Determination of lansoprazole and two of its metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography: application to measuring CYP2C19 activity

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method for the simultaneous determination of lansoprazole, a proton pump inhibitor and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human plasma. The test compounds were extracted from 1 mL of plasma using diethyl ether-dichloromethane (7:3, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 microm, 3.5 mm x 4.6 mm i.d.) for clean-up and column I (C(18) STR ODS-II analytical column, 5 microm, 150 mm x 4.6 mm i.d.) for separation. The peak was detected by a ultraviolet detector set at a wavelength of 285 nm, and the total time for a chromatographic separation was approximately 25 min. The method was validated for the concentration range from 3 to 5000 ng/mL. Mean recoveries were 74.0% for lansoprazole, 68.3% for 5-hydroxylansoprazole, and 79.4% for lansoprazole sulfone. Intra- and inter-day relative standard derivatives were less than 6.1 and 5.1% for lansoprazole, 5.8 and 5.8% for 5-hydroxylansoprazole, 4.4 and 5.9% for lansoprazole sulfone, respectively, at the different concentration ranges. This method is suitable for use in therapeutic drug monitoring and pharmacokinetic studies, and provides use tool for measuring CYP2C19 activity.     

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.     

Sensitive determination of omeprazole and its two main metabolites in human plasma by column-switching high-performance liquid chromatography: application to pharmacokinetic study in relation to CYP2C19 genotypes

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether-dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 microm, 150 mm x 4.6mm i.d.) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer-acetonitrile-methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was approximately 25 min. The validated concentration ranges of this method were 3-2000 ng/ml for omeprazole, 3-50 ng/ml for 5-hydroxyomeprazole and 3-1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.     

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.     

Enantioselective determination of tramadol and its main phase I metabolites in human plasma by high-performance liquid chromatography

A sensitive and relatively rapid reversed-phase HPLC method was applied to the enantiomeric separation of tramadol and its two main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in plasma samples. Chromatography was performed on an AGP column containing alpha1-acid glycoprotein as chiral selector with a mobile phase of 30 mM diammonium hydrogen phosphate buffer-acetonitrile-triethylamine (98.9:1:0.1, v/v), adjusted to pH 7 by phosphoric acid, and a flow rate of 0.5 ml/min. The fluorescence of analytes was detected at excitation and emission wavelengths of 200 and 301 nm, respectively. The sample preparation was a simple extraction with ethyl acetate using fluconazol as internal standard (IS). The enantiomers of all analytes and IS peaks eluted within 32 min, without any endogenous interference. The calibration curves were linear (r(2) > 0.993) in the concentration range of 2-200, 2.5-100 and 2.5-75 ng/ml for tramadol, M1, and M2 enantiomers, respectively. The within- and between-day variation determined by the measurement of quality control samples at four tested concentrations, showed acceptable values. The lower limit of quantitation was 2 ng/ml for tramadol enantiomers and 2.5 ng/ml for M1 or M2 enantiomers. Mean recoveries of enantiomers from plasma samples were > 81% for all analytes. The procedure was applied to assess the pharmacokinetics of the enantiomers of tramadol and its two main metabolites following oral administration of single 100-mg doses to healthy volunteers.     

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.     

Determination of modafinil, modafinil acid and modafinil sulfone in human plasma utilizing liquid-liquid extraction and high-performance liquid chromatography

An assay was developed to determine concentrations of modafinil (dl-2-[(diphenylmethyl)sulfinyl]acetamide; Provigil) and its two major circulating metabolites, modafinil acid and modafinil sulfone, in human plasma. The assay utilized liquid-liquid extraction of the analytes and an internal standard, (phenylthio)acetic acid, from plasma into a mixture of hexane-dichloromethane-glacial acetic acid (55:45:2, v/v). The analytes were resolved isocratically on a narrow-bore phenyl column at a mobile phase flow-rate of 0.3 ml/min and were monitored by UV detection at 235 nm. The method reported herein reduces the required sample volume of previously reported methods from 1.00 to 0.200 ml of plasma while lowering the limit of quantification (LOQ). The linear range of the assay was from 0.100 to 20.0 microg/ml for each of the three compounds.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

A sensitive and selective HPLC/ESI-MS/MS assay for the simultaneous quantification of 16-dehydropregnenolone and its major metabolites in rabbit plasma

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M(1)), 5-pregnene-3beta-ol-20-one (M(2)), 5-pregnene-3beta, 20-diol (M(3)), 5-pregnene-3beta-ol-16, 17-epoxi-20-one (M(4)) and 5,16-pregnadien-3beta, 11-diol-20-one (M(5)) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS-MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M(3) and M(5) where linearity was over the 3.13-400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.     

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C₁₈ column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC–MS–MS samples, an isocratic mobile phase consisting of [0.1% triethylamine–0.1% acetic acid (pH 7.1)]–acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.     

Sensitive quantification of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry

A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC-electrospray mass spectrometry. Following liquid-liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH(+) ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.     

HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 degrees C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5-500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were 0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(-)- and R-(+)-bufuralol from plasma was in the range 97-102% at 15-400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6-102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.     

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.     

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography

UMF-078, methyl (±)-[5-(α-amino-4-fluorobenzyl)benzimidazol-2-yl]carbamate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of ±20% at the lowest limit of quantitation, whereas at higher concentrations it was ±15%. The analytes showed stability up to two freeze–thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at −30°C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Simultaneous liquid chromatography-tandem mass spectrometric determination of albendazole sulfoxide and albendazole sulfone in plasma

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid-liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol-water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.     

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.     

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.     

High-throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma using a monolithic column

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50 mm x 4.6 mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid-liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23s. The isotope-labeled D(6)-bupropion and D(6)-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25-200 ng/ml (bupropion and threo-hydrobupropion), and 1.25-1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.