HPLC VARIABLES

Guard column: 10 X 3.2 5 |xm Partisil 0DS3

Column: 100 X 4.6 5 jxm Partisil 0DS3
Mobile phase: MeCN: buffer 25:75 (Buffer was 60 mM KH2PO4 adjusted to pH 3.0 with
phosphoric acid.)
Flow rate: 0.6-1
Injection volume: 10-100
Detector: UV 270
OTHER SUBSTANCES

Also analyzed: oxprenolol

REFERENCE
Palm, K.; Luthman, K.; Ungell, A.-L.; Strandlund, G.; Artursson, P. Correlation of drug absorption with
molecular surface properties. J.Pharm.Sci., 1996, 85, 32-39

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Chirex 3022 (Phenomenex)

Mobile phase: Hexane: 1,2-dichloroethane: EtOH/trifluoroacetic acid 60:35:5 (EtOH/
trifluoroacetic acid was premixed 20:1.)
Flow rate: 0.7-1
Injection volume: 20
Detector: UV 276
KEYWORDS

chiral; a = 1.08
REFERENCE
Cleveland, T. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical racemates. J.Liq.Chromatogr., 1995, 18, 649-671

SAMPLE

Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 1 mg/mL solution.
HPLCVARIABLES

Column: 250 X 4.6 10 ^m Chiralcel OD

Mobile phase: Hexane: isopropanol: diethylamine 80:20:0.1
Flow rate: 0.5
Injection volume: 20
Detector: UV 275
CHROMATOGRAM

Retention time: k' 0.69, 2.02 (enantiomers)

KEYWORDS

chiral
REFERENCE
Ekelund, J.; van Arkens, A.; Bronnum-Hansen, K.; Fich, K.; Olsen, L.; Peter sen, RV. Chiral separations
of (3-blocking drug substances using chiral stationary phases. J.Chromatogr.A, 1995, 708, 253-261

SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 200 |JLM solution in MeOH.
HPLCVARIABLES
Column: 100 X 4.7 7 Lim Hypercarb (Shandon)
Mobile phase: MeOH containing 5 mM N-benzyloxycarbonylglycyl-L-proline and 4.5 mM
NaOH
Column temperature: 17
Injection volume: 20
Detector: UV 270
CHROMATOGRAM
Retention time: k' 18 (first enantiomer)
KEYWORDS
chiral; a = 1.09
REFERENCE
Huynh, N.-H.; Karlsson, A.; Pettersson, C. Enantiomeric separation of basic drugs using N-benzyloxycarbonylglyclyl-L-proline as counter ion in methanol. J.Chromatogr.A, 1995, 705, 275-287

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine chemically bonded to silica (Regis)
Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: k' 2.72
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, antazoline, atenolol, betaxolol, bisoprolol, bopindolol,
bupranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cinnarizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, esmolol, famotidine, isothipendyl, ketotifen, metiamide, moxonidine, nadolol, naphazoline, nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol,
pizotyline (pizotifen), practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine,
triprolidine, tymazoline, UK-14,304
REFERENCE
Kaliszan, R.; Nasal, A.; Turowski, M. Binding site for basic drugs on a r acid glycoprotein as revealed by
chemometric analysis of biochromatographic data. Biomed.Chromatogr., 1995, 9, 211215

SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 fjim Supelcosil LC-DP (A) or 250 X 4 5 p,m LiChrospher 100 RP-8 (B)

Mobile phase: MeCN: 0.025% phosphoric acid .buffer 25:10:5 (A) or 60:25:15 (B) (Buffer
was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust
pH to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Injection volume: 25
Detector: UV 229
CHROMATOGRAM
Retention time: 7.12 (A), 4.19 (B)
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam,
bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine,
chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine,
chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal,
diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine,
fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,
labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol,
mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol,
metformin, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine,
methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline,
naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam,
prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine,
promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline,
thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide,
tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,
zopiclone
KEY WORDS
some details of plasma extraction
REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.

J.ChromatogrA, 1995, 692, 103-119

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 cellulose 3,5-dimethylphenylcarbamate/10-undecenoate bonded to
allylsilica

Mobile phase: Heptane: isopropanol: diethylamine 80:20:0.1

Flow rate: 1
Injection volume: 1000
Detector: UV 254
CHROMATOGRAM
Retention time: k' 1.61
KEYWORDS
chiral; a = 1.24
REFERENCE
Oliveros, L.; Lopez, P.; Minguillon, C; Franco, P. Chiral chromatographic discrimination ability of a
cellulose 3,5-dimethylphenylcarbamate/10-undecenoate mixed derivative fixed on several chromatographic matrices. J.Liq.Chromatogr., 1995, 18, 1521-1532

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 300 X 3.9 5 |xm Nova-Pak C18
Mobile phase: MeOH: buffer 30:70 (Buffer was pH 4.0 phosphate buffer (ionic strength =
0.1) containing 2.86 mM N, N-dimethyloctylamine, pH readjusted to 4.00 with 85% phosphoric acid.)
Column temperature: 30
Flow rate: 1
Injection volume: 100
Detector: UV 220
CHROMATOGRAM
Retention time: k; 2.81
OTHER SUBSTANCES
Also analyzed: acebutolol, bunitrolol, carazolol, celiprolol, esmolol, mepindolol, timolol
REFERENCE
Hamoir, T.; Verlinden, Y.; Massart, D.L. Reversed-phase liquid chromatography of (3-adrenergic blocking
drugs in the presence of a tailing suppressor. J.Chromatogr.Sci., 1994, 32, 14-20

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, aspirin, 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, flufenamic 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, Iidocaine, 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, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, 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, triprolidine, tropacocaine, tyramine, verapamil, vincamine,
warfarin, yohimbine, zoxazolamine
REFERENCE
Hill, D.W.; Kind, A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.ToxicoL,
1994, 18, 233-242

SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 10 |xm Chiralpak OD (Daicel)

Mobile phase: Carbon dioxide: MeOH: diethylamine 69.5:30:0.5

Column temperature: 30
Flow rate: 2
Detector: UV 223
CHROMATOGRAM
Retention time: 2, 3.1 (enantiomers)
KEYWORDS
SFC; chiral; pressure 200 bar
REFERENCE
Kot, A.; Sandra, P.; Venema, A. Sub- and supercritical fluid chromatography on packed columns: A
versatile tool for the enantioselective separation of basic and acidic drugs. J.Chromatogr.Sci., 1994,
32, 439-448

SAMPLE
Matrix: solutions
Sample preparation: Prepare a 25 |Jig/mL solution in MeCN: water 40:60, inject an
aliquot.
HPLCVARIABLES
Column: 250 X 4.6 3 |xm silica (Phenomenex)
Mobile phase: MeCN: 6.25 mM pH 3.0 phosphate buffer 40:60
Flow rate: 1
Injection volume: 50
Detector: UV 254
CHROMATOGRAM
Retention time: 7.53
OTHER SUBSTANCES
Also analyzed: atenolol, clonidine, diltiazem, nifedipine, prazosin, propranolol, verapamil
REFERENCE
Simmons, B.R.; Stewart, J.T. HPLC separation of selected cardiovascular agents on underivatized silica
using an aqueous organic mobile phase. J.Liq.Chromatogr., 1994, 17, 2675-2690

SAMPLE
Matrix: solutions
Sample preparation: Filter (0.22 jxm), inject a 10 \xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 internal surface reversed-phase silica (Pinkerton) (Regis Chemical)
Mobile phase: Isopropanol: 100 mM pH 6.8 KH2PO4 10:90
Flow rate: 1
Injection volume: 10
Detector: UV 232-274 (wavelength of maximum absorption used)
CHROMATOGRAM
Retention time: 26.0
OTHER SUBSTANCES
Simultaneous: acebutolol, alprenolol, atenolol, carteolol, oxprenolol, pindolol

REFERENCE
Ohshima, T.; Takagi, K.; Miyamoto, K.-I. High performance liquid chromatographic retention time of (Bblockers as an index of pharmacological activity. J.Liq.Chromatogr., 1993, 16, 3933-3939

SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4.9 Spherisorb S5W silica
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM
NaOH in MeOH, pH 6.7
Flow rate: 2
Injection volume: 20
Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM
Retention time: 2.1
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethane, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine,
benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol,
brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine,
butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine,
clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine,
deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine,
ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine,
ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol,
hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine,
methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine,
methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide,
metopimazine, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol,
oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate,
phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine,
phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen,
procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,
protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine,
theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene,
thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline

REFERENCE
Jane, L; McKinnon, A.; Flanagan, R. J. High-performance liquid chromatographic analysis of basic drugs
on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection. J.Chromatogr., 1985, 323, 191-225

SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 30 |xL 1 mg/mL ()-toliprolol in MeOH + 1 mL 2 M
potassium carbonate + I g NaCl, extract twice with 5 mL ethyl acetate. Remove the
organic layers and dry them over anhydrous sodium sulfate, evaporate to dryness under
a stream of nitrogen at 50, reconstitute the residue in 100 |xL 0.4% triethylamine in
MeCN:MeOH 50:50, add 100 |JLL 1% S-(-)-menthyl chloroformate in MeCN (prepare
weekly in MeCN dried over anhydrous sodium sulfate), let stand at room temperature
for 1 h, evaporate to dryness under a stream of nitrogen, reconstitute with 300 \xL MeOH,
inject a 10 |JLL aliquot.
HPLCVARIABLES
Guard column: 20 X 4 30 |xm Hypersil HP ODS
Column: 250 X 4.6 Hypersil 5 C18
Mobile phase: Gradient. A was 13.8 g NaH2PO4-H2O and 1.59 g propylamine hydrochloride
in 1 L water, pH adjusted to 3.2 with concentrated phosphoric acid. B was MeOH. A:B
from 25:75 to 15:85 over 15 min, maintain at 15:85 for 5 min, to 10:90 over 5 min,
maintain at 10:90 for 3 min.
Flow rate: 1
Injection volume: 10
Detector: F ex 223 em 340 (cut-off filter)
CHROMATOGRAM
Retention time: 19.7 (-), 20.6 (+)
Internal standard: toliprolol (23.3 (-), 24.5 (+))
Limit of detection: 5 ng
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
chiral; derivatization; pharmacokinetics
REFERENCE
Li, F.; Cooper, S.R; Cote, M. Determination of the enantiomers of metoprolol and its major acidic metabolite in human urine by high-performance liquid chromatography with fluorescence detection.
J.Chromatogr.B, 1995, 668, 67-75

SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 500 jxL 5 M NaOH + I g anhydrous sodium sulfate
+ 2 mL diethyl ether, shake mechanically for 15 min, centrifuge at 734 g for 5 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 60,
reconstitute the residue in 10 mL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: ixBondapak C18
Column: 300 X 3.9 10 |xm (xBondapak C18
Mobile phase: MeCN: water 40:60 containing 5 mM KH2PO4ZK2HPO4 adjusted to pH 6.5
with phosphoric acid or 1 M KOH
Column temperature: 30 0.2

Flow rate: 1.3

Injection volume: 20
Detector: E, EG&G Princeton Applied Research PAE Model 400, glassy carbon cell +1300
mV, d.c. mode, Ag/AgCl reference electrode (At the end of each day clean electrode with
MeOH as mobile phase and potential -600 mV for 1 min and +1500 mV for 10 min, repeat
3 times. If necessary, wipe with a tissue wetted with water then a tissue wetted with
MeOH.)
CHROMATOGRAM
Retention time: 5.42
Limit of quantitation: 400 ppb
OTHER SUBSTANCES
Extracted: alprenolol, nadolol, oxprenolol, timolol
Simultaneous: atenolol
REFERENCE
Maguregui, M.L; Alonso, R.M.; Jimenez, R.M. High-performance liquid chromatography with amperometric detection applied to the screening of (3-blockers in human urine. J.Chromatogr.B, 1995, 674,
85-91

SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 0 mg p-glucuronidase/arylsulfatase (Helix pomatia,
Sigma), heat at 37 overnight, add an equal volume of buffer, centrifuge at 2000 g for 5
min, inject an aliquot of the supernatant onto column A with mobile phase A and elute
to waste. After 2.5 min backflush the contents of column A onto column B with mobile
phase B, monitor the effluent from column B. For gradient elution, after 15 min re-equilibrate both columns for 12.5 min before the next injection. For isocratic elution, remove
column A from the circuit after 1.25 min, re-equilibrate column A for 1.5 min. (Buffer was
200 mM boric acid adjusted to pH 9.5 with 5 M NaOH.)
HPLCVARIABLES
Column: A 10 X 4.6 5 |xm Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120
(Shiseido)
Mobile phase: A water; B Gradient. MeCN: buffer from 3:97 to 30:70 over 30 min, to 40:
60 over 8 min (for screening) or isocratic 23:77 (Buffer was 3.4 mL/L phosphoric acid
adjusted to pH 3.0 with 5 M NaOH.)
Flow rate: A 1.25; B 1
Injection volume: 100
Detector: UV 220
CHROMATOGRAM
Retention time: 12 (gradient), 6 (isocratic)
Limit of detection: 250 ng/mL
OTHER SUBSTANCES
Extracted: metabolites (gradient), acebutolol (gradient), alprenolol (gradient), amphetamine (gradient), atenolol (gradient), bopindolol (gradient), codeine (gradient), ephedrine
(gradient), labetalol (gradient), morphine (gradient), nadolol (gradient), oxprenolol (gradient), pindolol (gradient), propranolol (gradient), timolol (gradient)
KEYWORDS
column-switching

REFERENCE
Saarinen, M.T.; Siren, H.; Riekkola, M.-L. Screening and determination of (3-blockers, narcotic analgesics
and stimulants in urine by high-performance liquid chromatography with column switching.
J.Chromatogr.B, 1995, 664, 341-346

ANNOTATED BIBLIOGRAPHY
Ghosh, T.K.; Adir, J.; Xiang, S.-L.; Onyilofur, S. Transdermal delivery of metoprolol II: In-vitro skin
permeation and bioavailability in hairless rats. J.Pharm.Sci., 1995, 84, 158-160 [rat; plasma; pindolol (IS); fluorescence detection; LOD 5 ng/mL; pharmacokinetics]
Welch, CJ.; Perrin, S.R. Improved chiral stationary phase for p-blocker enantioseparations.
J.Chromatogr. A, 1995, 690, 218-225 [chiral; also bufuralol]
Xie, H.G.; Zhou, H.H. Assay of metoprolol and alpha-hydroxymetoprolol in human urine by reversedphase liquid chromatography with direct-injection. Chung Kuo Yao Li Hsueh Pao, 1995, 16, 32-35
Bailey, C.J.; Ruane, R.J.; Wilson, LD. Packed-column supercritical fluid chromatography of (3-blockers.
J.Chromatogr.Sci., 1994, 23, 426-429 [SFC; simultaneous alprenolol, atenolol, labetalol, metoprolol,
oxprenolol, pindolol, practolol, propranolol, toliprolol, xamoterol]
Hermansson, J.; Grahn, A. Resolution of racemic drugs on a new chiral column based on silica-immobilized cellobiohydrolase. Characterization of the basic properties of the column. J.Chromatogr., 1994,
687, 45-59 [chiral; also acebutolol, atenolol, betaxolol, bisoprolol, carbuterol, cathinone, cimetidine,
dobutamine, dopropizine, epanolol, epinephrine, laudanosine, metanephrine, moprolol, norepinephrine, normetanephrine, octopamine, oxybutynine, pamatolol, practolol, prilocaine, propafenone, proxyphylline, sotalol, talinolol, tetrahydropapaveroline, tetramisole, timolol, tolamolol, toliprolol]
Armstrong, D.W.; Chen, S.; Chang, C; Chang, S. A new approach for the direct resolution of racemic
beta adrenergic blocking agents by HPLC. J.Liq.Chromatogr., 1992, 15, 545-556 [chiral; also alprenolol, atenolol, cateolol, labetolol, nadolol, oxprenolol, pindolol, propranolol, timolol]
Leloux, M.S. Rapid chiral separation of metoprolol in plasma-application to the pharmacokinetics/pharmacodynamics of metoprolol enantiomers in the conscious goat. Biomed.Chromatogr., 1992,
6, 99-105
Balmer, K.; Persson, A.; Lagerstrom, P.-O.; Persson, B.-A.; Schill, G. Liquid chromatographic separation
of the enantiomers of metoprolol and its alpha-hydroxy metabolite on Chiralcel OD for determination
in plasma and urine. J.Chromatogr., 1991, 553, 391-397 [plasma; urine; human; dog; extracted
metabolites; fluorescence detection; chiral; column temp 35; column temp 25; LOD 10 nM]
Shen, J.; Wanwimolruk, S.; Hung, C.T.; Zoest, A.R. Quantitative analysis of (3-bloekers in human plasma
by reversed-phase ion-pair high-performance liquid chromatography using a microbore column.
J.Liq.Chromatogr., 1991, 14, 777-793 [plasma; microbore; also atenolol, labetalol, pindolol, propranolol; oxprenolol (IS); fluorescence detection; UV detection; LOD 1-10 ng/mL]
Persson, B.A.; Balmer, K.; Lagerstrom, P.O.; Schill, G. Enantioselective determination of metoprolol in
plasma by liquid chromatography on a silica-bonded alpha 1-acid glycoprotein column.
J.Chromatogr., 1990, 500, 629-636
Bui, K.H.; French, S.B. Direct serum injection and analysis of drugs with aqueous mobile phases containing triethylammonium acetate. J.Liq.Chromatogr., 1989, 12, 861-873 [direct injection; serum;
plasma; dog; rat; fluorescence detection; UV detection; also antipyrine, atenolol, hexanophenone,
naproxen, propranolol]
Rutledge, D.R.; Garrick, C. Rapid high-performance liquid chromatographic method for the measurement of the enantiomers of metoprolol in serum using a chiral stationary phase. J.Chromatogr., 1989,
497, 181-190
Schuster, D.; Modi, M.W; Lalka, D.; Gengo, FM. Reversed-phase high-performance liquid chromatographic assay to quantitate diastereomeric derivatives of metoprolol enantiomers in plasma.
J.Chromatogr., 1988, 433, 318-325
Buhring, K.U.; Garbe, A. Determination of the new beta-blocker bisoprolol and of metoprolol, atenolol
and propranolol in plasma and urine by high-performance liquid chromatography. J.Chromatogr.,
1986, 382, 215-224
Schill, G.; Wainer, LW; Barkan, S.A. Chiral separation of cationic drugs on an al-acid glycoprotein
bonded stationary phase. J.Liq.Chromatogr., 1986, 9, 641666 [chiral; also atropine, bromdiphen-

hydramine, brompheniramine, bupivacaine, butorphanol, carbinoxamine, chlorpheniramine, clidinium, cocaine, cyclopentolate, dimethindene, diperidone, disopyramide, doxylamine, ephedrine, homatropine, labetalol, labetalol A, labetalol B, mepensolate, mepivacaine, methadone, methorphan,
methylatropine, methylhomatropine, methylphenidate, metoprolol, nadolol, nadolol A, nadolol B, oxprenolol, oxyphencyclimine, phenmetrazine, phenoxybenzamine, promethazine, pronethalol, propoxyphene, propranolol, pseudoephedrine, terbutaline, tocainide, tridihexethyl]
Harrison, P.M.; Tonkin, A.M.; McLean, A.J. Simple and rapid analysis of atenolol and metoprolol in
plasma using solid-phase extraction and high-performance liquid chromatography. J.Chromatogr.,
1985, 339, 429-433 [SPE]
Lennard, M.S. Quantitative analysis of metoprolol and three of its metabolites in urine and liver microsomes by high-performance liquid chromatography. J.Chromatogr., 1985, 342, 199-205
Gengo, F.M.; Ziemniak, M.A.; Kinkel, W.R.; McHugh, W.B. High-performance liquid chromatographic
determination of metoprolol and alpha-hydroxymetoprolol concentrations in human serum, urine,
and cerebrospinal fluid. J.Pharm.ScL, 1984, 73, 961-963
Godbillon, J.; Duval, M. Determination of two metoprolol metabolites in human urine by high-performance liquid chromatography J.Chromatogr., 1984, 309, 198-202
Lecaillon, J.B.; Godbillon, J.; Abadie, R; Gosset, G. Determination of metoprolol and its alpha-hydroxylated metabolite in human plasma by high-performance liquid chromatography. J.Chromatogr.,
1984, 305, 411-417
Johnston, G.D.; Nies, A.S.; Gal, J. Determination of metoprolol in human blood plasma using highperformance liquid chromatography. J.Chromatogr., 1983, 278, 204-208
Lennard, M.S.; Silas, J.H. Rapid determination of metoprolol and alpha-hydroxymetoprolol in human
plasma and urine by high-performance liquid chromatography. J.Chromatogr., 1983, 272, 205-209
Mehta, A.C. High-performance liquid chromatographic determination of oxprenolol hydrochloride and
metoprolol tartrate in tablets and injections. Analyst, 1982, 107, 1379-1382
Pautler, D.B.; Jusko, W.J. Determination of metoprolol and alpha-hydroxymetoprolol in plasma by highperformance liquid chromatography. J.Chromatogr., 1982, 228, 215-222
Rosseel, M.T.; Belpaire, F.M.; Bekaert, L; Bogaert, M.G. High-performance liquid chromatographic determination of metoprolol in plasma. J.Pharm.Sci., 1982, 71, 114115
Winkler, H.; Ried, W.; Lemmer, B. High-performance liquid chromatographic method for the quantitative
analysis of the aryloxypropanolamines propranolol, metoprolol and atenolol in plasma and tissue.
J.Chromatogr., 1982, 228, 223-234
Lefebvre, M.A.; Girault, J.; Fourtillan, J.B. ^-Blocking agents: Determination of biological levels using
high performance liquid chromatography. J.Liq.Chromatogr., 1981, 4, 483-500 [fluorescence detection; plasma; also acebutolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol, sotalol, timolol]