A dynamic multiple reaction monitoring strategy to develop and optimize targeted metabolomics methods: Analyzing bile acids in capecitabine-induced diarrhea.

OBJECTIVE: We sought to develop and optimize a targeted bile acids (BAs) metabolomics method based on a dynamic multiple reaction monitoring (dMRM) strategy and explored the dynamic alterations of BAs in diarrhea induced by capecitabine in a mouse model. METHOD: The targeted metabolomics method was developed using an Agilent 6460A triple quadrupole mass spectrometer, and 41 types of BAs were monitored in negative ionization mode. The mass spectrometer detection was optimized using dMRM to enhance the responses, separation, and peak shape and to shorten the analysis time. A mouse model of diarrhea was established by multiple administration of capecitabine, and plasma samples were collected at baseline and the end of drug administration for subsequent BAs analysis. RESULTS: The targeted BA metabolomics method achieved shorter chromatographic separation time (10 min) for 41 BAs, with good peak shapes and response increases of 3- to 10-fold after application of dMRM. The mouse model of capecitabine-induced diarrhea was established, and the three BAs 23-norcholic acid, isolithocholic acid, and isodeoxycholic acid in the baseline samples contributed the most to differentiating mice with diarrhea from those without diarrhea. For mice that ultimately developed diarrhea, apocholic acid, isodeoxycholic acid, and 7-ketodeoxycholic acid exhibited the largest change in concentrations compared with their baseline concentrations. CONCLUSION: The dMRM strategy has obvious advantages compared with common MRM. The results in model mice showed that a differentiated profile of BAs in the baseline may indicate biomarkers of diarrhea induced by capecitabine, and disturbed homeostasis may explain the metabolomic mechanism of diarrhea occurrence.

Simultaneous and Rapid Determination of Six Tyrosine Kinase Inhibitors in Patients with Non-Small Cell Lung Cancer Using HPLC-MS/MS.

OBJECTIVE: To develop a new method for quantitatively analyzing six tyrosine kinase inhibitors (gefitinib, erlotinib, icotinib, afatinib, osimertinib, and crizotinib) used in the treatment of non-small cell lung cancer (NSCLC) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The analytes were detected in the selected reaction monitoring mode on a triple quadrupole mass spectrometer with the positive ionization mode. Carbamazepine was utilized as the internal standard. The pretreatment of the plasma sample was completed based on protein precipitation with acetonitrile, and the analytes were separated on an Agilent Zorbax SB-C18 reversed-phase column (2.1 mm × 100 mm, 3.5 µm, Agilent, USA) using gradient elution. The mobile phase consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). The flow rate was 0.3 mL/min, and the injection volume was 5 µL. The column temperature was set and maintained at 35°C. RESULTS: The calibration curves were linear over the range from 5.0 to 1000.0 ng/mL for gefitinib, crizotinib, and osimertinib; from 50.0 to 4000.0 ng/mL for icotinib and erlotinib; and from 5.0 to 400.0 ng/mL for afatinib. Linear correlation coefficients were >0.990 for all regression curves. The intra- and interday accuracy and precision of the method were within ±15.0% and not more than 15.0%, respectively. The mean recovery of all the analytes ranged from 70.18% to 110.76%, the matrix effect was from 88.85% to 127.58%, and stability was within ±15.0%. CONCLUSION: This newly developed method was sensitive, simple, and robust and could be used in therapeutic drug monitoring of six tyrosine kinase inhibitors in NSCLC patients.

Simultaneous Quantification of Methotrexate and Its Metabolite 7-Hydroxy-Methotrexate in Human Plasma for Therapeutic Drug Monitoring.

OBJECTIVE: To establish and validate a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of methotrexate (MTX) and its major metabolite 7-hydroxy-methotrexate (7-OH-MTX) in human plasma. METHOD: The chromatographic separation was achieved on a Zorbax C18 column (3.5 µm, 2.1 × 100 mm) using a gradient elution with methanol (phase B) and 0.2% formic acid aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 3.5 min. Mass spectrometry detection was performed in a triple-quadruple tandem mass spectrometer under positive ion mode with the following mass transitions: m/z 455.1/308.1 for MTX, 471.0/324.1 for 7-OH-MTX, and 458.2/311.1 for internal standard. The pretreatment procedure was optimized with dilution after one-step protein precipitation. RESULTS: The calibration range of methotrexate and 7-OH-MTX was 5.0-10000.0 ng/mL. The intraday and interday precision and accuracy were less than 15% and within ±15% for both analytes. The recovery for MTX and 7-OH-MTX was more than 90% and the matrix effect ranged from 97.90% to 117.60%. CONCLUSION: The method was successfully developed and applied to the routine therapeutic drug monitoring of MTX and 7-OH-MTX in human plasma.

UHPLC-MS/MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients.

Carbamazepine (CBZ) was considered as the drug of choice in the treatment for various forms of epilepsy, yet the non-negligible adverse effects of CBZ suspend as the major concern for rational and efficient clinical medication. This study developed a method for the simultaneous determination of CBZ and its seven metabolites acridine (AI), iminostilbene (IM), acridone (AO), carbamazepine­10,11­epoxide (CBZ­EP), 10,11­dihydro­10,11­trans­dihy­droxy­carbamazepine (CBZ­DiOH), 2­hydroxycarbamazepine (2­OH­CBZ) and 3­hydroxycarbamazepine (3­OH­CBZ) with phenacetin as the internal standard (IS) using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma samples were purified with the one-step 96-well protein precipitation plate. Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 (1.8 µm, 2.1 mm × 50 mm) chromatography column carrying the mobile phrase of acetonitrile and 0.1% formic acid in a gradient elution at a flow rate 0.3 mL/min. Agilent G6460 MS/MS in the positive MRM mode using electrospray ionization (ESI) was applied for quantification of target compounds. CBZ and its seven metabolites showed good linear correlation coefficient (r > 0.990). Intra-day and inter-day precision (CV) were no >15%. This method was successfully accomplished within 5 min which was especially applicable for therapeutic drug monitoring (TDM) of CBZ and its metabolites in epileptic patients, and it provided insights for toxicological studies to achieve a rational and effective individualized administration in clinical practice.

One-Step Solid Extraction for Simultaneous Determination of Eleven Commonly Used Anticancer Drugs and One Active Metabolite in Human Plasma by HPLC-MS/MS.

Therapeutic drug monitoring for anticancer drugs could timely reflect in vivo drug exposure, and it was a powerful tool for adjusting and maintaining drug concentration into a reasonable range, so that an enhanced efficacy and declined adverse reactions could be achieved. A liquid chromatography-tandem mass spectrometry method had been developed and fully validated for simultaneous determination of paclitaxel, docetaxel, vinblastine, vinorelbine, pemetrexed, carboplatin, etoposide, cyclophosphamide, ifosfamide, gemcitabine, irinotecan, and SN-38 (an active metabolite of irinotecan) in human plasma from cancer patients after intravenous drip of chemotherapy drugs. One-step solid-phase extraction was successfully applied using an Ostro sample preparation 96-well plate for plasma samples pretreated with acetonitrile containing 0.1% formic acid. Chromatographic separation was achieved on an Atlantis T3-C18 column (2.1 × 100 mm, 3.0 µm) with gradient elution using a mobile phase consisting of acetonitrile and 10 mM ammonium acetate plus 0.1% formic acid in water, and the flow rate was 0.25 mL/min. The Agilent G6410A triple quadrupole liquid chromatography-mass spectrometry system was operated under the multiple reaction monitoring mode with an electrospray ionization in the positive mode. Linear range was 25.0-2500.0 ng for paclitaxel, 10.0-1000.0 ng for docetaxel and SN-38, 100.0-10000.0 ng for vinorelbine and pemetrexed, 10.0-10000.0 ng for vinblastine and irinotecan, 1.0-1000.0 ng for cyclophosphamide and ifosfamide, 50.0-5000.0 ng for carboplatin, etoposide, and gemcitabine. Linearity coefficients of correlation were >0.99 for all analytes. The intraday and interday accuracy and precision of the method were within ±15.0% and less than 15%. The mean recovery and matrix effect as well as stability of all the analytes ranged from 56.2% to 98.9% and 85.2% to 101.3% as well as within ±15.0%. This robust and efficient method was successfully applied to implement therapeutic drug monitoring for cancer patients in clinical application.

Comparison of LC-MS/MS vs chemiluminescent microparticle immunoassay in measuring the valproic acid concentration in plasma of epilepsy patients in a new perspective.

OBJECTIVES: This study was designed to compare the performance of LC-MS/MS with chemiluminescent microparticle immunoassay (CMIA) for determination of VPA in epilepsy patients in the perspective of metabolites' hepatotoxicity. METHOD: Samples were collected and then analyzed using both LC-MS/MS and CMIA. A LO2 cells (normal human hepatic cells) experiment was carried out to confirm VPA metabolites' hepatotoxicity using AST(Aspertate Aminotransferase, AST), ALT(Alanine aminotransferase, ALT) and LDH(lactate dehydrogenase, LDH) in supernate as index. RESULTS: The regression equation analysis showed as LC-MS/MS=1.0094CMIA-1.8937, with the concordance correlation coefficient of 0.9700, and the CUSUM test proved no significant deviation from linearity (P>.05). CMIA compared to LC-MS/MS gave a positive bias of 1.2 µg/mL. In LO2 experiment, VPA and its metabolites groups showed an obvious increment of AST, ALT, and LDH in supernate. CONCLUSION: The LC-MS/MS is largely consistent with the CMIA in analytical time and quantification ability for VPA, but the LC-MS/MS can simultaneously determinate VPA and its metabolites in plasma, and is also a higher cost-efficiency method in consideration of toxic metabolites monitoring. The overestimation of VPA by CMIA showed no clinical significance. The metabolites 3-OH-VPA and 5-OH-VPA damage the LO2 cells and the results presented a statistical significance (P<.05). It is vital to monitor the metabolites' concentrations for VAP's clinical safety application, and now is the occasion that laboratory and clinic consider the LC-MS/MS method as a more advantageous alternative to CMIA method in therapeutic monitoring of VPA.

Bioequivalence of two oseltamivir formulations in healthy Chinese volunteers
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BACKGROUND: The aim of this study was to compare the bioavailability of a new generic formulation of oseltamivir 75-mg capsule (test) and a branded formulation Tamiflu® (reference) to meet regulatory criteria for marketing the test product in healthy Chinese male volunteers. METHODS: This single-dose, randomized-sequence, open-label, two-period crossover study was conducted in fasted healthy Chinese male volunteers, who first received a single oral dose of the test or reference formulation with a 7-day washout period, and then the alternative formulation. The study drug was administered after a 10-hour overnight fast. Blood samples were collected at baseline and at 0.25, 0.5, 0.75, 1.0, 1.5, 2, 4, 6, 8, 10, 12, 24, and 36 hours after administration of the study drug. Plasma concentrations of the parent oseltamivir and its metabolite oseltamivir carboxylate were determined using an LC-MS/MS method. The formulations were considered bioequivalent if the 90% confidence intervals (CIs) for the log-transformed values were within the predetermined equivalence range (70 - 143% for Cmax, 80 - 125% for AUC) according to the guidelines of the State Food and Drug Administration of China. Adverse events (AEs) were monitored throughout the study based on clinical parameters and patient reports. RESULTS: Characteristics of the 20 male volunteers included were as follows: mean age 23 (± 0.7, SD) years (range 21 - 24 years); weight 69 (± 7.1) kg (range 60 - 88 kg); height 177 (± 5.9) cm (range 168 - 192 cm). All included subjects completed the study. The mean geometric ratio between the test and reference formulations of oseltamivir was 99.5% (90% CI), 86.3 - 114.8%) for Cmax, 104.4% (95.7 - 113.9%) for AUC0-t, and 104.4% (95.6 - 113.9%) for AUC0-∞. That of oseltamivir carboxylate was 103.7% (90% CI, 95.3 - 112.8%) for Cmax, 101.7% (96.6 - 107.1%) for AUC0-t, and 101.4% (96.5 - 106.5%) for AUC0-∞. There was no significant difference in pharmacokinetic parameters between the two groups. Only 1 AE (nausea) occurred in 1 subject who received the test formulation; the AE resolved without any treatment. CONCLUSIONS: The result of this single-dose study indicated that the test formulation of oseltamivir capsule met the Chinese regulatory criteria for bioequivalence vs. the reference formulation in fasted healthy Chinese male volunteers.
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Analysis of amino acids in human blood using UHPLC-MS/MS: Potential interferences of storage time and vacutainer tube in pre-analytical procedure.

OBJECTIVES: To investigate potential interferences of two pre-analytical variables, the storage time and the vacutainer tube, on the quantification of 20 amino acids using a UHPLC-MS/MS method. DESIGN AND METHODS: Blood samples from 25 apparently healthy subjects were collected into duplicate sets of EDTA-2K, EDTA-3K, coagulation, heparin and citrate tubes, and stored in capped vacutainer tubes at 4°C for 6h, 12h and 24h before sample analysis. A UHPLC-MS/MS method was established for simultaneous quantification of 20 amino acids. ANOVA for repeated measurement was conducted based on the model of Mauchly's test of Sphericity. Student's t-test was applied for comparison between amino acid concentrations obtained from different vacutainer tubes, and consistency of the results was checked through Bland-Altman difference plots and Passing-Bablok regression analysis. RESULTS: Most of the 20 amino acids showed a least concentration fluctuation with storage time in heparin plasma, followed by EDTA-3K and citrate plasma. The amino acid concentrations were significantly lower in citrate plasma and slightly higher in serum, compared with those in heparin plasma. No fixed bias was observed for amino acid concentrations in EDTA and heparin plasma, but the differences were mostly of statistical significance. Amino acid concentrations in EDTA-3K plasma achieved a good consistency with those in heparin plasma by UHPLC-MS/MS analysis. CONCLUSIONS: Storage time and vacutainer tube were important variables for amino acid analysis. They should draw researchers' attention and then be controlled in good laboratory practice to reduce pre-analytical errors.

Validation of an LC-MS/MS method for quantitative analysis of the 5 bioactive components of Wuzhi capsule in human plasma samples.

BACKGROUND: Wuzhi capsule (WZC) is an ethanol extract from the ripe fruit of Schisandra sphenanthera in traditional Chinese medicine that has long been used to treat viral and drug-induced hepatitis in China. The principal active components in WZC are Schisandra lignans. The clinical pharmacokinetics of these components in the form of an oral WZC preparation is unknown. To optimize the WZC dosage and develop WZC-related combination therapies, it is necessary to conduct a comprehensive pharmacokinetic study of the Schisandra lignans. METHODS: A method was developed for simultaneous quantification of multiple bioactive lignans in WZC in human plasma through liquid-liquid extraction followed by multiple reaction monitoring liquid chromatography/tandem mass spectrometry with positive-mode electrospray ionization. The 5 bioactive constituents were separated by isocratic elution using a mobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid at a flow rate of 0.3 mL/min. The total run time was 3.5 minutes. RESULTS: All analytes showed good linearity over a wide concentration range with a lower limit of quantification at 0.5 ng/mL. Using this method, we determined the 5 bioactive lignans in WZC from human plasma simultaneously and performed a pharmacokinetic study on the Schisandra lignans in healthy volunteers. CONCLUSIONS: Owing to simplicity, quickness, high sensitivity, and selectivity, and a sufficient lower limit of detection of the new liquid chromatography/tandem mass spectrometry method, it may be used as a routine technique for clinical monitoring of WZC, and for understanding interactions between herbal and conventional drug therapies.

Rapid and sensitive liquid chromatography coupled with electrospray ionization tandem mass spectrometry method for the analysis of paclitaxel, docetaxel, vinblastine, and vinorelbine in human plasma.

BACKGROUND: A rapid and sensitive analytical method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed for the determination of paclitaxel, docetaxel, vinblastine, and vinorelbine in human plasma. METHODS: A simple liquid-liquid extraction procedure was applied using only 100-µL plasma. Chromatographic separation of these anticancer drugs was achieved with an isocratic mobile phase consisting of acetonitrile/aqueous buffer (10 mmol/L ammonium acetate and 0.1% formic acid in 70:30, vol/vol) at a flow rate of 0.25 mL/min in a short time (4.5 minutes). RESULTS: The calibration curves for paclitaxel, docetaxel, vinblastine, and vinorelbine in spiked human plasma ranged from 25 to 2500, 10 to 1000, 10 to 1000, and 10 to 1000 ng/mL, respectively. The squares of the linear correlation coefficients were all more than 0.99. The intraday and interday relative standard deviations across 3 validation runs over the entire concentration range were less than 9.2%. CONCLUSIONS: The established method should be helpful for the pharmacokinetic monitoring of paclitaxel, docetaxel, vinblastine, and vinorelbine in the human plasma of non-small cell lung cancer patients.

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma.

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 µL samples by solid phase extraction using a ZORBAX SB-C8 column (3.5 µm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.

An liquid chromatography-tandem mass spectrometry assay for determination of trace amount of new antifungal drug iodiconazole in human plasma.

A simple, rapid and sensitive LC-MS/MS method for determination of trace amount of new antifungal drug iodiconazole in human plasma was developed (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[N-methyl-N-(3-chlor-benzyl)amino]-2-propanol), was used as internal standard (IS). The analytes were extracted by liquid-liquid extraction with n-hexane after internal standard spiked. The separation was performed by a ZORBAX SB-C(18) column (3.5 microm, 2.1 mm x 100 mm) with an isocratic mobile phase consisting of methanol-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The lower limit of quantification (LLOQ) was 0.10 ng/mL. This method was successfully used to determine the concentration of iodiconazole in human plasma following dermal administration.