Liquid Chromatography-tandem Mass Spectrometric Method Research Articles

High-throughput quantitation of serological dimethylarginines by LC/MS/MS: Potential cardiovascular biomarkers for rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by systemic inflammation of the joints and extra-articular tissues. The incidence of cardiovascular disease (CVD) remains the main cause of morbidity and mortality in patients with RA. Despite the development of new therapeutics targeting the articular manifestations, the relief of the cardiovascular burden is still an unmet medical need during the management of RA. So, the early prognosis of RA-associated CVD plays a crucial role in improving the clinical outcomes of RA patients. Recently, circulating dimethylarginines have gained attention as potential biomarkers for CVDs. Here, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of creatinine, arginine, and dimethylarginines in human serum within 2 mins by isotope dilution mass spectrometry. This method employed a protein precipitation method for rapid sample preparation, trichloroacetic acid (TCA)-based ion pairing chromatography for fast analyte separation, and multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (ISs) for simultaneous quantitation. To assure the quality, our method was validated against the FDA guidelines for lower limit of quantitation (0.2 µM), linearity (square of coefficient correlation>0.99), precision (intra-&inter-assay imprecision < 10 %), accuracy (intra-&inter-assay inaccuracy < 10 %), sample preparation recovery (recovery ≥ 90 %), stability (instability < 10 %), matrix effect (signal suppression < 55 %), and carryover ( < 0.01 %). Afterward, we applied the validated method to a retrospective cross-sectional study. We aimed to evaluate the utility of serological dimethylarginines as potential cardiovascular biomarkers in the development of RA-associated CVD. Our results revealed that the serological ratio of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), an indicator of physiological arginine methylation status, was significantly elevated in patients with RA. This finding might provide value in detecting CVD to improve clinical outcomes in RA management.

Development and validation of a sensitive assay for the quantification of arachidonoylcyclopropylamide (ACPA) in cell culture by LC–MS/MS

Synthetic and natural cannabinoid derivatives are highly investigated as drug candidates due to their antinociceptive, antiepileptic and anticancer potential. Arachidonoylcyclopropylamide (ACPA) is a synthetic cannabinoid with antiproliferative and apoptotic effects on non-small cell lung cancer and pancreatic and endometrial carcinoma. Thus, ACPA has a great potential for being used as an anticancer drug for epithelial cancers. Therefore, determining the levels of ACPA in biological fluids, cells, tissues and pharmaceutical dosage forms is crucial in monitoring the effects of various pharmacological, physiological and pathological stimuli on biological systems. However, the challenge in the quantification of ACPA is its short half-life and lack of UV signal. Therefore, we developed a liquid chromatography-tandem mass spectrometric (LC–MS/MS) method for sensitive and selective quantification of ACPA in cell culture medium and intracellular matrix. Multiple reaction monitoring in the positive ionization mode was used for detection with 344 → 203 m/z transitions. The separation of ACPA was performed on C18 column (50 × 3.0 mm, 2.1 μm) with the mobile phase run in the gradient mode with 0.1% formic acid (FA) in water and 0.1% FA in acetonitrile at a flow rate of 0.3 ml/min. The assay was linear in the concentration range of 1.8–1000 ng/mL (r = 0.999). The validation studies revealed that the method was linear, sensitive, accurate, precise, selective, repeatable, robust and rugged. Finally, the developed method was applied to quantify ACPA in cell culture medium and intracellular matrix.

Opportunities of Amlodipine as a Potential Candidate in the Evaluation of Drug Compliance during Antihypertensive Therapy.

Background and Objectives: Blood pressure measurement is essential evidence to establish that the chosen medicine and dosage are appropriate, and also indirectly indicates whether the medicine is being used at all. Therefore, current research compares adherence to the target blood pressure at home and in the hospital between different age groups, using similar combinations of the drugs prescribed by the doctor within ongoing antihypertensive therapy. Moreover, it is very important to develop a method for the determination of amlodipine and its metabolite, which would suitable for clinical applications, when the result is needed as quick as possible. Materials and Methods: This prospective study included patients aged ≥18 years who were diagnosed with hypertension. Subjects were divided into two age groups according to European Society of Cardiology (ESC) hypertension guidelines; older patients (≥65 years) and adult patients (<65 years). Assessment of adherence rate to antihypertensive medications was performed using a measurement of systolic blood pressure and comparing this to ESC hypertension guideline data. A simple liquid chromatography-tandem mass spectrometer (LC-MS/MS) method for determination of amlodipine and dehydroamlodipine was developed and validated according to the European Medicines Agency guideline on bioanalytical method validation at the Latvian Institute of Organic Synthesis. Results: A total of 81 patients with arterial hypertension were enrolled in this study. A significant number of patients were overweight (N = 33, 40.7%) and obese (N = 36, 44.4%). To control arterial hypertension, 70 (86.4%) patients used fixed-dose combinations, where one of the components was amlodipine. Practically, 36 (44.4%) hypertensive subjects were not able to comply with target blood pressure. Nonetheless, 38 (46.9%) patients who received fixed-dose combinations were able to comply with target blood pressure. Conclusions: Adherence to ESC hypertension guideline proposed target blood pressure was relatively low among hypertensive subjects even though a significant number of patients were taking fixed-dose combinations. Therefore, optimizing prevention, recognition, and care of hypertensive young adults require intensive educational interventions. Moreover, survey data suggest that therapeutic drug monitoring using the validated simple, sensitive LC-MS/MS method is pivotal for further understanding factors influencing adherence.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometric Method for the Quantification of Diclofenac in Human Plasma

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometric Method for the Quantification of Diclofenac in Human Plasma

Determination of 35 sulfonamides in pork by magnetic molecularly imprinted polymer-based dispersive solid-phase extraction and ultra-performance liquid chromatography photodiode array method.

Sulfonamide residues in foods of animal origin are potential risks to consumer health, so it is very important to inspect them. Among the previously reported instrumental methods, the best method can only be used to determine at most 22 sulfonamides. Thus, an instrumental method capable of determining more sulfonamide species is desirable. In this study, sulfadoxine was used as a template to synthesize a type of magnetic molecularly imprinted polymer that could recognize 35 sulfonamides. After characterization, this composite was used to develop a dispersive solid-phase extraction method for extraction and purification of the 35 sulfonamides in pork, followed by determination using an ultra-performance liquid chromatography photodiode array method. This composite exhibited high adsorption capacity (11.01-19.21 μg mg-1 ) and high recovery (>89.01%), and could be reused at least ten times. Due to the enrichment effect during sample preparation (enrichment factor 22-66), the limits of detection for determination of the 35 drugs in pork were in the range of 0.08-0.53 ng g-1 . The detection results for some real pork samples were consistent with a liquid chromatographic-tandem mass spectrometric method. After comparison, the present method showed generally better performances than the previously reported sample preparation methods and instrumental methods for detection of sulfonamides. The method developed in the present study could be used as a practical tool for routine detection of sulfonamide residues in pork samples. © 2022 Society of Chemical Industry.

The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) andhumanMDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

VALIDATION OF LC-MS/MS METHOD FOR DETERMINATION OF CYPROHEPTADINE IN DIETARY SUPPLEMENTS

Aims: A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was validated for determination of prohibited cyproheptadine hydrochloride (CP) in dietary supplements.&#x0D; Methods: CP was extracted by sonication in methanol for 30 min. The chromatography separation of CP took place on C18 column (100 mm x 2.1 mm, 1.8 µm) with gradient mobile phase of both of acetonitrile and water containing 0.1% formic acid. Multiple reaction monitoring (MRM) in the positive mode was used to quantify and confirm CP at m/z 288.2/191.1 and 288.1/96.0, respectively.&#x0D; Results: The method was validated according to the AOAC requirements. The linearity ranges were found from 0.1 to 50 ng. mL-1 of CP (R2 = 1). The limit of detection and limit of quantification were 1.5 ng/g or ng/mL and 5 ng/g or ng/mL, respectively. The accuracy was within the range from 92 to 99%, with the relative standard deviation (RSD%) of 2.0-5.9%.&#x0D; Conclusion: The validated parameters have met the requirement of Association of Official Agricultural Chemists (AOAC). This reliable method would be useful for the monitoring of cyproheptadine in dietary supplements.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of 25 psychoactive drugs in cerumen and its application to real postmortem samples.

In the present study, a method for the detection of 25 psychoactive substances in cerumen was developed and validated. This method targets opiates, cocaine, antidepressants, benzodiazepines, antipsychotics and antiparkinsons. Analysis was performed on a SCIEX Triple Quad 6500+ system after liquid-liquid extraction. Methanol with 1% acetic acid was chosen as the extraction solvent. After the addition of the solvent, samples were vortexed, sonicated, centrifuged and directly injected into the liquid chromatography-tandem mass spectrometry system. The method was found to be selective and sensitive (limit of detection: 0.017ng-0.33ng/mg), the assay was linear for all analytes with linear regression coefficient ranging 0.9911-1.00. The values for intra-assay precision was between 4.34 and 14.6% and inter-assay precision between 5.81 and 17.7%, with accuracy within the acceptable criteria for all analytes. All analytes in cerumen specimens were stable for 48h at 4°C and 72h at -20°C, whilst no significant matrix effect or carryover was observed. Applicability was proven by analyzing cerumen samples from 25 deceased with a history of drug abuse. All analytes were detected in real samples, thus confirming the sensitivity of the developed method. According to our knowledge, it is the first time that a method for the simultaneous detection of 25 psychoactive drugs in cerumen was developed, fully validated and finally applied to 25 postmortem samples.

Development of a Highly Sensitive and Rapid Liquid Chromatography-Tandem Mass Spectrometric Method Using a Basic Mobile Phase Additive to Determine the Characteristics of the Urinary Metabolites for Niemann-Pick Disease Type C.

As Niemann-Pick disease type C (NPC) is difficult to diagnose owing to its various clinical symptoms; biomarker tests have been developed. Previously, we revealed urinary sulfated cholesterol metabolites as noninvasive biomarkers for NPC. However, LC/tandem mass spectrometry (LC/MS/MS) requires long separation time and large urine volumes. Recently, a basic mobile phase was reported to increase the MS intensity. Thus, we developed a highly sensitive and rapid LC/MS/MS method for analyzing urinary cholesterol metabolites using a basic mobile phase additive. 3β-Sulfooxy-7β-N-acetylglucosaminyl-5-cholenic acid, its glycine and taurine conjugates, 3β-sulfooxy-7β-hydroxy-5-cholenic acid, and 7-oxo form were measured, with selected reaction monitoring in negative ion mode. Oasis HLB and L-column 3 were used for column-switching LC/MS/MS and urine diluted 10-fold was employed as the sample. After trapping, gradient separation was performed using solutions containing 1% (v/v) ammonium solution. On average, a 16-fold increase in peak areas was observed compared to that obtained at pH 5.5 with the mobile phases. Although the previous method needed 60 min for separation from interference peaks, we succeeded to separate them in 7 min with optimized LC condition. Further, all compounds showed good linearity from 0.3-1000 ng/mL, with satisfactory intra- and inter-day reproducibility. The developed method was applied to the urinalysis of healthy participants and NPC patients. Overall, the concentrations of metabolites correlated with those obtained using the previous method. Therefore, we succeeded to increasing MS intensity and shorten LC running time; and the method is useful for the noninvasive diagnostic screening of patients with NPC.

COVID-19 patients with altered steroid hormone levels are more likely to have higher disease severity.

PurposeThis study aims to evaluate the correlations between the severity of the disease and serum steroid levels by analyzing the serum steroid levels in COVID-19 patients with different levels of disease progression and the control group.MethodsMorning serum Aldosterone, 11-deoxycortisol, Androstenedione, 17-hydroxyprogesterone, Dihydrotestosterone (DHT), Dehydroepiandrosterone (DHEA), Corticosterone, Dehydroepiandrosterone sulfate (DHEAS), Estrone, Estradiol, Progesterone, 11-deoxycorticosterone, Cortisol, Corticosterone, Androsterone, Pregnenolone, 17-hydroxypregnenolone and 21-deoxycortisol levels were measured in 153 consecutive patients were grouped as mild, moderate, and severe based on the WHO COVID-19 disease severity classification and the control group. Steroid hormone levels were analyzed at once with a liquid chromatography-tandem mass spectrometric method (LC-MS/MS).ResultsIn our study, nearly all steroids were statistically significantly higher in the patients’ group than in the control group (p < 0.001). Also, DHEA was an independent indicator of the disease severity with COVID-19ConclusionsOur study reveals that the alteration in steroid hormone levels was correlated with disease severity. Also, steroid hormone levels should be followed up during COVID-19 disease management.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3′-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole) and KD85 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Development of a liquid chromatography mass spectrometry method for the determination of vitamin K1, menaquinone-4, menaquinone-7 and vitamin K1-2,3 epoxide in serum of individuals without vitamin K supplements.

Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations inthe blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research. We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake. The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06-9.0nmol/L for all analytes except for MK-7 with 0.04-6.16nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors: the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient. The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.

Simultaneous determination of novel β-lactamase inhibitor WCK 4234 and Meropenem in dog plasma by LC-MS/MS and its application to preclinical pharmacokinetic study.

A precise and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of WCK 4234 and meropenem (MEM) in dog plasma. Protein precipitation using acetonitrile was employed as a sample preparation approach. Cefepime was used as an internal standard. The developed method was selective, sensitive (limit of quantification, 0.075 μg/ml for both drugs), accurate (recovery > 90%), precise (CV <10%) and linear (r2 ≥0.99, concentration range 0.075-120 μg/ml for both analytes). The developed method was successfully applied for the determination of both drugs in plasma to assess the pharmacokinetics in beagle dogs. WCK 4234 + MEM in a 1:1 ratio at 15 + 15 and 30 + 30 mg/kg doses were administered by the intravenous route. The mean plasma concentration and area under the concentration-time curve of WCK 4234 ranged from 38.3 to 77.4μg/ml and from 47.8 to 77.1μg h/ml, respectively, and the values for MEM ranged from 52.2 to 115.3μg/ml and 70.5 to 133.6μg h/ml respectively. The elimination half-life of WCK 4234 and MEM was around 0.8h.

Detection of bazedoxifene, a selective estrogen receptor modulator, in human urine by liquid chromatography-tandem mass spectrometry.

Bazedoxifene, a selective estrogen receptor modulator, has been explicitly included in the prohibited list issued by the World Anti-Doping Agency (WADA) since January 2020. A high-resolution liquid chromatography-tandem mass spectrometric detection method was developed to identify bazedoxifene and its metabolites in human urine and to quantify bazedoxifene (free plus glucuronide) for doping control purposes. Bazedoxifene acetate (20 mg) was orally administered to seven male volunteers, and the urine samples collected were analyzed using the developed method. The linearity ranged from 0.5 to 200 ng/ml, and the limit of detection was <0.2 ng/ml. The interday precision (2.2% to 3.6%) and the interday accuracy (-10.0% to 1.9%) were adequate. Bazedoxifene, bazedoxifene-N-oxide, and bazedoxifene glucoconjugates were identified in the urine samples. The profiles of the urinary excretion indicated the presence of small amounts of free bazedoxifene and bazedoxifene-N-oxide, whereas bazedoxifene glucuronide was the predominant metabolite. The cumulative excretion amount of bazedoxifene (free form plus glucuronide conjugate) within 78 h after the administration was 0.7% to 1.3% of the total dose. In all subjects, bazedoxifene (free plus glucuronide) could be detected in urine up to 78 h after administration.

Development and validation of ultra-performance liquid chromatography-tandem mass spectrometric methods for simultaneous and rapid determination of contezolid and its major metabolite M2 in plasma and urine samples and its application to a study in subjects with moderate liver impairment

Contezolid is a novel oxazolidinone antibiotic with good antibacterial activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. For the purpose to further characterize the pharmacokinetics of contezolid and its major metabolite M2, accurate and rapid ultra-performance liquid chromatography-tandem mass spectrometric assays (UPLC-MS/MS) were developed and validated for simultaneous quantification of contezolid and M2 in human plasma and urine. The plasma samples were pretreated by liquid–liquid extraction. The automated solid phase extraction method was used to preprocess urine samples. ACQUITY UPLC® BEH C8 (2.1 mm × 100 mm, 1.7 µm) column was used to separate the analytes with a gradient mobile phase of acetonitrile and water at a flow rate of 0.4 mL/min. The calibration curves showed good linearity over the concentration ranges of 0.0100–5.00 µg/mL for contezolid in plasma and urine, 0.00200–1.00 µg/mL in plasma and 0.0200–10.0 µg/mL in urine for M2, respectively. For both plasma and urine assays, the intra- and inter-batch accuracy and precision were within 15% for all quality control levels, including the lower limit of quantitation. The methods were fully validated and successfully applied to a pharmacokinetic study of contezolid tablets in subjects with moderate hepatic impairment.

A rapid and sensitive UPLC–MS/MS method for quantification of erdosteine as bulk drug and in capsules as dosage forms

A rapid, sensitive, specific ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS-MS) method was developed for the determination of erdosteine (ERD) in pharmaceutical preparations. The chromatographic separation was achieved with 0.1% formic acid in combination with acetonitrile (25:75 v/v) using C18 UPLC column, 95Å, 2.1 x 50 mm, 1.8 µm. The flow rate was 0.15 mL/min and the total run time was 2.0 min. The column temperature was kept constant at 40 °C and the injection volume was 5 μL. Ibuprofen was used as internal standard (IS). The mass transitions of ERD and IS were m/z 249.9 → 231.8 and 205.1 → 161.0. Also, another product ion of ERD (m/z 249.80 → 231.80) was monitored as predictive ion during the analysis. The standard calibration curve shows determination coefficient (R2) greater than 0.996 with a range of 1-5000 ng/mL using the linear regression model. Within-run precision and between-run repeatability were expressed as relative standard deviation and were lower than 5%. The developed method was successfully applied in the analysis of ERD-containing capsule formulation indicating that the method could be used for routine quality control analyses. Keywords: erdosteine, UPLC-MS/MS, multiple reaction monitoring, pharmaceutical analysis, method validation

Simultaneous determination of periplocin, periplocymarin, periplogenin, periplocoside M and periplocoside N of Cortex Periplocae in rat plasma and its application to a pharmacokinetic study.

A sensitive and specific ultra-performance liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously determine periplocin, periplocymarin (PM), periplogenin (PG), periplocoside M (PSM) and periplocoside N (PSN) in rat plasma. Acetonitrile was employed to precipitate plasma with appropriate sensitivity and acceptable matrix effects. Chromatographic separation was performed using a Waters HSS T3 column with a gradient elution using water and acetonitrile both containing 0.1% formic acid and 0.1mm ammonium formate within 8min. Detection was performed in positive ionization mode using multiple reaction monitoring. The method was fully validated in terms of selectivity, linearity, accuracy, precision, recovery, matrix effects and stability. Using this method, the concentrations of periplocin, PM, PG, PSM and PSN were established after oral administration of Cortex Periplocae extract to rats, and the pharmacokinetic characteristics of periplocin, PM, PG, PSM and PSN were assessed. Generally, PM, PG, PSM and PSN were eliminated slowly and their half-lives were all >8h. In addition, the systemic exposure of PSM showed significant differences between genders with more than 10 times higher area under the concentration-time curve in female rats than in male rats. The findings of this study provide useful information for further research on Cortex Periplocae.

Determination of Acotiamide in Human Plasma by LC-MS/MS and its Application to a Pharmacokinetic Study

Background: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method had been developed for the quantification of acotiamide in rat plasma and been applied to pharmacokinetic studies. However, there was no LC-MS/MS method been developed for the determination of acotiamide in human plasma and its pharmacokinetic study. Objective: A simple and fast LC-MS/MS method was established and validated for the quantification of acotiamide in human Received: plasma and was applied to a pharmacokinetic study. Methods: Sample preparation was accomplished Revised: Accepted: through protein precipitation, and chromatographic separation was achieved on a Welch, Ultimate XB-C18 column (2.1×50 mm, 3 μm) with a security guard cartridge C18 using a binary gradient with DOI: mobile phase A (Methanol) and B (the solution of 10 mM Ammonium acetate with 0.1% Formic acid) at a flow rate of 400 μL/min. Results: The retention time of acotiamide and its internal standard, acotiamide-d6 was 1.78 min and 1.79 min, respectively. The total run time was 4.0 min. The method was developed and validated over the concentration range of 0.500-100 ng/mL for acotiamide, with correlation coefficient greater than 0.9987. The extraction recovery was more than 108.43% and the matrix effect was not significant. The inter- and intra-day precisions were below 5.80% and accuracies ranged from 92.7 to 103.0%. Acotiamide was demonstrated to be stable in human plasma under the tested conditions. Conclusion: The validated LC-MS/MS method was successfully applied to study the pharmacokinetic profiles of acotiamide in human plasma after oral administration and has achieved satisfactory results.

Toxicokinetic and bioavailability studies on retrorsine in mice, and ketoconazole-induced alteration in toxicokinetic properties.

Retrorsine (RTS) is a toxic retronecine-type pyrrolizidine alkaloid, which is widely distributed. The purpose of this study was to develop a high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum RTS determination in mice. Serum samples were deproteinated by acetonitrile, separated on a C18 -PFP column and delivered at 0.8ml/min with an eluting system composed of water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as mobile phases. RTS and the internal standard S-hexylglutathione (H-GSH) were quantitatively monitored with precursor-to-product transitions of m/z 352.1 → 120.1 and m/z 392.2 → 246.3, respectively. The method showed excellent linearity over the concentration range 0.05-50 μg/ml, with correlation coefficient r2 = 0.9992. The extraction recovery was >86.34%, and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <4.99%. The validated LC-MS/MS method was successfully applied to study the toxicokinetic profiles of serum RTS in mice after intravenous, oral administration and co-treated with ketoconazole, which showed that RTS displayed a long half-life (~11.05 h) and good bioavailability (81.80%). Co-administration of ketoconazole (KTZ) increased the peak serum concentration and area under the concentration-time curve and decreased the clearance and mean residence time. Summing up, a new standardized method was established for quantitative determination of RTS in sera.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of free and total dabigatran in human plasma and its application to a pharmacokinetic study

A high-performance liquid chromatography-tandem mass spectrometric method for the determination of free and total dabigatran in human plasma has been developed and validated using a stable labeled internal standard (IS) as dabigatran D4. The extraction of analyte and IS was accomplished by the solid-phase extraction technique. Chromatographic separations were achieved using Peerless basic C8 (150 × 4.6) mm, 5 µ column eluted at a flow rate of 1 mL/min with mobile phase Acetonitrile: 5 mM ammonium formate: Methanol and 0.2% formic acid (30:20:50, v/v/v). The run time of the method was about 2.5 min with elution times of dabigatran and dabigatran D4 at around 1.2 min. The multiple reaction monitoring transitions (Q1/Q3) were set at 472/289, 172 (m/z) for dabigatran, and 476/293 (m/z) for dabigatran D4. The calibration curves were linear (r 2 ≥0.99) over the range of 1.04–406.49 ng/mL. The presented method was successfully employed in the analysis of pharmacokinetic studies with the added advantage of demonstrating the effect of co-administration of dabigatran with the proton pump inhibitor pantoprazole on bioavailability and pharmacokinetic characteristics. Re-analysis of incurred sample resulted in >98% compliance indicating good assay precision of target analytes.