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In this study, various neurosteroids in brain and plasma were simultaneously determined using liquid chromatography-tandem mass spectrometry and their profile changes in a stress-induced rats were investigated. The investigated neurosteroids are as follows: progesterone (P4), 5α-dihydroprogesterone (5α-DHP), 5β-dihydroprogesterone, estrone, androstenedione (AE), cortisol, cortisone, corticosterone (CORT), dehydroepiandrosterone (DHEA), pregnanolone (3α,5β-THP), allopregnanolone (ALLO), 11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), and 5α/5β-tetrahydrodeoxycorticosterone (5α/5β-THDOC). Brain and plasma samples were processed using solid-phase extraction with methanol and acetic acid (99:1), and derivatized with a hydroxylamine reagent. Separation was achieved within 13 min at a flow rate of 0.4 mL/min with a C18 column (3.0 × 50 mm, 2.7 μm). The triple quadrupole mass spectrometer was operated in the positive electrospray ionization mode. Using this method, the neurosteroid level variation was quantitated and investigated in the brain and plasma upon immobilization stress in rats. As a result, AE, CORT, DOC, P4, 5α-DHP, 5α/5β-THDOC, DHEA, 3α,5β-THP, ALLO, and PREG levels were significantly altered in both the brain and plasma samples when stress was induced. These findings demonstrated that stress leads to the alteration of the GABAergic neurosteroid profile. The present results will be helpful for furthering an understanding of the role of neurosteroids in stressed conditions. [ABSTRACT FROM AUTHOR]

For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C 18 column (150 mm × 3.9 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8 mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m / z 368.9→ m / z 191.9 (fraxin), m / z 206.9→ m / z 191.8 (fraxetin) and m / z 339.0→ m / z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r ≥ 0.995) over a concentration range of 5.00–3000 ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from −5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50 mg/kg to rats. [ABSTRACT FROM AUTHOR]

Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the development of an analytical method for determination of both drugs in plasma with precision. A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of lenalidomide, ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide, ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 μl rat plasma was carried out by liquid–liquid extraction with ethyl acetate:dichloromethane (90:10) ratio. Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm × 4.6 mm, 5 μm) column under gradient conditions with acetonitrile:0.1% formic acid buffer as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.72–183.20 ng/ml for ibrutinib, 0.76–194.33 ng/ml for PCI-45227 and 1.87–479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227, lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze–thaw and long term stability was evaluated for all the analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples. [ABSTRACT FROM AUTHOR]

Highlights: [•] Highly selective method for the determination of ruxolitinib and nilotinib in plasma. [•] This is the first LC–MS/MS-based method for simultaneous determination of ruxolitinib and nilotinib in plasma. [•] Plasma sample clean-up involves a simple protein precipitation procedures. [•] The developed LC–MS/MS based method was applied successfully for pharmacokinetic study. [Copyright &y& Elsevier]

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1 × 50 mm, 1.8 μm) with a 0.05 % propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-C, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 μL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A simple and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitation of indirubin-5-nitro-3′-monoxime (5-NIO) in rat plasma using 5-chloro-5′-chloro-indirubinoxime as the internal standard. The sample preparation involved protein precipitation using acetonitrile. Separation was achieved on an Xterra column C18 (2.1 mm × 50 mm, i.d. 5 µm) using a mixture of 0.1% formic acid-water/acetonitrile (18:82, v/v) as the mobile phase at a flow rate of 300 µL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and multiple reaction monitoring (MRM) in negative ion mode. The total chromatographic run time was 2.5 min, and the calibration curves of 5-NIO were linear (correlation coefficient range: 0.9989–0.9999) over the concentration range of 0.2–2000 ng/mL in rat plasma. Also, the detection limit of the method was 0.05 ng/mL and it showed good accuracy and precision within the concentration range of 0.45–376 ng/mL. This validated method was successfully applied to estimating the pharmacokinetic parameters of 5-NIO following intravenous administration of 5-NIO to rats. [ABSTRACT FROM AUTHOR]

Abstract: A highly selective, sensitive, and robust liquid chromatography–tandem mass spectrometric method for the determination of 5-thio-d-glucose concentrations in rat and human plasma was developed and validated. The sample preparation procedure involved protein precipitation and solid phase extraction, which efficiently removed sources of interference present in the plasma. Chromatographic separation was obtained using an NH2-column with distilled water and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The selected reaction monitoring (SRM) transitions for 5-thio-d-glucose and an internal standard (5-thio-d-glucose-13C6) were m/z 195→ m/z 105 and m/z 201→ m/z 108, respectively. The correlation coefficients of the calibration curves ranged from 0.9997 to 0.9999 over a concentration range from 10 to 3000ng/mL plasma. The validated method was successfully applied to a pharmacokinetic study in rats. [Copyright &y& Elsevier]

Rumex acetosa (R. acetosa) has been used in folk remedies for gastrointestinal disorders and cutaneous diseases. Rumex species, in particular, contain abundant anthraquinones. Anthraquinone glycosides and aglycones show different bioactive effects. However, information on the pharmacokinetics of anthraquinone glycosides is limited, and methods to quantify anthraquinone glycosides in plasma are rarely available. A simple and sensitive liquid chromatography-tandem mass spectrometric bioanalytical method for the simultaneous determination of both anthraquinone glycosides and their aglycones, including emodin, emodin-8-O-β-d-glucoside, chrysophanol, chrysophanol-8-O-β-d-glucoside, physcion, and physcion-8-O-β-d-glucoside , in a low volume of rat plasma (20 µL) was established. A simple and rapid sample preparation was employed using methanol as a precipitating agent with appropriate sensitivity. Chromatographic separation was performed on HPLC by using a biphenyl column with a gradient elution using 2 mM ammonium formate (pH 6) in water and 2 mM ammonium formate (pH 6) in methanol within a run time of 13 min. The anthraquinones were detected on triple-quadrupole mass spectrometer in negative ionization mode using multiple-reaction monitoring. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability. The values of the lower limit of quantitation of anthraquinones were 1–20 ng/mL. The intra-batch and inter-batch accuracies were 96.7–111.9% and the precision was within the acceptable limits. The method was applied to a pharmacokinetic study after oral administration of R. acetosa 70% ethanol extract to rats at a dose of 2 g/kg. [ABSTRACT FROM AUTHOR]