Validated liquid chromatography–tandem mass spectrometry method for quantitative determination of dauricine in human plasma and its application to pharmacokinetic study

A highly sensitive and selective LC–MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid–liquid extraction and analyzed by LC–MS/MS. Chromatographic separation was performed on Agilent TC-C₁₈ column with a mobile phase of methanol–water–glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1–200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within ±9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

Rapid and simultaneous determination of tacrolimus (FK506) and diltiazem in human whole blood by liquid chromatography-tandem mass spectrometry: application to a clinical drug-drug interaction study

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI(+)). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7-->768.9 (m/z) for FK506, 415.5-->310.3 (m/z) for DTZ and 809.8-->757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5-200 ng/mL for FK506 and 2-250 ng/mL for DTZ. The recoveries of liquid-liquid extraction method were 58.3-62.6% for FK506 and 50.4-58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.     

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%.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid-liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8-->373.1, m/z 393.2-->147.1 and m/z 264.2-->58.1, respectively. The method was linear over the ranges 1.5-1000ng/mL for dexamethasone palmitate and 2.5-250ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100+/-7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy 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.     

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.     

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.     

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.     

Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: application to a bioequivalence study

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 microL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C(18) (50 mm x 4.6 mm, 5 microm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r>0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Chiral bioanalysis of torcetrapib enantiomers in hamster plasma by normal-phase liquid chromatography and detection by atmospheric pressure chemical ionization tandem mass spectrometry

A highly sensitive and enantioselective assay has been developed and validated for the estimation of torcetrapib (TTB) enantiomers [(+)-TTB and (-)-TTB] in hamster plasma with chiral liquid chromatography coupled to tandem mass spectrometry with an atmospheric pressure chemical ionization interface in the negative-ion mode. The assay procedure involves liquid-liquid extraction of TTB enantiomers and IS (DRL-16126) from 100 microL hamster plasma with acetonitrile. TTB enantiomers were separated using n-hexane:propanol (80:20, v/v) at a flow rate of 0.7 mL/min on a Chiralpak AD column. The MS/MS ion transitions monitored were 599.2-->340.2 for TTB and 623.2-->298.1 for IS. Absolute recovery was found to be between 64 and 68% for TTB enantiomers and >100% for IS. The standard curves for TTB enantiomers were linear (r(2)>0.995) in the concentration range 5-2500 ng/mL for each enantiomer with an LLOQ of 5 ng/mL for each enantiomer. The inter- and intra-day precisions were in the range of 10.5-12.4 and 9.15-11.5% and 3.75-12.9 and 5.16-12.5% for (+)-TTB and (-)-TTB, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 91.3-105 and 88.6-111% for (+)-TTB and (-)-TTB, respectively. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (-)-TTB.     

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.     

Determination of the quaternary ammonium compound trospium in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A highly sensitive, specific and evaporation free SPE extraction, LC–MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M⁺] cations, m/z 392–164 for trospium and m/z 400–172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05–10 ng/mL (r > 0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.     

Development and validation of a highly rapid and sensitive LC-MS/MS method for determination of SZ-685C, an investigational marine anticancer agent, in rat plasma--application to a pharmacokinetic study in rats

A sensitive and rapid method was developed and validated for the quantitative analysis of the novel anticancer agent SZ-685C in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in negative ion mode in order to support the following pre-clinical and clinical studies. SZ-685C and the internal standard (IS, emodin) were extracted from rat plasma by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on an Elite Hypersil BDS C18 column (100 mm × 2.1 mm i.d., 3 μm). Elution was carried out using methanol/acetonitrile/2mM ammonium formate (pH 4) (80:15:5 (v/v/v)) at a flow-rate of 0.3 mL/min with a run time of 2.5 min. This assay was linear over a concentration range of 50-10,000 ng/mL with a lower limit of quantification of 50 ng/mL. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 100, 1000 and 7500 ng/mL. These results indicate that the method was efficient with a short run time and acceptable accuracy, precision and sensitivity. This method was successfully applied to explore pharmacokinetics of SZ-685C in rats after oral and intravenous administration of this agent. The absolute bioavailability is about 54.8-66.8% and the t(1/2) is 5.7-9.2h, these results provide basic information for further comprehensive pre-clinical research.     

Stability evaluation and sensitive determination of antiviral drug,valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography–tandem mass spectrometry method

A simple, sensitive and high throughput liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of valacyclovir and acyclovir in human plasma using fluconazole as internal standard (IS). The method involved solid phase extraction of the analytes and IS from 0.5 mL human plasma with no reconstitution and drying steps (direct injection of eluate). The chromatographic separation was achieved on a Gemini C18 analytical column using isocratic mobile phase, consisting of 0.1% formic acid and methanol (30:70 v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for valacyclovir (m/z 325.2 → 152.2), acyclovir (m/z 226.2 → 152.2) and IS (m/z 307.1 → 220.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.0–1075 ng/mL and 47.6–10225 ng/mL for valacyclovir and acyclovir respectively. The mean recovery of valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was consistent and reproducible. The bench top stability of valacyclovir and acyclovir was extensively evaluated in buffered and unbuffered plasma. It was successfully applied to a bioequivalence study in 41 healthy human subjects after oral administration of 1000 mg valacyclovir tablet formulation under fasting condition.     

Simple quantification of lansoprazole and rabeprazole concentrations in human serum by liquid chromatography/tandem mass spectrometry

A rapid, simple and highly sensitive method was developed for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 microL of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1mM ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/mL. A good linear response was observed for each analyte (from 0.25 ng to 2.5 microg/mL). This method was useful for therapeutic drug monitoring and pharmacokinetic studies.     

Simultaneous quantitation of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in human plasma by liquid chromatography-tandem mass spectrometry and pharmacokinetics evaluation of "SHEN-FU" injectable powder

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of "SHEN-FU" injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/z 646.3-->586.0 for AC; m/z 632.4-->573.1 for MA; m/z 616.2-->556.1 for HA; m/z 604.2-->104.8 for BAC; m/z 590.1-->104.8 for BMA; m/z 574.1-->104.8 for BHA; m/z 585.2-->161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C(18) column (1.7 microm, 2.1 mm x 100 mm) and a gradient elution of methanol and 0.1% formic acid-water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient (r(2)) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of "SHEN-FU" injectable powder in phase I clinical trial.     

Development and validation of a rapid 96-well format based liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analysis method for ondansetron in human plasma

A rapid and sensitive LC-MS/MS method for the quantification of ondansetron was developed and validated. The plasma samples were treated by a semi-automated liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Ondansetron and the internal standard (IS) granisetron were analyzed by combined reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reactions monitoring (MRM). The statistical evaluation for this method reveals excellent linearity, accuracy and precision values for the range of concentrations 0.25-40.0 ng/mL. The proposed method enabled the reliable determination of ondansetron in bioequivalence studies after per os administration of a 4 or 8 mg tablet.