A rapid and highly sensitive UPLC-MS/MS method using pre-column derivatization with 2-picolylamine for intravenous and percutaneous pharmacokinetics of valproic acid in rats.

A rapid, highly sensitive and specific ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for the detection of valproic acid (VPA) in rat plasma following the topical application was developed and validated. This method was carried out with pre-column derivatization using 2-picolylamine (PA) which reacts with the carboxylic acid group of VPA. The derivatization was completed in 10min and the resulting PA-VPA derivative enabled the sensitive detection of VPA in selected reaction monitoring (SRM) mode. Sample preparation was done with simple liquid-liquid extraction and chromatographic separation was achieved within 5min on a C18 column using a gradient elution with the mobile phase of 2mM ammonium formate containing 0.1% formic acid and methanol. The standard curves were linear over the concentration range of 0.07-200µg/mL with a correlation coefficient higher than 0.99. The limit of detection (LOD) and the lower limit of quantification (LLOQ) was 0.03 and 0.07µg/mL, respectively with 100µL of plasma sample. The intra- and inter-day precisions were measured to be below 10.7% and accuracies were within the range of 94.1-115.9%. The validated method was successfully applied to the pharmacokinetics of VPA in the rat following topical and intravenous applications.

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method.

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 µm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).

Identification and evaluation of neutral sphingomyelinase 2 inhibitors.

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg(2+)-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg(2+)-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Evaluation of anti-platelet and anti-thrombotic effects of cilostazol with PFA-100® and Multiplate® whole blood aggregometer in vitro, ex vivo and FeCl3-induced thrombosis models in vivo.

We evaluate the anti-platelet and anti-thrombotic effects of cilostazol using Multiplate® and PFA-100® in vitro and ex vivo with freshly isolated rat whole blood and in vivo venous and arterial thrombosis models in the same species, in an effort to assess the sensitivity of the whole blood aggregometer assays without potential issues of species differences. In vitro assay of anti-platelet effects of cilostazol against collagen-induced aggregation using Multiplate® produced a graded dose-dependent inhibition curve with IC50 value of 75.4 ± 2.4 µM while it showed a highly sensitive and all-or-none type inhibition response from 25 µM in PFA-100®. Interestingly, cilostazol manifested anti-thrombotic effects in vivo at much lower plasma concentrations than the effective concentrations measured in ex vivo or in vitro aggregation tests using PFA-100® or Multiplate®. In addition, the tail bleeding time measurement demonstrated that rats have lower sensitivity to the anti-platelet effects of cilostazol than mice. These results suggest that the detailed comparative evaluation of whole blood aggregometer assays with anti-thrombotic effects in vivo should be preceded before the application of these methods for the pharmacodynamic studies of anti-thrombotic agents.