Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo((R)) C(18) column and the mobile phase was composed of acetonitrile:H(2)O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1-->308.3, 359.0-->308.3, 359.0-->111.2 and 347.0-->312.0 for triazolam, alpha-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05ng/mL for triazolam and 0.1ng/mL for alpha-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was -8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.     

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

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

Determination of levocetirizine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

We describe a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid-liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389>201 for I and m/z 502>467 for II. The limit of quantification and the dynamic range achieved were 0.5ng/mL and 0.5-500.0ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48h after oral administration of 5mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.     

Simultaneous determination of glycyrrhizin, a marker component in radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and alpha-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823--> 453 for GLY, 471--> 177 for GA and 752--> 456 for IS. The LC-MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.     

Sensitive quantification of rifaximin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-mass spectrometry method (LC-MS/MS) for the quantitative determination of rifaximin in human plasma was developed and validated. In the developed procedure, metoprolol was added to human plasma as an internal standard (IS) and acetonitrile was used to precipitate the plasma proteins before LC-MS/MS analysis. Chromatographic separation was obtained on a RESTEK Pinnacle C18 column (50 mm x 2.1mm, 5 microm) with a mobile phase consisted of ammonium acetate solution (15 mM, pH 4.32) as buffer A and methanol as mobile phase B. Quantification was performed in positive mode using multiple reaction monitoring (MRM) of the transitions m/z 786.1-->754.1 for rifaximin and m/z 268.3-->116.1 for the IS. The assay has been validated over the concentration range of 0.5-10 ng/ml (r=0.9992) based on the analysis of 0.2 ml of plasma. The assay accuracy was between 98.2% and 109%. The within-day and between-day precision was better than 3.9% and 8.9% at three concentration levels. The freeze-thaw stability was also investigated and it was found that both rifaximin and the IS were quite stable. This method provides a rapid, sensitive, specific and robust tool for the quantitative determination of rifaximin in human plasma, which is especially useful for the pharmacokinetic study of rifaximin.     

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC-MS/MS--application to a preclinical pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quanti?cation (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

Determination of rimonabant in human plasma and hair by liquid chromatography-mass spectrometry

Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50muL of plasma or 10mg of hair using diethylether. Chromatography was performed on a 150mmx2.1mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/z 463.1 for rimonabant and m/z 555.1 for IS. The most intense product ion of rimonabant (m/z 380.9) and IS (m/z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85-115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at -20 degrees C. This simple and robust LC-MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.     

Sensitive and specific LC-ESI-MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma

A LC-MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 microL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.     

High-throughput determination of fudosteine in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178-->91 and m/z 284-->91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r>0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.     

Development and validation of a liquid chromatography-tandem mass spectrometric assay for pitavastatin and its lactone in human plasma and urine

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with electrospray ionization (ESI) was developed and validated for the simultaneous determination of pitavastatin and its lactone in human plasma and urine. Following a liquid-liquid extraction, both the analytes and internal standard racemic i-prolact were separated on a BDS Hypersil C(8) column, using methanol-0.2% acetic acid in water (70: 30, v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 422.4-->m/z 290.3 for pitavastatin, m/z 404.3-->m/z 290.3 for pitavastatin lactone and m/z 406.3-->m/z 318.3 for the internal standard, respectively. Linear calibration curves of pitavastatin and its lactone were obtained in the concentration range of 1-200 ng/ml, with a lower limit of quantitation of 1 ng/ml. The intra- and inter-day precision values were less than 4.2%, and accuracies were between -8.1 and 3.5% for both analytes. The proposed method was utilized to support clinical pharmacokinetic studies of pitavastatin in healthy subjects following oral administration.     

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

A sensitive LC-MS/MS method for determination of levamisole in human plasma: application to pharmacokinetic study

A sensitive and rapid LC-MS/MS method was developed and validated for the determination of levamisole in human plasma. The assay was based on liquid-liquid extraction of analytes from human plasma with ethyl ether. Chromatographic separation was carried on an Agilent HC-C(8) column (150 mm × 4.6 mm, 5 μm) at 40°C, with a mobile phase consisting of acetonitrile-10 mM ammonium acetate (70:30, v/v), a flow rate of 0.5 mL/min and a total run time of 6 min. Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 205.1→178.2 for levamisole, and m/z 296.1→264.1 for mebendazole (internal standard). The assay was linear over a concentration range of 0.1-30 ng/mL with a lower limit of quantification of 0.1 ng/mL. The coefficient of variation of the assay precision was less than 8.5%. The assay was successfully used to analyze human plasma samples in a pharmacokinetic study where levamisole was administered as a liniment.     

Development and validation of a LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics

A new LC-ESI-MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C(18) 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH(3)COO](-)→179 and m/z 415 [M+CH(3)COO](-)→179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5-1000 ng/mL (n=7, r(2)≥0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N≥3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.     

Determination of memantine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of memantine (I) in human plasma is presented. Sample preparation consisted of the addition of amantadine (II) as internal standard (IS), liquid-liquid extraction in basic conditions using a mixture of diethyl ether-chloroform (7:3, v/v) as extracting solvent, followed by centrifugation, solvent evaporation and sample reconstitution in methanol. Both I and II (internal standard) were analyzed using a C18 column and a mobile phase composed of methanol-water-formic acid (80:20:0.1, v/v/v). Eluted compounds were monitored using positive mode electrospray (ES) tandem mass spectrometry. The analyses were carried out by selected reaction monitoring (SRM) using the parent to daughter combinations of m/z 180>163 (memantine) and m/z 152>135 (amantadine). The peak areas from the analyte and IS were used for quantification of I. The achieved limit of quantification (LOQ) was 0.1 ng/mL; the assay exhibited a linear dynamic range of 0.1-50.0 ng/mL with a determination coefficient (r2) of at least 0.98. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of samples taken up to 320 h after oral administration of 20mg (two 10mg capsules) of I in healthy volunteers demonstrated the applicability to bioequivalence studies.     

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.     

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid-liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 x 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/z 854.4--> 286.2 transition of paclitaxel is measured versus that of the m/z 808.5--> 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2-500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (< or = 15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy < or = 15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.