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Aspirin
Molecular formula: C9H8O4 Molecular weight: 180.2 CAS Registry No.: 50-78-2

SAMPLE Matrix: blood Sample preparation: Plasma. 200 (xL Plasma + 200 |xL 5 jjig/mL IS in 200 mM HCL: 200 mM orthophosphoric acid 50:50, vortex for 1-2 s, add 400 jxL MeCN, vortex, let stand at 4 for 15 min, centrifuge at 10500 g for 1 min. Remove the supernatant and add it to 100120 mg NaCl, vortex briefly, let stand at 4 for 10 min, vortex, centrifuge at 10500 g for 1 min, inject a 10 (xL aliquot of the upper organic layer. Whole blood. 200 (xL Lysed whole blood + 400 |xL 5 |xg/mL IS in 200 mM HCL: 200 mM orthophosphoric acid 50:50, vortex for 1-2 s, add 600 pX MeCN, vortex, let stand at 4 for 15 min, centrifuge at 10500 g for 1 min. Remove the supernatant and add it to 200 mg NaCl, vortex briefly, let stand at 4 for 10 min, vortex, centrifuge at 10500 g for 1 min, inject a 10 jxL aliquot of the upper organic layer. HPLCVARIABLES Column: 150 X 3.9 4 |xm Novapak C18 Mobile phase: MeCN: water: 85% orthophosphoric acid 18:74:0.09 (Before use prime column by recycling 200 mL mobile phase + 400 jxL di-n-butylamine overnight at 0.3 mL/min.) Column temperature: 30 Flow rate: 1 Injection volume: 10 Detector: UV 237 CHROMATOGRAM Retention time: 4.2 Internal standard: 2-methylbenzoic acid (8.9) Limit of quantitation: 100 ng/mL OTHER SUBSTANCES Extracted: metabolites, gentisic acid, salicylic acid, salicyluric acid KEYWORDS plasma; whole blood; pharmacokinetics REFERENCE
Kees, R; Jehnich, D.; Grobecker, H. Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography. J.Chromatogr.B, 1996, 677, 172-177

SAMPLE Matrix: blood Sample preparation: Add o-anisic acid to 1 mL plasma, acidify with HCl, extract with diethyl ether. Remove the organic layer and evaporate it to dryness, reconstitute the residue, inject an aliquot. HPLC VARIABLES Column: 150 X 4.6 5 |xm Supelcosil LC-8 Mobile phase: MeCN: 20 mM phosphoric acid 15:85 Flow rate: 1.6 Detector: UV 237

CHROMATOGRAM Internal standard: o-anisic acid Limit of quantitation: 20 ng/mL OTHER SUBSTANCES Extracted: salicylic acid KEYWORDS pharmacokinetics; plasma REFERENCE
Benedek, LH.; Joshi, A.S.; Pieniaszek, H.J.; King, S.-Y.R; Kornhauser, D.M. Variability in the pharmacokinetics and pharmacodynamics of low dose aspirin in healthy male volunteers. J.CHn. Pharmacol, 1995, 35, 1181-1186

SAMPLE Matrix: blood Sample preparation: Filter (0.22 |xm), inject a 2 JJLL aliquot of the filtrate. HPLCVARIABLES Column: 250 X 4 5 |xm LiChrospher 100 Diol Mobile phase: MeCN: 50 mM pH 3.0 phosphate buffer 1.5:98.5 Flow rate: 0.5 Injection volume: 2 Detector: UV 254 CHROMATOGRAM Retention time: 8 OTHER SUBSTANCES Extracted: salicylic acid KEYWORDS direct injection; serum REFERENCE
Nimura, N.; Itoh, H.; Kinoshita, T. Diol-bonded silica gel as a restricted access packing forming a binarylayered phase for direct injection of serum for the determination of drugs. J.Chromatogr.A, 1995, 689, 203-210

SAMPLE Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol: n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.) HPLCVARIABLES Column: 300 X 3.9 4 um NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30

Flow rate: 0.8 Injection volume: 50 Detector: UV 233

CHROMATOGRAM

Retention time: 3.40 Limit of detection: <120 ng/mL

OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amitriptyline, amodiaquine, amoxapine, astemizole, benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinoxamine, carpipramine, carteolol, celiprolol, cetirizine, chlorambucil, chlordiazepoxide, chlorophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, loperamide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, medifoxamine, mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapramine, methadone, methaqualone, methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine, mianserine, midazolam, minoxidil, moclobemide, moperone, nadolol, nalbuphine, nalorphine, naloxone, naproxen, nialamide, nicardipine, nifedipine, niflumic acid, nimodipine, nitrazepam, nitrendipine, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenylbutazone, pimozide, pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, promethazine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine, quinupramine, ramipril, reserpine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulindac, sulpride, suriclone, temazepam, tenoxicam, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine, tiaprofenic acid, ticlopidine, timolol, tioclomarol, tofisopam, tolbutamide, trazodone, triazolam, trifluoperazine, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: albuterol, amisulpride, atenolol, chlormezanone, codeine, lisinopril, metformin, naltrexone, phenobarbital, phenol, ranitidine, ritodrine, sultopride, terbutaline, tiapride, toloxatone KEYWORDS whole blood; plasma REFERENCE
Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic ScL, 1995, 40, 254-262

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 0.2 mL 1 M HCl + 1 0 mL diethyl ether, gently mix for 10 min, centrifuge at 1500 rpm for 4 min. Remove the organic phase, evaporate it to dryness at 0 under a stream of nitrogen, add 200 |xL mobile phase, vortex 90 s, inject a 5-100 |xL aliquot.

HPLCVARIABLES Guard column: 23 X 3.9 fxBondapak C18/Porasil B Column: 300 X 3.9 10 |xm jxBondapak C18 Mobile phase: MeOH: water: 1-butanol: orthophosphoric acid 270:720:10:0.13 Column temperature: 47 Flow rate: 1.8 Injection volume: 5-100 Detector: UV 234 CHROMATOGRAM Retention time: 5.6 Internal standard: m-anisic acid (9.6) Limit of quantitation: 10-15 ng/mL OTHER SUBSTANCES Simultaneous: salicylic acid Noninterfering: acetaminophen, albuterol, aminophylline, amitriptyline, atenolol, beclomethasone, bromazepam, caffeine, carbamazepine, chloral hydrate, chlordiazepoxide, cimetidine, clonazepam, codeine, desipramine, dexamethasone, dextropropoxyphene, diazepam, dicyclomine, digoxin, disopyramide, doxycycline, ergotamine, ethosuximide, furosemide, gentisic acid, haloperidol, hydrocortisone, imipramine, indomethacin, levodopa, lignocaine, lithium carbonate, meperidine, methdilazine, methylphenobarbitone, methylprednisolone, methysergide, metoclopramide, metoprolol, mexiletine, midazolam, naphthoxyacetic acid, nitrazepam, nitroglycerin, nortripyline, oxazepam, oxpranolol, pentobarbitone, pethidine, phenytoin, prednisolone, prednisone, primidone, procainamide, prochlorperazine, propranolol, quinidine, salicyluric acid, spironolactone, sulfamethoxazole, theophylline, trimethoprim, valproic acid, verapamil, warfarin Interfering: methyclothiazide KEYWORDS plasma; pharmacokinetics REFERENCE
Brandon, R.A.; Eadie, M.J.; Smith, M.T. A sensitive liquid chromatographic assay for plasma aspirin and salicylate concentrations after low doses of aspirin. Ther.Drug Monit., 1985, 7, 216-221

SAMPLE Matrix: blood Sample preparation: 50 \xL Serum + 50 \xL 15 jxg/mL p-hydroxyethyltheophylline in MeCN, mix for 30 s, centrifuge at 13000 g for 5 min. Inject the supernatant (about 20 |xL). HPLC VARIABLES Column: ixBondapak C18 Mobile phase: MeCN:buffer 9.75:90.25 (Buffer was 100 mM KH2PO4 adjusted to pH 4.0 with phosphoric acid.) (At the end of each day clean column with water for 20 min and MeOH for 30 min.) Flow rate: 2 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 12 Internal standard: p-hydroxyethyltheophylline (5.8) Limit of detection: 500 ng/mL

OTHER SUBSTANCES Extracted: salicylic acid Simultaneous: acetaminophen, acetylprocainamide, caffeine, dyphylline, procainamide, theophylline Noninterfering: benzoic acid KEYWORDS serum REFERENCE
Ou, C-N.; Frawley, V.L. Theophylline, dyphylline, caffeine, acetaminophen, salicylate, acetylsalicylate, procainamide, and N-acetylprocainamide determined in serum with a single liquid-chromatographic assay. Clin.Chem., 1982, 28, 2157-2160

SAMPLE Matrix: blood Sample preparation: Mix plasma with an equal volume of MeCN, vortex for 30 s, centrifuge at 900 g for 5 min, inject a 100 |xL aliquot of the supernatant. HPLCVARIABLES Column: 300 X 3.9 10 |xm (juBondapak C18 Mobile phase: MeOH: acetic acid: water 22:5:73 Flow rate: 2.6 Injection volume: 100 Detector: UV 280 CHROMATOGRAM Retention time: 4.75 Limit of quantitation: 2 fxg/mL OTHER SUBSTANCES Extracted: salicylic acid Noninterfering: acetaminophen, albuterol, aminophylline, amitriptyline, amoxicillin, ampicillin, amobarbital, beclomethasone, carbamazepine, carbenicillin, chlordiazepoxide, cimetidine, clonazepam, cyproheptadine, debrisoquine, dextropropoxyphene, diazepam, digoxin, dihydroxyanthraquinone, ergotamine, ethosuximide, fluphenazine, furosemide, gentamicin, gentisic acid, guaifenesin, haloperidol, heparin, hydrocortisone, indomethacin, methdilazine, methyclothiazide, methylphenobarbitone, methysergide, metoclopramide, naproxen, nitrazepam, nystatin, penicillin, pentobarbitone, phenytoin, phenytoin, pizotifen, prazosin, prednisone, prochlorperazine, propranolol, spironolactone, sulfamethoxazole, theophylline, trifluoperazine, trimethoprim, valproic acid KEYWORDS plasma REFERENCE
Cham, B.E.; Ross-Lee, L.; Bochner, R; Imhoff, D.M. Measurement and pharmacokinetics of acetylsalicylic acid by a novel high performance liquid chromatographic assay. Ther.Drug Monit., 1980, 2, 365-372

SAMPLE Matrix: blood, urine Sample preparation: Plasma. 500 fxL Plasma + 900 jxL 270 mM HCl + 100 JJLL 100 jjtg/mL a-phenylcinnamic acid in MeOH + 10 mL dichloromethane, shake at 125 cycles/min for 15 min, centrifuge at 750 g for 5 min. Remove the organic layer and evaporate it to

dryness, reconstitute the residue in 500 jxL MeOH, inject a 25 |xL aliquot. Urine. 2 mL Urine + 900 jxL 270 mM HCl + 100 JJLL 100 fjig/mL a-phenylcinnamic acid in MeOH + 10 mL hexane, shake at 125 cycles/min for 15 min, centrifuge at 750 g for 5 min. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 500 fxL MeOH, inject a 25 |xL aliquot. HPLCVARIABLES Column: 300 X 4 ixBondapak C18 Mobile phase: MeOH: 1% acetic acid 60:40 Flow rate: 2 Injection volume: 25 Detector: UV 280 CHROMATOGRAM Retention time: 2.5 Internal standard: a-phenylcinnamic acid (8.0) Limit of detection: 2 (xg/mL OTHER SUBSTANCES Extracted: salicylic acid (UV 300), salsalate (UV 300) KEYWORDS plasma; pharmacokinetics REFERENCE
Harrison, L.L; Funk, M.L.; Ober, R.E. High-pressure liquid chromatographic determination of salicylsalicylic acid, aspirin, and salicylic acid in human plasma and urine. J.Pharm.Sci., 1980, 69, 12681271

SAMPLE Matrix: bulk, formulations Sample preparation: Bulk. Prepare a 20 mg/mL solution of bulk aspirin in dichloromethane, inject a 10 |xL aliquot as soon as dissolution is complete. Tablets. Prepare a 20 mg/mL solution of ground aspirin tablets in dichloromethane, filter (0.45 |xm) immediately, immediately inject a 10 |xL aliquot of the filtrate. HPLC VARIABLES Column: 150 X 4.6 6 |xm Zorbax SIL Mobile phase: Hexane: chloroform: acetic acid 80:19:3 (Before first use pump 10 column volumes of dichloromethane: acetic acid: 2,3-dimethoxypropane 96:2:2 through column at 3 mL/min.) Flow rate: 3 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 3.6 OTHER SUBSTANCES Simultaneous: impurities, salicylic acid, salsalate KEYWORDS normal phase; tablets REFERENCE
Pfeiffer, CD.; Pankey, J.W. Determination of related compounds in aspirin by liquid chromatography. J.Pharm.Sci., 1982, 71, 511-514

SAMPLE Matrix: formulations Sample preparation: Weigh out powdered sample containing 68 mg aspirin, add 80 mL MeOH, sonicate for 10 min, dilute to 100 mL with MeOH, centrifuge. Remove a 5 mL aliquot of the supernatant and add it to 1 mL 2 mg/mL resorcinol, add 2 mL MeOH, make up to 20 mL with 50 mM pH 3.0 triethylamine phosphate, inject an aliquot. HPLCVARIABLES Column: 150 X 3.2 5 |xm Hypersil ODS Mobile phase: THF: 50 mM pH 3.0 triethylamine phosphate 12:88 Flow rate: 0.6 Injection volume: 20 Detector: UV 275 following post-column reaction. The column effluent flowed through a 10 m X 0.3 mm ID crocheted PTFE coil irradiated with an 8 W low-pressure mercury lamp at 254 nm to the detector. CHROMATOGRAM Retention time: 15 Internal standard: resorcinol (9) OTHER SUBSTANCES Simultaneous: acetaminophen (post-column irradiation gives little increase in peak height), caffeine (post-column irradiation gives little increase in peak height), propyphenazone (post-column irradiation gives a decrease in peak height) KEYWORDS post-column reaction REFERENCE
Di Pietra, A.M.; Gatti, R.; Andrisano, V; Cavrini, V. Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics. J.Chromatogr.A, 1996, 729, 355-361

SAMPLE Matrix: formulations Sample preparation: Condition a 500 mg Extract Clean silica SPE cartridge (Alltech stock no. 209250) with 2 mL hexane. Allow a solution of aspirin in 10 mM sorbitan trioleate in CFC-Il to evaporate, dissolve the residue in 5 mL hexane. Add 1 mL to the SPE cartridge, elute with two 2 mL portions of mobile phase, make up eluate to 5 mL with mobile phase, inject a 20 |JLL aliquot. HPLCVARIABLES Guard column: 20 X 2 30-40 jxm Perisorb RP-8 Pellicular (Upchurch) Column: 250 X 4.6 5 fxm Econosphere C8 Mobile phase: MeOH: THF: 1 M phosphoric acid: water 44:5:5:46 Flow rate: 1 Injection volume: 20 Detector: UV 275 CHROMATOGRAM Retention time: 5.6 OTHER SUBSTANCES Extracted: degradation products, salicylic acid

KEYWORDS

aerosols; SPE
REFERENCE
Blondino, RE.; Byron, RR. The quantitative determination of aspirin and its degradation products in a model solution aerosol. J.Pharm.Biomed.AnaL, 1995, 13, 111-119

SAMPLE

Matrix: formulations Sample preparation: Powder tablets, add 40 mg pyrimethamine, dissolve in 20 mL MeCN, add 40 mL mobile phase, filter (paper), wash filter with mobile phase, make up filtrate to 100 mL with mobile phase. Dilute a 5 mL aliquot to 50 mL with mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4 10 |xm Nucleosil C18 Mobile phase: MeOH:MeCN:water:triethylamine 55:5:40:0.1, pH adjusted to 4.0 with phosphoric acid Flow rate: 1.5 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 2.5 Internal standard: pyrimethamine (3.5) Limit of quantitation: 6 |xg/mL OTHER SUBSTANCES Simultaneous: dipyridamole
KEYWORDS

tablets
REFERENCE
Sane, R.T.; Ghadge, J.K.; Jani, A.B.; Vaidya, A.J.; Kotwal, S.S. Simultaneous high-performance liquid chromatographic determination of haloperidol with propantheline bromide, nalidixic acid with phenazopyridine hydrochloride, and dipyridamole with aspirin in combined dosage (forms). Indian Drugs, 1992, 29, 240-244

SAMPLE

Matrix: formulations Sample preparation: Oils. 1 mL Sample + 25 mL MeOH-.water 90:10, shake vigorously for 5 min, centrifuge, inject a 10 |xL aliquot of the supernatant. Tablets. Grind a tablet to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous). Make up 5 mL to 50 mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 |JLL aliquot of the remaining filtrate.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Zorbax ODS Mobile phase: MeOH: water 75:25 Flow rate: 1.5 Injection volume: 10 Detector: UV 240

CHROMATOGRAM Retention time: 2.5 Limit of detection: 5 |xg/mL OTHER SUBSTANCES Simultaneous: benzyl alcohol, benzyl benzoate, boldenone, calusterone, cortisone, dehydroepiandrosterone (UV 210), ethisterone, fluoxymesterone, mesterolone (UV 210), methandriol (UV 210), methandrostenolone, methenolone acetate, methyltestosterone, mibolerone, nandrolone, nandrolone acetate, nandrolone propionate, norethandrolone, norethindrone, norethindrone acetate, norgestrel, oxandrolone (UV 210), oxymetholone, stanozolol, testosterone, testosterone acetate, testosterone propionate, trenbolone acetate Interfering: caffeine, formebolone, testolactone KEYWORDS oils; tablets; suspensions REFERENCE
Walters, M.J.; Ayers, R.J.; Brown, D.J. Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry. J.Assoc.Off.Anal.Chem., 1990, 73, 904-926

SAMPLE Matrix: formulations Sample preparation: Place 5 tablets in MeCN: MeOH: 85% phosphoric acid 92:8:0.5, sonicate 15 min, shake 15 min, dilute to 250 mL. Centrifuge an aliquot in 50 mL tube at 2000 rpm for 15 min and filter supernatant (0.45 |xm). HPLCVARIABLES Column: 150 X 3.9 Resolve (Waters) Mobile phase: MeCN: water: 85% phosphoric acid 24:76:0.5 Column temperature: 35 Flow rate: 2 Injection volume: 10 Detector: UV 295 CHROMATOGRAM Retention time: 2.2 OTHER SUBSTANCES Simultaneous: acetaminophen, caffeine, salicylic acid KEYWORDS film coated tablets; tablets REFERENCE
Fogel, J.; Epstein, R; Chen, P. Simultaneous high-performance liquid chromatography assay of acetylsalicylic acid and salicylic acid in film-coated aspirin tablets. J.Chromatogr., 1984, 317, 507-511

SAMPLE Matrix: formulations Sample preparation: Grind tablets to a fine powder and add about 250 mg aspirin to 100 mL chloroform saturated with citric acid containing 500 |xL formic acid, add 500 mg solid citric acid, sonicate for 2 min, centrifuge or filter, inject an aliquot. (If buffers or antacid are present, add ground tablets equivalent to about 500 mg aspirin to 3 g acid-washed siliceous earth, mix, add 2 mL 6 M HCl, mix, add to a 200 X 25 column, dry wash

container with siliceous earth, add to column, elute column with chloroform saturated with citric acid at 10 mL/min. Collect 150 mL eluent, add 1 mL formic acid, make up to 200 mL with chloroform saturated with citric acid, add 500 mg citric acid, shake, inject an aliquot.) HPLC VARIABLES Column: 250 X 4.6 5jxm Zorbax-Sil Mobile phase: Chloroform: dichloromethane: acetonitrile: formic acid 700:300:30:4 (At the end of the day wash the column with 200 mL MeOH.) Flow rate: 2 Injection volume: 20 Detector: UV 300 CHROMATOGRAM Retention time: 8 OTHER SUBSTANCES Simultaneous: acetylsalicylic acid anhydride, acetylsalicylsalicylic acid, salicylic acid, excipients KEYWORDS normal phase; tablets; SPE REFERENCE
Galante, R.N.; Visalli, A.J.; Grim, W.M. Stabilized normal-phase high-performance liquid chromatographic analysis of aspirin and salicylic acid in solid pharmaceutical dosage forms. J.Pharm.Sci., 1984, 73, 195-197

SAMPLE Matrix: formulations Sample preparation: Pulverize tablets and weigh out 1 g, add 1 mL formic acid, add 25 mL MeOH, shake mechanically for 10 min, make up to 50 mL with methanol. Remove 10 mL and centrifuge. 5 mL Supernatant + 5 mL 0.0025% p-hydroxybenzoic acid in MeOH: water 20:80, make up to 25 mL with water, inject an aliquot. (Analyze within 1 h.) HPLCVARIABLES Column: 250 X 4.6 LiChrosorb RP8 Mobile phase: MeOH:200 mM pH 3.5 phosphate buffer:water 20:10:70 Flow rate: 1 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 24 Internal standard: p-hydroxybenzoic acid (18) OTHER SUBSTANCES Simultaneous: acetaminophen, O-acetyl-p-aminophenol, 2-O-acetylascorbic acid, 3-O-acetylascorbic acid, p-aminophenol, diacetyl-p-aminophenol (UV 280), saccharin, salicylic acid (UV 280), vitamin C KEYWORDS tablets

REFERENCE
Thomis, R.; Roets, E.; Hoogmartens, J. Analysis of tablets containing aspirin, acetaminophen, and ascorbic acid by high-performance liquid chromatography. J.Pharm.ScL, 1984, 73, 1830-1833

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.1 6 jjtm PolyEncap ODS (n-octadecylacrylate copolymerized with vinyl silica in heptane, carrier Ultrasep ES 100; preparation described in paper) Mobile phase: MeCN :pH 2.2 phosphate buffer 32:68 Flow rate: 1 Detector: UV 220 CHROMATOGRAM Retention time: 4 OTHER SUBSTANCES Simultaneous: diazepam, diphenhydramine, o-hydroxyhippuric acid, MPPH, niacin, toluene REFERENCE
Engelhardt, H.; Cunat-Walter, M.A. Polymer encapsulated stationary phases with improved efficiency. Chromatographia, 1995, 40, 657-661

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 8 |xm Unisphere-PBD (polybutadiene on alumina) (Biotage, Charlottesville, VA) Mobile phase: MeCN: water 20:80 Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: 5 OTHER SUBSTANCES Simultaneous: acetaminophen, phenacetin, salicylamide REFERENCE
Jedrejewski, RT.; Taylor, L.T. Comparison of silica-, alumina-, and polymer-based stationary phases for reversed-phase liquid chromatography. J.Chromatogr.Sci., 1995, 33, 438-445

SAMPLE Matrix: solutions HPLCVARIABLES Guard column: 30 X 3.2 7 |xm SI 100 ODS (not commercially available) Column: 150 X 3.2 7 |xm SI 100 ODS (not commercially available) Mobile phase: MeCN:buffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric acid in 1 L water, pH 2.3.) Flow rate: 0.5-1 Detector: UV 231

CHROMATOGRAM Retention time: 3.4 Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin (7.3) OTHER SUBSTANCES Also analyzed: carbamazepine, chlordiazepoxide, chlorprothixene, clonazepam, caffeine, diazepam, doxylamine, ethosuximide, furosemide, haloperidol, hydrochlorothiazide, methocarbamol, methotrimeprazine, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylamide, temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone REFERENCE
Below, E.; Burrmann, M. Application of HPLC equipment with rapid scan detection to the identification of drugs in toxicological analysis. J.Liq.Chromatogr., 1994, 17, 4131-4144

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210 OTHER SUBSTANCES Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, fiufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, na-

proxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine REFERENCE
Hill, D. W.; Kind, A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J'.AnaLToxicol., 1994, 18, 233-242

SAMPLE Matrix: solutions Sample preparation: Prepare a 50 jxg/mL solution in the mobile phase, inject a 10 |xL aliquot. HPLCVARIABLES Column: 250 X 4.6 7 ^m Lichrosorb RP 18 Mobile phase: MeOH: water 45:55 containing 1% acetic acid Flow rate: 1 Injection volume: 10 Detector: UV 230 CHROMATOGRAM Retention time: 5.09 OTHER SUBSTANCES Simultaneous: acetaminophen, phenacetin, salicylamide, salicylic acid REFERENCE
Nivaud-Guernet, E.; Guernet, M.; Ivanovic, D.; Medenica, M. Effect of eluent pH on the ionic and molecular forms of the non-steroidal anti-inflammatory agents in reversed-phase high-performance liquid chromatography. J.Liq.Chromatogr., 1994, 17, 2343-2357

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4 OmniPac PAX-500 (Dionex) Mobile phase: Gradient. A was MeCN: 10 mM sodium carbonate 18:82, B was MeCN: 50 mM sodium carbonate 33:67. A:B from 100:0 to 0:100 over 10 min. Flow rate: 1 Detector: UV 254

CHROMATOGRAM

Retention time: 3
OTHER SUBSTANCES

Simultaneous: carprofen, diflunisal, fenbufen, ibuprofen, indomethacin, naproxen, tolmetin

REFERENCE
Slingsby, R.W.; Rey, M. Determination of pharmaceuticals by multi-phase chromatography: Combined reversed phase and ion exchange in one column. J.Liq.Chromatogr., 1990, 13, 107134

SAMPLE

Matrix: solutions Sample preparation: Dissolve compounds in MeOH, inject a 1 |xL aliquot.
HPLCVARIABLES

Column: 150 X 1 3 u,m Hitachi-Gel 3011 porous polymer (Hitachi) Mobile phase: MeOH: ammonia 99:1 Flow rate: 0.03 Injection volume: 1 Detector: UV 254
CHROMATOGRAM Retention time: 3.52 OTHER SUBSTANCES

Also analyzed: acetaminophen, bucetin (3-hydroxy-p-butyrophenetidine), caffeine, dipyrone (sulpyrin), ethenzamide (o-ethoxybenzamide), mefenamic acid, phenacetin, salicylamide, salicylic acid, theobromine, theophylline
KEYWORDS

semi-micro; porous polymer

REFERENCE
Matsushima, Y.; Nagata, Y; Niyomura, M.; Takakusagi, K.; Takai, N. Analysis of antipyretics by semimicro liquid chromatography. J.Chromatogr., 1985, 332, 269-273

SAMPLE

Matrix: urine Sample preparation: Acidify 5 mL urine to pH 1 with 40% phosphoric acid, shake with two 5 mL aliquots of diethyl ether, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 2 mL MeOH, inject a 25 |xL aliquot.
HPLCVARIABLES

Column: jxBondapak C18 radial compression Mobile phase: MeCN: 0.085% phosphoric acid 20:80 Flow rate: 1.5 Injection volume: 25 Detector: UV 237
CHROMATOGRAM

Limit of detection: 500 ng/mL

KEYWORDS horse REFERENCE

Beaumier, P.M.; Fenwick, J.D.; Stevenson, A.J.; Weber, M.P.; Young, L.M. Presence of salicylic acid in standardbred horse urine and plasma after various feed and drug administrations. Equine.Vet.J., 1987, 19, 207-213

ANNOTATED BIBLIOGRAPHY

Smigol, V.; Svec, R; Frechet, J.M.J. Novel uniformly sized polymeric stationary phase with hydrophilized large pores for direct injection HPLC determination of drugs in biological fluids. J.Liq.Chromatogr., 1994, 17, 891-911 [plasma; cow; extracted salicylic acid] Wongyai, S. Synthesis and characterization of phenylpropanolamine bonded silica for multimode liquid chromatography of small molecules. Chromatographia, 1994, 38, 485-490 [simultaneous benzoic acid, salicylic acid] Hays, P.A.; Lurie, LS. Quantitative analysis of adulterants in illicit heroin samples via reversed phase HPLC. J.Liq.Chromatogr., 1991, 14, 3513-3517 [simultaneous acetaminophen, acetylcodeine, acetylmorphine, benzocaine, caffeine, chloroquine, diamorphine, diazepam, diphenhydramine, dipyrone, lidocaine, methaqualone, monoacetylmorphine, morphine, nicotinamide, noscapine, papaverine, phenacetin, phenobarbital, phenolphthalein, N-phenyl-2-naphthylamine, salicylic acid, strychnine] Shen, J.; Wanwimolruk, S.; Clark, CR.; Roberts, M.S. A sensitive assay for aspirin and its metabolites using reversed-phase ion-pair high-performance liquid chromatography. J.Liq.Chromatogr., 1990,13, 751-761 [simultaneous metabolites gentisic acid, salicyluric acid; LOD 50 ng/mL] Lam, S.; Malikin, G. An improved micro-scale protein precipitation procedure for HPLC assay of therapeutic drugs in serum. J.Liq.Chromatogr., 1989, 12, 1851-1872 [serum; also acetaminophen, amiodarone, caffeine, chloramphenicol, flecainide, pentobarbital, procainamide, pyrimethamine, quinidine, theophylline, tocainide, trazodone; fluorescence detection; UV detection] Lurie, LS.; McGuiness, K. The quantitation of heroin and selected basic impurities via reversed phase HPLC. IL The analysis of adulterated samples. J.Liq.Chromatogr., 1987, 10, 2189-2204 [UV detection; electrochemical detection; simultaneous acetaminophen, acetylcodeine, acetylmorphine, acetylprocaine, aminopyrene, amitriptyline, antipyrene, barbital, benztropine, caffeine, cocaine, codeine, diamorphine, diazepam, diphenhydramine, dipyrone, ephedrine, ethylmorphine, lidocaine, meconin, methamphetamine, methapyrilene, methaqualone, monoacetylmorphine, morphine, nalorphine (IS), niacinamide, noscapine, papaverine, phenacetin, phenmetrazine, phenobarbital, phenolphthalein, procaine, propanophenone, propoxyphene, pyrilamine, quinidine, quinine, salicylamide, salicylic acid, secobarbital, strychnine, tartaric acid, tetracaine, thebaine, tripelennamine, tropacocaine, vitamin B5, vitamin B3] Lau, A.H.; Chang, CW.; Schlesinger, PK. Evaluation of a potential drug interaction between sucralfate and aspirin. CHn.Pharmacol.Ther, 1986, 39, 151-155 [plasma; pharmacokinetics; extracted metabolites, salicylic acid, salicyluric acid; a-phenylcinnamic acid (IS); gradient; column temp 30] Mamolo, M.G.; Vio, L.; Maurich, V. High-pressure liquid chromatographic analysis of paracetamol, caffeine and acetylsalicylic acid in tablets. Salicylic acid quantitation. Farmaco.fPrat]., 1985, 40, 1 1 1 123 [tablets; simultaneous acetaminophen, caffeine, phenazone, salicylic acid] Pedersen, A.K.; FitzGerald, G.A. Preparation and analysis of deuterium-labeled aspirin: application to pharmacokinetic studies. J.Pharm.Sci., 1985, 74, 188-192 [stability; simultaneous salicylic acid] Bevitt, R.N.; Mather, J.R.; Sharman, D.C. Minimization of salicylic acid formation during preparation of aspirin products for analysis by high-performance liquid chromatography. Analyst, 1984, 109, 1327-1329 Sreenivasan, K.; Nair, PD.; Rathinam, K. A GPC method for analysis of low molecular weight drugs. J.Liq.Chromatogr., 1984, 7, 2297-2305 [GPC; SEC; simultaneous salicylic acid] Das Gupta, V. Simultaneous quantitation of acetaminophen, aspirin, caffeine, codeine phosphate, phenacetin, and salicylamide by high-pressure liquid chromatography. J.Pharm.Sci., 1980, 69, 110-113 Kirchhoefer, R.D. Simultaneous determination of aspirin and salicylic acid in bulk aspirin and in plain, buffered, and enteric-coated tablets by high-pressure liquid chromatography with UV and fluorescence detectors. J.Pharm.Sci., 1980, 69, 1188-1191