A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

Simultaneous determination of six C21 steroids of Xiao‐Ai‐Ping injection in rat plasma by LC‐MS/MS

Xiao‐Ai‐Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C₂₁ steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17β‐tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid–liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB‐C₁₈ column (3.5 µm, 150 × 3 mm i.d.) using methanol–water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00–2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r² > 0.99), 10.00–4000.00 ng/mL for 17β‐tenacigenin B and tenacigenoside A (r² > 0.99). Intra‐ and inter‐day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range ?6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Simple determination of plasma ibrutinib concentration using high‐performance liquid chromatography

Ibrutinib is an oral inhibitor of Bruton tyrosine kinase, which is one of the key drugs used for the treatment of chronic lymphocytic leukemia and mantle cell lymphoma. In this study, we aimed to develop a simple method for determining plasma ibrutinib concentration. The analysis required extraction of a 200 μL plasma sample and precipitation of proteins using solid‐phase extraction. Ibrutinib and nilotinib, which was used as an internal standard, were separated using high‐performance liquid chromatography (HPLC) using a mobile phase of acetonitrile–0.5% monopotassium phosphate (KH₂PO₄, pH 3.0; 52:48, v/v) on a Capcell Pack C₁₈ MG II (250 × 4.6 mm) monitored at 260 nm, at a flow rate of 1.0 mL/min. The calibration curve was linear at the plasma concentration range of 10–500 ng/mL with a coefficient of determination (r²) of 0.9999. The coefficients of intra‐day and inter‐day validation were 4.0–6.6 and 2.6–7.7%, respectively. The assay accuracy was ?4.4–8.6%, and the recovery was >84%. This HPLC method coupled with ultraviolet (UV) detection for determining ibrutinib plasma concentration has several advantages such as simplicity and applicability to routine therapeutic drug monitoring at hospital laboratories.     

A rapid and sensitive LC‐MS/MS method for evaluation of the absolute oral bioavailability of a novel c‐Met tyrosine kinase inhibitor QBH‐196 in rats

A sensitive, selective and high‐throughput UPLC‐MS/MS method was developed and validated for the determination of a novel c‐Met tyrosine kinase inhibitor, QBH‐196, in rat plasma. QBH‐196 and its analog BH357 (IS) were extracted from rat plasma using a mixture of dichloromethane and N‐hexane (2:3, v/v). The chromatographic separation was carried out on Phenomenex C₁₈ column (50 × 2.1 mm, 2.6 µm particle size) with a gradient mobile phase of methanol (A) and water containing 0.05% formic acid (B) at a flow rate of 0.2 mL/min. The assay was performed by positive electrospray ionization in multiple reaction monitoring mode using transitions of m/z 622.68 → 140.41 for QBH‐196 and m/z 591.19 →126.21 for the IS, respectively. Good linearity was obtained over the concentration range of 8.0–4000 ng/mL (r² > 0.99) for QBH‐196 and the lower limit of quantification was 8.0 ng/mL in rat plasma. Validations of the method, including its sensitivity, extraction recovery, matrix effect, intra‐ and inter‐day precision, accuracy and stability, were all within acceptable limits. The established method was successfully applied to determine absolute oral bioavailability of QBH‐196 in rats for the first time. The mean oral absolute bioavailability of QBH‐196 was found to be about 40.8% and the elimination half‐life was 40.0 ± 13.1 h. This result suggested that QBH‐196 exhibits good oral absorption in vivo, which is very important for the further development of QBH‐196 as a new oral anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of GDC‐0980 (apitolisib), a small molecule dual phosphatidylinositide 3‐kinase/mammalian target of rapamycin inhibitor in dog plasma by LC‐MS/MS to support a GLP toxicology study

An LC‐MS/MS method for the determination of GDC‐0980 (apitolisib) concentrations in dog plasma has been developed and validated for the first time to support pre‐clinical drug development. Following protein precipitation with acetonitrile, the resulting samples were analyzed using reverse‐phase chromatography on a Metasil AQ column. The mass analysis was performed on a triple quadruple mass spectrometer coupled with an electrospray interface in positive ionization mode. The selected reaction monitoring transitions monitored were m/z 499.3 → 341.1 for GDC‐0980 and m/z 507.3 → 341.1 for IS. The method was validated over the calibration curve range 0.250–250 ng/mL with linear regression and 1/x² weighting. Relative standard deviation (RSD) ranged from 0.0 to 10.9% and accuracy ranged from 93.4 to 113.6% of nominal. Stable‐labeled internal standard GDC‐0980‐d₈ was used to minimize matrix effects. This assay was used for the measurement of GDC‐0980 dog plasma concentrations to determine toxicokinetic parameters after oral administration of GDC‐0980 (0.03, 0.1 and 0.3 mg/kg) to beagle dogs in a GLP toxicology study. Peak concentration ranged from 3.23 to 84.9 ng/mL. GDC‐0980 was rapidly absorbed with a mean time to peak concentration ranging from 1.3 to 2.4 h. Mean area under the concentration–time curve from 0 to 24 hours ranged from 54.4 to 542 ng h/mL. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

Development of a LC‐MS/MS method for simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in rat plasma: application to the herb–drug interaction study of metoprolol and breviscapine

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α‐hydroxymetoprolol (HMT) and O‐desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC‐C₁₈ column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post‐column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42–7000 ng/mL for MET, 2.05‐4200 ng/mL for HMT and 1.95‐4000 ng/mL for DMT. The analytical method was successfully applied to herb–drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of tryptophan,kynurenine, kynurenic acid,xanthurenic acid and 5‐hydroxytryptamine in human plasma by LC‐MS/MS and its application to acute myocardial infarction monitoring

Reliable methods for the determination of tryptophan and its metabolites are vital to the monitoring of biochemical states during the initiation and progression of cardiovascular disease. In the present study, a single‐run liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of tryptophan (Trp) and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), xanthurenic acid (XA) and 5‐hydroxytryptamine (5‐HT), in human plasma. The plasma samples were prepared using a protein precipitation approach, and the chromatographic separation was performed by gradient elution on a C₁₈ column within a total analysis time of 3.5 min. The calibration ranges were 40–20,000 ng/mL for Trp, 4–2000 ng/mL for Kyn, 0.2–100 ng/mL for KA, 0.4–200 ng/mL for XA and 1–500 ng/mL for 5‐HT, and the precision and accuracy were acceptable. The evaluation of recovery and internal standard‐normalized matrix effect proved that the sample preparation approach was effective and the matrix effect could be negligible. The newly developed method was successfully applied to the analysis of plasma samples from healthy individuals and myocardial infarction patients. The findings suggested that the plasma concentrations of Trp, Kyn, 5‐HT as well as the concentration ratios of Kyn/Trp and Trp/5‐HT might serve as biomarkers for the monitoring of acute myocardial infarction.     

Fast quantitative determination of methylphenidate levels in rat plasma and brain ex vivo by MALDI‐MS/MS

This study presents a simple and sensitive high‐throughput matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐MS/MS) method for ex vivo quantification of methylphenidate (MPH) in rat plasma and brain. The common MALDI matrix alpha‐cyano‐4‐hydroxycinnamic acid was used to obtain an optimal dried droplet preparation. For method validation, standards diluted in plasma and brain homogenate prepared from untreated (control) rats were used. MPH was quantified within a concentration range of 0.1–40 ng/ml in plasma and 0.4–40 ng/ml in brain homogenate with an excellent linearity (R² ≥ 0.9997) and good precision. The intra‐day and inter‐day accuracies fulfilled the FDA's ±15/20 critera. The recovery of MPH ranged from 93.8 to 98.5% and 87.2 to 99.8% in plasma and homogenate, respectively. We show that MPH is successfully quantified in plasma and brain homogenate of rats pre‐treated with this drug using the internal standard calibration method. By means of this method, a linear correlation between plasma and brain concentration of MPH in rodents pre‐treated with MPH was detected. The simple sample preparation based on liquid‐liquid extraction and MALDI‐MS/MS measurement requires approximately 10 s per sample, and this significantly reduces analysis time compared with other analytical methods. To the best of our knowledge, this is the first MALDI‐MS/MS method for quantification of MPH in rat plasma and brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a liquid chromatography–isotope dilution mass spectrometry method for quantification of fentanyl in human plasma

Fentanyl is a potent analgesic drug in relieving chronic pain in patients. In this report, we present a simple, reliable and sensitive LC–ID/MS method for the quantification of fentanyl in human plasma. LC‐ID/MS analysis was carried out on a triple quadrupole mass spectrometer operated in positive electrospray ionization multiple‐reaction‐monitoring using the transitions m/z 337.6 → 187.9 for fentanyl and m/z 342.6 → 187.9 for the internal standard (D5‐fentanyl). The calibration curve covered the range 0.02–10 ng/mL. The intra‐ and inter‐batch precision were less than 6.739 and 3.126% for fentanyl and IS, with accuracy from 94.16 to 102.0%. The lower limit of quantification was identifiable and reproducible at 0.02 ng/mL. The validated method offered increased sensitivity and wide linear concentration range. This method was successfully adopted for the evaluation of bioequivalence of two fentanyl transdermal preparations after single dose administration to 20 Chinese pain‐patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of enzalutamide,an anti‐prostate cancer drug,in rat plasma using liquid chromatography–tandem mass spectrometry,and its application to a pharmacokinetic study

This report details a method using liquid chromatography–tandem mass spectrometry (LC‐MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC‐MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple‐reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001–1 µg/mL, as well as an intra‐ and inter‐assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Highly sensitive determination of Schisandrin and Schisandrin B in plasma of rats after administration of Wurenchun (Fructus Schisandrae Chinensis Extracts) preparations by LC‐ESI‐MS/MS

A sensitive and specific method based on liquid chromatography‐tandem mass spectrometry using electrospray ionization (LC‐ESI‐MS/MS) has been developed for the determination of Schisandrin and Schisandrin B in rat plasma. A 100 μL plasma sample was extracted by methyl tert‐butyl ether after spiking the samples with nimodipine (internal standard) and performed on an XTerra®MS‐C₁₈ column (150 mm × 2.1 mm, 3.5 μm) with the mobile phase of acetonitrile–water–formic acid (80:20:0.2, v/v) at a flow rate of 0.2 mL/min in a run time of 8.5 min. The lower limit of quantification of the method was 40 ng/mL for Schisandrin and 20 ng/mL for Schisandrin B. The method showed reproducibility with intra‐day and inter‐day precision of less than 13.8% RSD, as well as accuracy, with inter‐ and intra‐assay accuracies between 93.5 and 107.2%. Finally, the LC‐ESI‐MS/MS method was successfully applied to study the pharmacokinetics of Schisandrin and Schisandrin B in rats after administration of Wurenchun commercial formulations to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

Fat‐soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC–MS/MS method for determination of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple‐quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid–liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02–2 μg/mL for retinol, 0.5–20 μg/mL for α‐tocopherol, 5–100 ng/mL for 25‐hydroxyvitamin D2 and 2–100 ng/mL for 25‐hydroxyvitamin D3. Intra‐ and inter‐assay precision and accuracy of the method were satisfactory. Short‐ and long‐term stabilities of the analytes were determined. The HPLC‐MS/MS method was applied for the determination of the above fat‐soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.