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Metoprolol
Molecular formula: C15H25NO3
Molecular weight: 267.4
CAS Registry No.: 37350-58-6, 56392-17-7 (tartrate), 1 19637-66-0
(fumarate), 98418-47-4 (succinate)

SAMPLE
Matrix: blood
Sample preparation: Condition a Styrosorb cross-linked polystyrene (Biochrom, Moscow)
or Sep-Pak C18 SPE cartridge with two 3 mL portions of MeOH and two 3 mL portions
of water. Add 1 mL serum to the SPE cartridge, wash with two 3 mL portions of water,
elute with 600 |JLL MeOH: diethylamine 99.7:0.3. Evaporate the eluate to dryness under
a stream of air at 40, reconstitute with 50 jxL n-heptane: isopropanol: MeOH 83:13:4,
inject a 20 JULL aliquot onto a 250 X 4.6 10 |xm Silasorb-NH2 (Elsico, Moscow) column and
elute with n-heptane: isopropanol: MeOH 83:13:4 at 2.5 mL/min, collect the eluate containing metoprolol at about 4.1 min, evaporate to dryness, reconstitute with 30 |xL mobile
phase, inject a 10-20 JLJLL aliquot.
HPLCVARIABLES
Guard column: 50 x 4.6 10 |xm Chiralcel OD
Column: 250 X 4.6 10 |xm Chiralcel OD
Mobile phase: n-Heptane: isopropanol: MeOH 77:8:15
Flow rate: 1.3
Injection volume: 10-20
Detector: F ex 220 em 320 (cut-off filter)
CHROMATOGRAM
Retention time: 3.5 (R), 4.6 (S)
KEYWORDS
SPE; serum; silanize glassware; chiral
REFERENCE
Rumiantsev, D.O.; Ivanova, T.V. Solid-phase extraction of Styrosorb cartridges as a sample pretreatment
method in the stereoselective analysis of propranolol in human serum. J.Chromatogr.B, 1995, 674,
301-305

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 jxm 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 223
CHROMATOGRAM

Retention time: 4.20

Limit of detection: <120 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen,
aconitine, albuterol, alimemazine, alminoprofen, alpidem, alprenolol, amisulpride, amitriptyline, amodiaquine, amoxapine, aspirin, astemizole, atenolol, benazepril, benperidol,
benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine,
carbinoxamine, carpipramine, carteolol, cetirizine, chlorambucil, chlordiazepoxide, chlormezanone, ehlorophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, codeine, colchicine, cyamemazine, cyclizine, cyproheptadine,
cytarabine, dacarbazine, daunorubicin, demexiptiline, desipramine, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, diphenhydramine, dipyridamole, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam,
etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fiuoxetine, fluphenazine, flurbiprofen, fluvoxamine, glibenclamide, glibornuride,
glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychloroquine, hydroxyzine,
ibuprofen, imipramine, indomethacin, iproniazid, ketoprofen, labetalol, levomepromazine,
lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, loxapine, maprotiline,
medazepam, medifoxamine, mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, mepivacaine, metapramine, metformin, methadone, methocarbamol, methotrexate, metipranolol, metoclopramide, mexiletine, mianserine, midazolam,
moclobemide, moperone, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrexone, naproxen, nialamide, nicardipine, niflumic acid, nimodipine, nitrendipine, nizatidine,
nomifensine, nortriptyline, omeprazole, opipramol, oxprenolol, penbutolol, penfluridol,
phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone,
prazepam, prazosin, procainamide, procarbazine, proguanil, promethazine, propafenone,
propranolol, protriptyline, pyrimethamine, quinupramine, ramipril, ranitidine, reserpine,
ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulpride, sultopride, suriclone,
temazepam, tenoxicam, terbutaline, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine, tiapride, ticlopidine, timolol, tioclomarol, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin,
zolpidem, zorubicine
Interfering: ajmaline, alprazolam, celiprolol, clobazam, cycloguanil, debrisoquine, dextromethorphan, disopyramide, ketamine, lorazepam, mephentermine, methaqualone, metoprolol, minoxidil, nifedipine, nitrazepam, oxazepam, pentazocine, piroxicam, prilocaine,
quinidine, quinine, sulindac, tiaprofenic acid, tofisopam, yohimbine, zopiclone
KEYWORDS
whole blood; plasma
REFERENCE
Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic Sd.,
1995, 40, 254-262

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 5 |xg/mL R-propranolol in water + 50 \xL 100
mM NaOH + 4 mL chloroform, vortex for 30 s, centrifuge at 1800 g for 5 min. Remove

organic layer and evaporate it to dryness in vacuum, reconstitute in 200 |xL 0.05% S-(+)l-(l-naphthyl)ethyl isocyanate in chloroform, vortex for 30 s, inject a 75-150 JULL aliquot.
HPLCVARIABLES
Column: 250 mm long Whatman 5 |jim silica
Mobile phase: Hexane: chloroform: MeOH 85:14:1
Flow rate: 2
Injection volume: 75-150
Detector: F ex 220 no emission filter
CHROMATOGRAM
Retention time: 14.1 (R-(+)), 16.2 (S-(-))
Internal standard: R-propranolol (8.5)
Limit of quantitation: 5 ng/mL
KEYWORDS
plasma; normal phase; derivatization; chiral
REFERENCE
Bhatti, M.M.; Foster, R.T. Stereospecific high-performance liquid ehromatographic assay of metoprolol.
J.Chromatogr., 1992, 579, 361-365

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 JJLL 2 M NaOH + 4 mL dichloromethane, vortex
for 10 s, centrifuge at 3000 rpm for 10 min. Remove the organic layer and evaporate it
to dryness under a stream of nitrogen, reconstitute the residue in 100 JULL mobile phase,
inject an 80 jxL aliquot.
HPLCVARIABLES
Guard column: NewGuard C18 (Brownlee)
Column: 250 X 4.6 5 |xm Dynamax Microsorb C18
Mobile phase: MeCN: 0.1% triethylamine in water adjusted to pH 3.5 with 85% phosphoric
acid 20:80
Flow rate: 1
Injection volume: 80
Detector: F ex 215
CHROMATOGRAM
Retention time: 11.65
Internal standard: metoprolol
OTHER SUBSTANCES
Simultaneous: pindolol
KEYWORDS
serum; metoprolol is IS
REFERENCE
Chmielowiec, D.; Schuster, D.; Gengo, F. Determination of pindolol in human serum by HPLC.
J.Chromatogr.Sci., 1991, 29, 37-39

SAMPLE
Matrix: blood

Sample preparation: Condition an Analytichem 3 mL 200 mg SPE cartridge with 3 mL

MeOH and 3 mL water. 1 mL Plasma + 30 |xL 0.5 |xg/mL d-propranolol in MeOH, vortex
for 15 s, add to the SPE cartridge, wash with 3 mL water, wash with 1 mL MeOH: water
50:50, elute with two aliquots of 500 |JLL MeOH containing 0.1% triethylamine. Evaporate
the eluate to dryness under nitrogen at 30, reconstitute with 150 (xL mobile phase, vortex
for 30 s, inject a 100 JJLL aliquot.

HPLCVARIABLES
Guard column: Chiracel OD (Daicel Chemical Industries)
Column: 250 X 4.6 Chiracel OD (Daicel Chemical Industries)
Mobile phase: Hexane: EtOH: N,N-diethylamine 95:5:0.1
Flow rate: 0.5
Injection volume: 100
Detector: F ex 220 em 320
CHROMATOGRAM
Retention time: 19 (d), 22 (1)
Internal standard: d-propranolol (34)
Limit of detection: 4 ng/mL
KEYWORDS
plasma; SPE; chiral
REFERENCE
Herring, V.L.; Bastian, T.L.; Lalonde, R.L. Solid-phase extraction and direct high-performance liquid
chromatographic determination of metoprolol enantiomers in plasma. J.Chromatogr., 1991, 567,
221-227

SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with 2 mL MeCN and 1
mL MeCN: water 40:60. 995 |JLL Serum + 5 JULL 20 fxM oxprenolol in water, add 20 |JLL of
this serum to 500 [xL MeCN: water 40:60, vortex, add to SPE cartridge, wash with 2 mL
MeCN: water 40:60, elute with 1 mL MeOH: water 1:1 containing 0.05% trifluoroacetic
acid. Evaporate eluate to dryness, reconstitute in MeCN.water 20:80 containing 0.05%
trifluoroacetic acid, inject.
HPLCVARIABLES
Column: 150 X 4.6 5 [xm TSK gel ODS 80Tm (Tosoh)
Mobile phase: MeCN: water 31:69 containing 0.05% trifluoroacetic acid
Column temperature: 40
Injection volume: 20
Detector: F ex 230 em 300
CHROMATOGRAM
Internal standard: oxprenolol
KEYWORDS
serum; SPE
REFERENCE
Uzu, S.; Imai, K.; Nakashima, K.; Akiyama, S. Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,l,3benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the (3-blocker metoprolol in serum by high-performance liquid chromatography. Analyst, 1991, 116, 1353-1357

SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with 2 mL MeCN and 1
mL MeCN.water 40:60. Add 20 |ULL serum to 500 JJLL MeCN.water 40:60, vortex, add to
SPEf cartridge, wash with 2 mL MeCN: water 40:60, elute with 1 mL MeOH: water 1:1
containing 0.05% trifluoroacetic acid. Evaporate eluate to dryness under reduced pressure
at 40, reconstitute with 50 |xL 100 mM pH 9.0 borate buffer containing 2 mM EDTA,
add 50 [xL 50 mM DBD-F in MeCN, heat at 45for 8 h, add 100 |JLL 100 mM acetic acid
in MeCN: water 50:50, inject a 20 JJLL aliquot. (Synthesis of DBD-F is as follows. Dissolve
0.5 g magnesium sulfate heptahydrate and 6 g NaOH in 60 mL water, throughout the
reaction keep the flask at about 20with cold water cooling, add 15 mL 30% hydrogen
peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl peroxide in one go, stir for 10
min, pour into 150 mL 20% sulfuric acid, extract three times with 50 mL portions of
chloroform, determine peroxybenzoic acid concentration by iodometric titration (Tetrahedron 1967, 23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in chloroform to 7 g
2,6-difluoroaniline dissolved in 100 mL chloroform, stir at room temperature, when reaction is complete (iodometric titration) wash with 2% sodium thiosulfate, wash with 5%
sodium carbonate, wash with water, dry over anhydrous sodium sulfate, evaporate to
dryness under reduced pressure, recrystallize 2,6-difluoronitrosobenzene from EtOH (mp
108.5-109.5). Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO at room temperature
and add a solution of 3.91 g sodium azide in 85 mL DMSO dropwise, let stand for about
1 h, add to a large volume of water, extract with ether, dry the extracts over anhydrous
sodium sulfate, evaporate to dryness under reduced pressure and distil to give 4-fluoro2,1,3-benzoxadiazole as a colorless oil (bp 83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433).
Add 11 mL chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL
chloroform at 0-10 (use a calcium chloride drying tube), stir at room temperature for 1
h, reflux for 2 h, cool, slowly pour into ice water, remove the organic layer, extract the
aqueous layer with chloroform, combine the organic layer, wash, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, take up the residue in 5 mL benzene
(Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of silica gel
(100-200 mesh Kanto Chemical) with n-hexane: benzene 50:50, evaporate the appropriate
fractions to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow
needles (mp 64-66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at
0-10and add 1 g dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise,
adjust pH to 5 with 1 M HCl, concentrate to about 10 mL under reduced pressure, extract
three times with 200 mL portions of diethyl ether, wash with water, dry over anhydrous
magnesium sulfate, evaporate under reduced pressure, chromatograph on a 500 X 20
column of silica gel with chloroform, isolate the appropriate fraction and re-chromatograph on the same column with ethyl acetate: benzene 1:2 to give 4-(N, N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as white needles (mp 124-125) (yield
= 1% !). On a Merck no. 5714 60F254 tic plate eluted with chloroform DBD-F has Rf 0.32
and lies between two other reaction products (Analyst 1989, 114, 413). It is also reported
that DBD-F can be purchased from Tokyo Kasei.)
HPLCVARIABLES
Column: 250 X 4.6 5 \xm TSK gel ODS 80Tm (Tosoh)
Mobile phase: MeCN: THF: 50 mM pH 6.0 imidazole nitrate buffer 28:20:52

Column temperature: 40
Flow rate: 0.8
Injection volume: 20
Detector: Chemiluminescence following post-column reaction. The column effluent mixed
with the reagent pumped at 1.4 mL/min and the mixture flowed to the detector. (Reagent
was 0.25 mM bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO, Wako,
Osaka) and 37.5 mM hydrogen peroxide in MeCN: ethyl acetate 50:50.)
CHROMATOGRAM

Retention time: 9
Limit of detection: 0.8 ng/mL

KEYWORDS
SPE; derivatization; post-column reaction; serum
REFERENCE
Uzu, S.; Imai, K.; Nakashima, K.; Akiyama, S. Use of 4-(N,iV-dimethylaminosulphonyl)-7-fluoro-2,l,3benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the {3-blocker metoprolol in serum by high-performance liquid chromatography Analyst, 1991, 116, 1353-1357

SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 50 |xL 1 fxg/mL verapamil in MeOH + 500 JJLL 0.5
M HCl + 4 mL diethyl ether, vortex for 30 s, centrifuge at 2000 g for 5 min, discard
organic layer, add 100 jxL 2 M NaOH and 4 mL diethyl ether to the aqueous layer, vortex
for 1 min, centrifuge at 2000 g for 10 min. Remove the organic layer and dry it at 35
under a stream of helium. Reconstitute in 150 |xL mobile phase, inject a 25 JULL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Chiralcel OD (Diacel Chemical Industries)
Mobile phase: Hexane: isopropanol 90:10 which contained 10 mM octylamine
Flow rate: 1
Injection volume: 25
Detector: F ex 275 em 315
CHROMATOGRAM

Retention time: 6.13 (R), 14.34 (S)

Internal standard: verapamil (9.80)
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Simultaneous: procainamide
Noninterfering: diazepam, digoxin, furosemide, lidocaine, prochlorperazine, quinidine
KEYWORDS
serum; chiral
REFERENCE
Straka, R.J.; Johnson, K.A.; Marshall, RS.; Remmel, R.P. Analysis of metoprolol enantiomers in human
serum by liquid chromatography on a cellulose-based chiral stationary phase. J.Chromatogr., 1990,
530, 83-93

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M pH 9.9 carbonate buffer + 6 mL watersaturated diethyl ether, agitate for 15 min, centrifuge at 4200 g for 5 min. Remove 5 mL
of the organic layer and evaporate it to dryness under a stream of nitrogen at 35, reconstitute the residue in 250 (xL 142.4 mM triethylamine in dichloromethane, add 100 JULL
reagent, let stand for 15 min, evaporate to dryness under a stream of nitrogen at 35, add
2 mL 100 mM NaOH, agitate for 10 min, add 6 mL ether, extract for 15 min, centrifuge.
Remove 5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen
at 35, cool in an ice bath, reconstitute the residue in 250 |xL trifluoroacetic acid, let stand
at 0 for 10 min, add 2 mL 2 M NaOH, extract with 6 mL ether. Remove a 5 mL aliquot
of the organic layer and extract it with 100 p,L 100 mM phosphoric acid, inject a 94 JJLL
aliquot of the aqueous layer. (Purify triethylamine by drying it over NaOH pellets overnight, filter, add a volume of naphthylisocyanate equal to 2% of the volume of triethylamine, distill. Prepare reagent by dissolving 1 mmole N-tert-butoxycarbonyl-L-leucine (BOC-