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Aspirin

Molecular formula: C 9 H 8 O 4 Molecular weight: 180.2 CAS Registry No.: 50-78-2

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SAMPLE

Matrix: blood

Sampl e preparation : Plasma . 200 (xL Plasm a + 200 |xL 5 jjig/mL I S i n 200 mM HCL : 200 mM orthophosphoric acid 50:50 , vortex for 1-2 s, add 400 jxL MeCN, vortex, let stan d a t 4° for 15 min, centrifuge at 10500 g for 1 min. Remove the supernatant and add it to 100- 120 mg NaCl, vortex briefly, let stand at 4° for 10 min, vortex, centrifuge at 10500 g for

1 min , inject a 10 (xL aliquo t of th e uppe r organi c 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

1 min. Remove the supernatant and add it to 200 mg NaCl, vortex briefly, let stand at 4°

for 10 min, vortex, organic layer.

s, ad d 600 pX MeCN, vortex,

le t stan d a t 4° for 15 min , centrifuge a t 10500 g for

centrifuge a t 10500 g for 1 min, inject a 10 jxL aliquot of th e uppe r

HPLCVARIABLES

Column: 150 X 3.9 4 |xm Novapak C18

Mobile

18:74:0.09 (Before use prime col-

um n by recycling 200 mL mobile phas e + 400 jxL di-n-butylamine overnight a t 0.3 mL/min.)

phase: MeCN: water: 85% orthophosphoric acid

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 Flo w 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 phar- macokinetics 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 Flow rate: 0.5 Injection volume: 2 Detector: UV 254

CHROMATOGRAM Retention time: 8

OTHER SUBSTANCES Extracted: salicylic acid

KEYWORDS direct injection; serum

REFERENCE

buffer 1.5:98.5

Nimura, N.; Itoh, H.; Kinoshita, T. Diol-bonded silica gel as a restricted access packing forming a binary- layered 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,

ammonium chloride

solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)

inject a 50 |xL aliquot of th e supernatant . (Buffer was saturate d

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)) KH 2 PO 4 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, ami- triptyline, amodiaquine, amoxapine, astemizole, benazepril, benperidol, benzocaine, ben- zoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinoxamine, carpi- pramine, carteolol, celiprolol, cetirizine, chlorambucil, chlordiazepoxide, chlorophena- cinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, ci- cletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytar- abine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextrome- thorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihy- dralazine, diltiazem, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentia- zac, flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbipro- fen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, his- tapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, loper- amide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, medifoxam- ine, mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, me- phentermine, mepivacaine, metapramine, methadone, methaqualone, methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine, mianserine, midazo- lam, minoxidil, moclobemide, moperone, nadolol, nalbuphine, nalorphine, naloxone, na- proxen, nialamide, nicardipine, nifedipine, niflumic acid, nimodipine, nitrazepam, nitren- dipine, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenylbutazone, pimozide, pindolol, pi- pamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, pro- guanil, 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, tri- mipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vin- desine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: albuterol, amisulpride, atenolol, chlormezanone, codeine, lisinopril, metfor- min, naltrexone, phenobarbital, phenol, ranitidine, ritodrine, sultopride, terbutaline, tia- pride, 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 Flo w 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, beclo- methasone, bromazepam, caffeine, carbamazepine, chloral hydrate, chlordiazepoxide, ci- metidine, clonazepam, codeine, desipramine, dexamethasone, dextropropoxyphene, diaz- epam, dicyclomine, digoxin, disopyramide, doxycycline, ergotamine, ethosuximide, furosemide, gentisic acid, haloperidol, hydrocortisone, imipramine, indomethacin, levo- dopa, lignocaine, lithium carbonate, meperidine, methdilazine, methylphenobarbitone, methylprednisolone, methysergide, metoclopramide, metoprolol, mexiletine, midazolam, naphthoxyacetic acid, nitrazepam, nitroglycerin, nortripyline, oxazepam, oxpranolol, pen- tobarbitone, pethidine, phenytoin, prednisolone, prednisone, primidone, procainamide, prochlorperazine, propranolol, quinidine, salicyluric acid, spironolactone, sulfamethoxa- zole, 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 KH 2 PO 4 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, centri- fuge 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, am- picillin, amobarbital, beclomethasone, carbamazepine, carbenicillin, chlordiazepoxide, ci- metidine, clonazepam, cyproheptadine, debrisoquine, dextropropoxyphene, diazepam, di- goxin, dihydroxyanthraquinone, ergotamine, ethosuximide, fluphenazine, furosemide, gentamicin, gentisic acid, guaifenesin, haloperidol, heparin, hydrocortisone, indometha- cin, methdilazine, methyclothiazide, methylphenobarbitone, methysergide, metoclopram- ide, naproxen, nitrazepam, nystatin, penicillin, pentobarbitone, phenytoin, phenytoin, pizotifen, prazosin, prednisone, prochlorperazine, propranolol, spironolactone, sulfame- thoxazole, theophylline, trifluoperazine, trimethoprim, valproic acid

KEYWORDS

plasma

REFERENCE

Cham, B.E.; Ross-Lee, L.; Bochner, R; Imhoff, D.M. Measurement and pharmacokinetics of acetylsali- cylic acid by a novel high performance liquid chromatographic assay. Ther.Drug Monit., 1980, 2,

365-372

SAMPLE Matrix: blood, urine Sampl e preparation : Plasma . 50 0 fxL Plasm a + 90 0 jxL 270 m M HC l + 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 , reconstitut e th e residu e i n 500 jxL MeOH, inject a 25 |xL aliquot . Urine . 2 mL Urin e + 900 jxL 270 mM HCl + 100 JJLL 100 fjig/mL a-phenylcinnamic acid i n MeOH + 10 mL hexane, shake at 125 cycles/min for 15 min, centrifuge at 750 g for 5 min. Remove

50 0 fxL MeOH ,

th e organi c laye r an d evaporat e i t t o dryness , reconstitut e th e residu e i n 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 salicyl-

salicylic acid, aspirin, and salicylic acid in human plasma and urine. J.Pharm.Sci.,

1271

1980, 69, 1268-

SAMPLE Matrix: bulk, formulations Sample preparation: Bulk. Prepare a 20 mg/mL solution of bulk aspirin in dichlorome- thane, 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) immedi- ately, 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), propyphen- azone (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 chromatog- raphy with diode-array detection and on-line post-column photochemical derivatization to the deter- mination 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

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

X 2 30-40 jxm Perisorb RP-8 Pellicular (Upchurch)

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 phen- azopyridine 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, dehy- droepiandrosterone (UV 210), ethisterone, fluoxymesterone, mesterolone (UV 210), meth- andriol (UV 210), methandrostenolone, methenolone acetate, methyltestosterone, mibol- erone, 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 spectrophotom- etry. 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, son-

icate 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 acetyl- salicylic 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 saturate d 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 excipients

acid

anhydride,

KEYWORDS normal phase; tablets; SPE

acetylsalicylsalicylic

acid,

salicylic acid,

REFERENCE

Galante, R.N.; Visalli, A.J.; Grim, W.M. Stabilized normal-phase high-performance liquid chromato-

graphic analysis of aspirin and salicylic acid in solid pharmaceutical dosage forms. 1984, 73, 195-197

J.Pharm.Sci.,

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-ace- tylascorbic 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 as-

corbic acid by high-performance liquid chromatography. J.Pharm.ScL,

SAMPLE

Matrix: solutions

1984, 73, 1830-1833

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, toluene

diphenhydramine,

o-hydroxyhippuric

acid,

MPPH,

niacin,

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, Charlottes- ville, 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 KH 2 PO 4 and 4.8 g 85% phos- phoric 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, di- azepam, doxylamine, ethosuximide, furosemide, haloperidol, hydrochlorothiazide, meth- ocarbamol, methotrimeprazine, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylamide, temazepam, tetracaine, thiopental, triamterene, verapamil, zol- pidem, 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 trie- thylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL trie- thylamine 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, aprobar- bital, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chlo- ramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cin- chonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dex- tromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxy- late, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fen- proporex, fentanyl, flubendazole, fiufenamic acid, flunitrazepam, 5-fluorouracil, fluoxy- mesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glu- tethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, im- inostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproter- enol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lido- caine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, me- phentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesori- dazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldo- pamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, meto- prolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, na-

proxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, ox- ymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentaz- ocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phency- clidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, pi- perocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, qua- zepam, 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, the- obromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thiori- dazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolme- tin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, ver- apamil, 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 mo- lecular forms of the non-steroidal anti-inflammatory agents in reversed-phase high-performance liq- uid 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, tol- metin

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, 107—134

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, dipy- rone (sulpyrin), ethenzamide (o-ethoxybenzamide), mefenamic acid, phenacetin, salicy- lamide, salicylic acid, theobromine, theophylline

KEYWORDS

semi-micro; porous polymer

REFERENCE

Matsushima, Y.; Nagata, Y; Niyomura, M.; Takakusagi, K.; Takai, N. Analysis of antipyretics by sem- imicro 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

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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

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of aspirin products for analysis by high-performance liquid chromatography. Analyst,

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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, phe- nacetin, 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 fluores- cence detectors. J.Pharm.Sci., 1980, 69, 1188-1191