Resolution, quantification and confirmation of betamethasone and dexamethasone in equine plasma by liquid chromatography/tandem mass spectrometry

This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed in multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1-75 ng/mL for both analytes. Limit of detection (LOD) was 50 pg/mL and that of quantification (LOQ) was 100 pg/mL for both analytes. The limit of confirmation (LOC) for the presence of BTM or DXM by MRM was 0.5 ng/mL. The intra-and inter-day precisions expressed as coefficient of variation (CV) for quantification of DXM and BTM from 0.1 to 50 ng/mL were less than 7% and the accuracy was in the range of 97-105%. This method is capable of distinguishing BTM from DXM when both analytes are simultaneously present in equine plasma. Measurement uncertainty for both analytes was estimated at less than 16%. The method is rapid, specific, selective, sensitive, simple and reliable. The importance of this method is its usefulness in directly identifying and differentiating BTM from DXM without derivatization.     

Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Analytes were extracted from equine plasma by liquid-liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C(18) column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2 mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25-100 pg/mL and 100-200 pg/mL, respectively. The linear dynamic range of quantification was 100-10,000 pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100-500 pg/mL. The method is sensitive, robust, selective and reliably reproducible.     

Ultra‐performance liquid chromatography/tandem mass spectrometry in high‐throughput detection,quantification and confirmation of anabolic steroids in equine plasma

An ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast‐throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid‐liquid extraction using methyl tert‐butyl ether, separated on a 1.9 µm C₁₈ reversed‐phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screening results. Full product ion spectra of excellent quality for AAS, at 100 pg/0.5 mL in plasma, devoid of interfering spectra from impurities in plasma, were obtained. To our knowledge, this is the first report on the acquisition of full product ion spectra at such a low analyte concentration and plasma volume using a triple quadrupole instrument. In addition to product ion intensity ratios obtained from three SRM scans for identifying AAS in equine plasma, full product ion spectra were used as supporting evidence for confirmation. For quantification, deuterium‐labeled testosterone and stanozolol were used as internal standards (ISs). The limits of detection, quantification and confirmation were 6.25–12.5 pg/0.5 mL, 25 pg/0.5 mL and 50–100 pg/0.5 mL, respectively. There was no significant matrix effect on the analysis of all eight AAS. Intra‐day precision and accuracy were 2–15% and 91–107%, respectively. Inter‐day precision and accuracy were 1–21% and 94–110%, respectively. Total analysis time was 5 min. To date, the method has been successfully used in the analysis of >12 000 samples for AAS in plasma samples from racehorses competing in the State of Pennsylvania. The method is fast, selective, reproducible, and reliable. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of clenbuterol in equine plasma,urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry

Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method involved liquid-liquid extraction (LLE), separation by liquid chromatography (LC) on a short cyano column, and pseudo multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). At very low concentrations (picograms of CBL/mL), LLE produced better extraction efficiency and calibration curves than solid-phase extraction (SPE). The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CBL. The quantification range of the method was 0.013-10 ng of CBL/mL plasma, 0.05-20 ng/0.1 mL of urine, and 0.025-10 ng/g tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CBL in plasma, urine and various tissue samples, and in pharmacokinetic (PK) studies of CBL in the horse. CBL was quantified for 96 h in plasma and 288 h in urine post-administration of CLB (1.6 micro g/kg, 2 x daily x 7 days). This method is useful for the detection and quantification of very low concentrations of CBL in urine, plasma and tissue samples.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Simultaneous quantification of atorvastatin and active metabolites in human plasma by liquid chromatography-tandem mass spectrometry using rosuvastatin as internal standard

A simple, sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of atorvastatin and its active metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin in human plasma using rosuvastatin as internal standard (IS). Following simple liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 559/440 for atorvastatin, m/z 575/466 for ortho-hydroxyatorvastatin, m/z 575/440 for para-hydroxyatorvastatin and m/z 482/258 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for atorvastatin and its two metabolites in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin and the IS from spiked plasma samples were 54.2 +/- 3.2, 50.1 +/- 3.8, 65.2 +/- 3.6 and 71.7 +/- 2.7%, respectively. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on-line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre-treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospray ionization in multiple reaction monitoring mode. The retention times for EG and ES were 1.7 +/- 0.30 and 3.4 +/- 0.30 min, respectively. The internal standard used was d(5)-ethyl glucuronide with a retention time of 1.7 +/- 0.30 min. The entire separation was completed in <5 min. The limit of detection (LOD) and of quantification (LOQ) for both analytes were 100 ng/mL (S/N > or =3) and 500 ng/mL, respectively. The limit of confirmations (LOC) for EG and ES were 500 ng/mL and 1.0 microg/mL, respectively. The assay was linear over a concentration range of 0.5-100 microg/mL (r(2) > 0.995). Intra- and inter-day accuracy and precision were less than 15%. The analytes were stable in urine for 24 h at room temperature, 10 days at 4 degrees C and 21 days at -20 degrees C and -70 degrees C. Ion suppression or enhancement due to matrix effect was negligible. The measurement uncertainty was <14% for EG and <25% for ES. This method was successfully used for the quantification of EG and ES in urine samples following alcohol administration to research horses and for screening and confirmation of EG and ES in urine samples obtained from racehorses post-competition. The method is simple, rapid, inexpensive, and reliably reproducible.     

A rapid and highly sensitive UPLC-MS-MS method for the quantification of zolpidem tartrate in human EDTA plasma and its application to pharmacokinetic study

A rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the quantification of zolpidem in human EDTA plasma using ondansetron (IS) as an internal standard. The analyte and IS were extracted from human plasma using ethyl acetate and separated on a C18 column (Inertsil-ODS, 5 μm, 4.6 × 50 mm) interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase, which consisted of a mixture of methanol and 20 mM ammonium formate (pH 5.00 ± 0.05; 75:25 v/v), was injected at a flow rate of 0.40 mL/min. The retention times of zolpidem and IS were approximately 1.76 and 1.22. The LC run time was 3 min. The electrospray ionization source was operated in positive ion mode. Multiple reaction monitoring used the [M + H](+) ions m/z 308.13 → 235.21 for zolpidem and m/z 294.02 → 170.09 for the ondansetron, respectively. Five freeze-thaw cycles was established at -20 and -70°C.The linearity of the response/concentration curve was established in human EDTA plasma over the concentration range 0.10-149.83 ng/mL. The lower detection limit [(signal-to-noise (S/N) > 3] was 0.04 ng/mL and the lower limit of quantification (S/N > 10) was 0.10 ng/mL. This LC-MS-MS method was validated with intra-batch and inter-batch precision of 0.52-8.66.The intra-batch and inter-batch accuracy was 96.66-106.11. Recovery of zolpidem in human plasma was 87.00% and IS recovery was 81.60%. The primary pharmacokinetic parameters were T(max) (h) = (1.25 ± 0.725), C(max) (ng/mL) (127.80 ± 34.081), AUC(0→t), = (665.37 ± 320.982) and AUC(0→∞), 686.03 ± 342.952, respectively.     

Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies

A liquid chromatography/mass spectrometry method for simultaneous determination of paracetamol and dextropropoxyphene in human plasma is described. Paracetamol and dextropropoxyphene, together with their internal standards (tolbutamide and pyrroliphene), were extracted from 0.5 mL of plasma using solid-phase extraction. The chromatography was performed using a Thermo Hypersil APS-2 Amino column (250 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile and 0.4% glacial acetic acid in water (20:80). The total run time was 6 min for each sample. The triple-quadrupole mass spectrometer was operated in both positive (for detection of dextropropoxyphene and its IS pyrroliphene) and negative (for detection of paracetamol and its IS tolbutamide) modes using a polarity-switching technique. Multiple reaction monitoring was used for quantification. The method was linear over the concentration range of 0.1-20 microg/mL for paracetamol and 0.5-80 ng/mL for dextropropoxyphene. The intra- and inter-day precision were less than 10%, and the accuracy ranged from 92.2-110.9%. The lower limits of quantification were 0.1 microg/mL for paracetamol and 0.5 ng/mL for dextropropoxyphene. The present method provides a robust, fast and sensitive analytical tool for both paracetamol and dextropropoxyphene, and has been successfully applied to a clinical bioequivalence study in 14 subjects.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma

Artemisinin is a widely used antimalarial drug. To evaluate the pharmacokinetics of artemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of artemisinin in rat plasma. For detection, a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization (ESI) TurboIonSpray inlet in the positive ion multiple reaction monitoring (MRM) mode was used to monitor precursor ([M+NH4]+) --> product ions of m/z 300.4 --> 209.4 for artemisinin and m/z 316.4 --> 163.4 for artemether, the internal standard (IS). The plasma samples were pretreated by a simple liquid-liquid extraction with ether. The standard curve was linear (r > 0.99) over the artemisinin concentration range of 1.0-200.0 ng/mL in plasma. The method had a lower limit of quantification of 1.0 ng/mL for artemisinin in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of artemisinin. The intra- and inter-day precisions were measured to be within +/-5.3% and accuracy between -2.6% and 1.2% for all quality control samples, lower limit of quantification and upper limit of quantification samples. The extraction recoveries of artemisinin and the IS were 95.4 +/- 4.5% and 92.8 +/- 3.9%, respectively. This present method was successfully applied to the characterization of the pharmacokinetic profile of artemisinin in rats after oral administration.     

High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samples

A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2 mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36 min, respectively. In both plasma and tissue specimens the assay was linear in the range 50-5000 ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5 ng/mL and 20 ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations.     

Liquid chromatography/electrospray ionization mass spectrometry method for the quantification of valproic acid in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the quantification of valproic acid, an antiepileptic drug, in human plasma using benzoic acid as internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the single ion monitoring mode using the respective [M-H]- ions, m/z 143 for valproic acid and m/z 121 for the IS. The assay exhibited a linear dynamic range of 0.5-60 microg/mL for valproic acid in human plasma. The lower limit of quantification was 500 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of valproic acid and the IS from spiked plasma samples were 96.1+/-4.2 and 95.6+/-2.7%, respectively. A run time of 4.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

Simultaneous quantification of fexofenadine and pseudoephedrine in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: method development, validation and application to a clinical study

To support the pharmacokinetic and bioavailability study of a once-daily fexofenadine/pseudoephedrine combination, a high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous quantification of fexofenadine and pseudoephedrine was developed and validated with 500 microL human plasma using mosapride as an internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited linear dynamic ranges of 1-500 ng/mL and 2-1000 ng/mL for fexofenadine and pseudoephedrine, respectively, in human plasma. The lower limits of quantification were 1 and 2 ng/mL with a relative standard deviation of less than 10% for fexofenadine and pseudoephedrine, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time was 2 min and more than 400 human plasma samples could be analyzed in one day by running the system overnight. The method is precise and sensitive enough for its intended purpose.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Development and validation of high-performance liquid chromatography/electrospray ionization mass spectrometry for assay of madecassoside in rat plasma and its application to pharmacokinetic study

A rapid, sensitive and specific high-performance liquid chromatography/electrospray ionization mass spectrometric (LC-ESI-MS) method was developed and validated for the quantification of madecassoside, a major active constituent of Centella asiatica (L.) Urb. herbs, in rat plasma. With paeoniflorin as an internal standard (IS), a simple liquid-liquid extraction process was employed for the plasma sample preparation. Chromatographic separation was achieved within 6 min on a Shim-pack CLC-ODS column using acetonitrile and water (60:40, v/v) containing 0.1% (v/v) formic acid as the mobile phase. The detection was performed by MS with electrospray ionization interface in negative selected ion monitoring (SIM) mode. The linear range was 11-5500 ng/mL with the square regression coefficient (r(2) ) of 0.9995. The lower limit of quantification was 11 ng/mL. The intra- and inter- day precision ranged from 4.99 to 9.03%, and the accuracy was between 95.82 and 111.80%. The average recoveries of madecassoside and IS from spiked plasma samples were >92%. The developed method was successfully applied to the pharmacokinetic study of madecassoside in rats after an oral administration.     

Liquid chromatography/electrospray ionization tandem mass spectrometry validated method for the simultaneous quantification of sibutramine and its primary and secondary amine metabolites in human plasma and its application to a bioequivalence study

A high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the estimation of sibutramine and its two metabolites (M1 and M2). The extraction of sibutramine, its metabolites and imipramine (internal standard (IS)) from the plasma involved treatment with phosphoric acid followed by solid-phase extraction (SPE) using a hydrophilic-lipophilic balanced HLB cartridge. The SPE eluate without drying and reconstitution was analyzed by LC/MS/MS, equipped with a with turbo ion spray (TIS) source, operating in the positive and multiple reaction monitoring (MRM) acquisition mode. Sample preparation by this method yielded extremely clean extracts with quantitative and consistent mean recoveries; 95.12% for sibutramine, 92.74% for M1, 95.97% for M2 and 96.60% for the IS. The total chromatographic run time was 3.0 min with retention times of 2.51, 2.13, 2.09 min for sibutramine, M1, M2 and imipramine, respectively. The developed method was validated in human plasma matrix, with a sensitivity of 0.1 ng/mL (coefficient of variance (CV), 2.07%) for sibutramine, 0.1 ng/mL (CV, 3.59%) for M1 and 0.2 ng/mL (CV, 4.93%) for M2. Validation of the method for its accuracy, precision, recovery, matrix effect and stability was carried out especially with regard to real subject sample analysis. The response was linear over the dynamic range 0.1 to 8.0 ng/mL for sibutramine and M1, and 0.2 to 16.0 ng/mL for M2 with correlation coefficients of r > or = 0.9959 (sibutramine), 0.9935 (M1) and 0.9943 (M2). The method was successfully applied for bioequivalence studies in 40 human subjects with 15 mg capsule formulations.