Validation and application of a liquid chromatography‐tandem mass spectrometric method for the determination of GDC‐0152 in human plasma using solid‐phase extraction

A liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0152 in human plasma to support clinical development. The method consisted of a solid‐phase extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₇‐GDC‐0152 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 0.02–10.0 ng/mL for GDC‐0152. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 99.3% with a precision (%CV) of 13.9%. For quality control samples at 0.0600, 2.00 and 8.00 ng/mL, the between‐run %CV was ≤8.64. Between‐run percentage accuracy ranged from 98.2 to 99.6%. GDC‐0152 was stable in human plasma for 363 days at ?20°C and for 659 days at ?70°C storage. GDC‐0152 was stable in human plasma at room temperature for up to 25 h and through three freeze–thaw cycles. In whole blood, GDC‐0152 was stable for 12 h at 4°C and at ambient temperature. This validated LC‐MS/MS method for determination of GDC‐0152 was used to support clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography‐triple quadrupole mass spectrometric method for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma and its application to microdose clinical trial

A liquid chromatography–triple quadrupole mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma to support a microdose clinical trial. The method consisted of a liquid–liquid extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₃‐AGM‐130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10–2000 pg/mL for AGM‐130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between‐run accuracy ranged from 98.1 to 101.0%. AGM‐130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM‐130 was also stable in human plasma at room temperature for 6 h and through three freeze–thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC‐MS/MS method for determination of AGM‐130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous quantification of metformin and glipizide in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A selective, sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one‐step protein precipitation of 200 μL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol–10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10–656 ng/mL for metformin and 2.55–408 ng/mL for glipizide. The relative standard deviation of intra‐day and inter‐day precision was below 10% and the relative error of accuracy was between ?7.0 and 4.6%. The presented HPLC‐MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of two novel highly lipophilic anticancer drug candidates in rat plasma and tissues

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) method was developed and validated for determination of two highly lipophilic anticancer drug candidates, LG1980 and GH501, in rat plasma and tissues (liver, kidney and femur bones). LG1980 and GH501 were extracted from rat plasma and tissue homogenates using liquid–liquid extraction. The method provided a linear range of 1.0–200.0 ng/mL for GH501 in plasma and LG1980 in plasma and liver. For both analytes in other tissue homogenates the linear range was 2.0–400.0 ng/mL. The method was validated with precision within 15% relative standard deviation, accuracy within 15% relative error and a consistent recovery. This method has been successfully applied in two preclinical studies for LG1980 and GH501 to determine their concentrations in rat plasma, liver, kidney and bone over 24 h after intravenous injection of compounds.     

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

Rapid LC‐MS/MS determination and pharmacokinetic application of linarin in rat plasma

A sensitive, selective and robust liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the rapid determination of linarin in rat plasma. Separation of the analyte and warfarin as internal standard (IS) from 100 μL rat plasma was carried out by simple protein precipitation treatment. Chromatographic separation of the analyte was performed on a Diamonsil® C₁₈ column (150 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of methanol–0.5% formic acid (80:20, v/v). The flow rate was 0.6 mL/min and the total run time was not more than 4.0 min. The method was validated over a wide dynamic concentration range of 1.00–1000 ng/mL for linarin. The precision and accuracy values for linarin met the acceptance criteria according to US Food and Drug Administration guidelines. Linarin was stable in the stability studies including a long‐term test (?80°C for 43 days), a short‐term test (ambient for 2 h and autosampler for 8 h) and three freeze–thaw cycles (?80–25°C). The developed assay method was applied to the pharmacokinetic study in rats after a single intramuscular administration of 713 µg/kg linarin. Copyright © 2012 John Wiley & Sons, Ltd.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC‐MS/MS

A fast, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio‐active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile–0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050–0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio‐active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.     

A supported liquid extraction LC–MS/MS method for determination of concentrations of GDC‐0425, a small molecule Checkpoint kinase 1 inhibitor,in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC‐0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 μL) and the resulting samples were analyzed using reverse‐phase chromatography and mass spectrometry coupled with a turbo‐ionspray interface. The mass analysis of GDC‐0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00–1000 ng/mL using linear regression and 1/x² weighting. Within‐run relative standard deviation ranged from 0.8 to 5.1%, while between‐run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within‐run and from 94.0 to 100.0% of nominal for between‐run. Overall extraction recovery was 87.4% for GDC‐0425 and 87.9% for GDC‐0425‐d₉. Stability of GDC‐0425 was established in human plasma for 374 days at ?20 and ?70 °C and established in reconstituted sample extracts for 88 h when stored at 2–8 °C. Stable‐labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC‐0425 in cancer patients.     

Validated LC–ESI–MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study

A liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X‐Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC–MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A–D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r²) was >0.99 for all homologs with accuracy 90.7–107.4% and precision 0.74–15.1%. The recovery of homologs was 78.6–90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench‐top for 6 h, for up to three freeze–thaw cycles, in the injector for 24 h and for 1 month at ?80 ° C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.     

Validation and application of a liquid chromatography-tandem mass spectrometric method for the determination of GDC-0834 and its metabolite in human plasma using semi-automated 96-well protein precipitation

A liquid chromatographic–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0834 and its amide hydrolysis metabolite (M1) in human plasma to support clinical development. The method consisted of semi‐automated 96‐well protein precipitation extraction for sample preparation and LC‐MS/MS analysis in positive ion mode using TurboIonSpray® for analysis. D6‐GDC‐0834 and D6‐M1 metabolite were used as internal standards. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 1 – 500 ng/mL for both GDC‐0834 and M1 metabolite. The accuracy (percentage bias) at the lower limit of quantitation (LLOQ) was 5.20 and 0.100% for GDC‐0834 and M1 metabolite, respectively. The precision (CV) for samples at the LLOQ was 3.13–8.84 and 5.20–8.93% for GDC‐0834 and M1 metabolite, respectively. For quality control samples at 3, 200 and 400 ng/mL, the between‐run CV was ≤7.38% for GDC‐0834 and ≤8.20% for M1 metabolite. Between run percentage bias ranged from ?2.76 to 6.98% for GDC‐0834 and from ?6.73 to 2.21% for M1 metabolite. GDC‐0834 and M1 metabolite were stable in human plasma for 31 days at ?20 and ?70°C. This method was successfully applied to support a GDC‐0834 human pharmacokinetic‐based study. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated LC‐ESI‐MS/MS method for simultaneous quantitation of felodipine and metoprolol in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and specific LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of felodipine (FDP) and metoprolol (MPL) in rat plasma (50 μL) using phenacetin as an internal standard (IS) as per the FDA guidelines. Liquid–liquid extraction method was used to extract the analytes and IS from rat plasma. The chromatographic resolution of FDP, MPL and IS was achieved with a mobile phase consisting of 0.2% formic acid in water–acetonitrile (25:75, v/v) with a time program flow gradient on a C₁₈ column. The total chromatographic run time was 4.0 min and the elution of FDP, MPL and IS occurred at 1.05, 2.59 and 1.65 min, respectively. A linear response function was established for the range of concentrations 0.59–1148 and 0.53–991 ng/mL for FDP and MPL, respectively, in rat plasma. The intra‐ and inter‐day accuracy and precision values for FDP and MPL met the acceptance as per FDA guidelines. FDP and MPL were stable in battery of stability studies viz., bench‐top, auto‐sampler and freeze–thaw cycles. The validated assay was applied to a pharmacokinetic study in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Study the pharmacokinetics of AV-45 in rat plasma and metabolism in liver microsomes by ultra-performance liquid chromatography with mass spectrometry

An ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed and validated to determine AV‐45 in rat plasma. After the addition of the internal standard benzophenone, plasma samples were pretreated by protein precipitation. Chromatographic separation was achieved on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 µm) by gradient elution at a flow rate of 0.4 mL/min. Detection of analytes and internal standard (IS) was done by tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring mode. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect and stability study. The calibration curve showed good linearity over the concentration range 2.00–1000 ng/mL for AV‐45. Intra‐ and inter‐day precisions were less than 7.6%, and accuracy ranged from 100.6 to 107.8%. There was no matrix effect. The validated method was successfully applied to a pharmacokinetic study of AV‐45 in rats. Additionally, the metabolism of AV‐45 in rat liver microsomes was also studied by ultra‐performance liquid chromatography combined with time‐of‐flight mass spectrometry (UPLC/TOF‐MS). With the help of chromatographic behavior and accurate mass measurements, the metabolites were characterized. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of aripiprazole in rat plasma and brain using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Aripiprazole is an important antipsychotic drug. A simple, sensitive and rapid ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC‐ESI‐MS/MS) method was developed and validated for the simultaneous quantification of this compound in rat plasma and brain homogenate. The analyte was extracted from rat plasma and brain homogenate using a weak cation exchange mixed‐mode resin‐based solid phase extraction. The compound was separated on an Agilent Eclipse Plus C₁₈ (2.1 × 50 mm, 1.8 µm) column using a mobile phase of (A) 0.1% formic acid aqueous and (B) acetonitrile with gradient elution. The analyte was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, recoveries and stability were determined. The LOQ was 0.5 ng/mL for aripiprazole in plasma and 1.5 ng/g in brain tissue. The MS response was linear over the concentration range 0.5–100 ng/mL for aripiprazole in plasma and 1.5–300 ng/g in brain tissue. The precision and accuracy for intra‐day and inter‐day were better than 14%. The relative and absolute recoveries were above 72% and the matrix effects were low. This validated method was successfully used to quantify the rat plasma and brain tissue concentrations of the analyte following chronic treatment with aripiprazole. Copyright © 2012 John Wiley & Sons, Ltd.     

A high-sensitivity LC-MS/MS method for the determination of 4-methyl-piperazine-1-carbodithioc acid 3-cyano-3, 3-diphenylpropyl ester hydrochloride in rat plasma and its application to a pharmacokinetics study

4‐Methyl‐piperazine‐1‐carbodithioc acid 3‐cyano‐3, 3‐diphenylpropyl ester hydrochloride (TM208), a newly synthesized anticancer compound, was quantified using liquid chromatography–tandem mass spectrometry (LC‐MS/MS) for the first time. A simple, rapid and sensitive assay method using propranolol as internal standard (IS) after one‐step precipitation with acetonitrile was developed and validated to determine TM208 in rat plasma. Separation was achieved on a reverse‐phase C₁₈ column with a mobile phase composed of methanol–water (pH4.0) containing 5 m m ammonium acetate in gradient elution mode. A triple quadrupole tandem mass spectrometer with electrospray ionization source was used as detector and operated by multiple reaction monitoring in the positive ion mode. Calibration curves were linear (r > 0.99) between 0.2 and 500 ng/mL. The quantitative limit was 0.2 ng/mL; reliable precision and accuracy were validated by relative standard deviation values in the range 3.44–13.15% and relative error values between ?0.58 and ?9.78%. The method was successfully applied to preclinical pharmacokinetic studies of TM208. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification and pharmacokinetic study of entrectinib in rat plasma using ultra‐performance liquid chromatography tandem mass spectrometry

To characterize the preclinical plasma pharmacokinetics of entrectinib, a reproducible and precise assay is necessary. In this study, we developed and validated a simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the measurement of entrectinib using carbamazepine as the internal standard in rat plasma. Sample preparation was a simple protein precipitation with acetonitrile, then entrectinib was eluted on an Acquity UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 μm) using a gradient elution with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Detection was achieved using multiple‐reaction monitoring in positive ion electrospray ionization mode. The method showed good linearity over the concentration range of 1–250 ng/mL (r² > 0.9951). The intra‐ and inter‐day precision was determined with the values of 6.3–12.9 and 2.6–6.9%, respectively, and accuracy values of 0.5–11.6%. Matrix effect, extraction recovery, and stability data all met the acceptance criteria of US Food and Drug Administration guidelines for bioanalytical method validation. The method was successfully applied to a pharmacokinetic study. In this study, we developed the complete validated method for the quantification of entrectinib in rat plasma.     

Qualification and application of a liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the determination of trastuzumab in rat plasma

An liquid chromatography–quadrupole time‐of‐flight (QqTOF) mass spectrometric method was developed for the determination of humanized or human monoclonal antibodies in rat plasma at the early drug discovery stage. Trastuzumab was used as a model monoclonal antibody. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC‐TOF‐MS/MS analysis of specific signature peptides in the positive ion mode using electrospray ionization for analysis. A stable isotope‐labeled signature peptide was also used as internal standard. A quadratic regression (weighted 1/concentration²), with an equation y = ax² + bx + c, was used to fit calibration curves over the concentration range of 0.500–100 µg/mL for trastuzumab. Samples from a pharmacokinetic study in rat were analyzed by this qualified LC‐TOF‐MS/MS method and concentrations were compared with those generated by enzyme linked immunosorbent assays method. The LC‐TOF‐MS/MS method was accurate and precise, with quantitative results comparable with those of ELISA. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. Within‐run accuracy ranged from 1.53 to 9.20% with precision values ≤10.29%. This LC‐TOF‐MS/MS method approach could be used as a complementary method for humanized or human monoclonal antibodies at the early drug discovery stage. Copyright © 2015 John Wiley & Sons, Ltd.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry for pharmacokinetic study

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC‐MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 μm) was used for hydrophilic‐based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor–product ion pairs for multiple‐reaction monitoring were m /z 409.1 → 391.0 for eurycomanone and m /z 449.1 → 303.0 for IS. The linear range was 2–120 ng/mL. The intra‐ and inter‐day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The C _max and AUC_0–t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.