Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone

Methadone enantiomers and EDDP, the main metabolite of methadone, were separated (R(s) = 2.0 for methadone enantiomers) following liquid-liquid extraction from human serum and urine followed by reverse-phase high-performance liquid chromatography on a derivatized beta-cyclodextrin column and quantified at therapeutic concentrations with ultraviolet detection. Detector response was linear (r(2) > 0.98) to 1,000 and 2,500 ng x mL(-1) for methadone enantiomers and EDDP, respectively. The limit of quantification from a 1-mL biological sample was 2.5 and 5 ng x mL(-1) for methadone enantiomers and EDDP, respectively. Interday variation was <13% and intraday variation was <8% for the analytes of interest. The assay was applied to plasma protein and erythrocyte binding studies and a 96-h pharmacokinetic study in two healthy female volunteers following oral dosing with rac-methadone. The binding of methadone to plasma proteins was enantioselective with the active (-)-(R) enantiomer having the highest free fraction (mean +/- SD: 21.2+/-7.6% vs. 13.3+/-6.2% for (+)-(S)-methadone, n = 8). Binding of methadone to erythrocytes was not apparently enantioselective (38.6+/-1.3% and 38.1+/-1.4% bound for (-)-(R)- and (+)-(S)-methadone, respectively). The pharmacokinetic study revealed enantioselective disposition of methadone in one volunteer but not in the other. EDDP was observed in urine but was only in small or undetectable concentrations in serum. The method is applicable to in vitro and pharmacokinetic studies of rac-methadone disposition in humans.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levosulpiride in human plasma

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levosulpiride in human plasma was developed. Levosulpiride and internal standard, tiapride were extracted from human plasma with ethyl acetate at pH 11 and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile-ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.999) over the concentration range of 1.00-200 ng/ml. The lower limit of quantification for levosulpiride was 1.00 ng/ml using 100 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at three quality control (QC) levels were 3.8-9.1 and -2.9 to -0.1%, respectively. The recoveries of levosulpiride ranged from 80.5 to 87.4%, with that of tiapride (internal standard) being 84.6%. This method was successfully applied to the pharmacokinetic study of levosulpiride in humans.     

Gas chromatographic determination of methamphetamine and its metabolite amphetamine in human plasma and urine following conversion to N-propyl derivatives

A gas chromatographic method for the simultaneous determination of methamphetamine and its metabolite amphetamine in human plasma and urine is described. The method utilizes reductive alkylation with propionaldehyde and sodium borohydride to produce N-propyl derivatives, which have excellent chromatographic properties. Structural analogs of the analytes, p-methylmethamphetamine and p-methylamphetamine, are used as internal standards. The method has good precision and accuracy for concentrations ranging from less than 10 ng/ml to 5000 ng/ml and has been used to measure plasma concentrations as part of a pharmacokinetic/pharmacodynamic study of methamphetamine in humans.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine

Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography-electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5-1000 ng/ml; r > or = 0.9968), intra- and inter-batch precision and accuracy (< or =14.5%), and recoveries (94-106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.     

Simultaneous determination of piroxicam, meloxicam and tenoxicam in human plasma by liquid chromatography with tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of piroxicam, meloxicam and tenoxicam in human plasma was developed. Piroxicam, meloxicam, tenoxicam and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analyzed on a Sunfire column with the mobile phase of methanol:ammonium formate (15 mM, pH 3.0) (60:40, v/v). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring (MRM) mode. The standard curve was linear (r=1.000) over the concentration range of 0.50-200 ng/ml. The coefficient of variation (CV) and relative error (RE) for intra- and inter-assay statistics at three QC levels were 1.0-5.4% and -5.9 to 2.8%, respectively. The recoveries of piroxicam, meloxicam and tenoxicam ranged from 78.3 to 87.1%, with that of isoxicam being 59.7%. The lower limit of quantification for piroxicam, meloxicam and tenoxicam was 0.50 ng/ml using a 100 microl plasma sample. This method was successfully applied to a pharmacokinetic study of piroxicam after application of transdermal piroxicam patches to humans.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Determination of lefucoxib in rat plasma, urine, and feces by high-performance liquid chromatography with fluorescence detection: application in pharmacokinetic studies

A sensitive, specific, and reproducible high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of lefucoxib in rat plasma, urine, and feces. The method involved liquid-liquid extraction using methyl tert-butyl ether, and celecoxib was used as the internal standard. The chromatographic separation was performed on a Kromasil C18 column (250.0 mm x 4.6 mm, 5.0 microm) with a mobile phase gradient consisting of water and methanol at a flow rate of 1 ml min(-1). The assay was linear in the range of 5.0-1000.0 ng ml(-1) with a correlation coefficient (r) of 0.9994. The limit of quantification was 5.0 ng ml(-1). Inter- and intra-assay precisions were 相似文献   

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Enantiospecific determination of DL-methylphenidate and DL-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: application to human ethanol interactions

In humans, concomitant DL-methylphenidate (DL-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite DL-ethylphenidate (DL-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between D- and L-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic-pharmacodynamic correlations regarding the MPH-ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of D- and L-MPH as well as D- and L-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r(2)>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.     

The development of a radioimmunoassay for formoterol

The development of a radioimmunoassay (RIA) for the beta 2-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a 1-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and humans, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Development and validation of an LC-MS method with electrospray ionization for quantitation of digoxin in human plasma and urine: application to a pharmacokinetic study

A highly sensitive and specific LC-MS method was developed and validated for the quantification of digoxin in human plasma and urine using d5-dihydrodigoxin as internal standard (IS). The assay procedure involved extraction of digoxin and IS from human plasma with chloroform-isopropanol (95:5, v/v). Chromatogrphic separation was achieved on a Spherisorb ODS2 column using a gradient mobile phase with 5 mmol/L ammonium acetate in water with 1% acetic acid and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective [M+K](+) ions, m/z 819.4 for digoxin and m/z 826.4 for IS. The method was proved to be accurate and precise at linearity range of 0.12-19.60 ng/mL in plasma with a correlation coefficient (r(2)) of >or=0.9968 and 1.2-196.0 ng/mL in urine. The limit of quantification achieved with this method was 0.12 ng/mL in plasma and 1.2 ng/mL in urine. The intra- and inter-assay precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers following intravenous administration of digoxin.     

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C₁₈ columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.     

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane—2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5–100 ng/ml and 25–1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.