Quantitative determination of the novel anticancer drug E7070 (indisulam) and its metabolite (1,4-benzenedisulphonamide) in human plasma, urine and faeces by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

E7070 (indisulam) is a novel anticancer drug currently undergoing clinical investigation. We present a sensitive and specific method for the quantitative determination of E7070 and its metabolite M1 (1,4-benzenedisulphonamide) in human plasma, urine and faeces. The analytes and their tetra-deuterated analogues, which were used as internal standards, were isolated from the biological matrix by solid-phase extraction with OASIS cartridges (0.5 mL plasma or 1 mL urine) and by liquid-liquid extraction with ethyl acetate at pH 5 (1 mL faecal homogenate). The analytes were separated on a C8 reversed-phase chromatographic column and analyzed using electrospray ionization and tandem mass spectrometric detection in the negative ion mode. The validated concentration ranges in plasma were 0.1-20 microg/mL for E7070 and 0.01-2 microg/mL for M1. In urine and faecal homogenate, a concentration range from 0.05-10 microg/mL or microg/g, respectively, was validated for both analytes. Validation of the plasma assay was performed according to the most recent FDA guidelines. The assay fulfilled all generally accepted requirements for linearity (r > 0.99, residuals between -8 and 10%), accuracy (-13.5 to 1.4%) and precision (all less than 11%) in the tested matrices. We investigated recovery, stability (working solutions at -20 degrees C and at room temperature, biological matrices at -20 degrees C, room temperature and after 3 freeze/thaw cycles; final extracts at room temperature) and robustness. All these parameters were found acceptable. This method is suited for mass balance studies or therapeutic drug monitoring, as demonstrated by a case example showing plasma concentrations and cumulative excretion of E7070 and M1 in urine and faeces. Furthermore, we show the presence of E7070 metabolites in patient urine.     

Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.     

Determination of MDL 201,012 at femtomole/millilitre levels in human plasma by liquid chromatography with electrochemical detection.

A sensitive and selective liquid chromatographic method to quantitate MDL 201,012 in human plasma was developed and validated. MDL 201,012 (I), diethyl-MDL 201,012 (internal standard, II) and desmethyldiol-MDL 201,012 (masking agent, III) were isolated from basified plasma (2 mL) by solid phase extraction using Bond-Elut C-18 cartridges. Endogenous components were selectively removed prior to eluting the analytes from the sorbent. Components were separated using on-line LC column switching with a cyanopropyl precolumn and a phenyl analytical column. The analytical column effluent was monitored electrochemically at a glassy carbon electrode at a potential of +1025 mV vs. Ag/AgCl. Peak-height ratios were proportional to the amount of MDL 201,012 added to plasma over the range 125-7500 pg/mL MDL 201,012. Absolute recovery of MDL 201,012 from human plasma was > 94% across the calibration range. The minimum quantitation limit was 125 pg/mL. Assay precision (%RSD) ranged from 5.2 to 13% based on the analysis of quality control standards containing 125, 250, 500, 1000, 2500, 5000 and 7500 pg/mL MDL 201,012. Corresponding assay accuracy (% relative error) was +/- 8.5%. The method has been successfully used to quantitate MDL 201,012 in samples from acute dose tolerance studies in human volunteers.     

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.     

Quantitation of simvastatin and its beta-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry

A sensitive and reliable procedure for the simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite (SVA) in human plasma was developed and validated. The analytes were extracted simultaneously from 0.5 ml aliquots of human plasma samples by methyl tert-butyl ether (MTBE) via Chem Elut cartridge extraction [also called liquid-solid extraction (LSE) or liquid-liquid cartridge extraction (LLCE)], separated through a Kromasil C(18) column (50 x 2 mm i.d. 5 microm) and detected by tandem mass spectrometry with a turbo ionspray interface. Stable isotope-labeled SV and SVA, (13)CD(3)-SV and (13)CD(3)-SVA, were used as internal standards. SV and SVA were detected in positive and negative ion modes, respectively, via within-run polarity switching. The use of Chem Elut cartridges not only provided a simple and efficient means of plasma sample extraction but also successfully reduced the interconversion between SV and SVA to an undetectable (for lactonization of SVA) or negligible (<0.07%, for hydrolysis of SV) level. The method showed excellent reproducibility, with intra- and inter-assay precisions <4.5% (RSD), and intra- and inter-assay accuracy between 94% and 107% of nominal values, for both analytes. The extraction recoveries were 78% and 87% on average for SV and SVA, respectively. The analyte was found to be stable in plasma through three freeze (-70 degrees C)-thaw (4 degrees C) cycles and for at least 3 h under bench-top storage condition in an ice-bath (4 degrees C), and also in the reconstitution solution at 4 degrees C for at least 24 h. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1) for both analytes, and has proved to be very reliable for the analysis of clinical samples.     

Quantitation of oxcarbazepine and its metabolites in human plasma by micellar electrokinetic chromatography

A reliable micellar electrokinetic chromatographic method for the determination of oxcarbazepine and its two main metabolites, 10-hydroxycarbamazepine and 10,11-trans-dihydroxy-10,11-dihydroxycarbamazepine, in human plasma was developed. The separation and determination of the analytes was achieved using a system consisting of 60 mM SDS in phosphate buffer (30 mM, pH 8.0), to which 20% (v/v) methanol was added. Separation was carried out in an uncoated fused-silica capillary with a separation voltage of 25 kV and currents typically less than 40 microA. Spectrophotometric detection was at 205 nm. Isolation of oxcarbazepine and its metabolites from plasma was accomplished by a solid-phase extraction procedure. The mean extraction yield of the analytes from plasma was higher than 94%. The linear correlation coefficients were better than 0.994 for all analytes. The limit of detection was 0.05 microg/mL, the limit of quantitation 0.15 microg/mL. The repeatability for the spiked blank plasma samples was lower than 1.9% and the intermediate precision lower than 2.1%, both expressed as RSD%. The results obtained analysing real plasma samples from epileptic patients under therapy with Tolep were satisfactory in terms of precision, accuracy and detectability.     

Trace-level quantitation of iralukast in human plasma by microbore liquid chromatography/tandem mass spectrometry

Iralukast (CGP 45715A) is a potent peptido-leukotriene antagonist that is active in various in vitro and animal models for the treatment of asthma. An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 10 pg/mL for the analysis of iralukast when administered at low doses during clinical trials. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compound. First, iralukast appeared to be light sensitive and unstable at room temperature under acidic conditions. Second, a LLOQ of 10 pg/mL was needed to support several clinical trials. Third, positive electrospray ionization of iralukast did not yield the necessary sensitivity required for studies in humans. Consequently, LC/MS/MS conditions were optimized for the negative ion mode of detection. Fourth, sample preparation steps proved to be critical to reduce the possibility of microbore HPLC column (50 mm x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix-mediated electrospray ion suppression. While our validated method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, plasma concentration-time profiles for patients with moderate asthma after oral administration of 200, 500, 1000, and 5000 microgram/kg/day of iralukast were successfully obtained.     

Assessment of matrix effects and determination of niacin in human plasma using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry

A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.     

Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry

Docetaxel is an antineoplastic agent widely used in therapeutics. The objective of this study was to develop and validate a routine assay, using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), for the simultaneous quantification of docetaxel and its main hydroxylated metabolites in human plasma. A structural analogue, paclitaxel, was used as the internal standard. Determination of docetaxel and four metabolites (M1, M2, M3 and M4) was achieved using only 100 microL of plasma. Liquid-liquid extraction was used for sample preparation, with extraction efficiency of at least 90% for all analytes. Detection used positive-mode electrospray ionization in selected reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. The assay was linear in the calibration curve range 0.5-1000 ng/mL and acceptable precision and accuracy (<15%) were obtained with concentrations above the LLOQ. This method was sufficiently selective and sensitive for quantification of metabolites in plasma from cancer patients receiving docetaxel chemotherapy, and is suitable for routine analyses during pharmacokinetic studies.     

High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.     

Simultaneous quantitative determination of cyclosporine A and its three main metabolites (AM1, AM4N and AM9) in human blood by liquid chromatography/mass spectrometry using a rapid sample processing method

We have developed a sensitive and specific liquid chromatography/mass spectrometry (LC/MS) method for the simultaneous determination of cyclosporine A (CsA) and its three main metabolites (AM1, AM4N and AM9) in human blood. Following protein precipitation, supernatant was directly injected into the LC/MS system. Chromatographic separation was accomplished on a Symmetry C8 (4.6 x 75 mm, 3.5 microm) column with a linear gradient elution prior to detection by atmospheric pressure chemical ionization (APCI) MS using selected ion monitoring (SIM) in positive mode. This method can be applied to single mass equipment. The analytical range for each analyte was set at 1-2500 ng/mL using 100 microL of blood sample. The analytical method was fully validated according to FDA guidance. Intra-day mean accuracy and precision were 95.2-113.5% and 0.9-8.9%, respectively. Inter-day mean accuracy and precision were 95.8-107.0% and 1.5-10.7%, respectively. In blood all analytes were stable during three freeze/thaw cycles, for 24 h at room temperature and for 12 months at or below -15 degrees C. Stability was also confirmed in processed samples for 24 h at 10 degrees C and for 6 months at 4 degrees C in methanol. In addition, we confirmed the method could avoid matrix effects from transplant subjects' samples. This LC/MS technique provided an excellent method for simultaneous quantitative determination of CsA and its three metabolites for evaluation of their pharmacokinetic profiles.     

Quantitative analysis of 23-hydroxybetulinic acid in mouse plasma using electrospray liquid chromatography/mass spectrometry

23-Hydroxybetulinic acid is a newly isolated derivative of betulinic acid. The agent exhibits potential anti-tumor activity and functions in this regard via apoptosis. In support of pharmacokinetic and toxicological evaluations, a new assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of 23-hydroxybetulinic acid. Sample preparation consisted of extraction of the plasma by the addition of methylene chloride followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Molecules of 23-hydroxybetulinic acid and the internal standard limonin were detected using selected ion monitoring at m/z 471 and 469, respectively. The limit of detection of 23-hydroxybetulinic acid was 0.05 pg (0.11 fmol) injected on-column (10 pg/mL, 5 microL injection volume), and the limit of quantitation was 10 pg (21.19 fmol, 2 ng/mL, 5 muL injection volume). 23-Hydroxybetulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were 3.0 and 4.8%, respectively. The utility of the assay was demonstrated by measuring 23-hydroxybetulinicacid in mouse plasma following intragastric administration (IG) in vivo. Pharmacokinetic parameters were calculated using the 3P97 pharmacokinetic software package. A two-compartment, first-order model was selected for pharmacokinetic modeling. The result showed that after IG of 200 mg/kg 23-hydroxybetulinic acid, the plasma concentrations reached peaks at 2 h with C(max) of 3.1 microg/mL. The 200 mg/kg 23-hydroxybetulinic acid suspension IG doses were found to have long elimination half-lives of 25.6 h and low bioavailability of 2.3%. No interference was noted due to endogenous substances. These analytical methods should be of value in future studies related to the development and characterization of 23-hydroxybetulinic acid.     

Determination of midazolam and its hydroxy metabolites in human plasma and oral fluid by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

Midazolam (MDZ), a short-acting benzodiazepine, is a widely accepted probe drug for CYP3A phenotyping. Published methods for its analysis have used either therapeutic doses of MDZ, or, if employing lower doses, were mostly unable to quantify the two hydroxy metabolites. In the present study, a sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantitative determination of MDZ and two of its metabolites (1'-hydroxymidazolam (1'-OHMDZ) and 4-hydroxymidazolam (4-OHMDZ)) in human plasma and oral fluid. After liquid-liquid extraction with hexane/dichloromethane (73:27, v/v), the analytes were separated on a Luna C18(2) (100 x 2.1 mm) analytical column using gradient elution. Detection was achieved using tandem mass spectrometry on an ion trap mass spectrometer. Midazolam-d6 was used as internal standard for quantification. The calibration curves were linear (R2 >0.998) between 0.05 and 20 ng/mL for MDZ and both metabolites in both matrices. Using 1 mL samples, the limit of detection was 0.025 ng/mL and the limit of quantification was 0.05 ng/mL for MDZ and the hydroxy metabolites in both matrices. Intra- and inter-day accuracies, determined at three different concentrations, were between 92.1 and 102.3% and the corresponding coefficients of variation were <7.3%. The average recoveries were 90.6%, 86.7% and 79.0% for MDZ, 1'-OHMDZ and 4-OHMDZ in plasma and 95.3%, 96.6% and 86.8% for MDZ, 1'-OHMDZ and 4-OHMDZ, respectively, in oral fluid. The method was successfully applied to a pharmacokinetic study, showing that MDZ and its hydroxy metabolites can be determined precisely in in vivo samples obtained following a single oral or intravenous dose of 2 mg MDZ. The method appears to be useful for CYP3A phenotyping in plasma using sub-therapeutic MDZ doses, but larger studies are needed to test this assumption.     

Quantification of granisetron in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of granisetron in human plasma. Following 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 313/138 for granisetron and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for granisetron in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 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.     

Quantification of trandolapril and its metabolite trandolaprilat in human plasma by liquid chromatography/tandem mass spectrometry using solid-phase extraction

A sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous quantification of trandolapril and its metabolite trandolaprilat in human plasma using ramipril as an internal standard. 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 429/168 for trandolapril, m/z 401/168 for trandolaprilat and m/z 415/166 for the internal standard. The method exhibited a linear dynamic range of 20-10,000 pg/mL for both trandolapril and trandolaprilat in human plasma. The lower limit of quantification was 20 pg/mL for both trandolapril and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 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.     

Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

The aim of this study was to develop a reliable UPLC–MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C₁₈ column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20–10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2–1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias <15%. MPA and MPAG were stable at 25 °C up to 1 day in both heparin‐ and EDTA‐treated blood. In heparin‐ and EDTA‐plasma, MPA and MPAG were stable for at least 1 week at 25 and 4 °C, and 1 month at ?20 °C. However, 99% acyl MPA glucuronide degraded in both heparin‐ and EDTA‐blood as well as plasma when stored at room temperature for 1 day. All the analytes remained stable for at least 3 months in acidified EDTA‐plasma at ?80 °C. The assay was successfully applied on patients post hematopoietic stem cell transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in plasma by high-performance liquid chromatography with UV detection

A specific, accurate, precise and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in human plasma. The method employed a simple liquid-liquid extraction of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib and internal standard (IS, DRF-4367) from human plasma (500 microL) into acetonitirile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid (pH 3)-acetonitrile-methanol-water at a flow rate of 1.0 mL/min. The eluate was monitored using an ultraviolet (UV) detector set at 235 nm. The ratio of peak area of each analyte to IS was used for quantification of plasma samples. Nominal retention times of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide, IS and celecoxib were 15.63, 17.20, 21.66, 24.95, 26.27, 30.24 and 32.22 min, respectively. The standard curve for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib was linear (r2 > 0.999) in the concentration range 0.1-50 microg/mL and for nimesulide (r2 > 0.999) in the concentration range 0.5-50 microg/mL. Absolute recovery was >83% from human plasma for all the analytes and IS. The lower limit of quantification (LLOQ) of nimesulide was 0.5 microg/mL and for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib the LLOQ was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of QC samples, 0.1, 0.3, 15.0 and 40.0 microg/mL (for all analytes except nimesulide), were in the range 2.29-9.37% relative standard deviation (RSD) and 0.69-10.28% RSD, respectively. For nimesulide the inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.5, 1.5, 15.0 and 40.0 microg/mL, were in the range 3.21-7.37% RSD and 0.97-7.06% RSD, respectively. Accuracy in the measurement of QC samples for all analytes was in the range 91.03-106.38% of the nominal values. All analytes including IS were stable in the battery of stability studies, viz. bench top, autosampler and freeze-thaw cycles. Stability of all analytes was established for 21 days at -20 degrees C. The application of the assay in an oral pharmacokinetic study in rats co-administered with celecoxib and valdecoxib is described.     

An automated method for the determination of a new potential antiepileptic agent (CGP 33101) in human plasma using high performance liquid chromatography.

An automated analytical method utilizing laboratory robotics has been developed and validated for quantifying concentrations of a new antiepileptic drug candidate (CGP 33101) in human plasma. The robotic system aliquots the biological sample, adds the internal standard (CGP 23901) and pH 12 buffer, extracts the compounds from the basified matrix into an organic phase (methyl-t-butyl ether:dichloromethane, 2:1) and concentrates the extracts for reversed-phase, high performance liquid chromatographic (HPLC) analysis. The robotic system is directly interfaced with the HPLC system. Separation is achieved on a Hypersil 3 microns C18 column (4.6 x 50 mm) with ultraviolet detection of the analytes at 230 nm. Specificity was demonstrated by the lack of interfering peaks at the retention times for both the drug and internal standard. Recovery and reproducibility assessments indicated good accuracy (overall mean relative recovery of 102.7%) and precision (coefficient of variation of 4.4 to 7.7%) for CGP 33101 over the concentration range of 50-4000 ng/mL. The limit of quantification (LOQ) is 50 ng/mL. The method has been successfully applied to a clinical study in which normal volunteers received single oral doses of 400-1200 mg of this new drug candidate.     

Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1-20 microg/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 microg/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.     

Simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma by HPLC/ESI/MS: platform for the pharmacokinetic evaluation of total panax notoginsenoside, a typical kind of multiple constituent traditional Chinese medicine

A platform for the pharmacokinetic study of multiple constituent traditional Chinese medicine was developed and validated. An HPLC/ESI/MS method was employed for the simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma. After the addition of digoxin as an internal standard (IS), rat plasmas were extracted with n-butanol saturated with pure water and all analytes were separated on a reversed-phased C(18) column with a mobile phase of acetonitrile-water (0.5 mM ammonium chloride) and pumped at a flow rate of 0.2 mL/min. Analytes were determined in a single quadrupole mass spectrometer using an electrospray ionization source. HPLC/ESI/MS was performed in the selected-ion monitoring mode with the chlorinated adducts of molecular ions [M + Cl]( -) at m/z 967.75, 835.80, 981.80, 981.80, 1143.65 and 815.40 for R1, Rg1, Rd, Re, Rb1 and digoxin, respectively. The method showed excellent linearity over the concentration range 3.03-775.00 ng/mL (r(2) = 0.9994) for R1, 4.00-1025.00 ng/mL (r(2) = 0.9991, 0.9988, 0.9991) for Rg1, Rd and Re, respectively, and 2.77-710.00 ng/mL for Rb1 (r(2) = 0.9990). The low limit of quantification was 3.03, 4.00, 4.00, 4.00 and 2.77 ng/mL for R1, Rg1, Rd, Re and Rb1, respectively, with S/N > 10. The intra- and inter-day precisions were below 12.00% and the accuracy was between -2.31 and +4.43% for all analytes. The extract recoveries of analytes were from 67.47 to 94.18%. All analytes were stable in rat plasma after storage for 12 h at ambient temperature, at 4 degrees C for 12 h in the sample pool, at -20 degrees C for 4 weeks and at -20 degrees C for three thaw-freeze cycles. The HPLC/ESI/MS technique provided an excellent method for the simultaneous quantification of R1, Rg1, Rd, Re and Rb1 in rat plasma and was successfully applied to the pharmacokinetic study of a multiple-constituent traditional Chinese medicine, total panax notoginsenoside (Xuesaitong injection).