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.     

Rapid liquid chromatography-tandem mass spectrometry method for quantification of ziprasidone in human plasma

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ziprasidone (ZIP) in human plasma was developed. ZIP and N-methyl ziprasidone as internal standard (IS) were extracted from alkalinized plasma using tert- butyl methyl ether. Separation was performed isocratically on a C8 column with 90% acetonitrile containing 2 mmol/L ammonium acetate as a mobile phase with a total run time of 2.5 min. MS/MS transitions of m/z 413 --> 194 and m/z 427 --> 177 of the analyte and internal standard were used for quantification. Confirmatory ions of m/z 413 --> 177 and m/z 427 --> 180 were collected as well. The calibration curve based on peak-area ratio was linear up to at least 200 ng/mL with a detection limit of 0.1 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 5%. The method was successfully applied to the analysis of ZIP in spiked human plasma.     

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 --> 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 --> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1-20.0 ng/mL. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between -2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 +/- 2.73 ng/mL which occurred at 3.50 +/- 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 +/- 12.05 h and 609.3 +/- 122.2 ng . h/mL. LC/MS/MS is a rapid, sensitive and specific method for determining donepezil in human plasma samples.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM02734, a novel antineoplastic agent, in dog plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --> 212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --> 212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (< or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (< or =15% bias) and from 99-109% (< or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.     

Assessment of pharmacokinetics,bioavailability and protein binding of anacetrapib in rats by a simple high‐performance liquid chromatography–tandem mass spectrometry method

Anacetrapib is a potent and selective CETP inhibitor and is undergoing phase III clinical trials for the treatment of dyslipidemia. A simple and sensitive high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the quantification of anacetrapib in rat plasma was developed and validated using an easily purchasable compound, chlorpropamide, as an internal standard (IS). A minimal volume of rat plasma sample (20 μL) was prepared by a single‐step deproteinization procedure with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed using selected reaction monitoring modes at the mass/charge transitions m/z 638 → 283 for anacetrapib and m/z 277 → 175 for IS. The assay was validated to demonstrate the selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. The lower limit of quantification was 5 ng/mL. This LC‐MS/MS assay was successfully applied in the rat plasma protein binding and pharmacokinetic studies of anacetrapib. The fraction of unbound anacetrapib was determined to be low (ranging from 5.66 to 12.3%), and the absolute oral bioavailability of anacetrapib was 32.7%.     

Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionisation

A new method for simultaneous determination of amiloride and hydrochlorothiazide by liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) operated in positive and negative ionization switching mode was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation. The analytes were separated on a Phenomenex Curosil-PFP (250x4.6 mm, 5 microm) column by a gradient elution with a mobile phase consisting of 0.15% formic acid solution containing 0.23% ammonium acetate and methanol pumped at a flow rate of 1.0 mL.min(-1). Rizatriptan was used as the internal standard (IS) for quantification. The determination was carried out on a Waters Quattro-micro triple-quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode using the following transitions monitored simultaneously: positive m/z 230-->171 for amiloride, m/z 270-->158 for rizatriptan, and negative m/z 296-->205 for hydrochlorothiazide. The lower limits of quantification (LLOQs) were 0.1 and 1.0 ng.mL(-1) for amiloride and hydrochlorothiazide, respectively, which were lower than other published methods by using ultraviolet (UV), fluorimetric or mass spectrometric detection. The intra- and inter-day precision and accuracy were studied at three different concentration levels and were always better than 15% (n=5). This simple and robust LC/MS/MS method was successfully applied to the pharmacokinetic study of compound amiloride and hydrochlorothiazide tablets in healthy male Chinese volunteers.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM00104, a novel antineoplastic agent, in mouse, rat, dog, and human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM00104, in mouse, rat, dog, and human plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM00104 was established using PM00104 standards from 0.01-5.0 ng/mL in blank plasma. The selected reaction monitoring (SRM), based on the m/z 692.2 --> 218.2 transition, was specific for PM00104, and that based on the m/z 697.2 --> 218.2 transition was specific for PM00104 ((13)C(2),(2)H(3)) (the internal standard, IS); no endogenous materials interfered with the analysis of PM00104 and IS from blank plasma. The assay was linear over the concentration range 0.01-5.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9981-0.9999. The mean intra-day and inter-day accuracies for all calibration standards (n = 8) ranged from 97-105% (< or =5% bias) in human plasma, and the mean inter-day precision for all calibration standards was less than 8.5%. The mean intra- and inter-day assay accuracy for all quality control (QC) replicates in human plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 96-112% (< or =12% bias) and from 102-105% (< or =5% bias), respectively. The mean intra- and inter-day assay precision was less than 15.0 and 11.8% for all QC levels, respectively. For the QC samples prepared in animal species plasma, the %CV values of the assays ranged from 1.8-8.8% in mouse plasma, from 3.7-13.8% in rat plasma, and from 3.0-7.2% in dog plasma. The assay accuracies ranged from 92-102% (< or =8% bias) for all QC levels prepared in mouse plasma; ranged from 93-106% (< or =7% bias) in rat plasma; and ranged from 95-114% (< or =14% bias) in dog plasma. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies and is currently used to measure PM00104 plasma concentrations to support clinical trials.     

Development of an LC‐MS/MS method for quantification of kadsurenone in rat plasma and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method was developed and validated for the determination of kadsurenone in rat plasma using lysionotin as the internal standard (IS). The analytes were extracted from rat plasma with acetonitrile and separated on a SB‐C₁₈ column (50 × 2.1 mm, i.d.; 1.8 µm) at 30 °C. Elution was achieved with a mobile phase consisting of methanol–water–formic acid (65:35:0.1, v/v/v) at a flow rate of 0.30 mL/min. Detection and quantification for analytes were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 357.1 → 178.1 for kadsurenone, and m/z 345.1 → 315.1 for IS. Calibration curves were linear over a concentration range of 4.88–1464 ng/mL with a lower limit of quantification of 4.88 ng/mL. The intra‐ and inter‐day accuracies and precisions were <8.9%. The LC‐MS/MS assay was successfully applied for oral pharmacokinetic evaluation of kadsurenone using the rat as an animal model. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of docetaxel in rat plasma and its application in the comparative pharmacokinetics of Taxotere and SID530, a novel docetaxel formulation with hydroxypropyl‐β‐cyclodextrin

In this study, a sensitive, simple and reliable method for the quantification of docetaxel in rat plasma was developed and validated using liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The plasma samples were prepared by protein precipitation, and paclitaxel was used as an internal standard (IS). Chromatographic separation was achieved using a Gemini C₁₈ column (2.0 × 150 mm, 5 µm) with a mobile phase consisting of 0.1% formic acid–acetonitrile (30:70, v/v). The precursor–product ion pairs used for multiple reaction monitoring were m/z 808.5 → 527.5 (docetaxel) and m/z 854.2 → 286.5 (IS, paclitaxel). A calibration curve for docetaxel was constructed over the range 1–1000 ng/mL. The developed method was specific, precise and accurate, and no matrix effect was observed. The validated method was applied in a comparative pharmacokinetic study in which two docetaxel formulations, SID530, a new parenteral formulation of docetaxel with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), and Taxotere, were administered to rats at a dose of 5 mg/kg. For SID530 and Taxotere, the mean C₀ values were 1494 and 1818 ng/mL, respectively, and the AUC_last values were 837 and 755 h ng/mL, respectively. These two formulations did not show any statistical differences with regard to the pharmacokinetic parameters, thus establishing that the SID530 and Taxotere products are pharmacokinetically comparable in male rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive and rapid method to determine trimetazidine in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive, and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method, using electrospray ionization, was developed and validated to quantify trimetazidine in human plasma using propranolol hydrochloride as an internal standard (IS). Samples were prepared by solid-phase extraction and analyzed without drying and reconstitution. The analyte and IS were chromatographed on a C18 reversed-phase column under isocratic conditions using 2 mM ammonium acetate (pH 3.5)-acetonitrile (40 + 60, v/v) as the mobile phase with a run time of 2.0 min. Quantitation was done on a triple-quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring mode to detect parent --> product ion (m/z 267.2 --> 181.4) transition. The method was validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, and stability. Linearity in plasma was observed over the concentration range of 1.5-300 ng/mL. Lower limit of quantification achieved was 1.5 ng/mL with precision < 10% using 10 microL injection volume. The mean relative recovery of analyte (97.36%) and IS (99.93%) was consistent and reproducible. Interbatch and intrabatch precision was < 8.0% and the accuracy determined was within +/- 8% in terms of relative error.     

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.     

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.     

Development of a rapid and sensitive liquid chromatography/tandem mass spectrometry method for the determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel anti-cancer agent in rat plasma

A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed to determine 1, 2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel antineoplastic agent, in rat plasma. The analytes were separated on a C18 column with a mobile phase of methanol-water (75:25, v/v) and detected using a triple-quadrupole mass spectrometer in positive mode with the selective reaction monitoring. The characteristic ion dissociation transitions were m/z 603.0 --> 448.9 for derivatized BBSKE and m/z 631.0 --> 476.8 for derivatized internal standard. The assay was linear over a range of 1-1000 ng/mL with a lower limit of quantification of 1 ng/mL. Intra- and inter-day precisions were less than 9.6 and 5.0%, respectively, and the accuracy ranged from -5.2 to 4.0%. The validated method was successfully applied to the characterization of pharmacokinetic profile of BBSKE after oral administration in rats. Cop     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

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.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics,tissue distribution and excretion in the study of anemoside B4, a novel antiviral agent candidate,in rats

A simple, sensitive and reliable LC–MS/MS method was developed and validated for the quantification of anemoside B4, a potential antiviral constituent isolated from Pulsatilla chinensis in rat plasma, tissue, bile, urine and feces. All biological samples were prepared by protein precipitation method, and ginsenoside‐Rg1 was chosen as the internal standard (IS). The analyte and IS were separated using a C₁₈ column (2.1 × 50 mm, 1.8 μm) and a mobile phase consisting of 0.1% formic acid in water (v /v) and acetonitrile running at a flow rate of 0.2 mL/min for 5 min. The multiple reaction monitoring transitions were monitored at m /z 1219.5–749.5 for anemoside B4 and 845.4–637.4 for ginsenoside‐Rg1 in electrospray ionization negative mode. The calibration curve was linear in the range of 10–2000 ng/mL for all biological matrices with a lower limit of quantification of 10 ng/mL. The validated method was successfully applied to a pharmacokinetics, tissue distribution and excretion study. These preclinical data will be beneficial for further development of anemoside B4 in future studies.     

Effect of collision-activated dissociation gas and collision energy on the fragmentation of dipyridamole and its rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric determination in human plasma

The effect of nitrogen as the collision-activated dissociation (CAD) gas on the fragmentation of dipyridamole was investigated in the range of 10-90 eV collision energy. The results support the collision model reported elsewhere, that the degree of ion fragmentation increases with the increasing mass of the collision gas. A simple, sensitive and high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of dipyridamole, a platelet aggregation inhibitor in human plasma, using granisetron as internal standard (IS). The method involved liquid-liquid extraction of the analyte and IS from 0.5 mL human plasma with diethyl ether. The chromatographic separation was achieved under isocratic conditions and the ion transitions for dipyridamole (m/z 505.40 --> 429.60) and the IS (m/z 313.10 --> 138.20) were monitored on a triple quadrupole mass spectrometer, operating in positive ion multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.1-4499.1 ng/mL for dipyridamole. The method was rugged and rapid with a total run time of 1.2 min. It was successfully applied to a pivotal bioequivalence study in 67 healthy human subjects after oral administration of a 75 mg extended release formulation under fasting condition.