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In this work, we developed and validated a highly sensitive, rapid and stable LC-MS/MS method for determination of ibuprofen in human plasma with ibuprofen-d3 as stable isotopically labeled internal standard (SIL-IS). Human plasma samples were prepared by one-step protein precipitation. The chromatographic separation was achieved on a Poroshell 120 EC-C18 (2.1 x 50 mm, 2.7 μm). Water solution (containing 0.05% acetic acid and 5 mM NH4 Ac) and methanol were selected as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in negative ion mode. Multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.0→161.1 for ibuprofen and m/z 208.0→164.0 for SIL-IS, respectively. This method exhibited a linear range of 0.05-36 μg/mL for ibuprofen with correlation coefficient >0.99. Mean recoveries of ibuprofen in human plasma ranged from 78.4% to 80.9%. RSD of intra-day and inter-day precision were both < 5%. The accuracy was between 88.2% and 103.67%. The matrix effect was ignorable in human plasma, including lipidemia and haemolytic plasma. A simple, efficient and accurate LC-MS/MS method was successfully established and applied to a pharmacokinetics study in Chinese healthy volunteers after a single oral administration of ibuprofen granules.

Kansuinine A is a macrocyclic jatrophane diterpene isolated from the plant Euphorbia kansui Liou. It exhibits many pharmacological activities including cytoxic, antitumor, antiallergic and proinflammatory effects. In the present study, a simple and sensitive LC-MS/MS method was established and validated for the determination of kansuinine A in rat plasma. After methanol-mediated protein precipitation, chromatographic separation was achieved on an Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm) using acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. Kansuinine A and IS were quantified in negative multiple reaction monitoring mode with ion transitions at m/z 731.1-693.2 for kansuinine A and m/z 723.2-623.1 for IS. The method showed excellent linearity over the range 1-500 ng/ml. The intra- and inter-day precisions (relative standard deviation) were 2.13-4.28 and 3.83-7.67%, respectively, whereas accuracy (relative error) ranged from -4.17 to 3.73%. The extraction recovery, stability and matrix effect met the requirement of the regulations issued by the US Food and Drug Administration. The validated method was successfully applied to the pre-clinical pharmacokinetic study of kansuinine A in rats after oral administration (20 mg/kg) and intravenous administration (2 mg/kg). This study provides valuable reference for the further study of E. kansui liou, especially for the drug development and clinical application of kansuinine A.

Ginsenoside Rh3 (GRh3) is a bacterial metabolite of ginsenoside Rg5, which is the main component of hot-processed ginseng. A simple, efficient and sensitive method was developed and validated for the determination of GRh3 in rat plasma by LC-tandem mass spectrometry. After protein precipitation with methanol/acetonitrile (1:1, vol/vol) using propranolol as the internal standard, the target analytes were separated on an XDB C18 column, with methanol containing 0.1% formic acid and water containing 0.1% formic acid used as mobile phases for gradient elution. Mass spectrometry was performed in electrospray ion source-positive ion mode and multiple reaction monitoring mode, monitoring the transitions m/z 622.5 → 425.5 and m/z 260.1 → 116.1 for GRh3 and internal standard, respectively. The concentration range of GRh3 was 20-20,000 ng/mL and the correlation coefficient (r2 ) was greater than 0.99. The accuracy error and relative standard deviation were below 15%. The extraction recovery and matrix effect were 74.2% to 78.7% and 96.9% to 108.4%, respectively. Under different conditions, GRh3 was stable in the range of 1.8%-8.7%. This method has been successfully applied to study the pharmacokinetics of GRh3 with an oral dose of 10.0 mg/kg and an intravenous dose of 2.0 mg/kg in rats, respectively. The absolute bioavailability of GRh3 was 37.6%.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N1 -methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5 μm) with gradient elution within 7 min. Acetonitrile and 5 mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N1 -methylnicotinamide and N'-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N1 -methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N1 -methylnicotinamide were 5.000-160.0 and 2.500-80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N1 -methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.

Vanillic acid, a phenolic compound isolated from Angelica sinensis and green tea, exhibits excellent antioxidant and anti-inflammatory activities. In this study, a rapid and sensitive ultra-high-performance liquid chromatography tandem mass spectrometry method was established and validated for the determination of vanillic acid in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Vanillic acid and caffeic acid (internal standard, IS) were quantified by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with a correlation coefficient of >0.99. The carryover, matrix effect, extraction recovery, dilution effect, intra- and interday precision and accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of vanillic acid in rats. After oral administration at doses of 2, 5 and 10 mg/kg, the plasma concentration reached peaks of 0.42 ± 0.09, 0.73 ± 0.21 and 0.92 ± 0.28 μg/ml at the time of 0.55-0.64 h, respectively. The oral bioavailability was calculated as 25.3-36.2% in rat plasma. The result provided pre-clinical information for further application of vanillic acid.

An efficient and reliable method using LC-MS/MS was established and validated for the simultaneous quantification of meropenem and imipenem in rat plasma. An electronic spray ion source in the positive multiple reaction monitoring mode was used for the detection and the transitions were m/z 384.6 → m/z 141.2 for meropenem, m/z 300.1 → m/z 141.8 for imipenem and m/z 423.4 → m/z 207.1 for matrine (IS). The calibration curves of meropenem and imipenem were linear in the range of 0.50-200 μg/mL. Satisfactory separation was achieved with a total run time of 3.0 min, the injection volume was 3 μl. The retention times of meropenem, imipenem and IS were 1.19, 1.14 and 1.13 min, respectively. Meropenem and imipenem are easily hydrolyzed in plasma. HEPES was used as a stabilizer and added to the plasma samples immediately after centrifugation. Extractions of meropenem, imipenem and IS were carried out by protein precipitation with acetonitrile. The specificity, precision and accuracy, stability, recovery and matrix effects were within acceptance limits. This method was successfully applied to investigate the pharmacokinetics of intravenous injection of meropenem and imipenem single administration or combined with sulbactam in rats. We found that sulbactam has no influence on the pharmacokinetics behavior of meropenem or imipenem.

Previously compound 12 showed great anti-trypanosome activity without toxicity in the in vivo study. In the current study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to investigate the pharmacokinetics of compound 12 in mouse plasma. Protein precipitation method was applied to extract the compound, and it was then separated by using a Kinetex C(18) column with a mobile phase consisting of acetonitrile-0.1% formic acid water (50:50, v/v) at a flow rate of 300 μL/min. The analytes were detected with the multiple reaction monitoring (MRM) in negative electrospray ionization source for quantitative response of the compounds. Compound 12 was detected at m/z 477.0→367.2, while the internal standard compound 14 was detected at m/z 499.2→268.2. Precision of the inter- and intra-day was less than 5.22% and 2.79% respectively, while the accuracy range was within ±9.65%. The method was successfully applied to evaluate the pharmacokinetic of compound 12 in mouse plasma with two formulations (20% Cremophor EL or sesame oil) and drug administration routes (oral and intraperitoneal injection). We observed a better drug serum concentration with the Cremophor formulation, and the two different drug administration routes did not show significant difference to the drug distribution.

An ultra-high pressure liquid chromatography high-resolution mass spectrometric (UHPLC-HRMS) method was developed for the simultaneous and sensitive quantification of 10 beta-lactam antibiotics (cefepime, meropenem, amoxicillin, cefazolin, benzylpenicillin, ceftazidime, piperacillin, flucloxacillin, cefuroxime and aztreonam), linezolid and beta-lactamase inhibitors tazobactam and clavulanic acid in human plasma. Validation according to the EMA guidelines showed excellent within- and between-run accuracy and precision (i.e. between 1.1 and 8.5%) and high sensitivity (i.e. lower limit of quantification between 0.25 and 1 mg/L). The UHPLC-HRMS method enables a short turnaround time and high sensitivity and needs only a small amount of plasma, allowing appropriate routine therapeutic drug monitoring. The short turnaround time is obtained by speeding up the protocol on multiple levels, i.e. fast and workload-efficient sample preparation (i.e. protein precipitation and dilution), short (4 min) instrument run time, simultaneous measurement of all relevant beta-lactam antibiotics used in the intensive care unit and the use of the same instrument, column and mobile phases as for the other routine methods in our laboratory.

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 μL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 μL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 μg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges.

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572) and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a "dilute-and-inject" approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/in vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs' biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency-compliant initial testing (limits of detection 0.02-0.60 ng/ml) and confirmation procedures (limits of identification 0.18-0.89 ng/ml) for human urine and blood matrices. The obtained results allow extension of the test spectrum of doping agents in multitarget screening assays for growth hormone-releasing factors from human urine.

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid-2 H3 , voriconazole-2 H3 , itraconazole-2 H4 , and hydroxyitraconazole-2 H4 as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 μm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 μm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.

A simple, rapid, and selective ultra-performance liquid chromatography-tandem mass spectrometry method for determination of l-carnitine (LC) and acetyl-l-carnitine (ALC) in human serum was developed. Acetyl-l-carnitine-d3 (ALC-d3 ) was selected as internal standard (IS). After protein precipitation with acetonitrile-water (1 mL, 2:1, v/v), the analytes and IS were separated on a 2.5-μm XSelect HSS T3 C18 column by gradient elution with methanol-water (containing 0.01% ammonia water) as the mobile phase at a flow rate of 0.2 mL/min. Analytes were detected with multiple reaction monitoring using a positive scan mode with electrospray ionization. Good linearity (R2 > 0.999) was observed in the concentration range for LC and ALC. The inter- and intra-day values of relative error were -10.4% to 10.0% with CVs less than 9.84%. The average recoveries of the two analytes were 91.29%-98.23%. Blood samples containing LC and ALC were stable under various storage conditions. Normal, haemolytic, and hyperlipidaemic serum had no significant effect on the quantification of LC and ALC. This method was successfully applied to study the concentrations of endogenous LC and ALC in the serum of patients with first-episode depression.

Antibiotics are widely used in intensive care patients to treat severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum concentration of ABs is advisable. Therefore, in this study, we developed and validated a simple and fast high-performance liquid chromatography method with UV detection for the simultaneous determination of four β-lactam ABs (meropenem, imipenem, ceftazidime, and piperacillin) and two coadministered substances (cilastatin and tazobactam) in human serum. Sample preparation required a simple protein precipitation by methanol. The separation of the ABs occurred within a timeframe of 17 min. For this purpose, we used a Kinetex F5 column with a linear gradient of acetonitrile and phosphate buffer (pH 6.9). The UV detector recorded two separate chromatograms at 220 and 295 nm simultaneously. Validation has demonstrated that the method is linear, accurate, and precise within the clinically relevant range for each substance.

From the point of view of drug efficacy and safety, pharmacokinetic profiles of both In this work, a sensitive and reliable liquid chromatographic-tandem mass spectrometric method was established for simultaneous determination of sutetinib and N-oxide metabolite (SNO) in human plasma and further applied to a pharmacokinetic study. Analytes were extracted from plasma samples (100 μl) via acetonitrile-induced protein precipitation and separated on a C18 column using ammonium acetate with ammonium hydroxide and acetonitrile as the mobile phase. Positive electrospray ionization was carried out through multiple reaction monitoring with transitions of m/z 440.2 → 367.1 and 446.2 → 367.1 for sutetinib and SNO, respectively. The method was linear within the concentration range of 0.5-100 ng/ml for both analytes. The precision, accuracy, selectivity, recovery and matrix effect of this method all met the requirements of bioanalytical guidance. In addition, a plasma stability assessment demonstrated unexpected results. Sutetinib was prone to form covalent conjugates with plasma albumin in vitro. The degree of covalent binding increased with increasing temperature, resulting in a significant decrease in its plasma concentrations. However, SNO could not easily bind with albumin owing to steric hindrance or electronegativity. Furthermore, sutetinib and SNO remained stable when blood and plasma samples were kept on wet ice. The validated method was successfully employed for the pharmacokinetic evaluation of sutetinib in patients with advanced malignant solid tumors.

Hypertension is a major risk factor for atherosclerosis and ischemic heart disease. Most hypertensive patients need a combination of antihypertensive agents to achieve therapeutic goals. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of enalapril maleate (ENA) and its major metabolite enalaprilat (ENAT), nitrendipine (NIT) and its major metabolite dehydronitrendipine (DNIT), and hydrochlorothiazide (HCT) in human plasma using felodipine as an internal standard (IS). The drugs were extracted from plasma using one-step protein precipitation. Chromatographic separation was performed on a Symmetry C18 column, with water and acetonitrile (10:90, v/v) as mobile phase. The detection was carried out using multiple reaction monitoring mode and coupled with electrospray ionization source. Multiple reaction monitoring transitions were m/z 377.1 → 234.1 for ENA, m/z 349.2 → 206.1 for ENAT, m/z 361.2 → 315.1 for NIT, m/z 359 → 331 for DNIT, m/z 295.9 → 205.1 for HCT, and m/z 384.1 → 338 for felodipine (IS). The method was linear over concentration ranges of 1-200, 20-500, 5-200, 2-100, and 5-200 ng/mL for ENA, ENAT, NIT, DNIT, and HCT, respectively, with r2 ≥ 0.99. Method validation was performed according to U.S. Food and Drug Administration guidelines. The validated method showed good sensitivity and selectivity and could be applied for therapeutic drug monitoring and bioequivalence studies.

A simple LC-tandem mass spectrometry (MS/MS) method to determine ebastine and carebastine (active metabolite) in human plasma was developed and validated. Analytes and internal standards were precipitated by protein precipitation and separated on Synergi Hydro-RP 80A column (4 μm, 50 mm × 2.0 mm; Phenomenex) by gradient elution with mobile phase A comprising 0.1% formic acid in 5 mm ammonium acetate (NH4 Ac) and B comprising 100% methanol at a flow rate 0.4 mL/min. Ions were detected in positive multiple reaction monitoring mode, and they exhibited linearity over concentration range 0.01-8.0 and 1.00-300 ng/mL for ebastine and carebastine, respectively. A clinical pharmacokinetic study was conducted in healthy Chinese volunteers under fasting and fed conditions after a single oral administration of 10 mg ebastine. The maximum plasma concentration (Cmax ), time to Cmax (Tmax ) and elimination half-life for ebastine were 0.679 ± 0.762 ng/mL, 1.67 ± 1.43 h and 7.86 ± 6.18 h, respectively, whereas these for carebastine were 143 ± 68.4 ng/mL, 5.00 ± 2.00 h and 17.4 ± 4.97 h, respectively under fasting conditions; the corresponding values under fed conditions were 4.13 ± 2.53 ng/mL, 3.18 ± 1.09 h and 21.6 ± 7.77 h for ebastine and 176 ± 68.4 ng/mL, 6.14 ± 2.0 h and 20.0 ± 4.97 h for carebastine.

A simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed and validated for the determination of ARQ531, a Bruton's tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C18 column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5-1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from -13.50 to 11.35% and the precision was 85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1-10 mg/kg and good oral bioavailability (>50%).

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C18 , 150 mm × 2.0 mm, 5 µm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10-10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 µL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.

Canagliflozin (CNZ) is the first sodium-glucose co-transporter-2 inhibitor approved for treatment of type 2 diabetes mellitus. In the proposed work, a sensitive, rapid and validated high-performance thin-layer chromatography (HPTLC) method was established for the estimation of CNZ in human plasma for the first time. HPTLC analysis of CNZ and internal standard (sildenafil) was performed on glass coated silica gel 60 F254 HPTLC plates using a binary mixture of chloroform-methanol 9:1 (%, v/v) as the mobile phase. Densitometric detection was done at 295 nm. Retardation factor values were obtained as 0.22 and 0.52 for the CNZ and the IS, respectively. The linearity range of CNZ was obtained as 200-3,200 ng/ml. A simple protein precipitation method was used for the extraction of analyte from plasma using methanol. The proposed HPTLC technique was validated for linearity, accuracy, precision and robustness. The proposed HPTLC technique was successfully utilized for the assessment of pharmacokinetic profile of CNZ in rats after oral administration. After oral administration, the peak plasma concentration of CNZ was obtained as 1458.01 ng/ml in 2 h. The proposed HPTLC method could be applied to the study of the pharmacokinetic profile of pharmaceutical formulations containing CNZ.

A practical, sensitive, and robust UPLC-MS/MS method was developed and validated to quantify lacosamide in human plasma. A simple one-step protein precipitation was used to extract lacosamide and labeled lacosamide-13C, D3 as an internal standard (IS) from 150-μL plasma. The extracts were analyzed on an Eclipse Plus C18 column (50 × 2.1 mm, 1.8 μm) using 0.1% formic acid in water and methanol:acetonitrile (50:50, v/v) under gradient conditions. The extracts were quantified on LCMS-8040 using electrospray ionization source operated in positive ionization and multiple reaction monitoring modes. The method showed good linearity from 0.02 to 20 μg/mL, which was adequate to cover lacosamide concentration assayed in formulations with different strengths. The bioanalytical assay was fully validated as per current regulatory guidelines. The intra-batch and inter-batch precision values of lacosamide were less than 4.6%. Lacosamide was found to be stable at different storage conditions. The extraction recoveries and IS-normalized matrix factors for lacosamide ranged from 97.17 to 99.68% and from 0.973 to 1.012, respectively. The validated method was successfully applied to a pharmacokinetic study with three lacosamide formulations (50, 100, and 200 mg) in 36 healthy subjects. The assay reliability was determined by reanalysis of 81 subject samples.

A rapid and sensitive method for the quantitative detection of busulfan (BU) in children's hemolytic samples by HPLC-tandem mass spectrometry (MS/MS) was established. In this study, the sample preparation procedure involved a one-step protein precipitation with acetonitrile (ACN) solution, and the HPLC-MS/MS method used Hypersil GOLD C18 . The mobile phase consisted of 10 mM ammonium acetate solution (containing 0.1% formic acid) and ACN with a flow rate of 0.4 mL/min. Multiple reaction monitoring modes were used for quantitative analysis and the ion pairs of BU and BU-d8 were m/z 263.9 → 150.9 and 272.0 → 159.0, respectively. BU had a good linearity in the range of 0.01-10 μg mL-1 . The intra- and inter-day relative error was between -7.21% and 8.26%, and the coefficient of variation was less than 12.64%. The average extraction recovery rate in plasma samples was 99.76% ± 6.53%, and the matrix in normal plasma and hemolyzed plasma had no significant effect on the detection results. Normal and hemolytic samples could maintain good stability at 4, 25 and -40°C. As a result, this method is particularly suitable for determining BU in hemolytic samples from children with hematopoietic stem cell transplantation (HSCT), and this study provides the methodological basis for further research on the pharmacokinetics of BU in children with HSCT.

Turkish galls (TG) is a traditional Uygur medicine typically used in clinics for dental disease and chronic ulcerative colitis. In this study, a novel liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of gallic acid, methyl gallate, and 1,3,6-tri-O-galloyl-β-d-glucose in rat plasma, which are the major bioactive compounds of TG. After a feasible protein precipitation using acetonitrile for sample preparation, chromatographic separation was performed with a BDS Hypersil C18 column (2.1 × 100 mm, 5 μm) at 30°C, and water containing 10 mmol of ammonium acetate and acetonitrile was used as the mobile phase with a flow rate of 0.3 mL/min. The MS detector was operated in the selective reaction monitoring with negative-ionization mode. The results of the method validation, including selectivity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability of the compounds in the biosamples, were all within the current acceptance criteria. The established method was successfully applied to the pharmacokinetics study of three analytes in rats after an oral administration of TG extract and laid the foundation for studying the active components and mechanism of TG in vivo.

MMAE is a potent antimitotic drug used as payload of an antibody-drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography-high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography - quadrupole - time-of-flight - tandem mass spectrometry (LC-qTOF-MS/MS) analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2 ), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1.01-2,200 ng/mL for MMAE. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. Recovery was 42.84%. The dilution integrity was determined for 5-fold dilution and the accuracy and precision ranged within ±25%. The stability results indicated that MMAE was stable for the following conditions: short-term (4 h), long-term (4 weeks), freeze/thaw (3 cycles) and post-preparative stability (12 h). This qualified method was successfully applied to a pharmacokinetic study of MMAE in rat as a preclinical animal model. The PK results suggest that MMAE has moderate CL and low BA.Also, these results would be helpful in having a comprehensive understanding of the PK characteristics of MMAE and developing ADC in future.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 μl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 μm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5-2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.

Buparlisib is a selective phosphoinositide 3 kinase inhibitor currently evaluated in clinical trials. We developed and validated an LC-MS/MS coupled with a one-step protein precipitation extraction method for the quantitation of buparlisib in rat plasma. After protein precipitation with acetonitrile, the plasma sample was analyzed using a Cortecs UPLC C18 column, with acetonitrile-0.1% formic acid as the mobile phase system. Mass spectrometric detection was conducted in positive ionization mode, with target quantitative ion pair of m/z 411.2 → 367.2 for buparlisib. The calibration curve showed good linearity (1.0-3000 ng/ml), with acceptable accuracy (RE ranging from -6.2 to 5.9%) and precision (RSD within 8.2%) values at quality control concentrations. Extraction recovery from plasma was 80.9-88.7% and the matrix effect was negligible (92.6-95.2%). The validated method presented a simple quantification method of buparlisib in detail and utilized it for a pharmacokinetic study at three dose concentrations after oral administration in Wistar rats.

In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC-QToF-MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C18 column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2-5 μg ml-1 for ethambutol, 0.2-7.5 μg ml-1 for isoniazid, 1-40 μg ml-1 for pyrazinamide and 0.25-2 μg ml-1 for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC-QToF-MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.

Although bilobetin, a biflavone isolated from the leaves of Ginkgo biloba, represents a variety of pharmacological activities, to date there have been no validated determination methods for bilobetin in biological samples. Thus, we developed a liquid chromatographic method using a tandem mass spectrometry for the determination of bilobetin in rat plasma. After protein precipitation with acetonitrile including diclofenac (internal standard), the analytes were chromatographed on a reversed-phased column with a mobile phase of purified water and acetonitrile (3:7, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally deprotonated ions [M - H]- at m/z 551.2 → 519.2 for bilobetin and 296.1 → 251.7 for the IS. The accuracy and precision of the assay were in accordance with US Food and Drug Administration regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of bilobetin over time following intravenous administration in rats.

The aim of the present study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of olaparib in rat plasma. The plasma samples were processed using one-step protein precipitation with acetonitrile and then separated on Waters Acquity BEH C18 column (50 × 2.1 mm, particle size 1.7 μm) using water containing 0.1% formic acid and acetonitrile as mobile phase with optimized gradient elution. Mass spectrometric detection was carried out by selective reaction monitoring mode via positive ESI mode with precursor-to-product transitions of m/z 435.3 > 367.1 and m/z 443.1 > 375.2 for olaparib and 2 H8 -olaparib (internal standard). The method was linear over the concentration range 0.1-2000 ng/ml with correlation coefficient >0.9987. The lower limit of quantitation was 0.1 ng/ml. The method showed excellent accuracy and precision, negligible matrix effect and high extraction recovery. The validated method was subsequently utilized to determine the concentration of olaparib in rat plasma and further applied to the pharmacokinetic study of olaparib in rat plasma. Our results demonstrated that olaparib showed gender-dependent pharmacokinetics in rats. Compared with that in males, olaparib showed high plasma exposure, long half-life, low clearance and high bioavailability in females.

In the present study, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach was designed to measure the rat plasma levels of verubecestat with diazepam as the internal standard. Acetonitrile-based protein precipitation was applied for sample preparation, then the analyte verubecestat was subjected to gradient elution chromatography with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Verubecestat was monitored by m/z 410.1 → 124.0 transition for quantification by multiple reaction monitoring (MRM) in positive ion electrospray ionization (ESI) source. When the concentration of verubecestat ranged from 1 to 2500 ng/mL, the method exhibited good linearity. For verubecestat, the intra- and inter-day precision were determined with the values of 2.9-9.0% and 0.4-6.5%, respectively; and the accuracy ranged from -2.2% to 10.4%. Matrix effect, extraction recovery, and stability data were in line with the standard FDA guidelines for validating a bioanalytical method. The validity of the developed method was confirmed through the pharmacokinetic study.

Amitriptyline (AMI) has been in use for decades in treating depression and more recently for the management of neuropathic pain. A highly sensitive and specific LC-tandem mass spectrometry method was developed for simultaneous determination of AMI, its active metabolite nortriptyline (NOR) and their hydroxy-metabolites in human serum, using deuterated AMI and NOR as internal standards. The isobaric E-10-hydroxyamitriptyline (E-OH AMI), Z-10-hydroxyamitriptyline (Z-OH AMI), E-10-hydroxynortriptyline (E-OH NOR) and Z-10-hydroxynortriptyline (Z-OH NOR), together with their parent compounds, were separated on an ACE C18 column using a simple protein precipitation method, followed by dilution and analysis using positive electrospray ionisation with multiple reaction monitoring. The total run time was 6 min with elution of E-OH AMI, E-OH NOR, Z-OH AMI, Z-OH NOR, AMI (+ deuterated AMI) and NOR (+ deuterated NOR) at 1.21, 1.28, 1.66, 1.71, 2.50 and 2.59 min, respectively. The method was validated in human serum with a lower limit of quantitation of 0.5 ng/mL for all analytes. A linear response function was established for the range of concentrations 0.5-400 ng/mL (r2 > .999). The practical assay was applied on samples from patients on AMI, genotyped for CYP2C19 and CYP2D6, to understand the influence of metaboliser status and concomitant medication on therapeutic drug monitoring.

Monitoring the plasma concentrations of metformin and sodium-glucose cotransporter-2 inhibitors (canagliflozin, dapagliflozin and empagliflozin) is essential for pharmacokinetic and bioequivalence studies and therapeutic monitoring. The present work therefore aimed to develop and validate a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of these drugs in human plasma. The analyses were performed using an Agilent 1200 HPLC system coupled to an Applied Biosystems API 3200 triple quadrupole MS/MS with electrospray ionization in positive ion mode. After one-step protein precipitation of plasma with acetonitrile containing 0.1% formic acid, chromatographic separation was achieved on an Xbridge C18 column, with a mobile phase consisting of a gradient of water and acetonitrile, both containing 1 mm ammonium formate and 0.1% formic acid. Quantification was performed in multiple reaction monitoring mode using m/z 130.1 → 71.1 for metformin, m/z 462.0 → 191.2 for canagliflozin, m/z 426.1 → 167.1 for dapagliflozin and m/z 468.0 → 354.9 for empagliflozin. The proposed method was validated and demonstrated to be adequate for the quantification of metformin, canagliflozin, dapagliflozin and empagliflozin for clinical monitoring, pharmacokinetics and bioequivalence studies.

The aim of this study was to develop an analytical method to determine mequitazine in rat plasma and urine. Mequitazine was separated by UPLC-MS/MS equipped with a Kinetex core-shell C18 column (50 × 2.1 mm, 1.7 μm) using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.3 mL/min. Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring positive ion mode. Mass transitions were m/z 323.3 → 83.1 for mequitazine and 281.3 → 86.3 for imipramine as internal standard. Liquid-liquid extraction with ethyl acetate and protein precipitation with methanol were used for sample extraction. Chromatograms showed that the method had high resolution, sensitivity and selectivity without interference from plasma constituents. Calibration curves for mequitazine in rat plasma and urine were 0.02-200 ng/mL, showing excellent linearity with correlation coefficients (r2 ) >0.99. Both intra- and inter-day precisions (CV%) were within 4.08% for rat plasma and urine. The accuracies were 99.58-102.03%. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of mequitazine after oral and intravenous administration to rats.

A highly selective and efficient LC-MS/MS method was developed to determine the plasma concentration of magnolol, hesperidin, neohesperidin and geniposide following oral administration of Zhi-Zi-Hou-Po decoction in normal and depressed rats. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an XTerra® MS C18 column using a gradient elution with a mobile phase composed of acetonitrile-0.1% aqueous formic acid. The proposed method was validated to be specific, accurate and precise for the analytes determination in plasma samples. The calibration curves displayed good linearity over definite concentration ranges for the analytes. The intra- and inter-day precision of the proposed method at three different levels were all within

In this study, a more sensitive and reliable quantitative method based on ultra-high performance liquid chromatography coupled with Q-Exactive-Orbitrap-MS in full-mass scan was developed and validated for the determination of PF-04620110 in dog plasma. After protein precipitation with acetonitrile, the sample separations were carried out on an Acquity BEH C18 column with 1 mm ammonium acetate in water and acetonitrile containing 0.1% acetic acid as mobile phase, at a flow rate of 0.4 mL/min. The assay showed excellent linearity over the concentration range of 1-2000 ng/mL with correlation coefficient >0.9980 (r > 0.9980). The LLOQ was 1 ng/mL. The inter- and intra-day precision (RSD, %) was within 9.69% while the accuracy (RE, %) was in the range of -8.59-11.24%. The extraction recovery was >85.37% and the assay was free of matrix effects. PF-04620110 was demonstrated to be stable under various processing and handing conditions. The validated method was successfully applied to the pharmacokinetic study of PF-04620110 in dogs and the results revealed that PF-04620110 was slowly eliminated from plasma with a clearance of 60.81 ± 7.11 mL/h/kg for intravenous administration and 81.44 ± 25.79 mL/h/kg for oral administration. The oral bioavailability was determined to be 77.89% in dogs.

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C18 column, at 35°C, using a mobile phase composed of acetonitrile-methanol-2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r2 ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.

Therapeutic drug monitoring of antiepileptic drugs is widely practiced to achieve optimal efficacy and avoid adverse side effects. We describe an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method developed for the monitoring of four frequently prescribed antiepileptic drugs - lamotrigine, levetiracetam, oxcarbazepine and topiramate. The main pharmacologically active metabolite of oxcarbazepine (mono-hydroxy-derivative metabolite, MHD) was also quantified. After addition of internal standards and a simple stage of protein precipitation, plasmatic samples were analyzed on a C18 column. All antiepileptic drugs were separated and quantified in 6 min, without interference. A good linearity was observed all over the calibration range (r2 > 0.99), up to 20 μg/mL (40 μg/mL for MHD). The limit of quantification was 0.20 μg/mL (0.40 μg/mL for MHD) with precision and accuracy ranging from 1.0 to 2.1% and from 96.7 to 110.8%, respectively. Intra- and inter-day precision and accuracy values were within 15%. No significant matrix effect was observed for all analytes. Clinical application was successfully evaluated in 259 samples from patients treated for epilepsy or bipolar disorders. In conclusion, a rapid, specific and sensitive UHPLC/MS/MS method was developed and validated for simultaneous quantification of antiepileptic drugs, suitable for therapeutic drug monitoring in neurology and psychiatry.

Measurement of test article concentration in tissue samples has been an important part of pharmacokinetic study and has helped to co-relate pharmacokinetic/pharmacodynamic relationships since the 1950s. Bioanalysis of tissue samples using LC-MS/MS comes with unique challenges in terms of sample handling and inconsistent analyte response owing to nonvolatile matrix components. Matrix effect is a phenomenon where the target analyte response is either suppressed or enhanced in the presence of matrix components. Based on previous reports electrospray ionization (ESI) mode of ionization is believed to be more affected by matrix components than atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization. To explore the impact of ionization source with respect to bioanalysis of tissue samples, five structurally diverse compounds - atenolol, verapamil, diclofenac, propranolol and flufenamic acid - were selected. Quality control standards were spiked into 10 different biological matrices like whole blood, liver, heart, brain, spleen, kidney, skeletal muscle, eye and skin tissue and were quantified against calibration standards prepared in rat plasma. Quantitative bioanalysis was performed utilizing both APCI and ESI mode and results were compared. Quality control standards when analyzed with APCI mode were found to be more consistent in terms of accuracy and precision as compared with ESI mode. Additionally, for some instances, up to 20-fold broader dynamic linearity range was observed with APCI mode as compared with ESI mode. As phospholid interferences have poor response in APCI mode, protein precipitation extraction technique can be used for multimatrix quantitation, which is more amenable to automation. The approach of multiple biological matrix quantitation against a single calibration curve helps bioanalysts to reduce turnaround time. Copyright © 2016 John Wiley & Sons, Ltd.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 μg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 μg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDMPREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing 13C2H4‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDMPREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r 2 = 0.997) over the range 5.0–1250 pg/1 × 106 PBMCs. The inaccuracy was

A simple, rapid, and selective method for determination of plasma biotin was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After single-step protein precipitation with methanol, biotin and stable isotope-labeled biotin as an internal standard (IS) were chromatographed on a pentafluorophenyl stationary-phase column (2.1 × 100 mm, 2.7 μm) under isocratic conditions using 10 mm ammonium formate-acetonitrile (93:7, v/v) at a flow rate of 0.6 mL/min. The total chromatographic runtime was 5 min for each injection. Detection was performed in a positive electrospray ionization mode by monitoring selected ion transitions at m/z 245.1/227.0 and 249.1/231.0 for biotin and the IS, respectively. The calibration curve was linear in the range of 0.05-2 ng/mL using 300 μL of plasma. The intra- and inter-day precisions were all