Rapid Liquid Chromatography-tandem Mass Research Articles

Effects of Avitinib on CYP450 Enzyme Activity in vitro and in vivo in Rats

PurposeAvitinib is the first third-generation epithelial growth factor receptor (EGFR) inhibitor independently developed in China and is mainly used for treating non-small cell lung cancer. However, pharmacokinetic details are limited. This study explored the in vivo and in vitro effects of avitinib on cytochrome CYP450 enzymes metabolic activity.MethodsA rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining six probe substrates and their metabolites. Avitinib influence on activity levels of CYP isozymes was examined in vitro using human and rat liver microsomes (HLMs/RLMs). For in vivo studies, rats were pretreated with 30 mg/kg avitinib once daily for 7 days (avitinib multiple-doses group), 30 mg/kg avitinib on day 7 (avitinib single-dose group), or an equivalent amount of CMC-Na once daily for 7 days (control group), followed by intragastrical administration of the probe substrates (1 mg/kg tolbutamide and 10 mg/kg phenacetin, bupropion, chlorzoxazone, dextromethorphan, and midazolam). Plasma pharmacokinetics and IC50 values of the probe substrates were then compared. Pharmacokinetic parameters were determined using non-compartmental analysis implemented in a pharmacokinetic program.ResultsIn vitro experiments revealed different inhibitory effects of avitinib on the six probe substrates with various IC50 values (bupropion, 6.39/22.64 μM; phenacetin, 15.79/48.36 μM; chlorzoxazone, 23.15/57.09 μM; midazolam, 27.64/59.6 μM; tolbutamide, 42.18/6.91 μM; dextromethorphan, 44.39/56.57 μM, in RLMs and HLMs respectively). In vivo analysis revealed significant differences (P <0.05) in distinct pharmacokinetic parameters (AUC(0-t), AUC (0-∞), Cmax, MRT(0-t), MRT (0-∞), and CLz/F) for the six probe substrates after avitinib pretreatment.ConclusionA sensitive and reliable UPLC-MS/MS method was established to determine the concentration of six probe substrates in rat plasma. Avitinib had inhibitory effects on CYP450 enzymes, especially cyp2b1, cyp1a2 in RLMs, CYP2C9 in HLMs, and cyp1a2, cyp2b1, cyp2d1, and cyp2e1 in vivo. Our data recommend caution when avitinib was taken simultaneously with drugs metabolized by CYP450 enzymes.

A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy.

Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. To develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring serum methotrexate in patients on high-dose chemotherapy. Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 min. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2, respectively. Mean recovery was 111% for three different concentrations of methotrexate spiked into seven different patient samples, with ion suppression <1%. Between-batch and within-batch coefficient of variations were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, while mean bias compared to weighed in targets in external quality assessment samples was 1.6%. A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.

Pharmacokinetics and Tissue Distribution of Combined Triptolide and Paeoniflorin Regimen for Percutaneous Administration in Rats Assessed by Liquid Chromatography-Tandem Mass Spectrometry.

Triptolide (TP) has shown potential in rheumatoid arthritis (RA) treatment, but the narrow therapeutic window limits its clinical application. In clinical practice, the compatibility of Tripterygium wilfordii and Paeonia lactiflora is often used to attenuate the toxicity of TP, but its compatibility mechanism has not been fully elucidated. The aim of this study was to investigate the pharmacokinetics and tissue distribution of a combined regimen of TP and paeoniflorin (PF) after transdermal administration in male and female Sprague Dawley (SD) rats via a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The results showed that after percutaneous administration of TP and PF, there was no significant difference in AUC (0-t) (area under the curve) of TP, the peak concentration decreased by 58.17%, and the peak time was delayed. The AUC (0-t) of PF increased significantly (P < 0.01), the peak-reaching concentration and AUC (0-∞) increased, and the half-life and average retention time were shortened, indicating that TP absorption in rats may be delayed. After percutaneous administration of TP and PF, the content of TP in the heart, liver, spleen, lungs, and kidneys of male rats significantly decreased at 2 h (P < 0.05) and the drug concentration in the liver tissues significantly decreased at 2 h, 4 h, and 8 h (P < 0.05). The TP content in the spleen of female rats significantly decreased at 2 h and 4 h (P < 0.05) and also decreased in other tissues, but not significantly. After percutaneous administration of TP and PF, the PF content in the heart, liver, spleen, lungs, and kidneys of male and female rats had no significant difference. However, after percutaneous administration of TP and PF, the TP concentration in the skin increased, suggesting that the amount of TP retained in the skin increased, thereby reducing its content in blood and tissues, producing a reduction in toxicity effect.

Pharmacokinetics, tissue distribution and plasma protein binding rate of palmatine following intragastric and intravenous administration in rats using liquid chromatography tandem mass spectrometry

Palmatine is a natural isoquinoline alkaloid widely found in traditional Chinese medicines. In this study, a simple, sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantification of palmatine in the plasma and tissue samples in rats. Sample preparation involved a simple protein precipitation extraction technique using acetonitrile as the precipitating solvent. Chromatographic separation was accomplished on an ACQUITY UPLC BEH C18 column with a mobile phase of acetonitrile-5 mM ammonium acetate solution (70:30, v/v) at a flow rate of 0.3 mL/min. Coptisine was selected as the internal standard. The protonated analytes were determined with MRM in the positive ion mode. The assay exhibited a linear dynamic range of 1.0−1000 ng/mL for palmatine in each biological matrix and the low limit of quantification was 1.0 ng/mL. Non-compartmental pharmacokinetic parameters indicated that there is a significant difference in the apparent distribution volume and half-life between intragastric and intravenous administration modes. Palmatine could be detected in different tissues and the content in liver and kidney is relatively high, suggesting that liver and kidney might be the targeting organs of palmatine. The plasma protein binding rate test showed that the percent binding of palmatine is medium, and was found to be higher in human than in rats.

Pharmacokinetic and metabolism study of ginsenoside Rb2 in rat by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and rapid ultra-fast liquid chromatography tandem mass spectrometry method was established and validated to determine ginsenosides Rb2 in rat plasma. Acetonitrile-mediated protein precipitant was applied to the sample preparation. Chromatographic separation was carried out on an Acquity UPLC HSS T3 column (100 × 2.1mm, 1.8μm). The analytes were monitored using multiple reactions monitoring mode with precursor-to-product ion transitions at m/z 1077.4-945.3 and m/z 799.8 → 637.8 for ginsenoside Rb2 and internal standard, respectively. The mobile phase was composed of 0.1% formic acid aqueous solution and acetonitrile. The assay showed excellent linearity over the concentration range of 2-1,000 ng/ml, with correlation coefficient >0.995. The method was further validated for selectivity, precision, accuracy, recovery, and stability according to the US Food and Drug Administration guidelines. The validated method was successfully applied to pharmacokinetic and bioavailability studies of ginsenoside Rb2 in rat plasma. Based on the pharmacokinetic results, ginsenoside Rb2 showed slow clearance and low oral bioavailability (0.15%). In addition, the metabolites of ginsenoside Rb2 in rat urine and feces were characterized according to their accurate masses and fragment ions. The proposed metabolic pathway was deglycosylation.

Study on the Pharmacokinetic Profiles of Three Ingredients in Qilong Capsules and Their Potential Interactions in Rats by LC-MS/MS.

Qilong capsule (QLC) is a well-known Traditional Chinese Medicine, and it has a long history for the treatment of ischemic stroke. Its major ingredients are saponins, such as paeoniflorin, amygdalin and calycosin-7-glucoside, contributing to vasodilation function. In this study, a simple, rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed to determine three bioactive ingredients in rat plasma after oral administration of QLC. A simple acetonitrile precipitation method was introduced during the sample preparation. Chromatographic separation was performed on a Shiseido CAPCELL PAC MGIII-C18 column using a gradient elution with acetonitrile and water (0.1% formic acid) as a mobile phase, and the chromatographic separation was 5 min. The methodological evaluation showed that this method had a high sensitivity (the lower limit of quantification was 1 ng/mL for calycosin-7-glucoside, 5 ng/mL for paeoniflorin, 5 ng/mL for amygdalin), satisfactory accuracy (relative error ≤ ±15%) and precision (relative standard deviation ≤15%). Then, the analytical method was applied to the pharmacokinetic study in rats following oral administration of the extracts in QLC. Meanwhile, the pharmacokinetic parameters of complex system in QLC were analyzed and the potential interaction between ingredients was explored. The present quantification method and pharmacokinetic study will provide a meaningful reference for the formulas research of QLC in the treatment of ischemic stroke.

A simple LC-MS/MS method for simultaneous determination of cilostazol and ambroxol in Sprague-Dawley rat plasma and its application to drug-drug pharmacokinetic interaction study following oral delivery in rats

Recently, a combination of cilostazol and ambroxol has been used in the clinical treatment of stroke-associated pneumonia (SAP). However, the pharmacokinetic drug-drug interaction (DDI) of cilostazol and ambroxol has not been reported. In this paper, a rapid, reproducible and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of cilostazol and ambroxol in Sprague-Dawley (SD) rat plasma was established and validated for the first time. Domperidone was used as the internal standard (IS) and one-step liquid–liquid extraction (LLE) method was used to extract analytes and IS from plasma samples with methyl tert-butyl ether as extractant. A rapid chromatographic separation within 4.8 min was carried on an Ultimate ® XB-C18 column with a mobile phase consisting of methanol–acetonitrile-formic acid (0.1%) aqueous solution (90:2:8, v/v/v) at a flow rate of 500 μL/min. The quantitative detection of the analytes and IS were performed on a positive electrospray ionization mode (ESI), and scanned by multi-reaction monitoring (MRM) with the ion transitions m/z 370.3 → m/z 288.2 for cilostazol, m/z 378.8 → m/z 263.8 for ambroxol and m/z 426.2 → m/z 175.1 for domperidone (IS), respectively. It had good linearity in the range of 5.0–1000 ng/mL for cilostazol and 1.0–200 ng/mL for ambroxol in rat plasma. The methodology was fully validated with selectivity, linearity, lower limits of quantification, precision, accuracy, extraction recovery, matrix effect, stability and carry-over effect. The validated data have met the determination requirements of biological samples in FDA guideline. The method was successfully applied to the pharmacokinetics and DDI study of cilostazol and ambroxol in male SD rats. The current study found that the interaction between cilostazol and ambroxol may be caused by CYP3A4 and the pharmacological properties of cilostazol, which may be helpful for therapeutic drug monitoring, clinical dose reference and provide a valuable tool for drug-drug interactions.

LC-MS/MS method for determination of seneciphylline and its metabolite, seneciphylline N-oxide in rat plasma, and its application to a rat pharmacokinetic study.

A rapid and sensitive ultra-performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol-mediated precipitation. Chromatographic separation was achieved within 3min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1mm, i.d. 1.7 μm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1-1,000 ng/mL, with the lower limit of quantification of 1ng/mL. The intra- and inter-day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax , 0.23-0.32 h) and reached the maximum concentration of 0.82-1.75 μg/mL after oral administration. Both seneciphylline and seneciphylline N-oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43-10.31%) was related to the extensive metabolism in the liver and microflora.

A SELECTIVE AND SENSITIVE LC-MS/MS METHOD FOR QUANTIFICATION OF FOUR POTENTIAL GENOTOXIC IMPURITIES IN ATAZANAVIR SULFATE DRUG SUBSTANCE IN TRACE LEVEL

Objective: The main objective of current research work is to develop and validate a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the trace analysis of four potential genotoxic impurities in Atazanavir Sulfate drug substance.&#x0D; Methods: LC-MS/MS analysis of four potential genotoxic impurities was done on Acquity UPLC CSH C18 (100 mm × 2.1 mm, 1.7 μm) column. In this method, mobile phase A (10 mM ammonium acetate) mobile phase B (methanol: acetonitrile (90:10, v/v) with gradient run with the flow rate of 0.2 ml/min. The method was developed with the short run time of 13 min. Triple quadrupole mass detector coupled with positive electrospray ionization was used for the quantification of genotoxic impurities in multiple reaction monitoring (MRM) mode.&#x0D; Results: The method was linear in the range of 0.3 ppm to 4.5 ppm for BOC Hydrazine Acid impurity, BOC Epoxide and Keto impurity with a correlation coefficient not less than 0.9994. The accuracy of the method was in the range of 99.26% to 105.71% for all four potential genotoxic impurities (PGIs). No impurities were identified in the Atazanavir Sulfate active pharmaceutical ingredient sample.&#x0D; Conclusion: The proposed method is specific, linear, precise, accurate, robust and stable for the quantification of the four genotoxic impurities at very low levels.

Development and validation of liquid chromatography-tandem mass spectrometry method for screening six selective androgen receptor modulators in dietary supplements

ABSTRACT Selective androgen receptor modulators (SARMs) are compounds with specific androgenic properties that have been investigated for the treatment of conditions such as muscle wasting disease. The reported androgenic properties have resulted in their use by athletes, and consequently they have been on the World Anti–Doping Agency prohibited list for more than a decade. To minimise the chance of an unattended positive doping test and to avoid potential serious health problems, adequate screening methods for the detection of a wide range of SARMs in these supplements is necessary. In this study, a rapid and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated simultaneously to screen and quantify six SARMs in dietary supplements, with confirmation by liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-TOF/MS). The validated method was applied to 60 dietary supplements obtained by on-line and direct purchase from international vendors in 2020. Various SARMs were detected at high concentrations in 20 products which were advertised as having androgenic properties. For example, andarine was present at 7.2% in one product, and GW501516 was found at 3.49% in the another product. Furthermore, MK-677 and YK-11, not disclosed on the label, were detected in some products. YK-11 is easily hydrolysed in just a few hours. Although YK-11 is particularly unstable, such that the protonated ion [M + H]+ at m/z 431 for YK-11 was not detected, mass fragmentation, and a [M+ Na]+ ion at m/z 453.3 confirmed the presence of YK-11. Additionally, hydrolysed YK-11 under acidic conditions was confirmed by NMR spectral data, and 1H NMR and 13C NMR spectral data for YK-11 were in good agreement with literature data. This rapid and accurate LC-MS/MS method can therefore be successfully applied to screen and identify SARMs for the continuous control and supervision of dietary supplements.

Quantification and Confirmation of Zearalenone Using a LC-MS/MS QTRAP System in Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes

A simple, rapid, and efficient liquid chromatography tandem mass spectrometry (LC–MS/MS) method, operated in electrospray ionization (ESI) and quadrupole linear ion trap modes, has been developed for the identification and structural characterization of zearalenone (ZEN) in corn oil. Samples (5 g) were extracted with acetonitrile/water (80:20, v/v). After centrifugation and dilution, the extracts were separated on a C18 analytical column by gradient elution (acetonitrile/water) and analyzed by UPLC–MS/MS. The developed multiple reaction monitoring–information-dependent acquisition–enhanced product ion method enabled quantification and confirmation of the analyte in a single run. Enhanced product ion mode was used for qualitative analysis, while multiple reaction monitoring mode was used for quantitative analysis. An in-house library was constructed for identification. Calibration curve showed good linearity with correlation coefficients (r) higher than 0.995. Limit of detection was determined to be below 0.20 μg kg−1 for ZEN. The recovery for ZEN was in the acceptable range of 86.6 to 97.2%. 82.4 % of the samples were found to contain ZEN among the 51 samples.

Pharmacokinetic study of seven bioactive components of Xiaoyan Lidan Formula in cholestatic and control rats using UPLC-MS/MS

A rapid, sensitive, and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed to simultaneously determine the major bioactive components of Xiaoyan Lidan Formula (XYLDF) in rat plasma, using sulfamethoxazole as the internal standard (IS). The seven major bioactive components are andrographolide, dehydroandrographolide, enmein, 1-methoxicabony-β-carboline, 4,5-dimethoxy-canthin-6-one, 4-methoxy-5-hydroxy-canthin-6-one, and 1-hydroxymethyl-β-carboline. After pretreating by protein precipitation with methanol, separation was performed on a UPLC C18 column using gradient elution with a mobile phase consisting of acetonitrile and 0.1% formic acid at a flowing rate of 0.7 mL/min. Detection was performed on TSQ Quantum mass spectrometry set at the positive/negative ionization and multiple reaction monitoring (MRM) mode. The intra- and inter-day precision were less than 9.8%, whereas the intra- and inter-day accuracy were within ± 13.4%. The method was validated and applied to compare the pharmacokinetic profiles of the analytes in serum of Alpha-naphthylisothiocyanate (ANIT)-induced cholestasis and control rats after oral administration of XYLDF. The results showed remarkable differences in pharmacokinetic properties of the analytes between cholestatic (model) and control groups, thereby providing essential scientific information for better understanding of mechanism of XYLDF and a reference for its clinical applications.

Pharmacokinetic Study of Zhebeirine in Mouse Blood by Ultra- Performance Liquid Chromatography/tandem Mass Spectrometry

Introduction: In this study, a precise, rapid and accurate ultra-performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) method for the quantification of zhebeirine in mouse blood was developed, and pharmacokinetics of zhebeirine was studied for the first time after intravenous and oral administration. Methods: The mobile phase consisted of acetonitrile-0.1% formic acid, with a flow rate at 0.4 mL/m during 4 min run time. MRM modes of m/z 414.5→81.0 for zhebeirine and m/z 430.2→412.2 for 3- dehydroverticine (internal standard) were utilized to perform quantitative analysis. Protein in mouse blood was directly precipitated with acetonitrile for sample preparation. Results: The linear range was 1-3000 ng/mL with r&gt;0.995, and LLOQ was 1 ng/mL. The intra-and inter-day precision of zhebeirine in mouse blood was less than 13%. The accuracy ranged from 91.2% to 112.5%, while the matrix effects were between 84.8% and 106.4%. Conclusion: The UPLC-MS/MS was successfully applied to the pharmacokinetic study on zhebeirine after intravenous and oral administration, and the bioavailability was determined to be 22.8%.

Quantitative Determination of Methohexital in Human Whole Blood by Liquid Chromatography Tandem Mass Spectrometry.

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry method for the determination of methohexital in human whole blood was developed and validated. Ethyl acetate/n-hexane (9:1) was used as extraction solvent while aprobarbital was used as internal standard. Methohexital was recovered by liquid-liquid extraction from 100μL of human whole blood. The mobile phase was water-acetonitrile, and an ACQUITY BEH C18 (2.1×100mm, 1.7μm) column was adopted. Negative electrospray ionization source and multiply reaction monitoring mode were applied. The transitions of m/z were 261.2/42.2 and 261.2/119.0 for methohexital. The limit of detection was 0.5ng/mL, which was lower than the previous methods. Wide linear range (2-2,000ng/mL) with a good correlation coefficient (r>0.99) was also obtained. The intra- and inter- day precisions represented by relative standard deviation were <11.5%, and the recoveries were >79.67%. This analytical method involved small sample volume and had been proven to be rapid, easy, sensitive and specific. Therefore, it could be used for the clinical analysis of methohexital.

Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application

A rapid and easy liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed, validated, and used to quantify amlodipine besylate and metoprolol succinate in human plasma. Hydrochlorothiazide was used as the internal standard. LC–MS/MS with electrospray ionization in the positive ionization mode was performed under multiple reaction monitoring to examine analytes, which were drawn from human plasma via liquid–liquid extraction using a mixture of diethyl ether and dichloromethane (6:4). The extracts were dried under a nitrogen stream at 40°C, after which the samples were dissolved in a mixture of methanol and water (80:20). The re-dissolved samples were injected onto a C18 column and eluted using a mixture of methanol and 0.4% formic acid (80:20) as the mobile phase. Linear standard calibration was performed, and lower limits of quantification were determined for each analyte. The developedmethod was fully validated with respect to selectivity and carryover, as well as intra-and inter-day accuracy and precision in accordance with guidelines of the United StateFood and Drug Administration and the European Medicines Agency. Mean analyte recovery was between 71.72% and 97.55%, and matrix effects exerted only a minor influence on precision.The validated method was applied in aclinicalbioequivalence study to evaluate the in vivo bioequivalence of twocommer-cialproducts containing 5 mg amlodipine and 50 mg metoprolol. This randomized, single-dose, two-treatment, three-period, two-sequence, open-label, crossover study involved 14 healthy subjects, and a two-week washout period between the two phases of the research was implemented. Standard pharmacokinetic parameters were calculated to compare a test product with Selomax®as the reference. How to Cite this Article Pubmed Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. 2019; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Web Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. http://www.sysrevpharm.org/?mno=302644896 [Access: March 31, 2021]. doi:10.5530/srp.2019.2.01 AMA (American Medical Association) Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. 2019; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Vancouver/ICMJE Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. (2019), [cited March 31, 2021]; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Harvard Style Tuyen, N. T. L., Nghiem, . L. Q. & Tuan, . N. D. (2019) liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP, 10 (2), 1-7. doi:10.5530/srp.2019.2.01 Turabian Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. 2019. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and Reviews in Pharmacy, 10 (2), 1-7. doi:10.5530/srp.2019.2.01 Chicago Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. Systematic Reviews in Pharmacy 10 (2019), 1-7. doi:10.5530/srp.2019.2.01 MLA (The Modern Language Association) Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. Systematic Reviews in Pharmacy 10.2 (2019), 1-7. Print. doi:10.5530/srp.2019.2.01 APA (American Psychological Association) Style Tuyen, N. T. L., Nghiem, . L. Q. & Tuan, . N. D. (2019) liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and Reviews in Pharmacy, 10 (2), 1-7. doi:10.5530/srp.2019.2.01

A rapid and sensitive High-Performance Liquid Chromatography-tandem Mass Spectrometry method for determining apremilast in beagle dog plasma and urine: Application in a pharmacokinetic study

AbstractA rapid and sensitive High-Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC/MS/MS) method for determining apremilast in beagle dog plasma and urine samples was developed and validated using clopidogrel as the internal standard (IS). Apremilast was extracted from the plasma and urine samples by liquid–liquid extraction using methyl tert-butyl ether. Chromatographic separation was performed using a C8 column with gradient elution and a mobile phase containing methanol and 0.1% formic acid. Quantification was achieved in multiple reaction monitoring (MRM) mode with a transition of m/z 461.3→178.2 for apremilast and m/z 322.2→184.1 for clopidogrel (IS). This method was validated regarding its specificity, linearity, precision, accuracy, and stability. The lower limit of quantification (LLOQ) for this method was 5 ng/mL, and the calibration curve was linear over 5–1,000 ng/mL. The intra- and inter-run coefficients of variance (CV) of aprelimast in plasma samples were less than 12.92% and 10.64%, respectively, while in urine samples, the CV were less than 11.84% and 10.20%, respectively. The samples were stable under the tested conditions. This method was successfully applied to a pharmacokinetic study in beagle dogs following oral administration of 10 mg of apremilast.

Pharmacokinetic studies of ginsenosides Rk1 and Rg5 in rats by UFLC–MS/MS

A rapid ultra-fast liquid chromatography tandem mass spectrometry method was developed and validated to determine ginsenosides Rk1 and Rg5, a pair of isomers, in rat plasma, which was successfully applied to their pharmacokinetic studies. Two ginsenosides were given to male Sprague-Dawley rats via intragastrical and intravenous routes, respectively, and the impact of double bond position on the pharmacokinetic features of the two ginsenosides was elucidated in rats. Ginsenoside Rg3 was used as internal standard and ethyl acetate was applied to extract analytes and internal standard. Chromatographic separation was carried out on a reverse-phase UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm). The flow rate was set to 0.4 ml/min. The fragmentation transition was m/z 765.4 → m/z 101.1 for two ginsenosides. The mobile phases were composed of 0.1% formic acid aqueous solution and acetonitrile. The linear range was 2-1,000 ng/ml for the two ginsenosides. Intra- and inter-day precisions were <11.67%, and accuracy fluctuated from -7.44 to 6.78%. The extraction recovery, matrix effect and stability were within acceptable levels. After treatment with ginsenosides Rk1 and Rg5, some differences were found in their pharmacokinetic profiles in rats. The maximum plasma drug concentration and the area under the plasma drug concentration-time curve of ginsenoside Rg5 were about 5 times bigger than those of ginsenoside Rk1 after oral administration, and 3 times higher after intravenous administration. The oral bioavailabilities of ginsenosides Rk1 and Rg5 were 0.67 and 0.97%, respectively. The results indicated that ∆20(22) -ginsenosides showed better pharmacokinetic features than ∆20(21) -ginsenosides with the same glycosylation.

LC-MS/MS method for simultaneous quantification of dexamethasone and tobramycin in rabbit ocular biofluids

The complexity of Tobradex® ointment formulation (dexamethasone 0.1 wt% and tobramycin 0.3 wt%) and the high cost of pharmacokinetic (PK) studies in human aqueous humor may prevent generic drug companies from moving forward with a Tobradex®-equivalent product development. The in vitro drug release test would be an alternative approach for differentiating the generic formulations containing both dexamethasone (DEX) and tobramycin (TOB), and the results should be correlated with the in vivo ocular PK studies for further evaluation. To facilitate the in vivo ocular PK studies, a sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can simultaneously quantify both DEX and TOB in rabbit ocular matrices including tear, aqueous humor and cornea was established and validated. The lower limit of quantification (LLOQ) was 1.5 ng/ml for DEX and 3 ng/ml for TOB with good precision and accuracy. Both intra- and inter-batch precisions were within ±15%, and the accuracy for all QCs was within the range of 85–115%. This new method was successfully applied for a pilot pharmacokinetic analysis of DEX and TOB in rabbit tears after topical administration of Tobradex® ointment.

Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC-MS-MS.

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 μg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL for norfentanyl. The analytical range was 0.002-2 ng/mL for fentanyl, 0.005-5 ng/mL for alpha-methylfentanyl, 0.01-10 ng/mL for beta-hydroxyfentanyl and 0.04-40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%-92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC–MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00−1000 ng/mL for forsythin, 2.50−2500 ng/mL for KD-2-Glc, and 5.00−5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC–MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.