An LC-MS/MS assay for the quantitative determination of 2-pyridyl acetic acid, a major metabolite and key surrogate for betahistine, using low-volume human K2 EDTA plasma.

Betahistine is widely used for the treatment of vertigo. Owing to first-pass metabolism, 2-pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC-MS/MS method was developed and validated for quantitation of 2PAA using turbo-ion spray in a positive ion mode. A solid-phase extraction was employed for the extraction of 2PAA and 2PAA d6 (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 µm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile-methanol (90:10% v/v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m/z 138.1 to m/z 92.0 and m/z 142.1 to m/z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter- and intra-batch assays were <10%. The developed method was used to support clinical sample analysis.

Comparative bioavailability of betahistine tablet formulations administered in healthy subjects.

OBJECTIVE: To assess the comparative bioavailability of two formulations (16 mg tablet) of betahistine (CAS 5579-84-0) in healthy volunteers of both sexes. METHODS: The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Plasma samples were obtained for up to 36 h post dose. Plasma 2-pyridylacetic acid concentrations were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). From the 2-pyridylacetic acid plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained for AUCIast and Cmax. RESULTS: The limit of quantification was 4 ng/mL for plasma 2-pyridylacetic acid analysis. The geometric mean and 90% confidence interval (CI) of test/reference percent ratios were: 98.94% (92.21%-106.16%) for Cmax, 95.42% (91.74%-99.25%) for AUClast. CONCLUSION: Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the test formulation is bioequivalent to the reference for both the rate and the extent of absorption.

LC-MS-MS analysis of 2-pyridylacetic acid, a major metabolite of betahistine: application to a pharmacokinetic study in healthy volunteers.

1. A sensitive liquid chromatographic-tandem mas spectrometric assay was developed and validated to determine the major metabolite of betahistine, 2-pyridylacetic acid, in human plasma. 2. The analyte was extracted from plasma samples by liquid-liquid extraction and analysed using liquid chromatography-tandem mass spectrometry with an electrospray ionization interface. The method has a lower limit of quantitation of 1 ng ml(-1) fir a 0.5-ml plasma aliquot. The intra- and interday precision (relative standard deviation), calculated from quality control (QC) samples, was less than 10%. Accuracy as determined from QC samples was within +/-7%. 3. The validated method was successfully applied to a pharmacokinetic study of betahistine in healthy volunteers. After oral administration of a single dose of 24 mg betahistine mesylate to 20 healthy Chinese male volunteers, Cmax was 339.4 ng ml(-1) (range 77.3-776.4 ng ml(-1)). The t(1/2) was 5.2 h (range 2.0(-1)-11.4h). The AUC(0-t) obtained was 1153.5 ng ml(-1) h (range 278.5-3150.8 ng ml(-1)). The disposition of the metabolite exhibited a marked interindividual variation. 4. The plasma concentrations of the parent drug were less than 0.5 ng ml(-1), suggesting that it undergoes almost complete first-pass metabolism. The reported two active metabolites were not detected in the plasma of any volunteer. Although there is no evidence that the major metabolite has pharmacological activity, the clinical importance of 2-pyridylacetic acid in humans should be reinvestigated.

Mechanism for enhancement effect of lipid disperse system on percutaneous absorption.

To clarify the mechanism involved in the enhancement effect of lipid disperse systems (LDS) on percutaneous absorption, the effect of the LDSs of betahistine (BH), prepared using egg phosphatidylcholine (EPC, phase transition temperature, tau m, -15 to -17 degrees C) or hydrogenated soybean phosphatidylcholine (HSPC, tau m, 50 to 60 degrees C), cholesterol, and dicetylphosphate, on the percutaneous absorption of BH, the amount of skin lipids (ceramides, triglycerides, and phospholipids), the fluidity of skin lipids, and the partitioning of LDS-BH into the skin layers were investigated using Wistar and hairless rats. Also examined was whether the LDS penetrated through the stratum corneum (SC) or follicles, using a fluorescent probe (Nile Red). The plasma concentrations of BH were much higher and more sustained after application of a gel formulation containing EPC-LDS and D-limonene (prep. 2) than those after the non-LDS formulation containing D-limonene (prep. 1), whereas the plasma levels after application of a formulation containing HSPC-LDS (prep. 5) were not largely increased compared with those after prep. 1. The content of ceramides (intercellular lipids) and triglycerides (sebaceous gland lipides) in the SC were dramatically decreased by the treatment with prep. 1 and prep. 2, with the more decreased levels of these lipids by the treatment with prep. 2. The phospholipid content of the SC was enhanced by 2-fold following the prep. 2 treatment, indicating the extensive incorporation of LDS lipids into the SC. The histochemical examination of the skin, following application of EPC-LDS with a fluorescent probe, indicated that the LDS lipids penetrated rapidly through the SC and follicles into the viable skins. The fluidity of the SC lipids was dramatically increased following the treatment with the fluid EPC-LDS, whereas the fluidity was significantly decreased by the solid HSPC-LDS. The BH in each skin layer was also significantly increased by the treatment with prep. 2. These results surely demonstrated that the fluid LDS permeated rapidly into the SC and the viable epidermis through the intercellular domains and the follicles in intact vesicles or lipid mixtures, thus ensuring the facilitated transport of LDS-drug through the skin.

GLC determination of betahistine in serum.

A gas chromatographic method was developed for the determination of betahistine in serum in the range of 0.6-6.0 microgram/ml.