L-Leu) in 3 mL dichloromethane, add 2 mL 250 mM dicyclohexylcarbodiimide in

dichloromethane, let stand at 0 for 1 h, filter.)

HPLCVARiABLES

Column: 100 X 3.2 10 ^m jxBondapak C18

Mobile phase: MeCN: 100 mM pH 3.0 phosphate buffer 30:70
Flow rate: 0.5
Injection volume: 94
Detector: F ex 193 em (no cutoff filter)
CHROMATOGRAM

Retention time: 5 (S), 6.5 (R)

Limit of detection: 0.5 ng/mL
OTHER SUBSTANCES
Also analyzed: alprenolol
KEYWORDS

pharmacokinetics; plasma; chiral; derivatization

REFERENCE
Hermansson, J.; von Bahr, C. Determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol as
their diastereomeric derivatives in human plasma by reversed-phase liquid chromatography.
J.Chromatogr., 1982,227, 113-127

SAMPLE

Matrix: blood, urine

Sample preparation: Plasma. 1-2 mL Plasma + 100 ul, 4 M NaOH + 100 IJLL water + 5
mL dichloromethane, shake by hand for 10 s, vortex vigorously for 3 min, centrifuge at
1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream
of air at 37, reconstitute the residue in 100 |xL mobile phase, filter (0.45 |xm), inject a
40-60 JULL aliquot. Urine. 0.1-1 mL Urine + 100 JJLL 4 M NaOH + 100 JJIL water + 5 mL
dichloromethane, vortex vigorously for 1 min, centrifuge at 1500 g for 5 min. Remove the
organic layer and add it to 1 mL 100 mM phosphoric acid, vortex for 1 min, centrifuge at
1500 g for 5 min, inject a 40 |xL aliquot of the aqueous phase.
HPLCVARIABLES

Column: 250 X 4 Wakosil 5C18 (Wako)

Mobile phase: MeCN: water: triethylamine 18:81:1, pH adjusted to 3.0 with phosphoric
acid
Flow rate: 1
Injection volume: 40-60
Detector: UV 295
CHROMATOGRAM

Retention time: 8.69

Internal standard: metoprolol tartrate
OTHER SUBSTANCES

Simultaneous: acebutolol, N-acetylprocainamide, alprenolol, atenolol, bufetolol, bupranolol, carteolol, disopyramide, indenolol, lidocaine, nifedipine, pindolol, procainamide, propranolol, quinidine, timolol
Noninterfering: diltiazem, glycinexylidide, mexiletine, nicardipine, tocainide, verapamil
Interfering: nicainoprol
KEYWORDS

plasma; metoprolol is IS

REFERENCE
Kubota, K.; Nakamura, H.; Koyama, E.; Yamada, T.; Kikuchi, K.; Ishizaki, T. Simple and sensitive
determination of timolol in human plasma and urine by high-performance liquid chromatography
with ultraviolet detection. J.Chromatogr., 1990, 533, 255-263

SAMPLE
Matrix: blood, urine
Sample preparation: 500 |xL Plasma or 100-200 |xL urine + 100 (plasma) or 150 (urine)
IxL 10 (plasma) or 600 (urine) |xg/mL pindolol in MeOH + 0.5 m L I M NaOH + 3 mL
dichloromethane, vortex for 1 min, centrifuge at 1500 g for 10 min. Remove the organic
layer and evaporate it to dryness under a stream of air at 35, reconstitute the residue
in 100 (plasma) or 200 (urine) |xL mobile phase, inject a 20-30 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 pirn Zorbax ODS
Mobile phase: MeCN: water: triethylamine 25:74:1, adjusted to pH 4.0 with phosphoric
acid
Flow rate: 0.8
Injection volume: 20-30
Detector: F ex 230 em 300
CHROMATOGRAM
Retention time: 8.6
Internal standard: pindolol (6.0)
Limit of detection: 2 ng/mL (plasma)
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
plasma; pharmacokinetics
REFERENCE
Horai, Y; Ishizaki, T.; Kusaka, M.; Tsujimoto, G.; Hashimoto, K. Simultaneous determination of metoprolol and a-hydroxymetoprolol in human plasma and urine by liquid chromatography with a preliminary observation on metoprolol oxidation in Japanese subjects. Ther.Drug Monit., 1988, 10,
428-433

SAMPLE
Matrix: blood, urine
Sample preparation: Blood. Add MeCN to blood so that the ratio is 1:5. Remove a 1 mL
aliquot and add 1 mL water, adjust pH to 9.8-10.2 with 7-10 drops 100 mM NaOH, add
2 mL benzene (Caution! Benzene is a carcinogen!), shake on an automatic shaker for 30
min, centrifuge, remove the organic phase and extract the aqueous layer again with 2
mL benzene for 20 min. Combine the organic layers and evaporate them under reduced
pressure at 30, dissolve the residue in 50 (xL mobile phase, inject a 10-50 fxL aliquot.
Urine. 20-1000 JJLL Urine + 1 mL 200 mM pH 10.2 sodium borate buffer (Sorensen), adjust
pH to 9.8-10.2 with 100 mM NaOH (if necessary), add 500 mg NaCl, extract with 4 mL
benzene for 30 min, centrifuge. Remove the organic layer and evaporate it under reduced
pressure at 30, dissolve the residue in 50 |xL mobile phase, inject a 10-50 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 jjim jjiBondapak C18

Mobile phase: MeOH: water 48:52 containing 0.4% phosphoric acid and 0.2% heptanesulfonic acid
Flow rate: 2

Injection volume: 10-50

Detector: F ex 225 em 295
CHROMATOGRAM

Retention time: 4.6

Internal standard: metoprolol
OTHER SUBSTANCES
Simultaneous: dihydrolevobunolol
KEYWORDS
human; dog; metoprolol is IS
REFERENCE
Hengy, H.; Kolle, E.-U. Determination of levobunolol and dihydrolevobunolol in blood and urine by highperformance liquid chromatography using fluorescence detection. J.Chromatogr., 1985, 338,444-449

SAMPLE
Matrix: bulk
Sample preparation: Dissolve 10 ixmole compound (as free base or hydrochloride) in 500
[xL MeCN, add 250 |xL 5% sodium carbonate (for hydrochlorides only), add 500 jxL 100
mM reagent in MeCN5 vortex for 1 min, heat at 60for 2 h, add 100 ixmole L-proline, heat
at 60for 30 min. Remove a 100 |xL aliquot and dilute it with mobile phase, neutralize
with acetic acid, inject a 10 |JLL aliquot. (Prepare the reagent ((R, R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL
(lR,2R)-(-)-l,2-diaminoeyclohexane, 12 mL water, and 12 mL EtOH, heat the oil bath to
80, add 2.8 mL carbon disulfide drop wise (making sure that the product does not start
to precipitate), when addition is complete reflux for 1 h, acidify with 500 \xh 5 M HCl,
reflux for 12 h, cool, filter, wash the solid with a little cold EtOH to give trans-4,5-tetramethyleneimidazolidine-2-thione as a white fluffy solid (mp 148-150) (Tetrahedron 1993,
49, 4419). Stir 7.97 g 3,5-dinitrobenzoyl chloride in 30 mL dichloroethane at 50, add a
solution of 6 g trans-4,5-tetramethyleneimidazolidine-2-thione in 120 mL dichloroethane
containing a catalytic amount of 4-(dimethylamino)pyridine over 15 min, reflux for 2 h,
remove the crystals of (R, R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate by
filtration, evaporate the filtrate to dryness and dissolve the residue in 60 mL dichloroethane, reflux for 16 h to obtain more (R, R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate (mp >250, Ia]546 = -133 (c=l) in MeCN).)
HPLC VARIABLES
Column: 125 X 4 5 jxm Lichrospher 60 RP Select B
Mobile phase: MeCN: 20 mM ammonium acetate 55:45
Flow rate: 1
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: k' 5.33, k' 7.83 (enantiomers)
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, atenolol, carazolol, carvedilol, formoterol, methamphetamine, metipranolol, nifenanol, nitrilo atenolol, oxprenolol, pindolol, propranolol,
xamoterol
KEYWORDS
derivatization; chiral

REFERENCE
Kleidernigg, O.P.; Posch, K.; Lindner, W. Synthesis and application of a new isothiocyanate as a chiral
derivatizing agent for the indirect resolution of chiral amino alcohols and amines. J. Chromatogr.A,
1996, 729, 33-42

SAMPLE
Matrix: bulk
Sample preparation: Prepare a 2 mg/mL solution, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 |xm LiCHrospher RP-select B C8
Mobile phase: MeCN: buffer 17:83 (Buffer was 11.5 g ammonium dihydrogen phosphate
and 10 mL 1 M phosphoric acid made up to 2 L, pH 3.2.)
Flow rate: 1
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: impurities
REFERENCE
Erickson, M.; Karlsson, K.-E.; Lamm, B.; Larsson, S.; Svensson, L.A.; Vessman, J. Identification of a
new by-product detected in metoprolol tartrate. J.Pharm.Biomed.Anal., 1995, 13, 567-574

SAMPLE
Matrix: formulations
Sample preparation: Dissolve 2 mg tablet or capsule in 10 mL pH 10 solution, extract
twice with 2 mL ether, combine extracts, filter, inject a 20 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm (3-cyclodextrin bonded C18 (Advanced Separation Technologies)
Mobile phase: MeCN: MeOH: acetic acid: triethylamine 95:5:0.3:0.2
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 12, 13 (enantiomers)
OTHER SUBSTANCES
Simultaneous: atenolol, propranolol
KEYWORDS
capsules; tablets; chiral
REFERENCE
Tran, CD.; Dotlich, M. Enantiomeric separation of beta-blockers by high performance liquid chromatography. J.Chem.Educ, 1995, 72, 71-73

SAMPLE
Matrix: formulations
Sample preparation: Take up in mobile phase, inject an aliquot.

HPLCVARIABLES
Column: 250 X 4.6 10 |xm LiChrosorb C2
Mobile phase: MeCN.buffer 35:65 (1 mL 100 mM HCl + 1200 mL water + 5.84 g NaCl,
mix to dissolve, add 700 mL MeOH, make up to 2 L, apparent pH 4.5.)
Flow rate: 1.2
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 10.5
OTHER SUBSTANCES
Simultaneous: atenolol, nadolol, alprenolol, acebutolol, oxprenolol, pindolol, practolol, propranolol, sotalol, timolol
KEYWORDS
tablets
REFERENCE
Patel, B.R.; Kirschbaum, J. J.; Poet, R.B. High-pressure liquid chromatography of nadolol and other betaadrenergic blocking drugs. J.Pharm.Sci., 1981, 70, 336-338

SAMPLE
Matrix: saliva
Sample preparation: Condition a 100 mg 1 mL Bond-Elut C2 SPE cartridge with 1 mL
MeOH, 1 mL water, and 1 mL pH 9.0 borate buffer. Centrifuge a cotton roll soaked with
saliva at 1000 g for 5 min, remove the liquid supernatant. 1 mL Supernatant + 50 JJLL
10 |xg/mL alprenolol, add to the SPE cartridge, wash with 500 JULL water, wash with 500
|JLL MeCN, elute with two 500 \xL portions of acidified MeOH. Evaporate the eluate to
dryness under a stream of nitrogen at 60, reconstitute the residue in 50 fjuL mobile phase,
mix for 15 s, inject a 40 JJLL aliquot. (Acidified MeOH was 50 mL MeOH + 300 JJLL 96%
acetic acid.)
HPLCVARIABLES
Guard column: RCSS silica guard-pack (Waters)
Column: 250 X 4.6 Chiralcel OD-H
Mobile phase: n-Hexane: EtOH: diethylamine 91:8:1
Flow rate: 1
Injection volume: 40
Detector: F ex 225 em 320 cut-off filter
CHROMATOGRAM
Internal standard: (S)-alprenolol
KEYWORDS
SPE; chiral
REFERENCE
Hold, K.M.; de Boer, D.; Zuidema, J.; Maes, R.A.A. Evaluation of the Salivette as sampling device for
monitoring (3-adrenoceptor blocking drugs in saliva. J.Chromatogr.B, 1995, 663, 103-110

SAMPLE
Matrix: solutions
Sample preparation: Mix 20 |xL of a 1 mM solution in MeOH or water with 50 \xL pH 8
borate buffer and 50 (JLL 18 mM 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate in ace-

tone, vortex, let stand at room temperature for 30 min, add 100 |xL 10 mM trans-4hydroxy-L-proline in water, mix, let stand for 2 min, add 2 mL dichloromethane, vortex
for 30 s. Remove the organic layer and evaporate it to dryness under reduced pressure,
reconstitute the residue in 100 jxL mobile phase, inject an aliquot. (Prepare 2-(6-methoxy2-naphthyl)-l-propyl chloroformate as follows. Stir 1.5 mmoles lithium aluminum hydride
in THF, slowly add 2 mmoles (S)-naproxen in 20 mL anhydrous THF, reflux for 1 h,
evaporate most of the solvent, cautiously add water with stirring, acidify with 6 N HCl,
extract three times with diethyl ether. Combine the organic layers and dry them over
anhydrous sodium sulfate, evaporate to dryness, chromatograph on silica gel with
dichloromethane: MeOH 100:2 (flash chromatography), evaporate eluate to dryness, dry
under vacuum over KOH to give 2-(6-methoxy-2-naphthyl)propanol as a white solid (mp
92-3). Stir 0.5 mmoles 2-(6-methoxy-2-naphthyl)propanol and 0.5 mmoles triethylamine
in 10 mL dry toluene at 0, add 1 mL 20% phosgene in toluene (Caution! Phosgene is
highly toxic, perform reaction in a chemical fume hood!) (Fluka), stir for 4 h, filter, evaporate to dryness under reduced pressure, dry under vacuum to give 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate (mp 60). Store under vacuum over phosphorus pentoxide
at room temperature.)
HPLCVARIABLES
Column: 250 X 4 5 jim Zorbax-SIL
Mobile phase: n-Hexane: isopropanol 100:1.5
Flow rate: 1.5
Injection volume: 100
Detector: UV 230; F ex 270 em 365
CHROMATOGRAM
Retention time: k' 12.6 (S-(-)), k' 13.5 (R-(+))
Limit of detection: 0.2 ng/mL
OTHER SUBSTANCES
Simultaneous: flecainide, propafenone, tocainide
KEYWORDS
derivatization; chiral; normal phase
REFERENCE
Biischges, R.; Linde, H.; Mutschler, E.; Spahn-Langguth, H. Chloroformates and isothiocyanates derived
from 2-arylpropionic acids as chiral reagents: synthetic routes and chromatographic behaviour of
the derivatives. J.Chromatogr.A, 1996, 725, 323-334

SAMPLE
Matrix: solutions
Sample preparation: Mix 300 uX of a 30 |JLM solution in dichloromethane with 10 |xL 20
mM l-(6-methoxy-2-naphthyl)ethyl isothiocyanate in anhydrous dichloromethane and 50
|xL 0.1% triethylamine in dichloromethane, vortex thoroughly, heat at 50 for 1.5 h, inject
an aliquot. (Synthesize l-(6-methoxy-2-naphthyl)ethyl isothiocyanate as follows (protect
from light). Dissolve 500 mg (S)-(+)-naproxen in 50 mL dry toluene, slowly add 5 mL
freshly distilled thionyl chloride, reflux for 1 h, evaporate to dryness under vacuum, dry
the acyl chloride (mp 87.5) under vacuum over KOH for 2 days. Dissolve 0.5 mmoles acyl
chloride in 5 mL acetone, stir at 0, add 0.6 mmoles sodium azide dissolved in ice water,
stir at 0 for 30 min, add 10 mL ice-cold water, filter, dry solid in a desiccator under
vacuum. Dissolve the solid in 1 mL toluene or dichloromethane (dried over 3 A molecular
sieve), reflux for 10 min, evaporate, store resulting isocyanate (mp 51) under vacuum
over a desiccant. Dissolve 0.5 mmole isocyanate in 5 mL acetone, add 20 mL 8.5% phosphoric acid, heat to 80 for 1.5 h, adjust to pH 13, extract with diethyl ether: dichloromethane 4:1. Wash the organic layer twice with water, dry over anhydrous sodium sulfate, evaporate to dryness, dissolve in 1 mL toluene, evaporate to give the amine from