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Introduction Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. Experimental We analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC–MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18 weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21 days) and adult (18 weeks) rats. Hematocrit was determined concomitantly. Results Using these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. Discussion Our study provides correction factors for LC–MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis. [ABSTRACT FROM AUTHOR]

A specific and reliable liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed for the simultaneous determination of timosaponin H1 (TH1), timosaponin E1 (TE1), timosaponin E (TE), timosaponin B-II (TB-II), timosaponin B-III (TB-III) and anemarrhenasaponin I (AS-I) in rat plasma. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on a reverse phase ACQUITY™ BEH C 18 column (100 mm × 2.1 mm i.d., 1.7 μm) using a gradient mobile phase system of acetonitrile–water containing 0.05% formic acid and 5 mM ammonium formate. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for TH1, TE1, TE, TB-II, TB-III, AS-I and IS were m / z 1211.5 > 1079.6, 935.5 > 773.4, 935.4 > 773.5, 919.6 > 757.4, 901.5 > 739.3, 757.4 > 595.3 and 637.3 > 475.3, respectively. The method validation was conducted over the curve range of 0.5–400 ng/mL for the six saponins. The intra- and inter-day precisions (RSD%) were less than 9.4% and the average extraction recoveries ranged from 82.5% to 97.8% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The validated method was successfully applied for the first time to determine the concentrations of six main steroidal saponins in incurred rat plasma samples, after intragastric administration of the extract of Anemarrhena asphodeloides Bge. for a rat pharmacokinetic study. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed and validated a novel quantification method using HPLC–MS/MS. [•] It is for the first time we simultaneous determined six main steroidal saponins in Paris polyphylla in vivo. [•] We report the pharmacokinetics parameters of steroidal saponins in rat for the first time. [•] We report the steroidal saponins have a long MRT and low concentration. [•] We introduced the Accelerated Solvent Extraction in sample preparation. [ABSTRACT FROM AUTHOR]

Abstract: The analysis of stress-induced changes in the brain neurosteroid levels by liquid chromatography (LC)–electron capture atmospheric pressure chemical ionization-mass spectrometry (ECAPCI-MS) is described. In the present method, neurosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine (NFPH), to convert them to the corresponding hydrazones. The derivatized steroids showed over a 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured by positive atmospheric pressure chemical ionization (APCI)-MS. Application of this method to the analysis of rat brain samples confirmed the significant increase in the levels of pregnenolone (PREG), progesterone (PROG), 5α-dihydroprogesterone (DHPROG), allopregnanolone (3α-hydroxy-5α-pregn-20-one; AP), and epiallopregnanolone (3β-hydroxy-5α-pregn-20-one; EpiAP) in the fixated rats. The din stress, which we examined as a new short-term mental stress model, also elevated the brain neurosteroid levels. It is known that various types of stress lower the γ-aminobutyric acid type A (GABAA) receptor function and induce the neuronal overexcitation. The increase in the brain level of AP, a potent positive modulator of GABAA receptors, may be the defensive response against acute stress. The increase in the brain concentration of its precursors, PREG, PROG, and DHPROG, may be associated with the acceleration of the AP synthesis. Thus, the present studies suggest that changes in the brain levels of neurosteroids may play an important role in the homeostatic mechanisms that counteract the inhibitory effect of stress on the GABAA receptor function. [Copyright &y& Elsevier]