlorobenzene ...... 60 Sulfamerazine ...... 97, 154-157, 159 Oxazepam ...... 53-54 Sulfamethazine ...... 96, 154 Oxolinic Acid ...... 161 Sulfamethoxazole ...... 8, 156-157 Oxprenolol ...... 101, 134 Sulfanilamide ...... 97, 127, 144, 154 Oxycodone ...... 7, 114 ...... 97, 154-157

4 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Click on the application titles below to go directly to the desired application For a new search, click on the "List of Applications" bookmarks to the left List of Applications

Acephate in Drinking Water...... 9 Acetamide Herbicides and Metabolites in Drinking Water ...... 10 Acetaminophen and Codeine...... 11 Acetaminophen and Hydrocodone Bitartrate ...... 12 Acetaminophen in Serum ...... 13 Acidic Herbicides in Drinking Water ...... 14 Acyclovir ...... 15,16 Acyclovir in Plasma...... 17 Albuterol (Salbutamol) ...... 18 Albuterol (Salbutamol) in Serum...... 19 Alkaloids in Golden Seal...... 20 Amoxapine & Trazodone ...... 21 Amoxicillin ...... 22 Amoxicillin & Clavulanate Potassium Augmentin Tablet ...... 23 Amoxicillin in an Oral Suspension Formulation...... 24 Ampicillin ...... 25 Amphetamines ...... 26 Amphetamines in Human Urine...... 27 Anticonvulsants...... 28 Antidepressants in Plasma ...... 29 Antiepileptics & Metabolites ...... 30 Antipyrine & Its Metabolites ...... 31 Atrazine & Metabolites in Drinking Water...... 32 Barbiturates ...... 33 Barbiturates in Human Urine...... 34 Beclomethasone ...... 35 Benomyl & Bisphenol A...... 36 Betamethasone...... 37 Betamethasone in Syrup Formulation ...... 38 BSA Tryptic Digest ...... 39 BSA Tryptic Digest - Column Length Effects ...... 40 BSA Tryptic Digest - Flow Rate Effects ...... 41 BSA Tryptic Digest - Gradient Duration Effects ...... 42 Bupropion ...... 43 Busiperone ...... 44 Caffeine and Metabolites in Serum ...... 45 Captopril ...... 46 Carbadox...... 47 Catechins - 4.6 x 20 mm Intelligent Speed™ Separation ...... 48 Cefixime...... 49 Cefuroxime ...... 50 Cephalexin ...... 51 Chloramphenicol ...... 52

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Chlordiazepoxide & Metabolites...... 53 Chlordiazepoxide & Metabolites in Serum ...... 54 Chlorpheniramine...... 55 Cimetidine ...... 56-58 Clobetasol and Related Compound ...... 59 Clonazepam ...... 60 Cocaine ...... 61 Cocaine & Its Metabolites in Serum ...... 62 Codeine and Its Glucuronide Metabolite in Human Urine ...... 63 Codeine and Its Glucuronide Metabolite in Porcine Plasma ...... 64 Cyclizine & Chlorcyclizine ...... 65 Cytochrome c Tryptic Digest - Column Lifetime Study, Critical Pair Resolution ...... 66 Cytochrome c Tryptic Digest - Pore Size Effects ...... 67 Cytochrome c Tryptic Digest - Recovery Study ...... 68 Cytochrome c Tryptic Digest - Reproducibility Study ...... 69 Dextromethorphan ...... 70 Dextromethorphan in a Cough Syrup ...... 71 Diazepam in Serum - LC/MS ...... 72 Diclofenac Sodium ...... 73 Dihydroergotamine Mesylate ...... 74 Diltiazem HCL ...... 75 Diltiazem HCL -Isolation of Impurities, Transfer from Analytical to Prep ...... 76 Diphenoxylate HCL...... 77 Disopyramide ...... 78 Doxepin in Serum...... 79 Enalapril ...... 80 Endocrine Disruptors in Soil ...... 81 Enkephalins ...... 82 Etodolac...... 83 Flavanoids in Ginko ...... 84 Flunitrazepam ...... 85 Flunitrazepam and Related Compounds ...... 86 Four Drug Mixture: Warfarin, Dextromethorphan, Triprolidine, and Tetracaine ...... 87 Furosemide ...... 88 Hydrocortisone and Related Compunds...... 89 Ibuprofen and Naproxen in Serum ...... 90 Insulin Chain - QC Batch Test for Symmetry 300™ ...... 91 Insulin- Impurity Isolation, Transfer from Analytical to Prep ...... 92 Insulin- Isophase Suspension ...... 93 Ketoconazole...... 94 Lidocaine HCL ...... 95 Lifetime of 3.5 µm Symmetry® Column: 10,000 Injections of Sulfa Drugs ...... 96 Maprotiline HCL...... 97

6 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Click on the application titles below to go directly to the desired application For a new search, click on the "List of Applications" bookmarks to the left List of Applications

Meclizine HCL...... 98 Methadone in Human Urine ...... 99 Methylphenidate HCL ...... 100 Metoprolol Tartrate and Oxyprenolol...... 101 Metronidazole ...... 102,103 Morphine & Its Glucuronide Metabolites in Serum...... 104 Nadolol ...... 105 Naltrexone ...... 106 Naltrexone in Plasma ...... 107 Naltrexone in Plasma - LC/MS ...... 108 Naltrexone in Serum ...... 109 Naproxen ...... 110,111 Nicergoline ...... 112 Nifedipine...... 113 Oxycodone in Serum ...... 114 Oxytocin & Angiotensins in Serum ...... 115 Paclitaxel (Taxol) ...... 116 Paclitaxel (Taxol)- Resolution of Impurities...... 117 Papaverine HCL...... 118 Paracetamol ...... 119 Paraquat & Diquat in Tap Water ...... 120 Peptides- Alberta Peptide Mixture ...... 121 Pharmaceutical Test Mix - 2.1 x 20 mm Intelligent Speed™ Separation...... 122 Pharmaceutical Test Mix - 4.6 x 20 mm Intelligent Speed™ Separation...... 123 Phencyclidine in Human Urine ...... 124 Phenolics in Echinacea ...... 125 Prednisone...... 126 Procainamide in Serum ...... 127 Procainamides at pH 3.0 ...... 128 Procainamides at pH 6.0 ...... 129 Prochlorperazine- Isolation of Degradation Products, Transfer from Analytical to Prep ...... 130 Profens (Gradient) - 4.6 x 20 mm Intelligent Speed™ Separation ...... 131 Profens (Isocratic) - 4.6 x 20 mm Intelligent Speed™ Separation...... 132 Promethazine- Impurities Profile ...... 133 Propranolol in Human Urine...... 134 Propranolol in Serum...... 135 Pseudoephedrine HCL...... 136 Pseudoephedrine HCL & Chlorpheniramine ...... 137 Pseudoephedrine HCL & Triprolidine HCL ...... 138 Pseudoephedrine HCL & Codeine...... 139 Purine Alkaloids ...... 140 Quinidine Sulfate and Related Substance...... 141 Ranitidine...... 142,143

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Ranitidine in Serum ...... 144 Salicyclic Acid & Related Compounds in Serum ...... 145 Salicyclic Acid & Related Compounds...... 146 Speed Mix - 2.1 x 20 mm Intelligent Speed™ Separation ...... 147 Speed Mix - 4.6 x 20 mm Intelligent Speed™ Separation ...... 148 ...... 149 Steroids in Serum ...... 150 Steroids - 2.1 x 20 mm Intelligent Speed™ Separation ...... 151 Steroids - 4.6 x 20 mm Intelligent Speed™ Separation ...... 152 Succinimides ...... 153 Sulfa Drugs ...... 154,155 Sulfa Drugs - 2.1 x 20 mm Intelligent Speed™ Separation ...... 156 Sulfa Drugs - 4.6 x 20 mm Intelligent Speed™ Separation ...... 157 Sulfamethoxazole Oral Suspension ...... 158 Sulfamethoxazole and Trimethorprim in Serum ...... 159

® Symmetry C18 pH 3 Batch Test...... 160 Synthetic ...... 161 Tamoxifen ...... 162 Tamoxifen, Isolation of Impurities, Transfer from Analytical to Prep Columns ...... 163 Terfenadine ...... 164 Terfenadine and Related Compound ...... 165 Testosterone Esters in Serum ...... 166 Tetracyclines ...... 167 Tetracyclines in Serum ...... 168 Theophylline & Related Compounds ...... 169 Theophylline & Theobromine ...... 170 Trazodone...... 171 Triazine Herbacides in Drinking Water ...... 172 Trycyclic Antidepressants ...... 173 Trycyclic Antidepressants & Metabolites ...... 174 Trycyclic Antidepressants in Plasma ...... 175 Tripelennamine...... 176 Verapamil ...... 177 Verapamil and Norverapamil...... 178 Vitamins: Fat-Soluble ...... 179 Vitamins: Water-Soluble...... 180 Vitamins: Water-Soluble Folic Acid Mixture...... 181 Zidovudine (AZT) ...... 182

8 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Acephate in Drinking Water

HPLC Method Oasis® HLB Extraction Method Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg Part Number WAT094226 Column: SymmetryShield™ RP , 3.9 x 150 mm, 5 µm 8 Prepare Sample: Part Number: WAT200655 To 20 mL sample add 5 g NaCl Adjust to pH 3 with H PO Mobile Phase: H2O/ACN 96:4 3 4 Flow Rate: 1.0 mL/min Condition: Injection volume: 75 µL 3 mL methylene chloride Detection: UV @ 200 nm (0.02 AUFS) Rinse: 3 mL methanol Rinse: 3 mL H2O

Load: 20 mL sample @ 4 mL/min

Elute: 2 mL methylene chloride

Acephate Evaporate and Reconstitute: with 250 µL mobile phase O O H3CS P C H CO N CH 3 3 H % Recovery - contact and systematic Spike Level Spike Level 10 µg/L 5 µg/L organophosphorous insecticide 5 replicates 7 replicates - highly polar, water soluble 91.0% (16.3% RSD) 83.6% (15.0% RSD)

(Source water: chlorinated municipal tap water)

acephate

spiked tap water

nonspiked sample

blank cartridge

0105 MinutesMinutes 15

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HPLC Method Oasis® HLB Extraction Method Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg Part Number WAT094226 ® Column: Symmetry C8 3.9 x 150mm, 5µm Part Number: WAT046970 Condition: 3 mL methanol Mobile Phase A: 10mm phosphate buffer pH 6.8,/acetonitrile 70:30 Mobile Phase B: Acetonitrile Rinse: Gradient: Time Profile 2 mL H2O

(min) %A %B Load: 0 100 0 150 mL sample @ 4 mL/min 20 40 60 Wash: Flow Rate: 1.2 mL/min 1 mL H2O Injection volume: 80 µL Detection: UV @ 214 nm (0.02 AUFS) Elute: 1.2 mL methanol

Evaporate to 0.2 mL Reconstitute to 0.5 ml with reagent H2O

CH2CH3

CH2OCH3 N

COCH2Cl

CH2CH3

Alachlor

Compounds % Recovery Tap Water Tap Water Well Water 2.0 µg/L 200 ng/L 200 ng/L 5 replicates 5 replicates 5 replicates 1. Metolachlor-metabolite* 99.1 (10) 78.9 (22) 88.3 (3.3) 2. 2-Chloro-2,6-diethylacetanilide 98.5 (3.8) 109 (34) 105 (24) 3. 2,6-Diethylaniline 93.3 (11) 80.3 (23) 89.2 (7.8) 4. Alachlor 93.9 (3.2) 90.1 (21) 89.9 (1.2) spiked well water 5. Metolachlor 97.1 (6.6) 93.0 (16) 112 (3.3) (200 ng/L)

1 * 2-[(2-ethyl-6-methylphenyl)amino]1-propanol 3 4 2 5

nonspiked sample

blank cartridge

0 10 Minutes 20

10 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Acetaminophen and Codeine

HPLC Method

® Column: Symmetry C8, 3.9 x 150mm, 5 µm Part Number: WAT046970 Mobile phase: 10mM docusate sodium in methanol/water/THF/phosphoric acid 60:36:4:1 Flow rate: 0.75mL/min Injection volume: 15 µL of 55µg/mL acetaminophen and 35 µg of codeine sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.09 2. 1.04

1

1. Acetaminophen

2. Codeine

2

0246 81012

MinutesMinutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150mm, 5µm Part Number: WAT046980 Mobile phase: 50 mM phosphate, pH 5.8/acetonitrile 85:15 Flow rate: 1.0 mL/min Injection volume: 20 µL of 775µg/mL acetaminophen and 8 µg hydrocodone bitartrate sample Detection: UV @ 214 nm

1. Acetaminophen

2. Hydrocodone Bitartrate

Minutes

12 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Acetaminophen in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge

™ Part Number WAT094225 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part Numbers: Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 3.0/methanol 97:3) Load Flow rate: 1.0 mL/min 1 mL spiked porcine serum Injection volume: 50 µL of reconstituted porcine serum extract Wash Temperature: 30˚ C 1 mL 5% methanol in water Detection: UV @ 220 nm Elute 1 mL methanol Compounds: 1: Sulfanilamide (I.S.) Evaporate and Reconstitute 40 ˚C under nitrogen stream 2: Acetaminophen 200 µL of 50 µg/mL sulfanilamide (I.S.) in water

H N CH2 HO

O 1. Sulfanilamide (I.S) 2. Acetaminophen

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1

0.02 AU

2 Compound Concentration % Recovery %RSD µg/mL (n=6)

B Acetaminophen 0.100 107% 1.5% 0.500 101% 0.40% A

0 4 8 12 MinutesMinutes

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HPLC Method Oasis® HLB Extraction Method Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg Part Number WAT094226 Column: SymmetryShield™ RP 3.9 x 150mm, 5µm 8 Prepare Sample: 75 mL sample, Part Number: WAT200655 adjust to pH 2 with H3PO4 Mobile Phase A: 13 mM phosphate buffer, pH 3.4 Condition: Mobile Phase B: Acetonitrile 3 mL 10:90 methanol/MTBE*

Gradient: Time Profile Risnse: 2 mL methanol (min) %A %B Rinse: 2 mL H2O

08515 Load: 87030 75 mL sample @ 4 mL/min 15 70 30 Wash: 1 mL H O 30 40 60 2 35 10 90 Elute: 2 mL 10:90 methanol/MTBE* Flow Rate: 1.0 mL/min Evaporate to 0.2 mL Injection volume: 75 µL Reconstitute to 0.5 mL with H2O Detection: UV @ 230 nm (0.015 AUFS) * methyl t-butyl ether diethyl ether can be used as an alternative to MTBE

OH

O N Cl OH Cl Cl

Cl + O N N Cl N O O

OH 1. Picloram 2. Dicamba 3. Chloramben 4. 4-Nitrophenol

O Cl N S O Cl OH N O O O O O O O OH OH Cl

5. Bentazon Cl 6. 2,4-D Cl 7. MCPA 8. Dichloroprop 9. 2,4,5-T

O OH Compounds

OH

Cl 1 O 1. Picloram O 2. Dicamba Cl Cl 3. Chloramben 10. MCPP 11. 3,5-Dichlorobenzoic acid 4. 4-Nitrophenol 2 5. Bentazon 3 Cl OH spiked tap water Cl 6. 2,4-D O (400 ng/L) O Cl O 7. MCPA O Cl 8. Dichlorprop 4 5 Cl 9. 2,4,5-T 13 10 OH 6 9 12 15 12. 2,4-DB 13. 2,4,5-TP 7 8 11 14 10. MCPP 11. 3,5-Dichlorobenzoic Cl O OH nonspiked sample + 12. 2,4-DB O N O F 13. 2,4,5-TP O + NN F F O + O OOO O N 14. Acifluorfen O O 15. Dinoseb 0 10 20 MinutesMinutes 30 14. Acifluorfen 15. Dinoseb

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 0.1% glacial acetic acid (aq), pH 3.3 Flow rate: 1.0 mL/min Injection volume: 5 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1

1. Acyclovir

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM, pH 7.0 phosphate buffer/acetonitrile 95:5 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1

1. Acyclovir

Minutes

16 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Acyclovir in Plasma

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column 3.9 x 20 mm, 5 µm

Part numbers: Column - WAT200655, Guard - WAT200675 Condition Mobile phase: 20 mM ammonium acetate, pH 5 1 mL methanol/1 mL water Flow rate: 1.0 mL/min Injection volume: 50 µL Load 1 mL spiked plasma Detection: UV @ 250 nm

Wash Compounds: 300 µL water 1. Acyclovir 2. Guanosine hydrate Elute 500 µL 1% glacial acetic acid with 10% methanol

Evaporate and Reconstitute Dilute and add internal standard: 480 µL mobile phase and 20 µL internal standard

1. Acyclovir 2. Guanosine hydrate (I.S.)

Chromatogram of Plasma Extracts: A) Blank B) Spiked Sample

1 Compound Concentration % Recovery %RSD µg/mL (n=6)

Acyclovir 1.0 95.1% 3.4% 0.20 98.3% 5.0%

2

B

0.015AU A

0515 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 17 Albuterol (Salbutamol)

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: Water/methanol/glacial acetic acid 69:30:1 with 5 mM sodium 1-hexanesulfonate Flow rate: 1.0 mL/min Injection volume: 10 µL of 200 µg/mL salbutamol Detection: UV @ 276 nm

USP Tailing Factors 1. 1.2

1

HO

H N HO CH 3

CH 3 HO 1. Salbutamol

02468

Minutes

18 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Albuterol (Salbutamol) in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046980, Guard - WAT054250 Condition: 1 mL methanol/1 mL water Mobile phase: 5 mM hexanesulfonate and 1% glacial acetic acid in water/1% glacial acetic acid in methanol, 75:25 Load: Flow rate: 1.0 mL/min 1 mL spiked porcine serum with 1.0 µg/mL bamethan (I.S.) Injection volume: 20 µL of reconstituted porcine serum extract

Detection: Fluorescence, Ex = 275 nm, Em = 310 nm Wash: 1 mL 5% methanol in water Compounds: Elute: 1: Salbutamol 1 mL methanol 2: Bamethan (I.S.)

Evaporate and Reconstitute: 40 ˚C under nitrogen stream 200 µL mobile phase

HO

H N HO CH 3

CH 3 HO

1. Salbutamol 2. Bamethan (I.S.)

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample Compound Concentration % Recovery %RSD µg/mL (n=6)

Salbutamol 0.400 98.8% 3.4% 2.00 98.0% 3.5%

1 2

100 mV B

A

0 5 10 15 20 MinutesMinutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 19 Alkaloids in Goldenseal

HPLC Method Oasis® HLB method for alkaloids in Goldenseal Conditions for Oasis® HLB cartridge, 3 cc, 60 mg

® Part Number WAT094226 Column: Symmetry C18, 4.6 x 250 mm, 5 µm Part number: WAT054275 Condition/Equilibrate: Mobile Phase A: 0.1% phosphoric acid 1 mL methanol/1 mL 150 mM K2HPO4 Mobile Phase B: Acetonitrile Gradient: Time Profile Load: (min) %A %B 3 mL of diluted sample 09010 Wash: 13 78 22 1 mL 30% methanol/ 150 mM K2HPO4 40 60 40

Flow Rate: 1.5 mL/min Elute: Injection volume: 10 µL 3 mL methanol Temperature: 35° C Detection: UV @ 330 nm

Herbal tea extract Herbal tea extract after SPE cleanup before SPE cleanup using Oasis® MAX

Sample pre-preparation 100 mg sample is extracted with 25 mL of ethanol:water (70:30) 1 mL of the ethanolic extract is diluted 1:3 with water (~ pH 7)

• Recovery, measured with certified standards at 100 ppm in reagent water, was greater than 85 % for all compounds • All analyses gave results within ± 35 % of the expected values with the exception of the liquid supplement (see goldenseal analysis) • The selective SPE extraction and cleanup procedure provided a convenient analysis of echinacea phenolics in complex matrix such as in herbal tea

hydrastine berberine

0510 Minutes

20 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Amoxapine and Trazodone

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 20 mM potassium phosphate pH 7.0/ methanol 35:65 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL amoxapine and 100 µg/mL trazodone Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1 2. 1.4

1. Trazodone

2. Amoxapine

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 21 Amoxicillin

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium acetate, pH 4.0/acetonitrile 96:4 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sample Detection: UV @ 230 nm

USP Tailing Factors 1. 1.15

1. Amoxicillin

Minutes

22 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Amoxicillin and Clavulanate Potassium Augmentin Tablet

HPLC Method

® Column: Symmetry C8 ,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 56 mM sodium phosphate, pH 4.4/methanol 95:5 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL amoxicillin and 25 µg/mL clavulante extracted tablet sample at 10 to 39 µg/mL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.2 2. 1.1

1. Clavulanate

2. Amoxicillin

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium acetate, pH 4.0/acetonitrile 96:4 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sample Detection: UV @ 230 nm

USP Tailing Factors 1. 1.13

1. Amoxicillin

Minutes

24 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Ampicillin

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium acetate, pH 4.0/acetonitrile 92:8 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sample Detection: UV @ 230 nm

USP Tailing Factors 1. 1.07

1. Ampicillin

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 25 Amphetamines

HPLC Method

™ Column: SymmetryShield RP18, 3.9 x 150 mm, 5 µm, Part Number: 186000108 Mobile phase: 0.1% TFA in Water/acetontrile/methanol 91:7:2 Flow rate: 1.0 mL/min Injection volume: 50 µL Detection: UV @ 210 1. Ephedrine (I.S.)

USP Tailing Factors 1. 1.28 2. 1.32 3. 1.30

2. Amphetamine

3. Methamphetamine

Minutes

26 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Amphetamines in Human Urine

HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate, 30 mg/96-well ™ Part Number WAT058951 Column: SymmetryShield RP18, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column, 3.9 x 20 mm, 5 µm Part Numbers: Column - 186000108, Guard - 186000107 Conditioning: 1 mL methanol Mobile phase: 0.1% TFA in Water/Acetontrile 90:10 1 mL water Flow rate: 1.0 mL/min Injection volume: 50 µL Load: 2 mL of acidfied spiked human urine Temperature: 30˚ C Detection: UV @ 214 nm (0.14 AUFS) Wash 1: 1 mL 5% methanol with 2% ammonium hydroxide Compounds: Wash 2: 1. Ephedrine 1 mL 20% methanol with 2. Amphetamine 2% ammonium hydroxide

3. Methamphetamine Elute: 0.5 mL 20% methanol with 2% acetic acid

1. Ephedrine (I.S.) 2. Amphetamine 3. Methamphetamine

Chromatogram of A) Spiked Urine, B) Blank Urine

%Recovery (%RSD) Spike Levels: 0.7 µg/mL 3.5 µg/mL

Amphetamine (n=8) 99.3 (4.9) 104.7 (1.8)

FS

U 1 Methamphetamine (n=8) 105.5 (5.8) 100.9 (4.4) 4 A 2

0.1 Amphetamine Interday (n=16) 102.8 (2.7) 3 Amphetamine Interperson (n=16) 98.8 (6.2) 101.8 (3.3) Methamphetamine Interday (n=16) 98.5 (5.4) Methamphetamine Interperson (n=16) 96.4 (11.5) 99.4 (6.3) A Ephedrine (I.S.) (n=8) 89.5 (1.4) B

2 4 6 8 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 27 Anticonvulsants

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 100 mM potassium phosphate, pH 6.9/acetonitrile/water 20:30:50 Flow rate: 1.0 mL/min 1. Succinimide Injection volume: 10 µL of11-160 µg/mL sample Temperature: 30˚ C Detection: UV @ 214 nm

USP Tailing Factors 1. 1.41 2. 1.03 3. 1.11 4. 1.02 2. Primidone 5. 1.07

3. Phenobarbital

4. Mephenytoin

5. Mephobarbital

Minutes

28 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Antidepressants in Plasma

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part number: WAT200655 Condition: Mobile phase: 100 mM phosphate pH 7.0/acetonitrile/methanol 55:35:10 1 mL methanol/1 mL water

Flow rate: 1.0 mL/min Load: Injection volume: 100 µL acidified plasma sample solution 1 mL of acidified plasma sample solution Temperature: 30˚ C Detection: UV @ 230 nm Wash: 1 mL of 70% methanol, 2% ammonium hydroxide Compounds: Elute: 1. Nordoxepin 0.5 mL of 70% methanol, 2. Nortriptyline 2% acetic acid

3. Norverapamil Evaporate and Reconstitute: 4. Doxepin Not required 5. Amitriptyline 6. Verapamil 7. Methoxyverapamil (I.S.)

1. Nordoxepin 2. Nortriptyline 3. Norverapamil 4. Doxepin

5. Amitriptyline 6. Verapamil 7. Methoxyverapamil (I.S.)

Chromatogram of Plasma Extracts: A) Blank B) Spiked Sample Compound Concentration % Recovery %RSD

µg/mL (n=6) 1 Amitriptyline 1.46 99.9% 1.10% 4 5 0.002 AU 0.29 94.9% 0.60% 2 3 6 Doxepin 1.00 98.4% 0.69% 0.20 98.0% 2.30% 7 Nordoxepin 0.50 92.8% 0.91% 0.10 88.0% 1.33% Nortriptyline 0.62 95.3% 1.29% 0.12 91.1% 1.03% Verapamil 2.50 96.7% 0.63% B. 0.50 98.8% 0.70% A. Norverapamil 1.00 95.9% 0.59% 0.20 98.5% 1.04% 051015 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 29 Antiepileptics and Metabolites

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 100 mM potassium phosphate, pH 6.9/acetonitrile/methanol/water 17:25:5:53 Flow rate: 1.0 mL/min Injection volume: 5 µL of 25µg/mL sample Detection: UV @ 214 nm

USP Tailing Factors 1. NA 5. 1.10 2. NA 6. 1.17 3. 1.13 7. NA 4. 1.17 8. NA 1. 2-Phenyl-2ethylmalonamide 4. Phenobarbital

2. p-Hydroxyphenobarbital 5. Carbamezapine-10,11-epoxide

3. Primidone 6. Carbamezapine

7. 10,11-Dihydrocarbamezapine 8. Phenytoin

Minutes

30 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Antipyrine and Its Metabolite

HPLC Method

™ Column: SymmetryShield RP8,4.6 x 50 mm, 5µm Part Number: 186000224 Mobile phase: 0.1% glacial acetic acid/methanol 85:15 Flow rate: 1.0 mL/min Injection volume: 5 µL solution with antipyrine (240 ng) and 4-hydroxyantipyrine (29 ng) Detection: UV @ 254 nm

1. Antipyrine 1

2

2. 4-Hydroxyantipyrine

2 4

Minutes

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HPLC Method Oasis® MCX SPE Method Conditions for Oasis® MCX Cartridge, 6 cc, 150 mg

™ Part Number 186000256 Column: SymmetryShield RP18, 3.9 x 150 mm, 5 µm Part number: 186000108 Prepare Sample: Mobile Phase A: 20 mM Phosphate buffer, pH 6.8 adjust to pH 1.5 with HCI Mobile Phase B: Acetonitrile Condition: 2 mL methanol Gradient: Time Profile Rinse with 2 mL H2O (min) %A %B 0955 Load Sample 2955 Wash #1: 20 25 75 2 mL 0.1 N HCI Flow Rate: 0.8 mL/min Injection volume: 80 µL Wash #2: 2 mL methanol Detection: PDA (215 nm)

Elute: 3 mL methanol (4% NH4OH)

Evaporate and Reconstitute

Compounds (% Recovery - % RSD, n=5) 0.2 µg/L 1.0 µg/L 1. Hydroxydesisopropylatrazine 94 (3) 85 (3) 2. Desethyldesisopropylatrazine 75 (8) 76 (5) 3. Hydroxydesethylatrazine 89 (6) 76 (7) 4. Desisopropylatrazine 79 (4) 83 (2) Drinking water samples (100 mL) were spiked 5. Hydroxyatrazine 107 (7) 101 (2) with the herbicides and adjusted to pH 1.5. The 6. Desethylatrazine 79 (5) 83 (3) samples were then analyzed using 6 cc 7. Atrazine 89 (5) 77 (3) Oasis® MCX cartridges using the protocol for basic compounds

4 5 1

PDA (0.08 AUFS) 6 2 3

7 Spiked Sample

Blank Sample

0 10 20 MinutesMinutes

32 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Barbiturates

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 100 mM Potassium phosphate, pH 6.9/acetonitrile/water 20:30:50 Flow rate: 1.0 mL/min Injection volume: 10 µL of 25µg/mL sample Detection: UV @ 214 nm

USP Tailing Factors 1. 1.17 5. 1.11 2. 1.12 6. 1.11 3. 1.14 7. 1.09 1. Barbital 2. Allobarbital 4. 1.10 8. 1.09

3. Phenobarbital 4. Butabarbital

5. Butalbital 6. Amobarbital

7. Mephobarbital 8. Secobarbital

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 33 Barbiturates in Human Urine

HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate, 30 mg/96-well Part Number WAT058951 ™ Column: SymmetryShield RP18, 2.1 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column RP , 3.9 x 20 mm, 5 µm Condition: 18 1 mL of methanol Part numbers: Column - 186000111, Guard - 186000107 Mobile phase: 50 mM potassium phosphate, pH 7.0/acetonitrile 71: 29 Equilibrate: 1 mL water Flow rate: 1 mL/min Injection volume: 80 µL urine extract Load: 2 mL water of spiked human urine Temperature: 30 ˚C Detection: UV @ 214 nm (0.350 AUFS) Wash 1: 1 mL of 5% methanol

Wash 2: 1 mL 25% methanol with 2% acetic acid

Elute: 1 mL 35% methanol with 2% ammonium hydroxide

Evaporated: To dryness under N2 at 40˚C

Reconstituted: 300 µL of water

H O N O H C 3 NH

HC3 O

H3C 6. Secobarbital 1. Phenobarbital 2. Butabarbital 3. Butalbital 4. Amobarbital (I.S.) 5. Mephobarbital

Chromatogram of A) Blank Urine, B) Spiked Urine %Recovery (n=8) (%RSD) (n=8) Compound 0.2 µg/mL 1.0 µg/mL

1 1. Phenobarbital 114.3 (1.7) 106.5 (0.5) 2. Butabarbital 95.7 (1.3) 105.5 (0.7) 3. Butalbital 109.5 (0.9) 104.2 (0.9) 2 4. Amobarbital (I.S.) 86.3 (1.7) 3 5. Mephobarbital 92.5 (3.6) 92.4 (1.7) 6. Secobarbital 101.5 (5.2) 94.8 (2.2) 0.350 AUFS 4 5 6

B A

16 0 8 MinutesMinutes

34 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Beclomethasone

HPLC Method

® Column: Symmetry C18,4.6 x 100 mm, 3.5 µm Part number: WAT066220 Mobile phase: Acetonitrile/water 65:35 Flow rate: 1.4 mL/min Injection volume: 15 µL of 0.1 mg/mL of each beclomethasone-dipropionate and testosterone propionate (internal standard) Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1 2. 1.0

1

1. Beclomethasone Dipropionate

2

2. Testosterone Propionate (I.S.)

0 5 10

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 35 Benomyl and Bisphenol A

HPLC Method Oasis® SPE Method for Endocrine Disruptors Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg Part Number WAT106202 ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Prepare Sample: Part number: WAT046980 adjust to pH 3 Mobile Phase A: 10 mM phosphate, pH 6.8 Condition: Mobile Phase B: Methanol 3 mL MTBE*

Gradient: Time Profile Rinse: 3 mL methanol (min) %A %B Rinse: 3 mL H2O 06040 Load: up to 500 mL sample 20 0 100 Flow Rate: 1.0 mL/min Wash: 3 mL 5% methanol in H2O Injection volume: 100 µL Elute: Detection: PDA (283 nm extracted) 6 mL 10% methanol/MTBEt*

For GC analysis, dry For LC analysis, exchange extract over Na2SO4, to acetonitrile, then then adjust to 1 mL adjust to 1 mL

* methyl t-butyl ether diethyl ether can be used as an alternative to MTBE

1. Benomyl 2. Carbaryl

3. Atrazine 4. Bisphenol A 1 2 4 3 sample

Compounds % RECOVERY ± % RSD blank 1. Benomyl 65 ± 10 2. Carbaryl N/A 3. Atrazine N/A 0 5 10 4. Bisphenol A 83 ± 2

MinutesMinutes 100 ppb spike level in pear

36 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Betamethasone

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 mm x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Water/acetonitrile/phosphoric acid 63:37:0.1 Flow rate: 1.0 mL/min Injection volume: 10 µL of 50 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.12 2. 1.09

1. Betamethasone

2. Propylparaben (internal standard)

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Water/acetonitrile/phosphoric acid 63:37:0:1 Flow rate: 1.0 mL/min Injection volume: 10 µL of 150 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.14 2. 1.12

1. Betamethasone

2. Propylparaben (I.S.)

Minutes

Celestone is a registered trademark of Schering Corporation.

38 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. BSA Tryptic Digest

HPLC Method

™ Column: Symmetry300 C18, 4.6 x 50 mm, 5 µm Part number: WAT106209 Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 70 30 Flow rate: 1.0 mL/min Injection volume: 20 µL Detection: UV @ 214 nm

0.00 20.00 40.00

Minutes

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HPLC Method

™ Column A: Symmetry300 C18, 4.6 x 50 mm, 5 µm ™ Column B: Symmetry300 C18, 4.6 x 150 mm, 5 µm ™ Column C: Symmetry300 C18, 4.6 x 250 mm, 5 µm Part numbers: A: WAT106209 B: WAT106157 C: WAT106151 Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 70 30 Flow rate: 0.75 mL/min Injection volume: 20 µL (7 µL for 4.6 x 50 mm) Temperature: 35˚C Detection: UV @ 214 nm

A

0.00 20.00 40.00

Minutes

B

0.00 20.00 40.00

Minutes

C

0.00 20.00 40.00

Minutes

40 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. BSA Tryptic Digest - Flow Rate Effects

HPLC Method

™ Column: Symmetry300 C18, 4.6 x 50 mm, 5 µm Part number: WAT106209 Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 70 30 Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 214 nm

0.3 mL/min

0.00 20.00 40.00

Minutes

0.5 mL/min 0.00 20.00 40.00

Minutes

1.0 mL/min 0.00 20.00 40.00

Minutes

2.0 mL/min 0.00 20.00 40.00

Minutes

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HPLC Method

™ Column: Symmetry300 C18, 4.6 x 50 mm, 5 µm Part number: WAT106157 Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 70 30 Flow rate: 0.75 mL/min Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 214 nm

Gradient time: 15 min.

0.00 20.00 40.00

Minutes

Gradient time: 25 min. 0.00 20.00 40.00

Minutes

Gradient time: 50 min. 0.00 20.00 40.00

Minutes

Gradient time: 75 min.

0.00 20.00 40.00

Minutes

Gradient time: 150 min.

0.00 20.00 40.00

Minutes

42 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Bupropion

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 20 mM potassium phosphate, pH 7.0/methanol 35:65 Flow rate: 1.0 mL/min Injection volume: 10 µL of 20 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 105

1. Bupropion

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 55:45 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 240 nm

USP Tailing Factors 1. 1.08

1. Busiperone

Minutes

44 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Caffeine and Metabolites in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3.0 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT094243, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM ammonium acetate, pH 5/acetonitrile, 95:5 Load Flow rate: 0.6 mL/min 1 mL porcine serum with 0.2 µg ß-hydroxyethyltheophylline (I.S.) Injection volume: 20 µL of reconstituted porcine serum extract

Temperature: 25˚ C Wash Detection: UV @ 273 nm 1 mL water

Elute Compounds: 1 mL methanol 1: Theobromine 2: Paraxanthine Evaporate and Reconstitute 40 ˚C under nitrogen stream 3: Theophylline 200 µL mobile phase 4: β-hydroxyethyltheophylline (I.S.) 5: Caffeine

1. Theobromine 2. Paraxanthine 3. Theophylline

O CH2CH2OH

H3C N O CH2 N

H3C N N N O N

N CH3 O N

CH3

4. ß-hydroxyethyltheophylline (I.S.) 5. Caffeine

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD 1 µg/mL (n=6)

Theobromine 0.100 103.0% 3.4% 3 0.500 104.0% 2.4% 0.03 AU 2 Paraxanthine 0.100 101.0% 6.2% 0.500 98.9% 2.3% 5 Theophylline 0.100 100.0% 5.2% 0.500 101.0% 2.1% 4 Caffeine 0.100 92.6% 4.4% 0.500 97.5% 3.2% B

A

0 5 10 15

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 45 Captopril

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 0.1% phosphoric acid in water/methanol 60:40 Flow rate: 1.0 mL/min Injection volume: 10 µL of 200 µg/mL captopril and 20 µg/mL of captopril disulfide Temperature: 40˚ C Detection: UV @ 220 nm

USP Tailing Factors: 1. 1.0 2. 1.2

1. Captopril

Minutes

46 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Carbadox

HPLC Method

® Column: Symmetry C18, 4.6 x 100 mm, 3.5 µm Part number: WAT066220 Mobile phase: 20 mM ammonuim acetate, pH 5.0/Acetonitrile 85:15 Flow rate: 1.4 mL/min Injection volume: 15 µL of 0.025 mg/mL Temperature: 30˚ C Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1

1. Carbadox

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 47 Catechins - 4.6 x 20 mm Intelligent Speed™ Separation

HPLC Method

® ™ Columns: Symmetry C18, 4.6 x 20 mm, 3.5 µm IS ™ ™ SymmetryShield RP18, 4.6 x 20 mm, 3.5 µm IS Part numbers: Symmetry - 186002090, Shield - 186002092 Mobile phase A: 0.1% HCOOH in Water Mobile phase B: 0.1% HCOOH in MeOH Gradient: Time Profile (min) %A %B 0.0 100 0 4.0 60 40 Flow rate: 3.0 mL/min

Injection volume: 10 µL OH ˚ Temperature: 30 C O CH2 OH

Detection: UV @ 280 nm H3C N OH HO O OH N

Compounds: HO O O N OH O N OH OH 1. Caffeine O OH 2. Epicatechin CH3 OH OH 3. Epigallocatechin Gallate OH 1. Caffeine 2. Epicatechin 3. Epigallocatechin Gallate

1

™ SymmetryShield RP18

2 3 t0= 0.13 min Max psi= 2225

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes

1

® Symmetry C18 3 2

t0= 0.13 min Max psi= 2111

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes

48 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cefixime

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium acetate, pH 3.8/acetonitrile 80:20 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL extracted suspension sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.27

1. Cefixime

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 49 Cefuroxime

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 5 mM sodium acetate, pH 3.8/acetonitrile 80:20 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.44

1. Cefuroxime

Minutes

50 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cephalexin

HPLC Method

® Column: Symmetry C18, 3.0 x 150 mm, 5µm Part number: WAT054200 Mobile phase: 1.25% glacial acetic acid in water/methanol 88:12 Flow rate: 0.6 mL/min Injection volume: 400 µL of cephalexin (1 mg/mL) and 200 µL of 1-hydroxybenzotriazole (Internal standard, 1 mg/mL) Detection: UV @ 254 nm

USP Tailing Factors 1. 1.4 2. 1.2

1. 1-hydroxybenzotriazole (IS) 2

1

2. Cephalexin

0 10 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 51 Chloramphenicol

HPLC Method

® Column: Symmetry C8, 3.9 x 150mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Water/methanol/glacial acetic acid 55:45:0.1 Flow rate: 1 mL/min Injection volume: 10 µL of 100 µg/mL Detection: UV @ 280 nm

USP Tailing Factors 1. 1.1

1. Chloramphenicol

Minutes

52 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Chlordiazepoxide and Metabolites

HPLC Method

® Column: Symmetry C18,3.0 x 150 mm, 5 µm Part number: WAT054200 Mobile phase: 20 mM Potassium phosphate pH 7.0/methanol/acetonitrile 49:28:23 Flow rate: 0.59 mL/min Injection volume: 5 µL of 40 74 µg/mL Detection: UV @ 240 nm

USP Tailing Factors 1. 1.1 2. 1.2 3. 1.1 1. Norchlordiazepoxide 4. 1.2 5. 1.1

2. Oxazepam

1

3. Noradiazepam

3 4 5 4. Chlordiazepoxide 2

impurity

5 10

Minutes 5. Demoxepam

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 53 Chlordiazepoxide and Metabolites in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ® Part Number WAT094225 Column: Symmetry C18, 3.9 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition: 1 mL methanol/1 mL water Part numbers: Column - WAT046980, Guard - WAT054225 Mobile phase: 20 mM Potassium phosphate, pH 7.0/acetonitrile/methanol 56:21:23 Load: 1 mL spiked porcine serum Flow rate: 1.0 mL/min with 1.5 µg/mL demoxepam (I.S.) Injection volume: 20 µL of reconstituted porcine serum extract Wash: Detection: UV @ 240 nm 1 mL 5% methanol in water

Compounds: Elute: 1 mL methanol 1: Norchlordiazepoxide 2: Oxazepam Evaporate and Reconstitute: 40 ˚C under nitrogen stream 3: Nordazepam 200 µL of 20 mM potassium phosphate pH 4: Chlordiazepoxide 7/methanol 80:20 (v/v) 5: Demoxepam (I.S.)

H N O

Cl N

1. Norchlordiazepoxide 2. Oxazepam 3. Nordazepam 4. Chlordiazepoxide 5. Demoxepam

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1

Compound Concentration % Recovery %RSD µg/mL (n=6) 0.002 AU 1. Norchlordiazepoxide 0.200 97.8% 4.3% 5 2 3 0.040 96.5% 5.5% 4 2. Oxazepam 0.200 104.0% 2.6% 0.040 90.0% 2.5% 3. Nordazepam 0.200 101.0% 3.0% B 0.040 98.9% 1.2%

A 4. Chlordiazepoxide 0.200 90.0% 2.4% 0.040 100.0% 4.1% 5 10 15 20 MinutesMinutes

54 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Chlorpheniramine

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 50 mM potassium phosphate, pH 3.0/ acetonitrile 80:20 Flow rate: 1.0 mL/min Injection volume: 5 µL of 120 µg/mL sample Detection: UV @ 261 nm

USP Tailing Factors 1. 1.33

1. Chlorpheniramine Maleate

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 55 ™ Cimetidine- (SymmetryShield RP18)

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 250 mm, 5 µm Part number: 18600012 Mobile phase: 0.3 mL phosphoric acid, methanol/water 18:82 Flow rate: 1.8 mL/min Injection volume: 50 µL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.22

1. Cimetidine

01 23 Minutes

56 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cimetidine - -Pair Separation

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 6.6 mM sodium 1-hexanesulfanate, 0.04% phosphoric acid in water/methanol 76:24 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.2

1. Cimetidine

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 0.03% Phosphoric acid in water/methanol 90:10 Flow rate: 1.0 mL/min Injection volume: 10 µL injection of 10 µg/mL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.2

1

1. Cimetidine

1 2 3 4

Minutes

58 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Clobetasol and Related Compound

HPLC Method

™ Column: SymmetryShield RP18,4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: Acetonitrile/water/methanol 48:42:10 Flow rate: 1.0 mL/min Injection volume: 10 µL Detection: UV @ 240 nm

USP Tailing Factors 1. 1.07 2. 1.05

1. Clobetasol

2. Beclomethasone

Minutes

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HPLC Method

® Column: Symmetry C18, 4.6 x 250 mm, 5 µm Part number: WAT054275 Mobile phase: Water/methanol/acetonitrile 40:30:30 Flow rate: 1.5 mL/min Injection volume: 25 µL of sample with 40 µg/mL of clonazepam and 80 µL/mL of o-dichlorobenzene (internal standard) Detection: UV @ 254 nm

USP Tailing Factors 1. 1.08 2. 1.04

1. Clonazepam

2. O-dichlorobenzene (IS)

Minutes

60 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cocaine

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046980, Guard - WAT054225 Mobile phase: 20 mM potassium phosphate, pH 2.18/methanol 75:25 Flow rate: 1.0 mL/min Injection volume: 5 µL of 200 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.4

Me N O OMe

O O 1. Cocaine

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT046970, Guard - WAT054250 1 mL methanol/1 mL water Mobile phase A: 20 mM potassium phosphate, pH 7 Mobile phase B: Methanol Load Gradient: Time Profile 1 mL serum spiked porcine sample with 20 µL phosphoric acid (min) %A %B

07822 Wash 8.5 78 22 1 mL 5% methanol in water 8.6 42 58 30 42 58 Elute 1 mL methanol Flow rate: 1.0 mL/min

Injection volume: 1 mL of reconstituted porcine serum extract Evaporate and Reconstitute Detection: UV @ 235 nm 40 ˚C under nitrogen stream 250 µL water:methanol 80:20 (v/v)

Me Me N O N O OMe O

O O O O 1. Benzoylecgonine 2. Cocaine 3. Cocaethylene

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

2

3 1 Compound Concentration % Recovery %RSD µg/mL (n=6)

1. Benzoylecgonine 2.00 93.7% 1.7% 0.40 93.6% 2.2% 2. Cocaine 2.00 98.0% 1.1% 0.40 93.4% 2.9% 3. Cocaethylene 2.50 97.7% 2.8% 0.01. AU B 0.50 98.2% 5.1% A

5 10 15 20 25 Minutes

62 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Codeine and Its Glucuronide Metabolite in Human Urine

HPLC Method Oasis® MCX Extraction Method Oasis® MCX Extraction Cartridge, 3 cc/60 mg ® Column: Symmetry C18, 2.1 x 150 mm, 3.5 µm Part Number 186000254 Guard Column: Symmetry® C , 2.1 x 10 mm, 3.5 µm 18 Load Part numbers: Column - 186000174, Guard - 186000169 3 mL acidified spiked human urine Mobile phase: Methanol/Acetonitrile/0.05% TFA in water 5:5:90 Flow rate: 0.3 mL/min No Conditioning Injection volume: 80 µL urine extract Temperature: 30˚ C Wash 1 2 mL 0.1N Detection: UV @ 220 nm (0.04 AUFS)

Wash 2 Compounds: 2 mL methanol 1. Ranitidine (I.S.) Elute 2. Codeine-6-Glucuronide (C-6-G) 2 mL 5% ammonium hydroxide 3. Codeine with 95% methanol

After elution, the extract is diluted 1:3 with water for HPLC analysis

H3CO

CH3O

O H

N-CH3 O H O OH C O OH NCH3 O HO OH OH

1. Ranitidine 2. Codeine-6-Glucuronide 3. Codeine (C-6-G)

Compound % Recovery %RSD 0.12 µg/mL 0.6 µg/mL Chromatogram of Urine Extracts: A) Blank B) Spiked Sample Codeine (n=3) 88.5 (2.7) 99.5 (1.2) Codeine-6-Glucuronide (n=3) 99.3 (5.4) 98.7 (0.4) Codeine Interday (n=6) 102.5 (3.5) 105.4 (4.0) Codeine Interperson (n=9) 91.6 (7.1) 104.1 (5.4) 3 C-6-G Interday (n=6) 116.1 (9.4) 111.5 (3.3) C-6-G Interperson (n=9) 102.4 (7.7) 107.4 (7.3)

2 0.04 AUFS spiked sample

1

0 5 10 MinutesMinutes nonspiked sample

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 63 Codeine and Its Glucuronide Metabolite in Porcine Plasma

HPLC Method Oasis® MCX Extraction Method

® ® Oasis MCX Extraction Cartridge, 3 cc/60 mg Column: Symmetry C18, 2.1 x 150 mm, 3.5 µm ® Part Number 186000254 Guard Column: Symmetry C18, 2.1 x 10 mm, 3.5 µm Part numbers: Column - 186000174, Guard - 186000169 No Conditioning Mobile phase: Methanol/Acetonitrile/0.05% TFA in water 5:5:90

Flow rate: 0.3 mL/min Load Injection volume: 80 µL urine extract 3 mL acidified plasma sample Temperature: 30˚ C Detection: UV @ 220 nm (0.04 AUFS) Wash 1 2 mL 0.1N hydrochloric acid Compounds: 1. Ranitidine (I.S.) Wash 2 2. Codeine-6-Glucuronide (C-6-G) 2 mL methanol 3. Codeine Elute 2 mL 5% ammonium hydroxide with 95% methanol

After elution, the extract is diluted 1:3 with water for HPLC analysis

H3CO

CH3O

O H

N-CH3 O H O OH C O OH NCH3 O HO OH OH

1. Ranitidine 2. Codeine-6-Glucuronide 3. Codeine (C-6-G)

Chromatogram of Plasma Extracts: A) Blank B) Spiked Sample % Recovery(n=3) %RSD(n=3) Spike Level: 0.12 µg/mL 0.6 µg/mL

Codeine 95.4 (5.0) 103.4 (0.9) Codeine-6-Glucuronide 106.8 (1.9)1 04.6 (1.3)

Minutes

64 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cyclizine and Chlorcyclizine

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 7.4 mM sodium 1-heptanesulfonate in water/acetonitrile 65:35 Flow rate: 1.0 mL/min Injection volume: 8 µL of 100 µg/mL sample Detection: UV @ 230 nm

USP Tailing Factors 1. 1.28 2. 1.28

1. Cyclizine

2. Chlorcyclizine HCL

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 65 Cytochrome c Tryptic Digest - Column Lifetime Study, Critical Pair Resolution

HPLC Method

™ Column: Symmetry300 C18, 3.9 x 150 mm, 5 µm Part number: WAT106154 Mobile phase A: 0.1% TFA water/acetonitrile 90:10 Mobile phase B: 0.1% TFA water/acetonitrile 40:60 Gradient: Time Profile (min) %A %B 0 100 0 45 34 66 Flow rate: 0.75 mL/min Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 214 nm

Minutes Minutes

66 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cytochrome c Tryptic Digest - Pore Size Effects

HPLC Method

® Column: Symmetry C18, 4.6 x 150 mm, 5 µm, 100Å ™ Guard Column: Symmetry300 C18, 4.6 x 150 mm, 5 µm, 300Å Part numbers: Column - WAT045905, Guard - WAT106157 Mobile phase A: 0.1% TFA in Water Mobile phase B: 0.1% TFA in Acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 50 70 30 Flow rate: 0.75 mL/min Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 214 nm

® Symmetry C18, 4.6 x 150 mm, 5 µm, 100Å

0.00 20.00 40.00 Minutes

™ Symmetry300 C18, 4.6 x 150 mm, 5 µm, 300Å

0.00 20.00 40.00 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 67 Cytochrome c Tryptic Digest - Recovery Study

HPLC Method

™ Column: Symmetry300 C18, 4.6 x 50 mm, 5 µm Part number: WAT106209 Mobile phase A: 0.1% TFA in Water Mobile phase B: 0.1% TFA in Acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 70 30 Flow rate: 1.0 mL/min Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 214 nm

First Run

Second Run

Minutes TFA Blank Injection

68 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Cytochrome c Tryptic Digest - Reproducibility Study

HPLC Method

™ Column: Symmetry300 C18, 3.9 x 150 mm, 5 µm Part number: WAT106154 Mobile phase A: 0.05% TFA in Water Mobile phase B: 0.1% TFA in Acetonitrile Gradient: Time Profile (min) %A %B 0 100 0 45 72 28 Flow rate: 1.0 mL/min Injection volume: 20 µL Temperature: 35˚C Detection: UV @ 220 nm

Batch 107

Batch 106

Batch 104

Batch 103

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 69 Dextromethorphan

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 23 mM ammonium , pH 3.4/acetonitrile 30:70 Flow rate: 1.0 mL/min Injection volume: 20 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 0.98

1 1. Dextromethorphan

Minutes

70 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Dextromethorphan in a Cough Syrup

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 23 mM ammonium nitrate, pH 3.4/acetonitrile 30:70 Flow rate: 1.0 mL/min Injection volume: 20 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 0.95

1. Dextromethorphan

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 71 Diazepam in Serum - LC/MS

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30mg Extraction Cartridge ® Part Number WAT04225 Column: Symmetry C18, 2.1 x 100 mm, 3.5 µm

Part number: WAT058965 Condition: Mobile phase: 2 mM Ammonium Acetate/Acetonitrile/Formic Acid 65:35:0.1 1 mL methanol/1 mL water Flow rate: 200 µL/min

Injection volume: 10 µL Load: MS: Micromass Quattro LC 1 mL porcine serum Ion Mode: ES+ Cone Voltage: 45 V Wash: Collision Energy: 25 eV 1 mL 5% methanol in water

Elute: 1 mL methanol

Evaporate and Reconstitute: 40˚ C under nitrogen stream, 200 µL mobile phase 2.28 2.26 2.27 39 100 100 100 A B C Diazepam

CH3 N O

% % % Cl N

0 Time 0 Time 0 Time 1.00 2.00 3.00 4.00 1.00 2.00 3.00 1.00 2.00 3.00 4.00

Figure 4: MRM Chromatograms under optimum conditions of pure diazepam standard at (A) 0.2ng/mL (LOD) and (B) 5.0ng/mL and (C) a processed human plasma sample with a low concentration of diazepam (calculated as 0.75ng/mL) 285.1 100 Precursor (m/z) 285 % Compound 3 name: Diazepam 287.1 Coefficent of Determination: 0.998439 Calibration curve: 150.210* x + -0.398957 Response type: External Std., Area Curve type: Linear, Origin: Exclude, Weighting: 1/x Axis trans: None 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 Figure 1: Background-subtracted electrospray mass spectrum of pure diazepam standard (5µg), under optimum conditions. 771 Chromatographed as described above to remove contaminants.

154.1 100 Response 165 Product 285.4 (m/z) 193.4 222.4 Response 154 % 228.2 3.5 257.3

-0.399 ng/mL 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 182.0 -0.399 ng/mL 167.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 4.0 4.5 5.0 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 Figure 3: Calibration curve with triplicate injections for each point and demonstrating LOQ at 0.2ng/mL Figure 2: Background-subtracted electrospray product tion spectrum of pure diazepam standard (5µg), under optimum conditions. Chromatographed as described above to remove contaminants.

72 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Diclofenac Sodium

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 65:35 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 220 nm

USP Tailing Factor 1. 1.18

1 1. Diclofenac

0 1 2 3 4 5 6

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 73 Dihydroergotamine Mesylate

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM ammonium phosphate, pH 5.8/Acetonitrile 35:65 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL dihydroergotamine mesylate Detection: UV @ 281 nm

USP Tailing Factors 1. 1.1

1. Dihydroergotamine mesylate

Minutes

74 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Diltiazem HCL

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 100 mM sodium acetate, 5mM camphosulfonic acid, pH 6.2/acetonitrile/methanol 40:30:30 Flow rate: 1.0 mL/min Injection volume: 10 µL of 50 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.08

1. Dilitiazem HCL

Minutes

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HPLC Method

® Column: A. Symmetry C18, 3.9 x 150 mm, 5 µm ™ B. SymmetryPrep C18, 7.8 x 150 mm, 7 µm ™ C. SymmetryPrep C18, 19 x 150 mm, 7 µm Part numbers: A. WAT046980 B. WAT066288 C. WAT066240 Mobile phase: Acetonitrile/0.1% TFA in water 30:70 Flow rates: A. 0.7 mL/min B. 2.8 mL/min C. 16.6 mL/min Injection volume: Diltiazem: A. 0.5 mg B. 2.0 mg C. 11.9 mg Detection: UV @ 280 nm

® A. Symmetry C18 ,5 µm, 3.9 mm x 150 mm

1. Diltiazem Hydrochloride

™ B. SymmetryPrep C18 ,7 µm, 7.8 mm x 150 mm

™ C. SymmetryPrep C18 ,7 µm, 19 mm x 150 mm

Minutes

76 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Diphenoxylate HCL

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate, pH 6.0/acetonitrile 30:70 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL Diphenoxylate hydrochloride Detection: UV @ 210 nm

USP Tailing Factors 1. 1.3

1. Diphenoxylate HCL

Minutes

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HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate, pH 7.0/methanol 70:30 Flow rate: 1.0 mL/min Injection volume: 20 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.3

1. Disopyramide

Minutes

78 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Doxepin in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 7/methanol 38:62 Load Flow rate: 1.0 mL/min 1 mL spiked porcine serum Injection volume: 20 µL of reconstituted porcine serum extract Temperature: 30˚ C Wash 1 mL 5% methanol in water Detection: UV @ 254 nm Elute 1 mL methanol with 5 µg Compounds: butyl paraben (I.S.) 1: Butyl paraben (I.S.) 2: Doxepin Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL methanol

1. Butyl paraben 2. Doxepin

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample Compound Concentration % Recovery %RSD µg/mL (n=6) 1 Doxepin 0.200 95.8% 1.4% 2 4.00 96.6% 1.9%

0.001 AU

B A

0 4 8 12 MinutesMinutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 79 Enalapril

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 10 mM potassium phosphate, pH 2/acetonitrile 75:25 Flow rate: 2 mL/min Injection volume: 2 µL of 1.67 mg/mL Temperature: 80˚ C Detection: UV @ 215 nm

Compounds 1. Maleic Acid 2. Enalapril

1. Maleic Acid 2. Enalapril

Minutes

80 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Endocrine Disruptors in Soil

Oasis® SPE Method for Endocrine Disruptors HPLC Method Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg Part Number WAT106202 ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Prepare Sample: Part number: WAT046970 adjust to pH 3

Mobile Phase A: 10 mM phosphate pH 6.8 Condition: Mobile Phase B: Methanol 3 mL MTBE* Gradient: Time Profile Rinse: 3 mL methanol Rinse: 3 mL H2O (min) %A %B Load: 06040 up to 500 mL sample 20 0 100 Wash: Flow Rate: 1.0 mL/min 3 mL 5% methanol in H2O Injection volume: 100 µL Elute: Sample: 10 g potting soil extracted with 25 mL 6 mL 10% methanol/MTBEt* acetonitrile; then SPE on Oasis® HLB Detection: PDA (225 nm extracted, 0.04 AUFS) For GC analysis, dry For LC analysis, exchange extract over Na2SO4, to acetonitrile, then then adjust to 1 mL adjust to 1 mL

* methyl t-butyl ether diethyl ether can be used as an alternative to MTBE

1. Benomyl 2. Carbaryl 3. Atrazine 4. Bisphenol A

3

2

4 50 ppb spike level Compounds % Recovery ± % RSD 1. benomyl 62 ± 6 sample 2. carbaryl 91 ± 4 3. atrazine 84 ± 5 blank 4. bisphenol A 78 ± 6 1 Soil samples (5 g) were spiked with the appropriate compounds and extracted with 25 mL of acetonitrile (30 minutes on shaker). A 5 mL aliquot of the acetonitrile extract was diluted to 100 mL with reagent water (MilliQ) and then processed by SPE.

0 5 10

Minutes Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 81 Enkephalins

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase A: 0.1% TFA Mobile phase B: 0.1% TFA in acetonitrile/water 90:10 Gradient: Time Profile (min) %A %B 08020 30 40 60 Flow rate: 1.0 mL/min Injection volume: 5 µL of mixture with 500 µg/mL of each peptide Detection: UV @ 214 nm

USP Tailing Factors 1. 0.99 2. 0.97

Peptides

1: Met enkephalin: Tyr-Gly-Phe-Met

2: Leu enkephalin: Tyr-Gly-Gly-Phe-Leu

Minutes

82 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Etodolac

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 65:35 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.16

1. Etodolac

Minutes

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HPLC Method Oasis® HLB method for flavonoids in ginkgo ® Column: Symmetry® C , 4.6 x 250 mm, 5 µm Conditions for Oasis HLB cartridge, 3 cc, 60 mg 18 Part Number WAT094226 Part number: WAT054275 Condition/Equilibrate: Mobile Phase: 0.5% Phosphoric acid/Methanol 50:50 1 mL methanol/1 mL water Flow Rate: 1.5 mL/min Load: Injection volume: 10 µL 3 mL of diluted sample Temperature: 25° C Wash 1: Detection: UV @ 270nm 0.02 AUFS 2 mL 5% methanol/water

Wash 2: 2 ml methanol/ 50mM NH4OAc (30:70) pH 5

Dry Cartridge: air dry for 15 min with vacuum

Elute: 3 ml MTBE/methanol (80:20)

Evaporate and Reconstitute: 1 mL mobile phase

1. Quercetin 2. Kaempferol Sample pre-preparation 1 g sample is refluxed in 50 mL of ethanol: 3M HCl (70:30) for 2.5 hr The cooled sample is adjusted to exactly 100 mL 0.3 mL of the ethanolic extract is diluted 1:10 with water

• Recovery, measured with certified standards at 100 ppm in reagent water, was 82 % for quercitin

1 and > 90 % for the other compounds • All analyses gave results within ± 40 % of the expected values with the exception of the capsule (+ 60%) • The selective SPE extraction and cleanup procedure provided a convenient analysis of ginkgo flavonoids in a complex matrix (Herbal One with 16 herbal ingredients)

2 Compounds % RECOVERY 1. quercetin 82% 2. kaempferol > 90% 3. isorhamnetin > 90% total: 11 mg / tablet

3

10 10 20 MinutesMinutes

84 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Flunitrazepam

HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate pH 6.0/acetonitrile 60:40 Flow rate: 1.0 mL/min Injection volume: 10 µL of 30 µg/mL flunitrazepam Detection: UV @ 219 nm

USP Tailing Factors 1. 1.2

O N

N O2N F

1. Flunitrazepam

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate,30 mg 96-well ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT058951 Part number: WAT200655 Condition: Mobile phase: Water/Acetonitrile 70:30 1 mL methanol Flow rate: 1.5 mL/min

Injection volume: 100 µL Equilibrate: Temperature: Ambient 1 mL water Detector: UV @ 220 nm

Load: Compounds: 250 µL 1. 7-Amino Nitrazepam 2. 7-Amino Clonazepam Wash: 3. 7-Amino Flunitrazepam 1 mL, 10% methanol with acetic acid 4. Nitrazepam 5. Clonazepam Elute 1 mL, 40% methanol with ammonium hydroxide 6. Flunitrazepam

Data supplied by J. Vukovik, L. Schofield Waters Ltd., Canada

O N

N O2N F

Flunitrazepam

Compound % Recovery

7-Amino Nitrazepam 118 2 3 4 5 7-Amino Clonazepam 100

6 7-Amino Flunitrazepam 95.9

1 Nitrazepam 93.7

Clonazepam 101

Flunitrazepam 85.9

1 2 3 4 5 6 7 8 9 10 MinutesMinutes

86 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Four Drug Mixture- Warfarin, Dextromethorphan, Triprolidine, and Tetracaine

HPLC Method

™ Column: SymmetryShield RP18,3.9 x 150 mm, 5 µm Part number: 186000108 Mobile phase A: 25 mM Potassium phosphate, pH 7 Mobile phase B: Methanol Gradient: Time Profile (min) %A %B 08515 26 20 80 Flow rate: 1.0 mL/min Injection volume: 10 µL of mixture with 0.3 mg/mL triprolidine, 0.4 mg/mL dextromethorphan, 0.15 mg/mL tetracaine, and 0.05 mg/mL of warfarin Detection: UV @ 280 nm

USP Tailing Factors 1. 1.3 2. 1.6 3. 1.1 4. 1.2

1. Warfarin

2. Dextromethorphan HCL

3. Triprolidine HCL

4. Tetracaine Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 87 Furosemide

HPLC Method

™ Column: SymmetryShield RP18,4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 75:25 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.30

1. Furosemide

Minutes

88 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Hydrocortisone and Relate Compound

HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: Acetonitrile/water 35:65 Flow rate: 1.0 mL/min Injection volume: 10 µL of 10 µg/mL hydrocortisone and 30 µg/mL hydrocortisone-21-acetate Detection: UV @ 254 nm

USP Tailing Factors 1. 1.1 2. 1.1

1. Hydrocortisone 2. Hydrocortisone-21-Acetate

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 89 Ibuprofen and Naproxen in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.0 x 150 mm, 5 µm Part Number WAT094425 Guard Column: Sentry™ Guard column, 3.9 x 20 mm, 5 µm

Part Numbers: Column - WAT094243, Guard - WAT200675 Condition 1 mL methanol/1 mL water Mobile phase: 1.2% glacial acetic acid in water, Acetonitrile 50:50 Flow rate: 0.6 mL/min Load Injection: 20 µL of reconstituted porcine serum extract 1 mL spiked porcine serum acidified with Temperature: 25 ˚C 20 µL of phosphoric acid Detection: UV @ 254 nm Wash 1 mL 5% methanol in water Compounds:

1: Naproxen Elute 2: Ibuprofen 1 mL methanol

Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL of mobile phase

O CH3

OH H3C

1. Naproxen 2. Ibuprofen

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD µg/mL (n=6)

Naproxen 0.100 90.0% 4.0% 0.500 98.7% 1.9% 2.00 99.2% 0.85% Ibuprofen 0.500 86.8% 4.6% B 2.50 95.0% 3.7% A

051015 Minutes

90 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Insulin Chain - QC Batch Test for Symmetry300™ HPLC Columns

HPLC Method

™ Column: Symmetry300 C4 , 3.9 x 150 mm, 5 µm Part number: WAT106154 Mobile phases: A. 25% of 0.05% TFA in Acetonitrile / 75% of 0.05% TFA in Water B. 32% of 0.05% TFA in Acetonitrile/ 68% of 0.05% TFA in Water (Linear Gradient from 100% A to 100% B in 14 Min.) Flow rate: 1.0 mL/min Injection volume: 4 µg, 10 µL Detection: UV @ 220 nm

Compounds: 1. Insulin 2. Desamino 3. Deoxidized

Batch 3 Batch 2 Batch 1

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 91 Insulin- Impurity Isolation, Transfer from Analytical to Prep

HPLC Method

® Columns: A. Symmetry C18, 4.6 x 150 mm, 5 µm ™ B. SymmetryPrep C18, 7.8 x 300 mm, 7 µm ™ C. SymmetryPrep C18, 19 x 150 mm, 7 µm Part numbers: A. WAT045905 B. WAT066235 C. WAT066240 Mobile phases: A. 0.1% TFA in Water B. 0.1% TFA/acetonitrile 26% B to 33% B in 14 minutes adjusted for gradient delay on prep system Flow rates: A. 1.0 mL/min b. 5.76 mL/min c. 17mL/min Detection: UV @ 280 nm Sample Load: Bovine pancreas insulin, 10 mg/mL A. 0.07 mg b. 3 mg c. 8.5mg HPLC Systems: A. analytical system B. and C. prep system with 0.04 in. I.D. tubing

Compounds:

1. Insulin A and B chains with 51 residues, 5733 daltons 2. Impurity

® A. Symmetry C18 , 5 µm, 4.6 mm x 150 mm

™ B. SymmetryPrep C18 , 7 µm, 7.8 mm x 300 mm

™ C. SymmetryPrep C18 , 7 µm, 19 mm x 150 mm

Minutes

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HPLC Method

® Column: Symmetry C18, 4.6 x 150 mm, 5 µm Part number: WAT045905 Mobile phase A: 0.1% TFA in Water Mobile phase B: Acetonitrile with 0.1% TFA Gradient: Time Profile (min) %A %B 0 100 0 20 40 60 Flow rate: 1.0 mL/min Injection volume: 5 µL of sample with 150 µL of insulin suspension and 300 µL of 0.01N HCL Suspension contains -insulin crystals and protamine sulfate in buffered waters Detection: UV @ 280 nm

USP Tailing Factors 1. 1.1 2. 1.2 3. 1.2

Compounds: 1. Unknown 2. Insulin A and B chains with 51 residues, 5733 daltons 3. Unknown

Minutes

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HPLC Method

™ Column: SymmetryShield RP18,4.6 x 250 mm, 5 µm Part number: 186000112 Mobile phase: 70% diisopropylamine solution/30% ammonium acetate solution Diisopropylamine solution: 2 mL diisopropylamine in 1 L methanol Ammonium acetate solution: 1 g in 200 mL of water Flow rate: 1.5 mL/min Injection volume: 20 µL Detection: UV @ 225 nm

USP Tailing Factors 1. 1.03 2. 1.06

1. Ketoconazole

2. Terconazole

Minutes

94 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Lidocaine HCL

HPLC Method

™ Column: SymmetryShield RP18,4.6 x 250 mm, 5 µm Part number: 186000112 Mobile phase: 50 mL glacial acetic acid in 930 mL of water, pH adjusted to 3.4 with 0.1 N sodium hydroxide/70% buffer/30% acetonitrile Flow rate: 1.5 mL/min Injection volume: 20 µL Detection: UV @ 254 nm

USP Tailing Factors 1. 2.49 2. 0.99

1. Lidocaine HCL

2. Methylparaben

Minutes

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HPLC Method

® Column: Symmetry 3.5µm C18,4.6 x 100 mm Guard Column: Sentry™ Guard column, 3.9 x 20 mm, 5 µm Part numbers: Column - WAT066220, Guard - WAT054225 Mobile phase: Water/methanol/glacial acetic acid 79:20:1 Flow rate: 1.5 mL/min Injection volume: 10 µL of mixture of six sulfa drugs at 10 to 39 µg/mL Detection: UV @ 254 nm 1. Sulfanilamide

O

NH S NH2

N O N

2. Sulfadiazine

H2N

O S

S NH N O

3. Sulfathiazole

NH2

N O

S H C N NH 3 O

4. Sulfamerazine

5. Sulfamethazine

6. Succinylsulfathiazole

End Start

Minutes

96 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Maprotiline HCL

HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate pH 3.0/acetonitrile 65:35 Flow rate: 1.0 mL/min Injection volume: 5 µL of 100 µg/mL maprotiline hydrochloride Detection: UV @ 254 nm

USP Tailing Factors 1. 1.4

1. Maprotiline HCL

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 7.4mM sodium 1-heptanesulfonate in water/acetonitrile 58:42 Flow rate: 1.0 mL/min Injection volume: 20 µL of 25 µg /mL sample Detection: UV @ 230 nm

USP Tailing Factors 1. 1.36

1. Meclizine HCL

Minutes

98 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Methadone in Human Urine

HPLC Method Oasis® MCX Extraction Method Oasis® MCX Extraction Cartridge, 3 cc/60 mg ® Part Number 186000254 Column: Symmetry C18, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column, 3.9 x 20 mm, 5 µm No Conditioning Part numbers: Column - WAT046980, Guard - WAT054225 Mobile phase: 0.1% TFA in water/Methanol 60:40 Load Flow rate: 1 mL/min 3 mL acidified spiked human urine Injection volume: 100 µL urine extract Wash 1 Temperature: 30˚ C 2 mL 0.1M hydrochloric acid Detection: UV @ 210 nm Wash 2 2 mL methanol Compounds: 1. EDDP (0.2 µg/mL) Optional Wash 3 2 mL of 5% ammonium hydroxide 2. Estazolam (I.S.) (0.2 µg/mL) with 60% methanol 3. Methadone (0.2 µg/mL) Elute 2 mL 5% ammonium hydroxide with 95% methanol

Each elution diluted 1:1 with water for HPLC analysis

N N N CH3 CH O 3 CH N 3 N + N Cl CH3 CH3 CH3

CH3 1. EDDP (Methadone Metabolite) 2. Estazolam 3. Methadone

Chromatogram of Human Urine: A) Spiked Sample, B) Blank

3 %Recovery (%RSD) Compound n=3 n=9 1 (1 Subject) (3 Subject) (3 SPE Washes) (2 SPE Washes)

2 Methadone (0.5 µg/mL) 95.2 (1.0) 97.4 (3.0) Methadone (0.1 µg/mL) 98.0 (4.8) 114.7 (8.2) EDDP (0.2 µg/mL) 93.3 (1.0) 92.9 (2.4) 0.01 AU EDDP (0.4 µg/mL) 98.4 (2.9) 100.5 (5.8) Estazolam (IS) 94.4 (3.3) 91.2 (91.2)

A B

0 5 10 15

MinutesMinutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 20 mM potassium phosphate, pH 2.18/methanol 80:20 Flow rate: 1.0 mL/min Injection volume: 15 µL of 200 µg/mL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.5

1. Methylphenidate HCL

Minutes

100 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Metoprolol Tartrate and Oxprenolol

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 250 mm, 5 µm Part number: 186000112 Mobile phase: 1.92 g of 1-pentansulfonic acid sodium monohydrate, 0.27 g of sodium acetate trihydrate and 1.14 mL glacial acetic acid in 940 mL of water; 54% methanol/46% buffer Flow rate: 1.0 mL/min Injection volume: 30 µL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.29 2. 1.98

1. Metoprolol tartrate

2. Oxprenolol

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 95:5 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.03

1. Metronidazole

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: Water/Methanol 90:10 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL metronidazole Detection: UV @ 254 nm

USP Tailing Factors: 1. 1.2

1. Metronidazole

Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 103 Morphine and Its Glucuronide Metabolites in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT094225 Part number: WAT200655 Condition Mobile phase: 20 mM potassium phosphate, pH 6.4 1 mL methanol Flow rate: 1.0 mL/min Injection volume: 100 µL of porcine serum extract Equilibrate Detection: Fluorescence; ex 280 nm, em 355 nm 1 mL water

Load Compounds: 1 mL spiked porcine serum 1: Morphine-D3-glucuronide 2: Morphine-D6-glucuronide Wash 3: Morphine 1 mL water

Elute 0.5 mL 3% triethylamine

No Evaporation Sample injected directly

HO

COOH HO O O O OH H HO NCH3 OH COOH O H O H O O NCH3 OH NCH3 HO HO OH HO 1. Morphine-D3-glucuronide 2. Morphine-D6-glucuronide 3. Morphine

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD 6 3 µg/mL (n=6)

Morphine-D3-glucuronide 0.48 90.7 2.0 0.097 100 3.1 1 Morphine-D6-glucuronide 2.4 92.2 2.8

2 0.49 93.1 2.5 Morphine 3.6 102 3.6 mV 0.73 102 3.2

B A

05 10 15

MinutesMinutes

104 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Nadolol

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 250 mm, 5 µm Part number: 186000112 Mobile phase: Methanol/water 35:65, 2.93 g of sodium chloride, 1.0 mL of 0.1 N hydrochloric acid Flow rate: 1.0 mL/min Injection volume: 20 µL Detection: UV @ 220 nm

USP Tailing Factors 1. 1.19

1. Nadolol

Minutes

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HPLC Method

™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part number: WAT200655 Mobile phase: 100 mM Ammonium acetate, pH 5.0/acetonitrile 88:12 Flow rate: 1.0 mL/min Injection volume: 35 µL (500 µL eluate) Detection: UV @ 281 nm

USP Tailing Factors 1. 1.05 2. 1.04

1. Naloxone (I.S.)

HO

O OH N O

2. Naltrexone

Minutes

106 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Naltrexone in Plasma

HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate, 30 mg/96-well ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT058951 Part number: WAT200655 Mobile phase: 100 mM Ammonium acetate pH 5/acetonitrile 88:12 Condition 1 mL methanol/1 mL water Flow rate: 1.0 mL/min

Injection volume: 1 mL spiked plasma with naloxone (I.S.) Load Temperature: 8˚ C 1 mL spiked plasma with naloxone (I.S.) Detection: UV @ 281 nm

Wash Compounds: 1) 1 mL 5% methanol containing 2% ammonium hydroxide 1. Naloxone (I.S.) 2) 1 mL 20% methanol containing 2. Naltrexone 2% ammonium hydroxide

Elute 0.5 mL of 25% methanol containing 2% acetic acid

Evaporate and Reconstitute Not required

HO

O OH N O

1. Naloxone (I.S.) 2. Naltrexone

Chromatogram of Plasma Extracts: A) Blank B) Spiked Sample

1

2

0.001 AU

Compound Concentration % Recovery %RSD µg/mL (n=6) B A Naltrexone 3.3 108% 4.2%

02 4 6 8 10

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate, 30 mg/96-well ® Part Number WAT058951 Column: Symmetry C8, 2.1 x 30 mm, 5 µm

Part number: WAT058977 Condition 1 mL of methanol/1mL water Mobile phase: 10 mM NH4OH, pH 5/Acetonitrile 88:12 Flow rate: 200 µL/min Loading Injection volume: 50 µL plasma extract 1 mL of spiked plasma ™ MS: Micromass Quattro II Wash 1 Ion Source Temp: 120˚ C 1 mL of 5% methanol/ 2% ammonium hydroxide CID: 1.8e-3 mBar, 20 eV Desolvation Temp: 400 ˚C Wash 2 1 mL of 20% methanol/ Nebulizer: 40 L/hr 2% ammonium hydroxide Drying gas: 400 L/hr Elution Cone volt: 56 V 0.5 mL 25% methanol/ 1% acetic acid

Dilution 0.5 mL water CID mass spectra

100 HO 324 A Analyte: -H O 2 Naltrexone O OH 324 N O Conc Average % Std. Dev (RSD%) (rel. error) ng/mL n=8 0 0 310 0.5 0.6 0.09 16 15 100 B HO 1 0.9 0.06 6 -14 Int. Std.: -H2O Naloxone O OH 5 5.1 0.2 4 3 N 328 10 10.1 0.4 4 1 O Relative Abundance (%) 0 20 19.7 0.8 4 -2 0 100 200 300 400 50 49.1 3.9 8 -2 Mass/Charge (m/z) 100 101.1 3.7 4 1 Naltrexone m/z 342 > 324 Naloxone m/z 328 > 310

HPLC-MS/MS (MRM Analysis at 0.5 ng/mL and 100 ng/mL 5.35 0.6

1.38 100100 1.38 0.5 ng/mL 342.1>324.0 % A 0

0.38 0 10 0.12 0.58 1.12 1.80 779 0

0 1.19 100 Response 100 1.19 328.0>310.0 % B 2.14e4 0 Time 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 0 1.34 100 1.34 100 100 ng/mL 342.1>324.0 0.0 50.0 100.0

% C 8.90e4 Conc. (ng/mL)

0

0 1.17 100 Coefficient of Determination: 0.996457 100 1.17 328.0>310.0 % D Calibration Curve: 0.0508208* x + 0.00306776 2.42e4 0 Time Response type: Internal Std (20 ng/mL Naloxone), 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 0.5 1.0 1.5 2.0 Area * ( IS Conc. / IS Area) Curve type: Linear, Origin: Exclude, Weighting: 1x, Axis trans: None Time (min)

108 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Naltrexone in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge

™ Part Number WAT094225 Column: SymmetryShield RP8, 3.0 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT094243, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM ammonium acetate, pH 5/acetonitrile, 90:10 Load Flow rate: 0.6 mL/min 1 mL spiked porcine serum with 0.2 µg naloxone (I.S.) Injection volume: 20 µL of reconstituted porcine serum extract

Temperature: 25˚ C Wash Detection: UV @ 215 nm 1 mL 5% methanol in water

Elute Compounds: 1 mL methanol 1: Naloxone (I.S.) Evaporate and Reconstitute 2: Naltrexone 40 ˚C under nitrogen stream 200 µL mobile phase

HO

O OH N O

1. Naloxone (I.S.) 2. Naltrexone

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD µg/mL (n=6)

Naltrexone 0.200 98% 3.2% 1 1.00 100% 3.5%

2

B

0.05 AU A

0 5 10 15 Minutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 109 ® Naproxen (Symmetry C8)

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 mm x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Acetonitrile/water/glacial acetic acid 50:49:1 Flow rate: 1.0 mL/min Injection volume: 5 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.15

1. Naproxen

110 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. ™ Naproxen (SymmetryShield RP18)

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 250 mm, 5 µm Part number: 186000112 Mobile phase: Acetonitrile/water 46:54, 2.0 mL of glacial acetic acid in 1 L of mobile phase Flow rate: 1.0 mL/min Injection volume: 20 µL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.09 2. 1.13

1. Naproxen

O

2. Butyrophenone

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: Acetonitrile/100 mM sodium acetate, pH 5.0 40:60 Flow rate: 1.0 mL/min Injection volume: 10 µL of 30 µg/mL nicergoline Detection: UV @ 227 nm

USP Tailing Factor 1. 1.2

1. Nicergoline

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 40:60 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 240 nm

USP Tailing Factors 1. 1.03

1. Nifedipine

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3 x 150 mm, 5 µm ™ Guard Column: Sentry RP8, guard column, 3.9 x 20 mm, 5 µm Condition: Part Numbers: Column - WAT094243, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM ammonium acetate, pH 5/acetonitrile, 90:10

Flow rate: 0.6 mL/min Load: Injection volume: 20 µL of reconstituted porcine serum extract 1 mL spiked porcine serum with 0.2 µg naloxone (I.S.) Temperature: 25˚ C Detection: UV @ 215 nm Wash: 1 mL 5% methanol in water Compounds: 1: Oxycodone 2: Naltrexone (I.S.) Elute: 1 mL methanol

Evaporate and Reconstitute: 40˚ C under nitrogen stream, 200 µL mobile phase

H3CO HO

O OH O OH NCH3 N O O 1. Oxycodone 2. Naltrexone (I.S.)

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD µg/mL (n=6)

Oxycodone 0.200 96.0% 6.4% 1.00 97.9% 1.0%

1

2 0.05 AU

B A

0 5 10 15 MinutesMinutes

114 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Oxytocin and Angiotensins in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 4.6 x 100 mm, 3.5 µm Part Number WAT094225 Guard Column: Sentry™ guard column 3.9 x 20 mm, 3.5 µm Condition Part numbers: Column - WAT094226, Guard - 186000704 1 mL methanol/1 mL water Mobile phase A: 0.1% TFA in water

Mobile phase B: Acetonitrile/water 90:10 with 0.1% TFA Load 15 to 60% B in 22 minutes 1 mL spiked porcine serum with 20 µL of phosphoric acid Gradient: Time Profile (min) %A %B Wash 08515 1 mL 10% acetonitrile in 0.1% TFA 22 40 60 Flow rate: 1.4 mL/min Elute 1 mL acetonitrile/water 80:20 Injection volume: 30 µL of reconstituted porcine extract (with or without 0.1% TFA-no impact) Temperature: 30˚ C Detection: UV @ 214 nm Evaporate and Reconstitute 40 ˚C under nitrogen stream 1 mL 0.1% aqueous TFA (for greater Compounds: sensitivity use less volume 1. Oxytocin for reconstituting sample) 2. Angiotensin II 3. Angiotensin I (I.S.)

1. Oxytocin 2. Angiotensin II 3. Angiotensin I

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

3 2 B.

1 A.

0.05

Compound Concentration % Recovery %RSD µg/mL (n=6)

Oxytocin 5.0 103.7% 1.9% Angiotensin I 5.0 97.8% 2.0% Angiotensin II 5.0 99.2% 4.3%

46MinutesMinutes 8

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 115 Paclitaxel (Taxol)

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 20 mM ammonium acetate, pH 5.0/acetonitrile/methanol 50:40:10 Flow rate: 1 mL/min Injection volume: 5 µL of 100 µg/mL Detection: UV @ 227 nm

USP Tailing Factors 1. 1.0

1. Paclitaxel (Taxol)

Minutes

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HPLC Method

® Column: Symmetry C18, 4.6 x 100 mm, 3.5 µm Part number: WAT066220 Mobile phase: Water/acetonitrile/methanol 57:34:9 Flow rate: 1.4 mL/min Injection volume: 25 µL of paclitaxel, 1 mg/mL in methanol Detection: UV @ 230 nm

1. Paclitaxel (Taxol)

Minutes

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HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 10 mM sodium phosphate pH 3.0, 2.88 g/L sodium lauryl sulfate/acetonitrile 50:50 Flow rate: 1.0 mL/min Injection volume: 10 µL of 20 µg/mL papaverine hydrochloride Detection: UV @ 254 nm

USP Tailing Factor 1. 1.1

1. Papaverine HCL

Minutes

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HPLC Method

™ Column: SymmetryShield RP18,4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 85:15 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 254 nm

USP Tailing Factor 1. 1.06

1. Paracetamol

Minutes

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HPLC Method Oasis® MCX Method for Quaternary Amines in Drinking Water Conditions for Oasis® MCX Cartridge, 3 cc, 60 mg Column: Symmetry® C , 3.9 x 150 mm, 5 µm 18 Part Number 186000254 Part number: WAT046980

Mobile Phase: 25 mM Octanesulfonate, pH 3.0/methanol 45:55 Condition: 3 mL methanol Flow Rate: 0.75 mL/min Rinse with 3 mL H2O Injection volume: 80 µL Sample: 200 mL spiked tap water processed Load: 250 mL sample on Oasis® MCX cartridge Detection: PDA, 257 nm for Paraquat, 308 nm for Diquat Wash: 3 mL 50% methanol/ H2O

Elute: 2 mL of 1 M NH4CI in 50% methanol/ H2O

For LC analysis dilute the eluent to 5 mL with 50 mM sodium octanesulfonate

+ + N+ NCHN CH3 3 N +

1. Paraquat 2. Diquat

Compounds 2 mg/L Spike Level % Recovery, n=5 0.5 µg/L 2.0 µg/L 1 1. Paraquat 111 + 7110+5 2. Diquat 91 + 4 96 + 4 2

257 nm PDA (0.08 AUFS)

308 nm

2 4 6 Minutes

120 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Peptides- Alberta Peptide Mixture

HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient: Time Profile (min) %A %B 09010 30 60 40 35 40 60 Flow rate: 0.8 mL/min Injection volume: 9 µL of peptide mixture; lyophilized sample dissolved in 1 mL mobile phase Temperature: 35˚ C Detection: UV @ 214 nm

USP Tailing Factors 1. 1.3 2. 1.2 3. 1.2 4. 1.2 5. 1.3 Peptides: Ac-Arg-Gly-x-x-Gly-Leu-Gly-Leu-Gly-Lys-amide where x-x= S1 Ala=Gly with free N alpha amino group S2 Gly-Gly S3 Ala-Gly S4 Val-Gly S5 Val-Val

Minutes

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HPLC Method

® ™ Columns: Symmetry C18,2.1 x 20 mm, 3.5 µm IS ™ ™ SymmetryShield RP18,2.1 x 20 mm, 3.5 µm IS Part numbers: Symmetry - 186002066 O OH SymmetryShield - 186002068 N NH O Mobile phase A: 0.1% HCOOH in Water Br HO OH N N NH2 Mobile phase B: 0.1% HCOOH in ACN HO O H OH

O Gradient: H Time Profile H H F NH H H H3C O (min) %A %B OH H 0 100 0 1. 8-Bromoguanosine 2. Acetanilide 3. Triamcinolone 40 100 OH

Flow rate: 0.6 mL/min O O HO OH Injection volume: 5 µL OH H Temperature: 30˚ C H HH Detection: UV @ 254 nm H H O O 5. 2-Amino-7-chlro-5-oxo-5H-[1]benzopyrano 4. Hydrocortisone [2,3-b]pyridine-3 carbonitrile 6. 6a-Methyl-17a-hydroxyprogesterone

NH2

Br

7. 3-Aminofluorathene 8. 2-Bromofluorene 9. Perylene 10. Naptho(2,3-a)pyrene

4 ® 2 Symmetry C18, 3.5µm 1 t0 = 0.20 min 3 5 6 8 7 9 Max PSI = 1224 10

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Compounds: 1. 8-Bromoguanosine MiMinutesnutes 2. Acetanilide 3. Triamcinolone 4 ™ 2 SymmetryShield RP18, 3.5µm 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H-[1] benzopyrano[2,3-b]pyridine-3-carbonitrile 1 8 6 6. 6λMethyl-17λ-hydroxyprogesterone 7. 3-Aminofluoranthene 379 5 10 8. 2-Bromofluorene

t0 = 0.20 min 9. Perylene Max PSI = 1424 10. Naphtho(2,3-a)pyrene

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

MiMinutesnutes

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HPLC Method

® ™ Columns: Symmetry C18,4.6 x 20 mm, 3.5 µm IS ™ ™ SymmetryShield RP18,4.6 x 20 mm, 3.5 µm IS O OH

N Part numbers: Symmetry - 186002090 NH O Br SymmetryShield - 186002092 HO OH N N NH 2 H OH Mobile phase A: 0.1% HCOOH in Water HO O O H H H F Mobile phase B: 0.1% HCOOH in ACN NH H H H3C O Gradient: Time Profile OH H 1. 8-Bromoguanosine 2. Acetanilide 3. Triamcinolone (min) %A %B OH 0 100 0 O 40 100 O HO OH OH Flow rate: 3.0 mL/min H H

Injection volume: 10 µL HH H H O Temperature: 30˚ C O Detection: UV @ 254 nm 5. 2-Amino-7-chlro-5-oxo-5H-[1]benzopyrano 4. Hydrocortisone [2,3-b]pyridine-3 carbonitrile 6. 6a-Methyl-17a-hydroxyprogesterone

NH2

Br

7. 3-Aminofluorathene 8. 2-Bromofluorene 9. Perylene 10. Naptho(2,3-a)pyrene

® 2 Symmetry C18, 3.5µm 4 t0 = 0.14 min 1 Max PSI = 1169 6 8 3 5 9 7 10

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 MiMinutesnutes

Compounds: SymmetryShield™ RP , 3.5µm 18 1. 8-Bromoguanosine 2 2. Acetanilide 4 3. Triamcinolone 4. Hydrocortisone 5. 2-Amino-7-chloro-5-oxo-5H-[1] benzopyrano[2,3-b]pyridine-3-carbonitrile 1 68 6. 6λMethyl-17λ-hydroxyprogesterone t0 = 0.17 min 9 7. 3-Aminofluoranthene Max PSI = 1122 7 3 5 10 8. 2-Bromofluorene 9. Perylene 10. Naphtho(2,3-a)pyrene

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

MiMinutesMinutesnutes

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 123 Phencyclidine in Human Urine

HPLC Method Oasis® MCX Extraction Method Oasis® MCX Extraction Plate, 30 mg/96-well ™ Column: SymmetryShield RP18, 3.9 x 150 mm, 5 µm Part Number 186000248 ™ Guard Column: Sentry Guard Column RP18, 3.9 x 20 mm, 5 µm Load Part numbers: Column - 186000108, Guard - 186000107 1 mL x 4 of acidified spiked human Mobile phase: 50 mM Phosphate buffer, pH 7/Acetonitrile 70:30 urine (4 mL Total Sample) Flow rate: 1 mL/min Wash 1 Injection volume: 20 µL urine extract 1 mL 0.1N hydrochloric acid Temperature: 30˚ C Detection: UV @ 210 nm (0.01 AUFS) Wash 2 1 mL methanol Compounds: 1. Phencyclidine Elute 2. Ketamine HCI 1 mL 5% ammonium hydroxide with 95% methanol

CI O

N NH

CH3 1. Phencyclidine (PCP) 2. Ketamine (I.S.)

Chromatogram of A) Spiked Urine, B) Blank Urine

Compound % Recovery (n=6) %RSD (n=6) 0.3 µg/mL 1.5 µg/mL 2 Phencyclidine (PCP) 101.7 (2.3) 100.2 (0.8) 1 Ketamine (I.S.) 86.5 (1.0) 0.01 AUFS

A

B

0 7 14

MinutesMinutes

124 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Phenolics in Echinacea

HPLC Method Oasis® MAX method for phenolics in echinacea Conditions for Oasis® MAX Cartridge, 3 cc, 60 mg Part Number 186000367 ® Column: Symmetry C18, 4.6 x 250 mm, 5 µm Condition/Equilibrate: Part number: WAT054275 1 mL methanol/1 mL water Mobile Phase A: 0.1% phosphoric acid Load: Mobile Phase B: Acetonitrile 3 mL of diluted sample Gradient: Time Profile Wash 1: (min) %A %B 2 mL of 50 mM sodium acetate 09010 13 78 22 Wash 2: 2 mL of methanol 40 60 40 Flow Rate: 1.5 mL/min Dry Cartridge: air dry for 15 min with vacuum Injection volume: 10 µL Temperature: 35° C Elute: 3 mL MTBE/methanol/TFA (49:49:2) Detection: UV @ 330 nm Evaporate and Reconstitute: 1 mL mobile phase

Herbal tea extract Herbal tea extract after SPE cleanup before SPE cleanup using Oasis® MAX

Sample pre-preparation 100 mg sample is extracted with 25 mL of ethanol:water (70:30) 1 mL of the ethanolic extract is diluted 1:3 with water (~ pH 7)

• Recovery, measured with certified standards at 100 ppm in reagent water, 3 was greater than 85 % for all compounds • All analyses gave results within ± 35 % of the expected values with the exception of the liquid supplement (see goldenseal analysis) • The selective SPE extraction and cleanup procedure provided a convenient analysis of echinacea phenolics in complex matrix such as in herbal tea

1. cafteric acid 2. chlorogenic acid 3. cichoric acid 1 total: 5 mg / teabag

Tea bag extract 2

0 10 20 MinutesMinutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 18600109 Mobile phase: 20 mM Phosphate buffer, pH 7/Acetonitrile 65:35 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 254 nm

USP Tailing Factor 1. 1.02

1. Prednisone

Minutes

126 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Procainamide in Serum

HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 3.0/methanol 97:3 Load Flow rate: 1.0 mL/min 1 mL spiked porcine serum Injection volume: 50 µL of reconstituted porcine serum extract

Temperature: 30˚ C Wash Detection: UV @ 220 nm 1 mL 5% methanol in water

Compounds: Elute 1 mL methanol 1: Procainamide

2: Sulfanilamide (I.S.) Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL of 50 µg/mL sulfanilamide (I.S.) in water

O CH2 CH3 N H N N 2 H CH2 CH3

1. Procainamide 2. Sulfanilamide

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD µg/mL (n=6)

Procainamide 0.100 93.8% 1.8% 0.500 98.1% 2.1%

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 50 mM potassium phosphate, pH 3.0/methanol 85:15 Flow rate: 1.0 mL/min Injection volume: 10 µL of 40 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factors 1. 1.2 2. 1.0 3. 1.0

1. Procainamide

2. N-Acetylprocainamide

3. N-Propionylprocainamide

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 20 mM potassium phosphate, pH 6.0/acetonirtrile 90:10 Flow rate: 1.0 mL/min Injection volume: 10 µL of 40 µg/mL each compound Detection: UV @ 254 nm

USP Tailing Factors 1. 1.4 2. 1.4 3. 1.4

1. Procainamide

2. N-Acetylprocainamide

3. N-Propionylprocainamide

Minutes

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HPLC Method

® Columns: A. Symmetry C18, 3.9 x 150 mm, 5 µm ™ B. SymmetryPrep C18, 7.8 x 150 mm, 7 µm Part numbers: A. WAT046980, B. WAT066288 Mobile phase A: 0.1% TFA in Water Mobile phase B: Acetonitrile Gradient: Time Profile (min) %A %B 09010 50 40 60 Flow rate: A. 0.7 mL/min B. 2.8 mL/min Injection volume: Prochlorperazine edisylate A. 0.8 mg B. 3.2mg Detection: UV @ 280 nm

1. Prochlorperazine Edisylate

® 1. Symmetry C18 , 5 µm, 3.9 mm x 150 mm

™ 2. SymmetryPrep C18 , 7 µm, 7.8 mm x 150 mm

Minutes

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HPLC Method

® ™ O CH Columns: Symmetry C18,4.6 x 20 mm, 3.5 µm IS 3 ™ ™ O SymmetryShield RP18,4.6 x 20 mm, 3.5 µm IS Part numbers: Symmetry - 186002090 OH SymmetryShield - 186002092 Mobile phase A: 0.1% HCOOH in Water Mobile phase B: 0.1% HCOOH in ACN 1. Ketoprofen Gradient: Time Profile (min) %A %B

06535 O 26040 Flow rate: 3.0 mL/min O OH

Injection volume: 10 µL CH3 Temperature: 30˚ C 2. Fenoprofen Detection: UV @ 220 nm O OH Compounds:

1. Ketoprofen CH3 2. Fenoprofen 3. Flurbiprofen F 4. Ibuprofen 3. Flurbiprofen

CH3 2 ® Symmetry C18, 3.5µm O CH3

OH H3C 1 4 3 4. Ibuprofen

t0 = 0.15 min Max PSI = 1111

0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Minutes

™ SymmetryShield RP18, 3.5µm 2

1 3 4 t0 = 0.19 min Max PSI = 1090

0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Minutes

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HPLC Method

O CH3 Columns: Symmetry® C ,4.6 x 20 mm, 3.5 µm IS™ 18 O ™ ™ SymmetryShield RP18,4.6 x 20 mm, 3.5 µm IS Part numbers: Symmetry - 186002090 OH SymmetryShield - 186002092 1. Ketoprofen Mobile phase A: 0.1% HCOOH in Water Mobile phase B: 0.1% HCOOH in ACN Isocratic: 65% A, 35% B Flow rate: 3.0 mL/min O Injection volume: 10 µL O OH Temperature: 30˚ C CH Detection: UV @ 220 nm 3 2. Fenoprofen

Compounds: O OH 1. Ketoprofen 2. Fenoprofen CH 3. Flurbiprofen 3 4. Ibuprofen

F

3. Flurbiprofen

CH3

O 2 CH3

OH H3C ® 1 Symmetry C18, 3.5µm

4. Ibuprofen t0 = 0.15 min 3 Max PSI = 1111 4

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes

2

™ SymmetryShield RP18, 3.5µm 1 t0 = 0.18 min Max PSI = 1082 3 4

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes

132 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Promethazine- Impurities Profile

HPLC Method

™ Column: SymmetryShield RP18, 3.0 x 150 mm, 5 µm Part number: 186000692 Mobile phase A: 20 mM potassium phosphate, pH 7.0 Mobile phase B: Acetonitrile Gradient: Time Profile (min) %A %B 0955 30 20 80 Flow rate: 1.0 mL/min Injection volume: 10 µL of 0.5 mg/mL Detection: UV @ 254 nm

1. Promethazine HCL

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HPLC Method Oasis® MCX Extraction Method Oasis® MCX Extraction Cartridge, 3 cc/60 mg ™ Part Number 186000254 Column: SymmetryShield RP18, 3.9 x 150 mm, 5 µm ™ Guard Column: SymmetryShield RP18, 3.9 x 20 mm, 5 µm Load Part numbers: Column - 186000108, Guard - 186000107 3 mL acidified spiked human urine Mobile phase: 0.1% TFA in water/Acetonitrile 80:20 No Conditioning Flow rate: 1 mL/min Injection volume: 50 µL Wash 1 Temperature: 30˚ C 2 mL 0.1M hydrochloric acid Detection: UV @ 275 nm Wash 2 2 mL of 100% methanol Compounds: Wash 3 1. Metoprolol 2 mL of 50% methanol with 2. Oxprenolol (I.S.) 5% ammonium hydroxide 3. Propranolol Elute 2 mL 95% methanol with 5% ammonium hydroxide

Neutralize each elution with 100 µL of acetic acid. Evaporate une N2 at 40 ˚C and reconstitute with 300 µL of water

CH OH 3 H H NCH O 3 O N CH3 O N CH3 H OH CH CH2 CH 3 O 3 H3CO

1. Metoprolol 2. Oxprenolol (I.S.) 3. Propranolol

Chromatogram of A) Spiked Urine, B) Blank Urine

3 2 Compound %Recovery (%RSD) 0.08 µg/mL 0.4 µg/mL 1 Propranolol (n=3) 105.8 (2.7) 98.5 (0.7) Metoprolol (n=3) 101.0 (3.4) 99.6 (0.4) Propranolol Interday (n=6) 99.0 (0.9)

0.0080 AUFS Propranolol Interperson (n=9) 100.4 (6.4) Metoprolol Interday (n=6) 100.3 (0.9) Metoprolol Interperson (n=9) 97.3 (2.6)

B Oxprenolol (I.S.) 91.1 (3.2) A

0 481216 MinutesMinutes

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Oasis® HLB Extraction Method HPLC Method Oasis® HLB 1 cc/30mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 7/methanol 38:62 Flow rate: 1.0 mL/min Load Injection volume: 20 µL of reconstituted porcine serum extract 1 mL spiked porcine serum Temperature: 30˚ C Wash Detection: UV @ 254 nm 1 mL 5% methanol in water

Compounds: Elute 1: Propranolol 1 mL methanol with 5 µg butyl paraben 2: Butyl paraben (I.S.)

Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL methanol

OH N O H

1. Propranolol 2. Butyl paraben (I.S.)

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

2

1

0.002 AU

B Compound Concentration % Recovery %RSD µg/mL (n=6) A Propranolol 0.800 85.9% 2.1% 4.00 96.4% 2.4%

0 4 8 12

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 50mM potassium phosphate, pH 3.0/acetonitrile 85:15 Flow rate: 1.0 mL/min Injection volume: 5 µL of 2.88 µg/mL sample Detection: UV @ 261 nm

USP Tailing Factors 1. 1.65

1. Pseudoephedrine HCL

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase A: 50 mM potassium phosphate, pH 3.0 Mobile phase B: Acetonitrile Gradient: Time Profile (min) %A %B 08515 18515 15 50 50 Flow rate: 1.0 mL/min Injection volume: 5 µL of 1.8 mg/mL pseudoephedrine and 120 µg/mL chlorpheniramine sample Detection: UV @ 261 nm

USP Tailing Factors 1. 1.7 2. 1.2

1. Pseudoephedrine HCL

2. Chlorpheniramine Maleate

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase A: 50 mM potassium phosphate, pH 3.0 Mobile phase B: Acetonitrile Gradient: Time Profile (min) %A %B 08515 18515 15 50 50 Flow rate: 1.0 mL/min Injection volume: 5 µL of 2.88 µg/mL pseudoephedrine and 120 µg/mL triprolidine extracted tablet sample Detection: UV @ 261 nm

USP Tailing Factors 1. 1.53 2. 1.16

1. Pseudoephedrine HCL

2. Triprolidine HCL

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HPLC Method

™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number: WAT200655 Mobile phase A: 50 mM potassium phosphate, pH7 Mobile phase B: Acetonitrile Gradient: Time Profile (min) %A %B 08515 15 50 50 Flow rate: 1 mL/min Injection Volume: 5 µL of 2 mg/mL pseudoephedrine HCL and 0.2 mg/mL codeine Detection: UV @ 254 nm

USP Tailing Factors 1. 1.8 2. 1.1

1. Pseudoephedrine HCL

2. Codeine

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Part Number WAT094225 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition 1 mL methanol/1 mL water Part Numbers: Column - WAT094243, Guard - WAT200675 Mobile phase: 20 mM ammonium acetate, pH 5/acetonitrile, 95:5 Load 1 mL porcine serum with 0.2 µg Flow rate: 0.6 mL/min ß-hydroxyethyltheophylline (I.S.) Temperature: 25˚ C Wash Injection volume: 20 µL of reconstituted porcine serum extract 1 mL water Detection: UV @ 273 nm Elute 1 mL methanol Compounds: Evaporate and Reconstitute 1: Theobromine 40 ˚C under nitrogen stream 2: Paraxanthine 200 µL mobile phase 3: Theophylline 4: b-hydroxyethyltheophylline (I.S.) 5: Caffeine

1. Theobromine 2. Paraxanthine 3. Theophylline

O CH2CH2OH O CH2

H3C H3C N N N N

N N O N O N

CH3 CH3

4. ß-hydroxyethyltheophylline (I.S.) 5. Caffeine

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1

3 Compound Concentration % Recovery %RSD 0.03 AU 2 µg/mL (n=6) Theobromine 0.100 103.0% 3.4% 5 0.500 104.0% 2.4% Paraxanthine 0.100 101.0% 6.2% 4 0.500 98.9% 2.3% Theophylline 0.100 100.0% 5.2% B 0.500 101.0% 2.1% A Caffeine 0.100 92.6% 4.4% 0.500 97.5% 3.2% 0 5 10 15

140 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Quinidine Sulfate and Related Substance

HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 250 mm, 5 µm Part Number: 186000112 Mobile phase: 860 mL water, 20 mL methanesulfonic acid sloution, 20 mL diethylamine solution, 100 mL acetonitrile; pH adjusted to 2.6 with diethylamine solution Diethylamine solution: 10 mL diethylamine in 100 mL water Methanesulfonic acid solution: 14 mL methanesulfonic acid and 8 mL glacial acetic acid in 200 mL water Flow rate: 1.0 mL/min Injection volume: 50 µL Detection: UV @ 235 nm

USP Tailing Factor 1. 1.76 2. 1.33

1. Quinidine sulfate 2. Dihydroquinidine sulfate

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part Number: 18600109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile, 85:15 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 254 nm

USP Tailing Factor 1. 1.07

1

1. Ranitidine

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 100 mM sodium acetate, 5mM camphosulfonic acid, pH 6.2/ acetonitrile/methanol 80:10:10 Flow rate: 1.0 mL/min Injection volume: 5 µL of 100 µg/ml sample Detection: UV @ 254 nm

USP Tailing Factor 1. 1.12

1. Ranitidine

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Part numbers: Condition Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 3.0/methanol 97:3

Flow rate: 1.0 mL/min Load Injection volume: 50 µL of reconstituted porcine serum extract 1 mL spiked porcine serum Temperature: 30˚ C Wash Detection: UV @ 220 nm 1 mL 5% methanol in water

Compounds: Elute 1: Sulfanilamide (I.S.) 1 mL methanol 2: Ranitidine Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL of 50 µg/mL sulfanilamide (I.S.) in water

H3C H O H N N N S CH3

H3C CHNO2 1. Sulfanilamide 2. Ranitidine

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD µg/mL (n=6)

Ranitidine 0.100 104% 1.9% 0.500 102% 1.7%

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.0 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT094243, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 1.2% glacial acetic acid/acetonitrile 75:25 Flow rate: 0.6 mL/min Load Injection volume: 10 µL of reconstituted porcine serum extract 1 mL spiked porcine serum acidified with 20 µL phosphoric acid Temperature: 40˚ C

Detection: UV @ 236 nm Wash 1 mL 5% methanol in water Compounds: 1: Benzoic acid Elute 1 mL methanol 2: Nalidixic acid (I.S.)

3: Salicylic acid Evaporate and Reconstitute 40 ˚C under nitrogen stream 1 mL 5 µg/mL nalidixic acid in mobile phase

1. Benzoic acid 2. Nalidixic acid (I.S.) 3. Salicylic acid

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1 Compound Concentration % Recovery %RSD µg/mL (n=6)

2 Benzoic Acid 0.100 84.8% 3.8% 0.500 87.1% 3.0% Salicylic Acid 0.100 92.3% 5.0% 3 0.500 91.3% 3.3%

0.035 AU B

A

0 5 10 15 MinutesMinutes

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HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate pH 3.0/acetonitrile 70:30 Temperature: 40˚ C Flow rate: 0.7 mL/min Injection volume: 6 µL of phthalic acid (0.5 mg/mL), benzoic and salicylic acids (1 mg/mL) Detection: UV @ 254 nm

USP Tailing Factors 1. 1.7 2. 1.1 3. 1.4

1. Phthalic Acid

2. Salicylic Acid

3. Benzoic Acid

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HPLC Method

® ™ O Columns: Symmetry C18,2.1 x 20 mm, 3.5 µm IS O ™ ™ SymmetryShield RP18,4.6 x 20 mm, 3.5 µm IS NH H3C Part numbers: Symmetry - 186002066 O 3. Acetanilide SymmetryShield - 186002068 1. Theophylline 2. 2-Acetylfuran Mobile phase A: 0.1% HCOOH in Water O Mobile phase B: 0.1% HCOOH in ACN O O

Gradient: Time Profile CH3 (min) %A %B 0 100 0 4. Aceophenone 5. Propiophenone 6. Butyrophenone 40 100 Flow rate: 0.6 mL/min O O O Injection volume: 5 µL Temperature: 30˚ C Detection: UV @ 254 nm 7. Benzophenone 8. Valerophenone 9. Hexanophenone

O O

10. Heptanophenone 11. Octanophenone

® Symmetry C18, 3.5µm Compounds: 7 1. Theophylline 2 4 2. 2-Acetylfuran 3. Acetanilide 6 8 4. Acetophenone t0 = 0.17 min 1 3 5 91110 5. Propiophenone Max PSI = 1247 6. Butyrophenone 7. Benzophenone 8. Valerophenone 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 9. Hexanophenone 10. Heptanophenone Minutes 11. Octanophenone

™ 7 SymmetryShield RP18, 3.5µm

2 4 6 1 8 11 5 9 10 t0 = 0.17 min 3 Max PSI = 1452

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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HPLC Method

® ™ O Columns: Symmetry C18,4.6 x 20 mm, 3.5 µm IS O SymmetryShield™ RP ,4.6 x 20 mm, 3.5 µm IS™ 18 NH H3C Part numbers: Symmetry - 186002090 O SymmetryShield - 186002092 1. Theophylline 2. 2-Acetylfuran 3. Acetanilide Mobile phase A: 0.1% HCOOH in Water O Mobile phase B: 0.1% HCOOH in ACN O O

Gradient: Time Profile CH3 (min) %A %B 0 100 0 4. Aceophenone 5. Propiophenone 6. Butyrophenone 40 100 Flow rate: 3.0 mL/min Injection volume: 10 µL O O O Temperature: 30˚ C Detection: UV @ 254 nm 7. Benzophenone 8. Valerophenone 9. Hexanophenone

O O

10. Heptanophenone 11. Octanophenone

® Symmetry C18, 3.5µm Compounds: 7 1. Theophylline 2. 2-Acetylfuran 2 4 3. Acetanilide 4. Acetophenone 6 11 8 9 5. Propiophenone 1 5 6. Butyrophenone t0 = 0.15 min 3 10 7. Benzophenone Max PSI = 1162 8. Valerophenone 9. Hexanophenone 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 10. Heptanophenone 11. Octanophenone Minutes

™ SymmetryShield RP18, 3.5µm 7

2 4 1 6 11 3 8 9 5 10 t0 = 0.16 min Max PSI = 1124

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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HPLC Method

® Column: Symmetry C8 ,3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Acetonitrile/water 50:50 Flow rate: 1.0mL/min Injection volume: 5µL of 10 µg/mL sample Detection: UV @ 254nm

USP Tailing Factors 1. 1.08 Vo: Uracil 2. 1.18 3. 1.10 4. 1.31

1. Prednisolone

2. Betamethasone

3. Prednisolone Acetate

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4. Betamethasone Valerate

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part Number WAT094225 ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part numbers: Column - WAT200655, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 20 mM potassium phosphate, pH 7/methanol 41:59 Load Flow rate: 1.0 mL/min 1 mL spiked porcine serum Injection volume: 5 µL of reconstituted porcine serum extract Detection: UV @ 254 nm Wash 1 mL 5% methanol in water

Compounds: Elute 1 mL methanol, added 50 µL of 1: Prednisolone 0.1 mg/mL butyl paraben (I.S.) 2: Betamethasone 3: Butyl Paraben (I.S.) Evaporate and Reconstitute 40 ˚C under nitrogen stream 4: Betamethasone Valerate 200 µL acetonitrile

1. Prednisolone 2. Betamethasone 3. Butyl Paraben (I.S.) 4. Betamethasone valerate

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

3 2 0.05 AU 1 4

Compound Concentration % Recovery %RSD µg/mL (n=6)

Prednisolone 1.00 93.8% 4.7% 5.00 92.0% 1.4%

Betamethasone 1.00 85.7% 2.3% 5.00 91.3% 1.5% B Betamethasone 4.00 95.4% 2.2% A Valerate 20.00 90.6% 1.3% 0481216 MinutesMinutes

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HPLC Method

® ™ Column: Symmetry C18,2.1 x 20 mm, 3.5 µm IS Part number: 186002066 Mobile phase A: Water Mobile phase B: ACN Gradient: Time Profile (min) %A %B 08020 43664 Flow rate: 0.6 mL/min Injection volume: 5 µL Temperature: 30˚ C Detection: UV @ 220 nm

O O OH CH3 CH HO 3 OH O OH CH3 H CH3 H

H H H H O O

1. Prednisolone 2. Cortisone 3. Corticosterone

H3C O O CH3 CH3 OH CH3 CH H H 3 H H H H H H H O HO HO

4. Estradiol 5. Estrone 6. Progesterone

® Symmetry C18, 3.5µm 2 Compounds: 1 3 4 5 1. Prednisolone 6 2. Cortisone 3. Corticosterone t0 = 0.33 min Max PSI = 1280 4. Estradiol 5. Estrone 6. Progesterone 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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HPLC Method

® ™ Columns: Symmetry C18,4.6 x 20 mm, 3.5 µm IS ™ ™ SymmetryShield RP18,4.6 x 20 mm, 3.5 µm IS Part numbers: Symmetry - 186002090 SymmetryShield - 186002092 Mobile phase A: Water Mobile phase B: ACN Gradient: Time Profile O (min) %A %B O OH CH3 CH HO 08020 3 OH O OH 43664 CH3 H CH3 H Flow rate: 3.0 mL/min H H Injection volume: 10 µL H H O Temperature: 30˚ C O Detection: UV @ 220 nm 1. Prednisolone 2. Cortisone 3. Corticosterone

H3C O O CH3 CH3 OH CH3 CH H H 3 H H H H H H H O HO HO

4. Estradiol 5. Estrone 6. Progesterone

23 1 5 ® 6 Symmetry C18, 3.5µm 4

t0 = 0.14 min Max PSI = 1190

Compounds 1. Prednisone 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 2. Cortisone 3. Corticosterone Minutes 4. Estradiol 5. Estrone 23 6. Progesterone 1 5 ™ 6 SymmetryShield RP18, 3.5µm 4

t0 = 0.14 min Max PSI = 1132

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 100 mM potassium phosphate, pH 6.9/methanol/water 20:30:50 Flow rate: 1.0 mL/min Injection volume: 10 µL of 40-500µg/mL sample Detection: UV @ 214 nm

USP Tailing Factors 1. 0.96 2. 0.92 3. 0.91

1. Ethosuximide

2

1 2. Phensuximide

3

3. Methsuximide

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5µm Part numbers: Column - WAt046970, Guard - WAT054250 Mobile phase: Water/methanol/glacial acetic acid 79:20:1 Flow rate: 1.0 mL/min Injection volume: 10 µL of sample with components at 10 to 39 µg/mL Detection: UV @ 254 nm

USP Tailing Factors O NH S NH 1. 1.27 2 N O 2. 1.19 N 3. 1.18 4. 1.15 1. Sulfanilamide 2. Sulfadiazine 5. 1.12 6. 1.11

NH2

N O H2N S O S H C N NH 3 O S NH N O

3. Sulfathiazole 4. Sulfamerazine

5. Sulfamethazine 6. Succinylsulfathiazole

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part Number WAT094225 Part number: WAT046980 Condition Mobile phase: 1% glacial acetic acid and 4% methanol in water 1 mL methanol/1 mL water Flow rate: 1.0 mL/min Injection volume: 10 µL of reconstituted porcine serum extract Load 1 mL spiked porcine serum Temperature: 25˚ C with 10 µg/mL sulfathiazole (I.S.), Detection: UV @ 254 nm 20 µL phosphoric acid

Compounds: Wash 1 mL 5% methanol in water 1: Sulfadiazine

2: Sulfathiazole (I.S.) Elute 3. Sulfamerazine 1 mL methanol

Evaporate and Reconstitute 40 ˚C under nitrogen stream 1 mL mobile phase

O

NH NH S NH2 2 H2N N O N O O S N S S H3C N NH NH N O O

1. Sulfadiazine 2. Sulfathiazole (I.S.) 3. Sulfamerazine

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

Compound Concentration % Recovery %RSD 1 µg/mL (n=6)

Sulfadiazine 2.00 98.4% 2.7% 10.0 96.9% 0.65% Sulfamerazine 2.00 93.5% 1.9% 2 10.0 96.4% 0.74% 3

0.004 AU

B

A

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HPLC Method

® ™ Column: Symmetry C18,2.1 x 20 mm, 3.5 µm IS Part number: 186002066 Mobile phase A: 0.1% HCOOH in Water Mobile phase B: 0.1% HCOOH in MeOH Gradient: Time Profile (min) %A %B 0 100 0

45050 O

H N NH Flow rate: 0.6 mL/min NH S NH2 2 2 S Injection volume: 10 µL N O O N O N S S Temperature: 30˚ C N NH H3C N NH Detection: UV @ 270 nm O O 1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine Compounds: H3C N NH2 H C 1. Sulfadiazine 3 O CH3 NH2 2. Sulfathiazole O O N O O H C 3. Sulfamerazine S 3 NH S NH2 S N NH H3C N NH O 4. Sulfadimidine O O 5. Sulfamethoxazole 4. Sulfadimidine 5. Sulfamethoxazole 6. Sulfisoxazole 6. Sulfisoxazole 7. Sulfadimethoxine

H3C O NH2

N N O

H3C S O NH O

7. Sulfadimethoxine

® Symmetry C18, 3.5µm 1 5 2 3 67 4 t0 = 0.17 min Max PSI = 2050

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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HPLC Method

® ™ Column: Symmetry C18,4.6 x 20 mm, 3.5 µm IS Part number: 186002090 Mobile phase A: 0.1% HCOOH in Water Mobile phase B: 0.1% HCOOH in MeOH Gradient: Time Profile (min) %A %B 0 100 0

45050 O

Flow rate: 3.0 mL/min H N NH NH S NH2 2 2 Injection volume: 10 µL S N O O N O Temperature: 30˚ C N S S N NH H3C N NH Detection: UV @ 270 nm O O 1. Sulfadiazine 2. Sulfathiazole 3. Sulfamerazine Compounds:

H3C 1. Sulfadiazine NH2 CH N 3 H3C NH2 O 2. Sulfathiazole O O N O O 3. Sulfamerazine S H C S N NH 3 NH S NH2 4. Sulfadimidine H3C N NH O O O 5. Sulfamethoxazole 5. Sulfamethoxazole 6. Sulfisoxazole 6. Sulfisoxazole 4. Sulfadimidine 7. Sulfadimethoxine

H3C O NH2

N N O

H3C S O NH O

7. Sulfadimethoxine

1 ® Symmetry C18, 3.5µm

2 3 5 6 7 4

t0 = 0.15 min Max PSI = 2280

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: Water/methanol/glacial acetic acid 79:20:1 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sulfamethoxazole and 30 µg/mL trimethroprim Detection: UV @ 254 nm

USP Tailing Factors 1. 1.5 2. 1.1

1. Trimethoprim

2. Sulfamethoxazole

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5 µm Part number: Column - WAT046980 Condition 1 mL methanol/1 mL water Mobile phase: 25 mM ammonium acetate, pH 5/methanol 82:18 (v/v)

Flow rate: 1.0 mL/min Load Injection volume: 20 µL of reconstituted porcine serum extract 1 mL spiked porcine serum with 20 µL phosphoric acid and Detection: UV @ 290 nm 2.5 µg/mL sulfamerazine (I.S.)

Compounds: Wash 1: Sulfamerazine (I.S.) 1 mL 5% methanol in water 2: Trimethoprim 3: Sulfamethoxazole Elute 1 mL methanol

Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL mobile phase

1. Sulfamerazine (I.S.) 2. Trimethoprim 3. Sulfamethoxazole

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

3 Compound Concentration % Recovery %RSD 1 µg/mL (n=6)

Sulfamethoxazole 1.20 90.4% 1.3% 0.240 95.4% 3.4% Trimethoprim 1.50 96.7% 1.1% 2 0.300 99.3% 3.2% 00.02 AU

B.

A.

`51015 20 Minutes

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HPLC Method

® Column: Symmetry C18,3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 50 mM potassium phosphate pH 3.0/acetonitrile 80:20 Flow rate: 1.0 mL/min Injection volume: 20 µL of mixture with chlorpheniramine, maleate, toluamide and propranolol Detection: UV @ 230 nm

Compounds 1. Maleate 2. Toluamide 3. Chlorpheniramine 4. Propranolol

1. Maleate

2. Toluamide

3. Chlorpheniramine

CH3

O N CH3 H OH

4. Propranolol

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 0.1% phosphoric acid in water/methanol 62:38 Flow rate: 1.0 mL/min Injection volume: 10 µL of mixture with 30 µg/mL furazolidone and oxolinic acid and 100 µg/mL nolidixic acid Detection: UV @ 254 nm

USP Tailing Factors 1. 1.2 2. 1.1 3. 1.2

1. Furazolidone

2. Oxolinic Acid

3. Nalidixic Acid

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 50 mM potassium phosphate, pH 3/acetonitrile 55:45 Flow rate: 1.0 mL/min Injection volume: 5 µL of 100 µg/mL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.3

1. Tamoxifen Citrate

Minutes

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HPLC Method

® Columns: Symmetry C18, 3.0 x 150 mm, 5 µm ™ SymmetryPrep C18, 7.8 x 300 mm, 7 µm Part numbers: Symmetry - WAT054200 SymmetryPrep - WAT066235 Mobile phase: Acetonitrile/50 mM potassium phosphate buffer, pH 3.0 40:60 Flow rate: A. 0.4 mL/min B. 5.6 mL/min Injection volume: 5 mg/mL tamoxifen citrate A. 15 mg B. 101mg Detection: UV @ 254 nm

1. Tamoxifen Citrate

1

® 1. Symmetry C18 , 5 µm, 3.0 mm x 150 mm

™ 1. SymmetryPrep C18 , 7 µm, 7.8 mm x 300 mm

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 4.6 x 150 mm, 5 µm Part number: 186000109 Mobile phase: 20 mM phosphate buffer, pH 7.0/acetonitrile 25:75 Flow rate: 1.0 mL/min Injection volume: 5 µL Detection: UV @ 220 nm

USP Tailing Factor 1. 1.04

1. Terfenadine

Minutes

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HPLC Method

® Column: Symmetry C8, 4.6 x 250 mm, 5 µm Part number: WAT054270 Mobile phase: 100 mM triethylammonium phosphate, pH 7.0/methanol 20:80 Flow rate: 1.0 mL/min Injection volume: A. 15 µL of mixture with 0.6 mg/mL terfenadine and 0.15 mg/mL of terfenadine related compound ARS B.15 µL of 8mg/mL crushed tablet ( equivalent to 0.6mg/mL terfenadine) Detection: UV @ 235 nm

Compounds: 1. Terfenadine 2. Terfenadine Related Compound ARS

USP Tailing Factor 1. 1.0

Tablet:

1. Terfenadine

Standard:

Minutes

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HPLC Method Oasis® HLB Extraction Method ® Oasis HLB 1 cc/30 mg Extraction Cartridge ® Part Number WAT094225 Column: Symmetry C8, 4.6 x 75 mm, 3.5 µm ™ Guard Column: Sentry guard column, 3.9 x 20 mm, 5 µm Condition Part Numbers: Column - WAT066224, Guard - WAT064225 1 mL methanol/1 mL water

Mobile phase: Acetonitrile/water 60:40 Load Flow rate: 1 mL sample with 800 µL spiked porcine 1.5 mL/min serum and 200 µL of methanol Injection Volume: 20 µL of reconstituted porcine serum extract Detection: UV @ 254 nm Wash 1 mL 5% methanol in water

Compounds: Elute 1 mL methylene chloride: 1: methanol 50:50 (v/v) 2: Testosterone propionate Evaporate and Reconstitute 3: Testosterone benzoate 40 ˚C under nitrogen stream 500 µL methanol with 5 µg/mL 4: Testosterone enanthate (I.S.) of testosterone enanthate

1. Testosterone acetate 2. Testosterone propionate 3. Testosterone benzoate 4. Testosterone enanthate (I.S.)

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1

2

3 Compound Concentration % Recovery %RSD 4 µg/mL (n=6)

Testosterone 1.00 98% 1.2% Acetate 0.200 94% 3.7% Testosterone 1.00 100% 1.9% 0.008 AU Propionate 0.200 94% 4.3% B Testosterone 1.00 94% 1.6% A Benzoate 0.200 87% 3.2%

0 10 20 MinutesMinutes

166 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Tetracyclines

HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium phosphate, pH 6.25/methanol 45:55 Flow rate: 1.0 mL/min Injection volume: 20 µL of 500 µg/mL sample Detection: UV @ 280 nm

USP Tailing Factors 1. 1.05 2. 1.29 3. 1.14

1. Doxycycline

2. Minocycline

3. Tetracycline

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB 1 cc/30 mg Extraction Cartridge ™ Column: SymmetryShield RP8, 3.0 x 150 mm, 5 µm Part Number WAT094225 Guard Column: Sentry™ guard column, 3.9 x 20 mm, 5µm Condition Part numbers: Column - WAT094243, Guard - WAT200675 1 mL methanol/1 mL water Mobile phase: 0.1% TFA in Water, Methanol, Acetonitrile 91:2:7 Flow rate: 0.90 mL/min Load 1 mL spiked porcine serum Injection volume: 20 µL of reconstituted porcine serum extract with 20 µL phosphoric acid and Detection: UV @ 350 nm 5.0 µg/mL demeclocycline (I.S.)

Compounds: Wash 1 mL 5% methanol in water 1: Minocycline 2: Tetracycline Elute 3: Demeclocycline (I.S.) 1 mL methanol

Evaporate and Reconstitute 40 ˚C under nitrogen stream 200 µL mobile phase

1. Minocycline 2. Tetracycline 3. Demeclocycline (I.S.)

Chromatogram of Serum Extracts: A) Blank B) Spiked Sample

1

2

3 Compound Concentration % Recovery %RSD µg/mL (n=6)

0.006 Minocycline 2.50 96.1% 1.3% 0.500 88.0% 1.6%

B Tetracycline 2.50 101.0% 0.5% A 0.500 97.8% 1.4%

10 20 30

Minutes

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HPLC Method

™ Column: SymmetryShield RP18, 3.0 x 150 mm, 5 µm Part number: 186000692 Mobile phase: 5 mM potassium phosphate, pH 2.0/acetonitrile 95:5 Flow rate: 0.6 mL/min Injection volume: 5 µL injection of solution containing theobromine (0.22 mg/mL), theophylline (0.26 mg/mL), and 7-(2-hydroxyethyl) theophylline (0.13 mg/mL) Detection: UV @ 270 nm

1. Theobromine

2. Theophylline

3. 7-(2-hydroxyethyl) Theophylline

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM potassium phosphate, pH7.0/ methanol 76:24 Flow rate: 1.0 mL/min Injection volume: 20 µL of 10 µg/mL sample Detection: UV @ 280 nm

USP Tailing Factors 1. 1.18 2. 1.03 3. 1.01

1. Theobromine

2. Theophylline

O CH2

H3C N N

N O N

CH3

3. Caffeine

Minutes

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HPLC Method

™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part number: 186000108 Mobile phase: 50 mM phosphate, pH7.0/methanol 36:64 Flow rate: 1.0 mL/min Injection volume: 25 µL Detection: UV @ 250 nm

USP Tailing Factors 1. 1.31 2. 1.21 3. 1.12

1. 1-(3-chlorophenyl)peperazine

2. Maprotiline (I.S.)

3. Trazodone

Minutes

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HPLC Method Oasis® HLB Extraction Method Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg

™ Part Number WAT094226 Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part number: WAT0200655 Condition: 3 mL methanol Rinse: 3 mL H2O Mobile Phase A: 5 mM phosphate buffer, pH 6.7/Acetonitrile 85:15 Mobile Phase B: Acetonitrile Load: 75 mL sample @ 4 mL/min Gradient: Time Profile (min) %A %B Wash: 1 mL H O 0 100 0 2 2 100 0 Elute: 25 30 70 1.2 mL methanol Flow Rate: 1.0 mL/min Evaporate to 0.2 mL Injection volume: 75 µL Detection: UV @ 214 nm (0.02 AUFS) Reconstitute to exactly 1.0 mL with H2O

1. Desisopropylatrazine 2. Hydroxyatrazine 3. Desethylatrazine

4. Simazine 5. Cyanazine 6. Atrazine

spiked well water Compounds % Recovery Tap Water Tap Water Spike Level Spike Level 500 µg/L 200 ng/L 5 replicates 7 replicates 1. Desisopropylatrazine 98.4 (5.0) 95.6 (5.8) 3 5 6 2. Hydroxyatrazine 132 (1.3) 109 (11) 4 3. Desethylatrazine 106 (5.1) 104 (4.0) 2 4. Simazine not determined 97.7 (3.9) 5. Cyanazine not determined 93.1 (3.7) 1 6. Atrazine 101 (5.0) 101 (4.4) nonspiked sample

blank cartridge

05MinutesMinutes 10 15

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 20 mM potassium phosphate, pH 7/methanol 35:65 Flow rate: 1.0 mL/min Injection volume: 20 µL of 100 µg/mL sample 1. Protriptyline HCL Detection: UV @ 254 nm

USP Tailing Factors 1. 1.7 2. 1.7 3. 1.8 4. 1.5 5. 1.4 6. 1.2 2. Nortriptyline HCL

3. Doxepin HCL

4. Imipramine HCL

5. Amtriptyline HCL

6. Trimipramine Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part number: WAT046980 Mobile phase: 20 mM potassium phosphate, pH 7.0/methanol 29:71 Flow rate: 1.0 mL/min Injection volume: 5 µL of 1 to 1.16 mg/mL Temperature: 35˚ C Detection: UV @ 254 nm

USP Tailing Factors 1. 1.5 2. 1.5 3. 1.3 4. 1.2

1. Nordoxepin HCL

2. Doxepin HCL

3. Nortriptyline HCL

4. Amtriptyline HCL

Minutes

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HPLC Method Oasis® HLB Extraction Method Oasis® HLB Extraction Plate, 30 mg/96-well ™ Part Number WAT058951 Column: SymmetryShield RP8, 4.6 x 75 mm, 3.5 µm

Part number: WAT094263 Condition Mobile phase: 50 mM phosphate, pH 7/methanol 26:74 1 mL methanol/1 mL water Flow rate: 1.4 mL/min Load Injection volume: 1 mL spiked plasma plus 20 µL concentrated phosphoric acid, 100 µL injection 1 mL spiked plasma plus 20 µL concentrated phosphoric acid Temperature: 29˚ C

Detection: UV @ 254 nm Wash A. 1 mL 2% ammonium hydroxide in 5% methanol Compounds: B. 1 mL 2% ammonium hydroxidein 65% methanol Spiked at 500 ng/mL versus Blank: C. 1 mL 2% acetic acid in 5% methanol 1. Nordoxepin (I.S.)

2. Nortriptyline Elute 3. Doxepin 600 µL 65% methanol 4. Imipramine Add internal standard (I.S.) 5. Amitriptyline 60 µL 36 µg/mL nordoxepin in 6. Trimipramine 10% ammonium hydroxide

1. Nordoxepin 2. Nortriptyline 3. Doxepin

4. Imipramine 5. Amitriptyline 6. Trimipramine

Chromatogram of Plasma Extracts: A) Blank B) Spiked Sample

500 ng/mL 100 ng/mL

Compound Recovery RSD Recovery RSD 1 (n=96) (n=95) 4 2 3 Nortriptyline 92.3% 1.4% 90.8% 5.7% Doxepin 90.6% 1.4% 90.4% 4.7% 5 Imipramine 92.2% 1.7% 86.4% 5.3% 6 Amitriptyline 90.2% 1.6% 85.3% 5.8% Trimipramine 90.3% 1.9% 89.8% 6.1%

0.02 AU A B

01234 Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20 mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 50 mM potassium phosphate, pH 3.0/acetonitrile 85:15 Flow rate: 1.0 mL/min Injection volume: 10 µL of 200 µg/mL sample Detection: UV @ 261 nm

USP Tailing Factor 1. 1.6

1. Tripelennamine

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: 10 mM sodium acetate with 3.3% glacial acetic acid, pH 2.6/acetonitrile 70:30 Flow rate: 1.0 mL/min Injection volume: 10 µL of 100 µg/mL sample Detection: UV @ 278 nm

USP Tailing Factor 1. 1.27

1. Verapamil

Minutes

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HPLC Method

™ Column: SymmetryShield RP8, 3.9 x 150 mm, 5 µm Part number: WAT200655 Mobile phase: 50 mM phosphate pH 7.0/acetonitrile/methanol 41:37:22 Flow rate: 1.0 mL/min Injection volume: 40 µL Detection: UV @ 230 nm

USP Tailing Factors 1. 1.10 2. 1.06 3. 1.07

1. Norverapamil

2. Verapamil

3. Methoxyverapamil (I.S.)

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Guard Column: Sentry™ Guard Column 3.9 x 20mm, 5 µm Part numbers: Column - WAT046970, Guard - WAT054250 Mobile phase: Methanol/acetonitrile/water 45:45:10 Flow rate: 1.0 mL/min Injection volume: 10 µL of 25 µg/mL sample Detection: UV @ 280 nm

USP Tailing Factors 1. 1.33 2. 1.20 3. 1.16 Vo. Uracil

1. Trans-Retinol (Vitamin A)

2. Cholecalciferol (Vitamin D3)

3. Alpha-Tocopherol (Vitamin E)

Minutes

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part numbers: Column - WAT046980, PIC B6 - WAT084199 1. Niacinamide Mobile phase: 20 mM PIC B6 (hexane sulfonic acid) and 0.1% phosphoric acid, pH 2.2/methanol 75:25 Flow rate: 0.7 mL/min Injection volume: 10 µL of mixture with component 1 at 11 µg/mL, 2 at 133 µg/mL, 3 at 17 µg/mL, 4 at 6 µg/mL, 5 at 25 µg/mL, 6 at 34 µg/mL, and 7 at 67 µg/mL 2. Calcium Pantothenate Detection: UV @ 210 nm

USP Tailing Factors 1. n.c. 2. n.c. 3. 1.4 4. 1.1 5. 1.2 3. Pyridoxal 6. 1.1 7. 1.2

4. Pyrodoxine Hydrochloride

5. Pyridoxamine Dihydrochloride

6. Riboflavin

Minutes 7. Thiamine Hydrochloride

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HPLC Method

® Column: Symmetry C18, 3.9 x 150 mm, 5 µm Part numbers: Column - WAT046980, PIC B6 - WAT085140 Mobile phase: 20 mM PIC B6 (hexane sulfonic acid) and 0.1% phosphoric acid, pH 2.2/methanol 75:25 Flow rate: 0.7 mL/min Injection volume: 10 µL of mixture with 0.02 to 0.17 mg/mL Temperature: 40˚ C Detection: UV @ 280 nm

USP Tailing Factors 1. 1.6 2. 1.4 3. 1.2 1. Nicergoline 4. 1.2

2. Folic Acid

3. Riboflavin

4. Thiamine Hydrochloride

Minutes

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HPLC Method

® Column: Symmetry C8, 3.9 x 150 mm, 5 µm Part number: WAT046970 Mobile phase: 50 mM potassium phosphate, pH 4.6/methanol 85:15 Flow rate: 1.0 mL/min Injection volume: 20 µL of 100 µg/mL sample Detection: UV @ 254 nm

USP Tailing Factor 1. 1.2

1. Zidovudine

Minutes

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® ® ™ Symmetry Analytical Columns Symmetry C18 PicoFrit Columns

Particle Part No. Part No. Dimension Particle Size Part Number Dimension Size C18 C8 75 µm x 50 mm 3.5 µm 186001407 1.0 x 50 mm 3.5 µm WAT106056 WAT106052 75 µm x 100 mm 3.5 µm 186001408 1.0 x 150 mm 3.5 µm WAT248059 WAT248072 75 µm x 100 mm 5 µm 186001409 2.1 x 30 mm 3.5 µm WAT058973 WAT058977 75 µm x 150 mm 5 µm 186001410 2.1 x 50 mm 3.5 µm WAT200650 WAT200624 2.1 x 100 mm 3.5 µm WAT058965 WAT058961 2.1 x 150 mm 3.5 µm WAT106005 WAT106011 ® ™ 3.0 x 100 mm 3.5 µm 186000696 186000698 Symmetry C18 IntegraFrit Columns 3.0 x 150 mm 3.5 µm 186000695 186000697 4.6 x 30 mm 3.5 µm 186000271 186000270 Dimension Particle Size Part Number 4.6 x 50 mm 3.5 µm WAT200625 WAT200620 4.6 x 75 mm 3.5 µm WAT066224 WAT066200 75 µm x 50 mm 3.5µm 186001403 4.6 x 100 mm 3.5 µm WAT066220 WAT066204 75 µm x 100 mm 3.5 µm 186001404 4.6 x 150 mm 3.5 µm WAT200632 WAT200630 75 µm x 100 mm 5 µm 186001405 75 µm x 150 mm 5µm 186001406 2.1 x 50 mm 5 µm 186000206 186000212 2.1 x 150 mm 5 µm WAT056975 WAT056955 Symmetry® Column and Cartridge Column 3.0 x 150 mm 5 µm WAT054200 WAT054230 3.0 x 250 mm 5 µm 186000690 186000691 Method Validation Kits 3.9 x 150 mm 5 µm WAT046980 WAT046970 Three Columns from Three Different Batches to Test Reproducibility. 4.6 x 50 mm 5 µm 186000207 186000213 4.6 x 150 mm 5 µm WAT045905 WAT045995 Particle Part No. Part No. 4.6 x 250 mm 5 µm WAT054275 WAT054270 Dimension Type Size C18 C8 4.6 x 150 mm Column 3.5 µm WAT094240 WAT094237 ® Symmetry Cartridge Columns (Requires endfittings, see Page 186) 2.1 x 150 mm Column 5 µm WAT094234 WAT094231 3.0 x 150 mm Column 5 µm WAT054446 WAT054434 Particle Part No. Part No. 3.9 x 150 mm Column 5 µm WAT047210 WAT046955 Dimension Size C18 C8 4.6 x 150 mm Column 5 µm WAT054448 WAT054435 4.6 x 250 mm Column 5 µm WAT054450 WAT054438 2.1 x 20 mm 3.5 µm 186000269 186000268 2.1 x 50 mm 3.5 µm 186000152 186000149 3.9 x 150 mm Cartridge* 5 µm WAT054452 WAT054440 2.1 x 100 mm 3.5 µm 186000151 186000153 4.6 x 150 mm Cartridge* 5 µm WAT054454 WAT054442 2.1 x 150 mm 3.5 µm 186000150 186000148 4.6 x 250 mm Cartridge* 5 µm WAT054456 WAT054444 4.6 x 75 mm 3.5 µm WAT066260 WAT066210 * Require Endfittings, see Page 13 4.6 x 100 mm 3.5 µm WAT066265 WAT066215 3.9 x 50 mm 5 µm WAT054220 WAT054240 SymmetryPrep™ Columns for Purification and Isolation 3.9 x 150 mm 5 µm WAT054205 WAT054235 4.6 x 150 mm 5 µm WAT054210 WAT054255 4.6 x 250 mm 5 µm WAT054215 WAT054245 Particle Part No.Part No. Dimension Size C18 C8

® ™ 7.8 x 10 mm 5 µm 186000711 186000712 Symmetry Sentry Guard Columns (2 per pack) 7.8 x 50 mm 5 µm 186000208 186000214 (Requires Sentry™ Guard Holders, see Page 186) 7.8 x 100 mm 5 µm 186000209 186000215 19 x 10 mm 5 µm 186000715 186000716 Particle Part No. Part No. 19 x 50 mm 5 µm 186000210 186000216 Dimension Size C18 C8 19 x 100 mm 5 µm 186000211 186000229 2.1 x 10 mm 3.5 µm WAT106127 WAT106128 30 x 50 mm 5 µm 186000235 186000237 3.9 x 20 mm 5 µm WAT054225 WAT054250 30 x 100 mm 5 µm 186000236 186000238 7.8 x 10 mm 7 µm 186000713 186000714

® 7.8 x 150 mm 7 µm WAT066288 WAT066285 Symmetry Capillary Columns 7.8 x 300 mm 7 µm WAT066235 WAT066225 19 x 10 mm 7 µm 186000717 186000718 Particle Part No. Part No. 19 x 150 mm 7 µm WAT066240 WAT066228 19 x 300 mm 7 µm WAT066245 WAT066230 Dimension Size C18 C8 0.32 x 50 mm 3.5 µm 186000945 186000946 Purification and Isolation Cartridge Column Holders 0.32 x 100 mm 3.5 µm 186000951 186000952 7.8 x 10 mm 5, 7 µm — 186000708 0.32 x 150 mm 3.5 µm 186000957 186000958 19 x 10 mm 5, 7 µm — 186000709 0.32 x 50 mm 5 µm 186000929 186000930 Replacement o-ring 7.8 mm, 2 pkg 700001019 0.32 x 100 mm 5 µm 186000935 186000936 Replacement o-ring 19 mm, 2 pkg 700001020 0.32 x 150 mm 5 µm 186000347 186000941

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SymmetryShield™ Columns SymmetryShield™ Column and Cartridge Column Method Validation Kits Particle Part No. Part No. Three Columns from Three Different Batches to Test Reproducibility. Dimension Size RP18 RP8 1.0 x 50 mm 3.5 µm 186000175 WAT106060 Particle Part No. Part No. 1.0 x 150 mm 3.5 µm 186000176 WAT106048 Dimension Type Size RP18 RP8 2.1 x 30 mm 3.5 µm 186000171 WAT106001 2.1 x 150 mm Column 3.5 µm 186000182 — 2.1 x 50 mm 3.5 µm 186000172 WAT094257 4.6 x 150 mm Column 3.5 µm 186000181 WAT094278 2.1 x 100 mm 3.5 µm 186000173 WAT058969 2.1 x 150 mm Column 5 µm 186000100 WAT094254 2.1 x 150 mm 3.5 µm 186000174 WAT106008 3.0 x 150 mm Column 5 µm - WAT094251 3.0 x 100 mm 3.5 µm 186000700 186000703 3.9 x 150 mm Column 5 µm 186000104 WAT210594 3.0 x 150 mm 3.5 µm 186000699 186000702 4.6 x 150 mm Column 5 µm 186000103 WAT210588 4.6 x 50 mm 3.5 µm 186000177 WAT094260 4.6 x 250 mm Column 5 µm 186000102 WAT210591 4.6 x 75 mm 3.5 µm 186000178 WAT094263 4.6 x 100 mm 3.5 µm 186000179 WAT094266 3.9 x 150 mm Cartridge* 5 µm 186000105 WAT210582 4.6 x 150 mm 3.5 µm 186000180 WAT094269 4.6 x 150 mm Cartridge* 5 µm 186000101 WAT210585 4.6 x 250 mm Cartridge* 5 µm 186000114 WAT210579 2.1 x 50 mm 5 µm 186000217 186000223 2.1 x 150 mm 5 µm 186000111 WAT094245 * Require endfittings, see Page 186 3.0 x 150 mm 5 µm 186000692 WAT094243 3.0 x 250 mm 5 µm 186000693 186000694 SymmetryShield™ Columns for Purification and Isolation 3.9 x 150 mm 5 µm 186000108 WAT200655 4.6 x 50 mm 5 µm 186000218 186000224 4.6 x 150 mm 5 µm 186000109 WAT200662 Particle Part No. Part No. 4.6 x 250 mm 5 µm 186000112 WAT200670 Dimension Size RP18 RP8 19 x 10 mm 5 µm 186001835 186001841 ™ 19 x 50 mm 5 µm 186001836 186001842 SymmetryShield Cartridge Columns (Requires endfittings, see Page 186) 19 x 100 mm 5 µm 186001837 186001843 19 x 150 mm 5 µm 186001838 186001844 Particle Part No. Part No. 19 x 150 mm 7 µm 186001839 186001845 Dimension Size RP RP 18 8 19 x 300 mm 7 µm 186001840 186001846 2.1 x 50 mm 3.5 µm 186000168 186000147 Purification and Isolation Cartridge Column Holders 2.1 x 100 mm 3.5 µm 186000167 186000146 2.1 x 150 mm 3.5 µm 186000166 186000145 7.8 x 10 mm 5, 7 µm — 186000708 4.6 x 75 mm 3.5 µm 186000183 WAT094272 19 x 10 mm 5, 7 µm — 186000709 4.6 x 100 mm 3.5 µm 186000170 WAT094275 Replacement o-ring 7.8 mm, 2 pkg 700001019 3.9 x 50 mm 5 µm — WAT094248 Replacement o-ring 19 mm, 2 pkg 700001020 3.9 x 150 mm 5 µm 186000106 WAT200658 4.6 x 150 mm 5 µm 186000110 WAT200665 4.6 x 250 mm 5 µm 186000113 WAT200661 Symmetry300™ Columns and Cartridges SymmetryShield™ Sentry™ Guard Columns (2 per pack) (Requires Sentry™ Guard Holders, see Page 186) Symmetry300™ Analytical Columns Particle Part No. Part No. Dimension Size RP18 RP8 Particle Part No.Part No. Dimension Size C C 2.1 x 10 mm 3.5 µm 186000169 WAT106129 18 4 3.9 x 20 mm 3.5 µm 186000701 186000704 1.0 x 150 mm 3.5 µm 186000185 186000276 3.9 x 20 mm 5 µm 186000107 WAT200675 2.1 x 50 mm 3.5 µm 186000187 186000277 2.1 x 100 mm 3.5 µm 186000188 186000278 SymmetryShield™ Capillary Columns 2.1 x 150 mm 3.5 µm 186000200 186000279 4.6 x 50 mm 3.5 µm 186000201 186000280 4.6 x 75 mm 3.5 µm 186000189 186000281 Particle Part No. Part No. 4.6 x 100 mm 3.5 µm 186000190 186000282 Dimension Size RP RP 18 8 4.6 x 150 mm 3.5 µm 186000197 186000283 0.32 x 50 mm 3.5 µm 186000947 186000948 2.1 x 150 mm 5 µm WAT106172 186000285 0.32 x 100 mm 3.5 µm 186000953 186000954 3.9 x 150 mm 5 µm WAT106154 186000286 0.32 x 150 mm 3.5 µm 186000959 186000960 4.6 x 50 mm 5 µm WAT106209 186000287 0.32 x 50 mm 5 µm 186000931 186000932 4.6 x 150 mm 5 µm WAT106157 186000288 0.32 x 100 mm 5 µm 186000937 186000938 4.6 x 250 mm 5 µm WAT106151 186000289 0.32 x 150 mm 5 µm 186000942 186000943

184 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. Symmetry300™ Cartridge Columns (Requires endfittings, see Page 186) Symmetry300™ Column and Cartridge Column Method Validation Kits Particle Part No. Three Columns from Three Different Batches to Test Reproducibility. Dimension Size C18 2.1 x 50 mm 3.5 µm 186000199 Particle Part No. Part No. 2.1 x 100 mm 3.5 µm 186000191 Dimension Type Size C18 C4 2.1 x 150 mm 3.5 µm 186000196 4.6 x 75 mm 3.5 µm 186000192 2.1 x 150 mm Column 3.5 µm 186000194 186000290 4.6 x 100 mm 3.5 µm 186000193 4.6 x 150 mm Column 3.5 µm 186000195 186000291 3.9 x 150 mm 5 µm WAT106169 2.1 x 150 mm Column 5 µm WAT106193 186000292 4.6 x 150 mm 5 µm WAT106163 3.9 x 150 mm Column 5 µm WAT106187 186000293 4.6 x 250 mm 5 µm WAT106160 4.6 x 150 mm Column 5 µm WAT106190 186000294 4.6 x 250 mm Column 5 µm WAT106184 186000295

3.9 x 150 mm Cartridge* 5 µm WAT106181 — ™ ™ Symmetry300 Sentry Guard Columns (2 per pack) 4.6 x 150 mm Cartridge* 5 µm WAT106175 — (Requires Sentry™ Guard Holders, see Page 186) 4.6 x 250 mm Cartridge* 5 µm WAT106178 —

* Require endfittings, see Page 186 Particle Part No. Part No. Dimension Size C18 C4 2.1 x 10 mm 3.5 µm 186000198 186000275 Symmetry300™ Columns for Purification and Isolation 3.9 x 20 mm 5 µm WAT106166 186000284 Particle Part No. Dimension Size C18 Symmetry300™ Capillary Columns 19 x 10 mm 5 µm 186001847 19 x 50 mm 5 µm 186001848 Particle Part No. Part No. 19 x 100 mm 5 µm 186001849 Dimension Size C18 C4 19 x 150 mm 5 µm 186001850 0.32 x 50 mm 3.5 µm 186000949 186000950 Purification and Isolation Cartridge Column Holders 0.32 x 100 mm 3.5 µm 186000955 186000956 7.8 x 10 mm 186000708 0.32 x 150 mm 3.5 µm 186000961 186000962 19 x 10 mm 186000709 0.32 x 50 mm 5 µm 186000933 186000934 Replacement o-ring 7.8 mm, 2 pkg 700001019 0.32 x 100 mm 5 µm 186000939 186000940 Replacement o-ring 19 mm, 2 pkg 700001020 0.32 x 150 mm 5 µm 186000348 186000944

™ ™ Symmetry IS™ Columns Opti-Pak Symmetry300 C18 trap columns (5 per pack)

Particle Symmetry300™ Particle Symmetry® Symmetry® Dimension Size C18 Dimension Size C18 C8 0.35 x 5 mm 5 µm 186001492 2.1 x 20 mm 3.5 µm 186002066 186002067 2.1 x 20 mm 3.5 µm 186002070 186002071 3.0 x 20 mm 3.5 µm 186002074 186002075 3.0 x 20 mm 5 µm 186002078 186002079 3.9 x 20 mm 5 µm 186002082 186002083 3.9 x 20 mm 5 µm 186002086 186002087 4.6 x 20 mm 5 µm 186002090 186002091 4.6 x 20 mm 5 µm 186002094 186002095

SymmetryShield IS™ Columns

Particle Symmetry® Symmetry® Dimension Size RP18 RP8 2.1 x 20 mm 3.5 µm 186002068 186002069 2.1 x 20 mm 3.5 µm 186002072 186002073 3.0 x 20 mm 3.5 µm 186002076 186002077 3.0 x 20 mm 5 µm 186002080 186002081 3.9 x 20 mm 5 µm 186002084 186002085 3.9 x 20 mm 5 µm 186002088 186002089 4.6 x 20 mm 5 µm 186002092 186002093 4.6 x 20 mm 5 µm 186002096 186002097

Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. 185 Cartridge and Guard Columns

Waters Cartridge Column Waters Sentry™ Guard Column and Universal Guard Holder Waters cartridge system requires re-useable endfittings. These cartridge endfit- tings need to be ordered when purchasing a Waters HPLC cartridge column Waters offers a line of Sentry™ Guard columns that contain the same high for the first time. These cartridge columns should be connected to your HPLC performance packing materials as all of the Waters analytical columns. instrument using Waters standard connectors. Developed for customers needing increased capacity for biological fluid clean- up and/or temperature control, the Sentry™ Guard column is available in two holder designs--one to be used as an integrated part of the Waters steel Connector C-Clip Endfitting cartridge column with reusable endfitting, the other for use with any HPLC WAT037525 column. Both are installed without tools.

Cartridge Sentry™ Guard Column Retaining Groove Nut

Disposable Cartridge Integrated Guard Holder

Integrated Sentry™ Guard Holder to go with cartridge end-fittings (see above) Waters Cartridge Column Universal Guard Holder 3.0 x 20 mm, 3.9 x 20 mm – use Part Number WAT046905 Reusable End Connectors Universal Sentry™ Guard Holder (Use on any column) Cartridge End-Fittings 2.1 x 10 mm use Part Number WAT097958 2.1 x 50 mm, 2.1 x 100 mm, 2.1 x 150 mm, 2.1 x 250 mm use Part Number 700000117 2.1 x 20 mm use Part Number 186000262 3.0 x 50 mm, 3.0 x 100 mm, 3.0 x 150 mm, 3.0 x 250 mm use Part Number WAT037525 3.0 x 20 mm use Part Number WAT046910 3.9 x 50 mm, 3.9 x 100 mm, 3.9 x 150 mm, 3.9 x 250 mm use Part Number WAT037525 3.9 x 20 mm use Part Number WAT046910 Sentry™ Guard Column 4.6 x 50 mm, 4.6 x 100 mm, 4.6 x 150 mm, 4.6 x 250 mm use Part Number WAT037525 4.6 x 20 mm use Part Number WAT046910

186 Waters, Symmetry, SymmetryShield, Sentry, SymmetryPrep, Platform LC, Quattro Ultima, IS, Oasis and Alliance are trademarks of Waters Corporation. ©2003 Waters Corporation. visit us on the internet: http://www.waters.com

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