Triple-quadruple Tandem Mass Spectrometer Research Articles

A sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide in human plasma and pharmacokinetic studies

Pomalidomide is an immunomodulatory agent (IMiD) that has been approved by the US Food and Drug Administration (FDA) for clinical treatment of patients with multiple myeloma. In this work, we developed a sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide over the range of 1.006–100.6 ng/mL (R2 = 0.9991) in human plasma and pharmacokinetic studies. A liquid-liquid extraction method using ethyl acetate was applied to extract pomalidomide and afatinib (as an internal standard, IS) from human plasma. Chromatographic separation was performed on a Hedera ODS column (150 mm × 2.1 mm, 5 µm) with security guard C18 column (4 mm × 2.0 mm) at 40 °C. Methanol and 10 mmol/L aqueous solution of ammonium acetate containing 0.1% formic acid were used as a gradient elution mobile phase, and the flow rate was 0.4 mL/min. A triple quadruple tandem mass spectrometer using multiplex reaction monitoring mode (MRM) with electrospray ionization (ESI) positive ionization was employed. The precursor to product ion transitions for the quantitative analysis of pomalidomide and the IS were m/z 274.2→163.1 and m/z 486.1 → 371.1, respectively. This established method has been validated according to regulatory guideline, and the results were all within the acceptance criteria. The validated LC-MS/MS method was successfully applied to analyze samples obtained from clinical pharmacokinetics study after oral administration of pomalidomide (4 mg) capsules in human.

Development and validation of an LC-MS/MS method for quantifying nine antimicrobials in human serum and its application to study the exposure of Chinese pregnant women to antimicrobials.

BackgroundTo study the prevalence of the exposure of pregnant women to antimicrobials, a sensitive and reliable liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated to determine nine antimicrobials, namely sulfadimidine, sulfapyridine, sulfadiazine, sulfathiazole, ofloxacin, ciprofloxacin, norfloxacin, tetracycline, and lincomycin, in human serum.MethodsThe sample preparation procedure included protein precipitation followed by a cleanup step with solid phase extraction (SPE). Separation was carried out using a CORTECS T3 column (100 × 2.1 mm, 2.7 µm) by gradient elution with a runtime of 8.0 min. Detection was performed on a triple quadruple tandem mass spectrometer with scheduled multiple reaction monitoring (sMRM) in positive ion scan mode.ResultsThe calibration curves were linear over the concentration range of 0.5–50 ng/ml, and the limit of quantitation was between 0.01 and 0.2 ng/ml. For each level of quality control samples, the inter‐ and intra‐assay precision values were less than 12.0%, and the accuracy ranged from 86.1% to 109.0%. No significant matrix effect or carryover was observed. The antimicrobials of interest were stable under all investigated conditions. The validated method was applied to analyze clinical samples from pregnant women in China, and 10 out of 500 samples showed the presence of antimicrobial residues. Moreover, compared with the time‐resolved fluoro‐immunoassay (TRFIA) method, the developed method showed greater sensitivity and specificity.ConclusionThis study provides a simple and rapid LC‐MS/MS method for the simultaneous measurement of nine antimicrobials in serum samples, which could be a useful tool in clinical utilization.

Measurement of plasma 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in population of patients with cardiovascular disease by UPLC-MS/MS method

Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25-hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC–MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid–liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triple-quadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (<20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status.

Development and validation of a LC-MS/MS method for simultaneous determination of six glucocorticoids and its application to a pharmacokinetic study in nude mice

In this study, a simple, rapid and sensitive LC–MS/MS analytical method for simultaneous determination of six glucocorticoids including 21-hydroxy deflazacort (21-OH DFZ), prednisolone (PNL), betamethasone (BET), beclomethasone (BEC), triamcinolone acetonide (TCA), budesonide (BUD) in nude mice plasma was developed and validated. Using testosterone as internal standard, the plasma samples were prepared by precipitation with acetonitrile and separated using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase composed of acetonitrile and 0.1 % (v/v) formic acid aqueous solution by gradient elution at a flow rate of 0.3 mL/min. Quantitation was performed on a triple quadruple tandem mass spectrometer by multiple reaction monitoring in positive electrospray ionization mode. Calibration curves were developed over the range of 1−400 ng/mL for TCA, 5−2000 ng/mL for 21-OH DFZ, BET, BEC as well as BUD, and 10−2000 ng/mL for PNL. The accuracy, precision, matrix effect, recovery and stability were validated to be within acceptable criteria. The method was successfully applied to a preclinical pharmacokinetic (PK) study of the six GCs on female nude mice after a single oral dose of 1 mg/kg. The PK profiles of all the six GCs were described by two-compartment model with first-order absorption rate.

Development of a novel LC-MS/MS method for detection and quantification of tramadol hydrochloride in presence of some mislabeled drugs: Application to counterfeit study.

Counterfeiting of pharmaceuticals has become a serious problem all over the world, particularly in developing countries. In the present work, a highly sensitive LC-MS/MS method was developed for simultaneous determination of tramadol hydrochloride in the presence of some suspected mislabeled drugs such as alprazolam, diazepam, chlorpheniramine maleate, diphenylhydramine and paracetamol. The prepared samples were analyzed on an API 4000 mass spectrometer using an Eclipse C18 column (3.5 μm, 4.6 × 100 mm). The mobile phase consisting of 0.01% formic acid, acetonitrile and methanol (60:20:20 v/v/v) was pumped with an isocratic elution at a flow rate of 0.7 mL min-1 . The detection was achieved on a triple quadruple tandem mass spectrometer in multiple reaction monitoring mode. The proposed method was successfully validated according to International Conference on Harmonization guidelines with respect to accuracy, precision, linearity, limit of detection and limit of quantitation. The calibration linear range for tramadol hydrochloride, alprazolam, diazepam, chlorpheniramine maleate, diphenylhydramine and paracetamol was 5-500 ng mL-1 . The results revealed that the applied method is promising for the differentiation of genuine tramadol tablets from counterfeit ones without prior separation.

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.

Quantitative determination of levonorgestrel in beagle dog plasma after vaginal administration of intravaginal ring by high-performance liquid chromatography-tandem mass spectrometry.

A specific and sensitive high-performance liquid chromatography-tandem mass spectrometry method that could be used to determine the concentration of levonorgestrel (LNG) in beagle dog plasma was developed. Specifically, terfenadine was used as the internal standard (IS). The separation was achieved on a Kinetex-C18 110A column (3 × 30 mm i.d., 2.6 um, Phenomenex) and a gradient mobile phase consists of methanol (0.1% formic acid) and water (0.1% formic acid) was used. The flow rate was 0.8 mL/min and the injection volume was 10 μL. The detection was performed on a triple-quadruple tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. Quantitative analysis was carried out at m/z 313.0 → 108.9 and m/z 472.6 → 436.2 for LNG and IS respectively. This method demonstrated the linearity of LNG over a concentration range of 0.5-50 ng/mL with a coefficient correlation (r) of 0.9973. The lower limit of quantification was 0.5 ng/mL. The intra- and inter-assay precisions were 2.9 and 11.2%, with an accuracy range from 94.8 to 108.4%. The stability data indicated that sample preparation and storage process had little effect on the concentration of LNG QC sample. The validated method was successfully applied to study the pharmacokinetics of LNG in beagle dog after vaginal administration of intravaginal ring.

A pilot data analysis of a metabolomic HPLC-MS/MS study of patients with COPD.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous condition with multiple clinical faces. Metabolomic profiling studies small molecules present in biological samples by combined use of chromatography with mass spectrometry. The goal of our work was to perform a high performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) metabolomic study to compare the concentrations of metabolites in COPD patients and in controls. Participants were recruited at the University Hospital, Hradec Králové, Czech Republic, with the approval of the ethics committee. The analysis of blood samples was performed at Health Sciences Center (HSC) in Kuwait. The blood samples were analyzed for concentrations of acylcarnitines and amino acids by high performance liquid chromatography (Waters 2690 HPLC; Waters, Milford, USA) and a triple-quadruple tandem mass spectrometer (Quattro LC, Micromass, Manchester, United Kingdom). Groups of 10 subjects with COPD and 10 healthy controls were analyzed. Carnitine analysis showed that the free carnitine to acylcarnitine ratio (C0/AC ratio) was significantly lower in COPD (0.58 μM/L) compared to the controls (0.73 μM/L; p = 0.002). The mean C8/C2 ratio in the COPD group was significantly higher (0.03 μM/L) - in the control group it was 0 μM/L (p = 0.03). Amino acid analysis showed lower levels of phenylalanine in the COPD group (22.05 μM/L) compared to the controls (30.05 μM/L; p = 0.008). The alanine concentrations were significantly lower in the COPD group (173 μM/L) than in the control group (253 μM/L; p = 0.001). The pyroglutamate levels were higher in COPD (1.58 μM/L) than in the controls (1 μM/L; p = 0.040). The carnitine and acylcarnitine levels in COPD subjects in this study possibly indicate a predisposition to atherosclerosis as a result of inadequate β-oxidation of fatty acids and show the presence of oxidative stress. Furthermore, the high sensitivity to changes in circulating amino acid levels may allow us to detect subclinical malnutrition and take early preventative interventions such as nutritional supplementation and patient education.

Determination of Riluzole in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and its Application to a Pharmacokinetic Study

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method was developed and validated for the estimation of Riluzole in human plasma using Olanzapine as an internal standard. Riluzole and Internal standard were extracted from 0.5 mL plasma by solid phase extraction method. The analytical separation was carried out in a reverse liquid chromatography by using C18 (50 x 4.6mm 1.8,) 10mMAmmonium Acetate: Methanol (10:90) v/v at 0.5 mL/min at isocratic mode . The detection was performed on a triple quadruple tandem mass spectrometer by multiple reactions monitoring (MRM) mode via electro spray ionization (ESI) source. Analytes were monitored in multiple reactions monitoring mode using the respective [M+H] +ions, m/z 234.84/137.63 for Riluzole and m/z 313.15/256.14 for the internal standard, respectively. The proposed method was validated with linear range of 5 -1000ng/ml for Riluzole with a runtime 2.5 minutes. The %R.S.D of intra-day and inter-day assay was lower than 15%. For its sensitivity and reliability, the proposed method is particularly suitable for pharmacokinetic studies.

Method Development Challenges and Regulatory Expectation in Nifedipine

Aim: A novel and accurate high-throughput tandem mass spectroscopic method has been developed and validated for determination of nifedipine, a calcium-channel blocker. Materials &amp; methods: Nifedipine was detected on a triple quadruple tandem mass spectrometer by multiple reactions monitoring mode via ESI source. Results: The selective and sensitive method was linear in the concentration range of 0.501 to 360.100 ng/ml and reported no matrix effect. The percent accuracy of the method was found to be between 99.7 and 102.80%, while percentage of RSD was in range of 1.77 to 4.73%. Conclusion: Validated method was used for bioavailability studies under fasting and fed conditions.

Determination of Ginkgolides A, B, C, J and Bilobalide in Plasma by LC-ESI (-)/MS/MS (QQQ) and its Application to the Pharmacokinetic Study of Ginkgo Biloba Extract in Rats.

A simple, rapid, and specific high-performance liquid chromatograph coupled with a tandem mass spectrometry method has been developed and validated for the quantification of ginkgolides in rat plasma, and the main pharmacokinetic parameters of ginkgolides after oral administration of Ginkgo biloba extract (GBE) was acquired. Methods: Plasma samples were pretreated with ethyl acetate extraction. Sulfamethoxazole was used as the internal standard (IS). Chromatographic separation was achieved on an Eclipse XDB-C18 column (2.1 mm×150 mm, 5 μm) with a mobile phase consisting of methanol/0.1% formic acid water (gradient elution: 0~25 min (77:23)→(60:40), V/V) at a flow rate of 0.3 mL·min-1. The detection was performed on a triple quadruple tandem mass spectrometer using an electrospray ionization (ESI) source for 25 min. The detection was operated by multiple reaction monitoring(MRM) under negative ionization mode of the transitions of m/z 325→163 for BB, 469→423 for GJ, 439→125 for GC, 453→351 for GA, 423→367 for GB and of m/z 252→156 for sulfamethoxazole (IS) respectively. Results: The pharmacokinetic properties of BB, GJ, GA, GB and GC were in line with the open 2-compartment model after oral administration of GBE in rats; The pharmacokinetic parameters of various lactones were calculated, and drugs-time curve and the curve fitting diagram of 5 ginkgolides were drew; The absorption and distribution rate of BB, GJ, GA, GB and GC were fast in rats in vivo, and half-life of absorption was less than 3 h. Conclusion: The developed LC-ESI (-)/MS/MS (QQQ) method was successfully applied to assess the pharmacokinetic parameters and oral bioavailability of ginkgolides in rats after administration of GBE, which can provide basis for further clinical efficacy studies.

Clinical development of imatinib: an anticancer drug.

Background:A novel and accurate high-throughput tandem mass spectroscopic method has been developed and validated for determination of imatinib, a protein-tyrosine kinase inhibitor against chronic myeloid leukemia.Materials & methods:Chromatographic separation was carried on XTerra® RP18 column (150 mm × 4.6 mm, 5 µm particle size) manufactured by Waters Corporation, MA, USA. The detection was performed on a triple quadruple tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization source.Results:The selective and sensitive method was linear in the concentration range of 9.57–4513.29 ng/ml and reported no matrix effect.Conclusion:The mean Cmax was found to be 10–15% lower in European subjects as compared with Indian subjects.

A HPLC–MS/MS method for the quantitation of free, conjugated, and total HDND-7, a novel hesperetin derivative, in rat plasma and tissues: Application to the pharmacokinetic and tissue distribution study

A sensitive and reliable HPLC–MS/MS method was developed and validated for the determination of free (unconjugated), glucuronidated, sulfated, and total (free and conjugated) HDND-7 in rat plasma and tissues. Plasma and tissues samples were treated prior to and after the enzyme hydrolysis. Chromatographic separation was achieved on a Phenomenex Luna C18 column (150×4.6mm, 3μm), using isocratic mobile phase consisting of 0.1% formic acid–acetonitrile (50:50, v/v) at a flow rate of 300μl/min. The detection was performed on a triple quadruple tandem mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 5.0min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 429.3→223.9 for HDND-7 and 272.9→152.9 for naringenin (IS), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The calibration curves for plasma and tissues were linear over a wide concentration range of 0.02–40μg/ml with a lower limit of quantification (LLOQ) of 0.02μg/ml. Mean extraction recoveries in plasma and tissues ranged from 87.4 to 97.1% and from 54.2 to 70.5%, respectively. The intra- and inter-day precision values were below 15% and the accuracy was within ±15%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study following oral doses of 25, 50 and 100mg/kg and intravenous dose of 25mg/kg, and tissue distribution study following oral dose of 50mg/kg.

An ultra high performance liquid chromatography with tandem mass spectrometry method for plasma and cerebrospinal fluid pharmacokinetics of rhein in patients with traumatic brain injury after administration of rhubarb decoction.

Damage of blood-brain barrier is a common result of traumatic brain injury. This damage can open the blood-brain barrier and allow drug passage. An ultraperformance liquid chromatography with tandem mass spectrometry method was established to determine the concentration of rhein in the biofluids (plasma and cerebrospinal fluid) of patients with a compromised blood-brain barrier following traumatic brain injury after rhubarb administration. Furthermore, the pharmacokinetic profiles were analyzed. A triple-quadruple tandem mass spectrometer with electrospray ionization was used for rhein detection. The mass transition followed was m/z 283.06→239.0. The calibration curve was linear in the concentration range of 10-8000 ng/mL for the biofluids. The intra- and interday precisions were less than 10%. The relative standard deviation of recovery was less than 15% in biological matrices. The pharmacokinetic data showed that rhein was rapidly transported into biofluids, and exhibited a peak concentration 1 h after rhubarb administration. The elimination rate of rhein was slow. The AUCcerebrospinal fluid /AUCplasma (AUC is area under curve) of rhein was approximately 17%, indicating that portions of rhein could pass the impaired blood-brain barrier. The method was successfully applied to quantify rhein in the biofluids of all patients. The data presented can help to guide clinical applications of rhubarb for treating traumatic brain injury.

A validated UPLC–MS/MS assay using negative ionization mode for high-throughput determination of pomalidomide in rat plasma

In this study, a sensitive UPLC-MS/MS assay was developed and validated for high-throughput determination of pomalidomide in rat plasma using celecoxib as an internal standard (IS). Liquid liquid extraction using dichloromethane was employed to extract pomalidomide and IS from 200μL of plasma. Chromatographic separation was carried on Acquity BEH™ C18 column (50mm×2.1mm, 1.7μm) using an isocratic mobile phase of acetonitrile: 10mM ammonium acetate (80:20, v/v), at a flow rate of 0.250mL/min. Both pomalidomide and IS were eluted at 0.66±0.03 and 0.80±0.03min, respectively, with a total run time of 1.5min only. A triple quadruple tandem mass spectrometer using electrospray ionization in negative mode was employed for analyte detection. The precursor to product ion transitions of m/z 272.01→160.89 for pomalidomide and m/z 380.08→316.01 for IS were used to quantify them respectively, multiple reaction monitoring mode. The developed method was validated according to regulatory guideline for bioanalytical method validation. The linearity in plasma sample was achieved in the concentration range of 0.47-400ng/mL (r(2)≥0.997). The intra and inter-day precision values were ≤11.1% (RSD, %) whereas accuracy values ranged from -6.8 to 8.5% (RE, %). In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of pomalidomide in rats.

A simple, rapid and reliable liquid chromatography-mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring.

A simple, rapid and reliable liquid chromatography-electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile-water (70:30, v/v), methotrexate (MTX) and p-aminoacetophenone (used as internal standard, IS) were separated on a Column C18 column (50 × 2.1 mm, 3 µm; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5 mL/min. The total chromatographic runtime was 5 min for each injection. Quantification detection was performed in a triple-quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m/z 455.3 → 308.3 for MTX and m/z 136.1 → 94.4 for IS. The calibration curve was linear over the range of 0.05-25.0 µmol/L with a lower limit of quantification of 0.05 µmol/L. The intra- and interday precisions were <5.2%, the accuracy varied from -4.1 to 4.5%. The recovery was >94%. The LC-MS/MS method showed an excellent agreement with the existing HPLC-UV method using Passing-Bablok regression and Bland-Altman difference plot analysis. The validated LC-MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories.

Trace level quantification of 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine HCl genotoxic impurity in trazodone using LC–MS/MS

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantitative determination of 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine HCl (CCP HCl) a process related impurity in trazodone. The method provided excellent sensitivity at a typical target analyte level of <0.1ppm, when the API samples were prepared at 5.0mg/mL. The CCP HCl sample was analyzed on a C18 symmetry (100mm×4.6mm, 3.5μm) column interfaced with a triple quadruple tandem mass spectrometer operated in a multiple reaction monitoring (MRM) mode. Positive electro spray ionization (ESI) was employed as the ionization source and the mobile phase used was 5.0mM ammonium acetate–acetonitrile (30:70, v/v). The injection precision of the lowest concentration standards was excellent with %RSD-1.42%. The calibration curve showed good linearity over the concentration range of 0.03–1.5ppm with a correlation coefficient of >0.9996. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.01 and 0.03ppm, respectively. The developed method was validated as per ICH guidelines in terms of LOD, LOQ, linearity, precision, accuracy, specificity and robustness.

Determination of zofenopril and its active metabolite in human plasma using high-performance liquid chromatography combined with a triple-quadruple tandem mass spectrometer.

A simple, selective and sensitive LC-MS-MS method has been developed and validated to simultaneously quantify zofenopril and its active metabolite zofenoprilat in human plasma, using diazepam as internal standard. 1,4-Dithiothreitol was used as a reducer to release and stabilize the thiol group of zofenoprilat from dimer and mixed forms with endogenous thiols in the treatment of plasma samples. After a liquid-liquid extraction with methyl tert-butyl ether under acidic conditions, the post-treatment samples were analyzed on an Agilent ZORBAX Eclipse XDB-C8 column interfaced with a triple-quadruple tandem mass spectrometer using positive electrospray ionization. A solution of methanol and 0.1% formic acid solution (85 : 15, v/v) was used as the isocratic mobile phase with a flow rate of 0.2 mL/min. The method was validated to demonstrate the specificity, lower limit of quantitation, accuracy and precision of measurements. The validated LC-MS-MS method has been successfully applied to study the pharmacokinetics of zofenopril calcium in healthy Chinese volunteers.

An HPLC-MS/MS method for the quantitative determination of platycodin D in rat plasma and its application to the pharmacokinetics of Platycodi Radix extract

AimsTo develop an HPLC-MS/MS method for the quantification of platycodin D (PD) in rat plasma, and to acquire the main pharmacokinetic parameters of PD after oral administration of pure PD or of Platycodi Radix extract (PRE) containing PD. MethodPlasma samples were pretreated with solid-phase extraction using an Oasis® HLB SPE cartridge. Madecassoside was used as the internal standard (IS). Chromatographic separation was achieved on an ODS column (100 mm × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of acetonitrile/water (30 : 70, V/V) containing 0.1 mmol·L−1 ammonium acetate at a flow rate of 0.25 mL·min−1. The detection was performed on a triple quadruple tandem mass spectrometer using an electrospray ionization (ESI) source with a chromatographic run time of 3.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 1 223.6→469.2 for PD and of m/z 973.6→469.2 for madecassoside (IS), respectively. ResultsThe calibration curve was linear from 5 to 2 000 ng·mL−1 (r2 >0.99) with a lower limit of quantification (LLOQ) of 5 ng·mL−1. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was from −15% to +15% at three quality control (QC) levels. Plasma concentrations of PD were determined for 24 h after i.v. administration of PD, and oral administration of PD and PRE, respectively. The absolute oral bioavailability of PD in rats was found to be (0.48 ± 0.19)% when administered PD, and to be (1.81 ± 0.89)% when administered PRE. ConclusionThe developed HPLC-MS/MS method was successfully applied to assess the pharmacokinetic parameters and oral bioavailability of PD in rats after administration of PD and Platycodi Radix extract.

Determination of xanthatin by ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry: Application to pharmacokinetic study of xanthatin in rat plasma

A sensitive, specific and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been established to study pharmacokinetic properties of xanthatin. Xanthatin, a compound which belongs to sesquiterpene lactone group, was determined in rat plasma with psoralen as internal standard. Chromatographic separation was performed on an Agilent Zorbax Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 μm) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase was composed of methanol and 0.1% formic acid water solution. Analysis was performed under a triple-quadruple tandem mass-spectrometer with an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM) mode to determine xanthatin at [M+H](+)m/z 247.3→m/z 205.2 and that of IS at [M+H](+)m/z 187.1→m/z 143.0 within 5 min. The assay method exhibited good separation of xanthatin from the interference of endogenous substances. The lower limit of quantification (LLOQ) was 1 ng/mL, with a good linearity within the concentration range of 1-5000 ng/mL (r=0.9990). Intra-day and inter-day precision RSD was less than 9.27%; intra-day and inter-day accuracy was 88.48% and 102.25% respectively. The extraction recoveries of xanthatin range from 82.12% to 89.55%, and the extraction RSD was less than 9.01%. The established LC-ESI-MS/MS method is rapid and sensitive, which has been successfully applied to quantify xanthatin in rat plasma for the first time.