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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 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 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 col-
umn 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 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 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 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,
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, 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
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, 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 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, 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
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 salicyl-
salicylic acid, aspirin, and salicylic acid in human plasma and urine. J.Pharm.Sci., 1980, 69, 1268-
1271

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 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 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 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 chromato-
graphic 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-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, 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, 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 KH2PO4 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
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, ace-
tylmorphine, benzocaine, caffeine, chloroquine, diamorphine, diazepam, diphenhydramine, dipyrone,
lidocaine, methaqualone, monoacetylmorphine, morphine, nicotinamide, noscapine, papaverine, phe-
nacetin, 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 ther-
apeutic drugs in serum. J.Liq.Chromatogr., 1989, 12, 1851-1872 [serum; also acetaminophen, amio-
darone, caffeine, chloramphenicol, flecainide, pentobarbital, procainamide, pyrimethamine, quini-
dine, 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 detec-
tion; electrochemical detection; simultaneous acetaminophen, acetylcodeine, acetylmorphine, acetyl-
procaine, 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 metab-
olites, 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, caf-
feine 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, 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
Astemizole
Molecular formula: C28H31FN4O
Molecular weight: 458.6
CAS Registry No.: 68844-77-9

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 |xL 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 |xm 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 278

CHROMATOGRAM
Retention time: 5.73
Limit of detection: <120 ng/mL

OTHER SUBSTANCES
Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen,
aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alpren-
olol, amisulpride, amitriptyline, amodiaquine, amoxapine, aspirin, atenolol, benazepril,
benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bromazepam, bromphenira-
mine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, car-
binoxamine, carpipramine, carteolol, celiprolol, chlorambucil, chlordiazepoxide, chlorme-
zanone, chlorophenacinone, chloroquine, chlorpromazine, chlorpropamide, cicletanine,
clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, clozapine, co-
caine, codeine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytara-
bine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextrome-
thorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac,
dihydralazine, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, dox-
ylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac,
flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen,
fluvoxamine, glibenclamide, glipizide, glutethimide, haloperidol, histapyrrodine, hydrox-
ychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine,
ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, lor-
atadine, lorazepam, loxapine, maprotiline, medazepam, mefenamic acid, mefenidramine,
mefloquine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapra-
mine, metformin, methadone, methaqualone, methocarbamol, methotrexate, metiprano-
lol, metoclopramide, metoprolol, mexiletine, mianserine, midazolam, minoxidil, moclo-
bemide, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrexone, naproxen,
nialamide, nifedipine, niflumic acid, nimodipine, nitrazepam, nitrendipine, nizatidine,
nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, pen-
 fluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide,
pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procar-
bazine, proguanil, promethazine, propafenone, propranolol, protriptyline, pyrimethamine,
quinidine, quinine, quinupramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital,
sotalol, strychnine, sulfinpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam,
tenoxicam, terbutaline, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine,
thioridazine, tianeptine, tiapride, tiaprofenic acid, ticlopidine, timolol, tioclomarol, tofi-
sopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, tri-
mipramine, triprolidine, tropatenine, verapamil, viloxazine, vincristine, vindesine, war-
farin, yohimbine, zolpidem, zopiclone, zorubicine
Interfering: bisoprolol, cetirizine, chlorpheniramine, cibenzoline, diltiazem, glibornuride,
ioprazolam, medifoxamine, moperone, nicardipine, vinblastine

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, tissue
Sample preparation: Plasma. 2 mL Plasma + 100 jxL 1 jxg/mL IS in MeOH + 2 mL 50
mM borax solution + 4 mL heptane: isoamyl alcohol 95:5, rotate at 10 rpm for 10 min,
centrifuge at 1000 g for 5 min. Remove upper organic layer and repeat extraction of
aqueous layer. Combine extracts, add 3 mL 50 mM sulfuric acid, extract. Remove acidic
aqueous phase and make it alkaline with concentrated ammonia, extract twice with 2 mL
heptane: isoamyl alcohol 95:5, combine organic layers and evaporate them to dryness
under a stream of nitrogen at 55°, take up in 50 |xL MeOH, inject the whole sample.
Tissue. Grind tissue in a Waring blender, homogenize 1:4 in water. 2 mL homogenate +
100 |xL 1 |xg/mL IS in MeOH + 2 mL 50 mM borax solution + 4 mL heptane: isoamyl
alcohol 95:5, rotate at 10 rpm for 10 min, centrifuge at 1000 g for 5 min. Remove upper
organic layer and repeat extraction of aqueous layer. Combine extracts, add 3 mL 50 mM
sulfuric acid, extract. Remove acidic aqueous phase and make it alkaline with concen-
trated ammonia, extract twice with 2 mL heptane: isoamyl alcohol 95:5, combine organic
layers and evaporate them to dryness under a stream of nitrogen at 55°, take up in 50
|xL MeOH, inject the whole sample.

HPLCVARIABLES
Column: 150 X 2.1 5 |xm Alltech RSiL C18HL
Mobile phase: MeCN.water 50:50 + 0.05% diethylamine
Flow rate: 0.6
Injection volume: 50
Detector: UV 254

CHROMATOGRAM
Retention time: 5.8
Internal standard: l-[(4-fluorophenyl)methyl]-N-{l-[2-(4-ethoxyphenyl)ethyl]-4-piperidi-
nyl}-lH-benzimidazol-2-amine (R 44 180) (8.3)
Limit of detection: 1 ng/mL (plasma); 5 ng/g (tissue)

OTHER SUBSTANCES
Extracted: metabolites, desmethylastemizole

KEYWORDS
plasma; human; dog
 Next page

REFERENCE
Woestenborghs, R.; Embrechts, L.; Heykants, J. Simultaneous determination of astemizole and its de-
methylated metabolite in animal plasma and tissues by high-performance liquid chromatography
J.Chromatogr., 1983, 278, 359-366

SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in MeOH.

HPLCVARIABLES
Column: 150 X 4.6 5 p,m MicroPack MCH-5
Mobile phase: MeOH: 30 mM pH 3.0 phosphate buffer 85:15
Flow rate: 1.5
Injection volume: 20
Detector: UV 276

REFERENCE
Fernandez Otero, G.C.; Lucangioli, S.E.; Carducci, CN. Adsorption of drugs in high-performance liquid
chromatography injector loops. J.Chromatogr.A, 1993, 654, 87-91