Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Sensitive semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681, the active parent drug of a new oral carbapenem antibiotic,DZ-2640, in human plasma and urine using a column-switching system as sample clean-up procedure

DZ-2640 is a new oral carbapenem antibiotic having a dihydro-pyrroloimidazole ring as a side chain and a pivaloyloxymethyl (POM) ester prodrug of DU-6681, the active parent compound. A simple and sensitive column-switching semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681 in human plasma and urine has been developed. Human plasma was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was filtered through an Ultrafree C3GV. The resulting filtrate was injected without further cleanup onto the HPLC system. Human urine was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was directly injected onto the HPLC system. The analyte was detected by monitoring the column effluent with UV light at a wavelength of 300 nm, which resulted in the limit of quantitation of 0.008 μg/ml of plasma and 0.32 μg/ml of urine. Calibration curves were linear in the range of 0.008 to 5.85 μg/ml in plasma and 0.32 to 104.4 μg/ml in urine. The present methods showed greatly increased sensitivity for DU-6681 compared to conventional HPLC methods and also showed satisfactory recovery, selectivity, precision, and accuracy. Stability studies showed that 1 M MOPS buffer (pH 7.0) acted as a stabilizer. In plasma and urine diluted with equal volume of the buffer, DU-6681 showed good stability at −80°C for up to 4 weeks with no significant loss of the drug.     

Determination of paclitaxel and metabolites in mouse plasma,tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1–0.2 g/ml) in bovine serum albumin (40 g/I) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200–1000 μl of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut column. 2′-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 ot 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 μl of sample. In tissues, faeces and urine the LLQs are 25–100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 μl (faeces: 200 μl) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25–100 000 ng/ml for paclitaxel and 25–500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite (III, H117/04) with the basic metabolites α-hydroxymetoprolol (II, H119/66), metoprolol (I) and guanoxan (IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C₁₈ solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C₁₈ radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r²≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% (n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.     

Simple and sensitive high-performance liquid chromatographic method for the determination of 1,5-benzodiazepine clobazam and its active metabolite N-desmethylclobazam in human serum and urine with application to 1,4-benzodiazepines analysis

A HPLC–UV determination of clobazam and N-desmethylclobazam in human serum and urine is presented. After simple liquid–liquid extraction with dichloromethane the compounds and an internal standard diazepam were separated on a Supelcosil LC-8-DB column at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:540:20:0.4, v/v and 360:580:60:0.4, v/v for serum and urine, respectively). The detection was performed at 228 nm with limits of quantification of 2 ng/ml for serum and 1 ng/ml for urine. Relative standard deviations for intra- and inter-assay precision were found below 8% for both compounds for all the tested concentrations. The described procedure may be easily adapted for several 1,4-benzodiazepines.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

High-performance liquid chromatographic assay for CI-980, a novel 1-deaza-7,8-dihydropteridine anticancer agent,in human plasma and urine

CI-980, a 1-deaza-7,8-dihydropteridine, is a novel anticancer agent that is a potent mitotic inhibitor acting as a tubulin binder similar to the vinca alkaloids. CI-980 has shown equivalent or superior anticancer activity in vitro compared to vincristine and retains full activity against vincristine resistant tumors in vitro. A high-performance liquid chromatographic (HPLC) assay was developed and validated for human plasma and urine to support Phase 1 clinical trials. CI-980 and PD 080658, internal standard, were isolated from 2-ml samples of human plasma and urine by solid-phase extraction with Bond-Elut C₁₈ cartridges. Urine samples must be pretreated with bovine serum albumin (BSA) to minimize the binding of CI-980 to glass and some plastics. The eluate from the cartridges for both matrices was evaporated to dryness and taken up in mobile phase. Zorbax RX C₁₈ columns, mobile phase buffer of 10 mM ammonium dihydrogen phosphate at pH 7.5 and a flow--rate of 0.75 ml/min were used for both matrices. Column dimensions, column temperature and mobile phase acetonitrile-buffer ratio were 300 mm × 4.6 mm I.D., 30°C and 38:62 (v/v), respectively, for the plasma assay and 250 mm × 4.6 mm I.D., 35°C and 40:60 (v/v), respectively, for the urine assay. Column effluent was monitored fluorometrically for the plasma method using excitation and emission wavelengths of 388 nm and 473 nm, respectively. Ultraviolet detection at 380 nm was used for the urine method. Peak-area ratios were proportional to CI-980 concentrations from 0.2 to 25 ng/ml and 1 to 100 ng/ml for plasma and urine, respectively. CI-980 in water will bind to glass and plastics but not PTFE or stainless steel. Urine calibration standards were frozen prior to use in order to compensate for loss of CI-980 due to freezing in this matrix. The accuracy of the assay was within 4.7%, with a precision of 5.6% for both matrices. Recoveries ranged from 93.8 to 102% and 90.7 to 92.3% for plasma and urine, respectively. CI-980 was stable in plasma and urine for at least 275 and 217 days, respectively, when stored at −70°C. The assay is suitable for studying the clinical pharmacokinetics of CI-980.     

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C₁₈ column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.     

Isolation,identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and Rhesus monkey by high-performance liquid chromatography

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by β-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N₄-acetylsulfadiazine (N₄), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 μg/ml (S 0.31, N₄ 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 μg/ml). In urine it varies between 0.6 and 1.1 μg/ml (S 0.75, N₄ 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 μg/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.     

Determination of a new oral cephalosporin,S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C₁₈ analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C₁₈ analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09–9 μg/ml and 0.5–100 μg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.     

Stereoselective determination of R(−)- and S(+)-MK-571, a leukotriene D4 antagonist,in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(−)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D₄ antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine—acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine—pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r²>0.999) for concentrations of enantiomers of 1 from 0.05 μg/ml, the lowest quantitation limit, up to 2.5 μg/ml. Intra-day coefficients of variation at 0.05 μg/ml were 2.4% for the R(−)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(−)- and S(+)-1 were 2.6 and 3.6%, respectively. The utilit of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Stereospecific determination of amisulpride,a new benzamide derivative,in human plasma and urine by automated solid-phase extraction and liquid chromatography on a chiral column application to pharmacokinetics

Amisulpride, a drug belonging to the benzamide series, demonstrates antischizophrenic and antidepressant (antidysthymic) properties in man. For the pharmacokinetic studies of the racemic drug in man, a method of determination based on solid-phase extraction (SPE) from plasma and HPLC on a stereoselective column was developed. For this aim, one millilitre of plasma, after the addition of the internal standard, tiapride or metoclopramide, is diluted with a borate buffer at pH 9, then automatically loaded onto a SPE C₁₈ 100-mg column. The column is washed with different solvents, then eluted with 0.5 ml of methanol. After evaporation of the eluted fraction, the residue is reconstituted in 0.25 ml of eluent mixture. An aliquot is injected onto the HPLC column, a Chiralpak AS, equilibrated with an eluent mixture constituted by n-hexane-ethanol, (67:33, v/v) containing 0.2% (v/v) of diethylamine (DEA) or n-heptane-ethanol, (70:29.8, v/v) containing 0.2% of DEA and connected to a UV detector set at 280 nm or to a fluorimetric detector set at λ_ex = 280 nm and λ_em = 370 nm. The limit of quantitation (LOQ) in human plasma is 2.5 ng ml⁻¹ for both S-(−)- and R-(+)-amisulpride isomers with both detection methods. The method has been demonstrated to be linear in the range 2.5–320 ng ml⁻¹ for both R-(+)- and S-(−)-amisulpride in human plasma with both UV and luorescence detection. Absolute recovery of S(−)- and R-(+)-amisulpride enantimers from human plasma, as well as selectivity, precision and accuracy have been demonstrated to be satisfactory for pharmacokinetics in man and equivalent for both the proposed methods that have been cross-validated on real dosed human plasma samples. The methods have been used for clinical pharmacokinetic studies allowing pharmacokinetic parameters for amisulpride enantiomers in agreement with those obtained for the racemate to be obtained. After dilution with water, urinary samples from subjects treated with amisulpride racemate can be analysed according to the method used for plasma.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.     

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(−) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C₁₈ column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ_ex=280 nm and λ_em=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.