A novel approach to the quantification of urinary aryl-propionamide-derived SARMs by UHPLC–MS/MS

A simple and sensitive procedure for the quantification of two commonly abused aryl-propionamide-derived selective androgen receptor modulators (SARMs), namely S-4 (GTx-007, andarine) and S-22 (GTx-024, MK-2866, ostarine, enobosarm), has been described. Urine samples were prepared for analysis by means of a dispersive liquid–liquid microextraction using methanol and chloroform as dispersive and extracting solvents, respectively. Factors that might influence the extraction process as well as their optimum conditions were evaluated by Box–Benken and central composite designs. After extraction, the analytes were quantified by UHPLC–MS/MS. The proposed procedure was validated on human urine samples. As a result, for both SARMs the detection limits were observed at 0.05 ng/mL and calibration curves were linear in the concentration range of 0.25–50 ng/mL with the coefficient of determination of 0.998.     

Quantification of fenoprofen in human plasma using UHPLC–tandem mass spectrometry for pharmacokinetic study in healthy subjects

A rapid, simple and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method has been developed to quantify fenoprofen, a nonsteroidal anti-inflammatory drug in human plasma for a pharmacokinetic study in healthy subjects. Owing to high levels of protein binding, protein precipitation followed by solid-phase extraction was employed for the extraction of fenoprofen and fenoprofen-d3 (used as internal standard) from 200 μL human plasma. Separation was performed on a BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using methanol−0.2% acetic acid in water (75:25, v/v) under isocratic elution. Electrospray ionization was operated in the negative mode for sample ionization. Ion transitions used for quantification in the selected reaction monitoring mode were m/z 241/197 and m/z 244/200 for fenoprofen and fenoprofen-d3, respectively. Under the optimized conditions, fenoprofen showed excellent linearity in the concentration range 0.02–20 μg/mL (r² ≥ 0.9996), adequate sensitivity, favorable accuracy (96.4–103.7%) and precision (percentage coefficient of variation ≤4.3) with negligible matrix effect. The validated method was successfully applied to a pharmacokinetic study of fenoprofen in healthy subjects. The significant features of the method include higher sensitivity, small plasma volume for processing and a short analysis time.     

Quantification and pharmacokinetic property of verubecestat an BACE1 inhibitor in rat plasma

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.     

Comparative bioavailability of two zolpidem hemitartrate formulations in healthy human Brazilian volunteers using high-performance liquid chromatography coupled to tandem mass spectrometry

To assess the bioequivalence of two zolpidem hemitartrate formulations in 30 healthy volunteers. Plasma samples were obtained over a 24 h period. Plasma concentrations of zolpidem were analyzed by liquid chromatography coupled to tandem mass spectrometry with positive ion electrospray ionization using multiple reaction monitoring. Values of peak concentration (C_max), area under curve (AUC), half-life, elimination constant, volume of distribution and clearance showed statistically significant differences when comparing women (604.34 ng h/ml, 127.36 ng/ml, 4.4 h, 0.18 1/h, 50.56 L and 8.55 L/h, respectively) and men (276.1 ng h/ml, 70.9 ng/ml, 3.3 h, 0.26 1/h, 91.42 L and 24.34 L/h, respectively), receiving the same dose (5 mg), respectively. The geometric means with corresponding 90% confidence interval for Test/Reference percentage ratios were 99.73% (CI 93.69–106.16) for C_max, 97.44% (90% CI = 91.85–103.37%) for area under curve of plasma concentration until the last concentration observed (AUC_last) and 98.30% (90% CI = 92.48–104.49) for the area under curve between the first sample (pre-dosage) and infinity (AUC_0–inf). Since the 90% CI for AUC_last, AUC_0–inf and C_max ratios were within the 80–125% interval proposed by the US Food and Drug Administration, it was concluded that zolpidem hemitartrate formulation (5 mg orodispersible tablet) is bioequivalent to the zolpidem hemitartrate formulation (Patz SL 5 mg sublingual tablet) with regard to both the rate and the extent of absorption. A new formulation of zolpidem 2.5 mg may be useful in women for the same clinical benefits as the 5 mg formulation in men.     

LC–MS/MS analysis of puerarin and 18β-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18β-glycyrrhetinic acid (18β-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18β-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography–electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18β-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18β-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

Quantitative determination of bilobetin in rat plasma by HPLC–MS/MS and its application to a pharmacokinetic study

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.     

Validated LC–MS/MS method for quantitation of a selective JAK1 inhibitor,filgotinib in rat plasma,and its application to a pharmacokinetic study in rats

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C₁₈ column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 → 291.3 and m/z 313.2 → 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78–1924 ng/mL. No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze–thaw cycles: on bench-top up to 6 h, in an autosampler up to 21 h, and at −80 ° C for 1 month. This novel method has been applied to a pharmacokinetic study in rats.     

Residues,dissipation and risk evaluation of spiroxamine in open-field-grown strawberries using liquid chromatography tandem mass spectrometry

The dissipation dynamic and residues of spiroxamine in open-field-grown strawberries were determined using liquid chromatography tandem mass spectrometry (LC–MS/MS). Spiroxamine application was performed according to Egyptian good agricultural practices recommendation. A QuEChERS-based extraction method along with direct analysis with an LC–MS/MS analytical method were optimized and validated, and the specificity of the techniques used was considered satisfactory. Good linearity (R² > 0.999) was obtained for spiroxamine within the range of 0.001–0.1 μg/ml. The mean recoveries varied between 97.1 and 108.2%, with inter- and intra-day precision (RSD) <4.9%. The limit of quantitation for spiroxamine was 0.001 mg/kg. The results indicated that spiroxamine degradation in strawberry followed first–order kinetics (R² > 0.9929) with an estimated half-life value of 4.71 days. Considering the Australian maximum residue limit (0.05 mg/kg) in strawberry and based on the results from residue trials with a preharvest interval of 14 days for strawberry, compliance can be expected. The present results could provide guidance to fully evaluate the risks of spiroxamine residues, preventing any potential health risk to consumers.     

UPLC–ESI–Q-TOF–MS/MS analysis of anticancer fractions from Ophiocordyceps xuefengensis and Ophiocordyceps sinensis

Ophiocordyceps xuefengensis (O. xuefengensis), a new species of caterpillar fungus, has been identified as the sister taxon of Ophiocordyceps sinensis (O. sinensis). The aims of the present study are to evaluate the anticancer activity and to qualitatively analyze the potential bioactive chemical constituents of O. xuefengensis and O. sinensis, comparatively. An MTT assay was used to evaluate the in vitro anticancer activities of different fractions from O. xuefengensis and O. sinensis. The results show that ethyl acetate fractions of O. xuefengensis and O. sinensis have significant in vitro anticancer activity. These two bioactive fractions were analyzed by ultra-performance liquid chromatography–electrospray ionization with quadrupole–time of flight tandem mass spectrometry technology. A total of 82 compounds and 101 compounds were identified or tentatively characterized in the bioactive fractions of O. xuefengensis and O. sinensis, respectively. Among these compounds, 68 existed in both O. xuefengensis and O. sinensis. A total of 67 compounds were reported in O. xuefengensis and 8 compounds were reported in caterpillar fungus for the first time. This is the first detailed comparative analysis of the in vitro anticancer activity and chemical ingredients between O. xuefengensis and O. sinensis. The application of this work will provide reliable fundamental pharmacological substances for the use of O. xuefengensis by Yao people.