Automated On-Line Microdialysis Sampling Coupled with HPLC for Synchronous Determination of Puerarin in Subcutaneous Tissue and Plasma Following Topical Administration.

BACKGROUND: Studies on transdermal administration have shown that puerarin can permeate rat skin rapidly with long-term drug delivery, but there are no reports demonstrating whether topical use of puerarin can provide a steady plasma concentration to produce therapeutic effects. The aim of the study is to evaluate the percutaneous penetration and plasma concentration of puerarin after transdermal administration in experimental rats. METHODS: The skin and plasma concentration of puerarin was quantified by microdialysis, and the recovery was determined by retrodialysis. Puerarin microdialysate concentrations were measured by on-line high-performance liquid chromatography (HPLC). Puerarin release from gels was determined by analysis of the amount of remaining drug after dermal application to hairless skin. RESULTS: The average recoveries of puerarin in the skin and plasma over an 8-hour period were 31.49% and 15.5%. Puerarin was rapidly absorbed with transdermal administration, with the C(max) values of 30.64 µg/mL and 3.53 µg/mL, the AUC0 t-values of 11.60 and 1.48 µg/mL per minute, for skin and plasma, respectively. CONCLUSIONS: The results indicate that the automated on-line microdialysis technique can be used to detect the skin and plasma pharmacokinetics of puerarin and that the use of skin gel can provide an effective means of puerarin administration.

[Pharmacokinetics of scopolamine hydrobromide oral disintegrative microencapsule tablets in Beagle dogs determined with LC-MS/MS].

The study aims to elucidate the characteristics of pharmacokinetics of scopolamine hydrobromide oral disintegrative microencapsule tablets in healthy Beagle dogs. Chromatographic separation was performed on a C18 column (100 mm x 3.0 mm, 3.5 microm) with methanol - 2 mmol x L(-1) ammonium formate (25 : 75) as the mobile phase. A trip-quadrupole tandem mass spectrum with the electrospray ionization (ESI) source was applied and positive ion multiple reaction monitoring mode was operated. Six Beagle dogs were randomly devided into two groups. They received oral single dose of scopolamine hydrobromide oral disintegrative microencapsule tablets 0.6 mg (test tablet) or scopolamine hydrobromide normal tablets (reference tablet). Plasma samples were collected at designed time. Plasma concentration of scopolamine hydrobromide was determined by LC-MS/MS and pharmacokinetic parameters were calculated. The pharmacokinetic parameters of test tablet vs reference tablet were as follows: C(max): (8.16 +/- 0.67) ng x mL(-1) vs (3.54 +/- 0.64) ng x mL(-1); t1/2: (2.83 +/- 0.45) h vs (3.85 +/- 0.82) h; t(max): (1.25 +/- 0.27) h vs (0.42 +/- 0.09) h; AUC(0-12h): (25.06 +/- 3.75) h x ng x mL(-1) vs (9.59 +/- 1.02) h x ng x mL(-1); AUC(0-infinity): (26.30 +/- 3.92) h x ng x mL(-1) vs (10.80 +/- 1.45) h x ng x mL(-1); MRT(0-12h): (3.38 +/- 0.34) h vs (3.86 +/- 0.26) h; MRT(0-infinity): (3.98 +/- 0.63) h vs (5.37 +/- 1.00) h. The absorption rate and AUC of test tablet is different from that of reference tablet. The bioavailability of test tablet is better than those of reference tablet.

[Enzyme kinetics of ligustilide metabolism in rat liver microsomes].

To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.

Assessment of in vitro and in vivo recovery of sinomenine using microdialysis.

BACKGROUND/PURPOSE: For microdialysis studies in the skin, laboratory-made linear probes are often used. The application of the microdialysis technique to the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of the linear probes' performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of sinomenine. METHODS: Using different lengths of the dialysis membrance and different perfusion flow rates, a flow rate of 2 microL/min with 20-min sampling intervals was selected for the subsequent studies, based on the analysis of sinomenine from the microdialysis probe. In vitro recovery of sinomenine from the microdialysis probe was independent of concentration, stable over an 8-h period. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of sinomenine from rat subcutaneous tissue. RESULTS: The performance of the microdialysis system was stable over an 8-h study, resulting in a mean in vitro recovery of 51.91+/-1.29%. Recovery obtained using the ZNF plot was 52.43%. In vivo recovery of sinomenine was 34.46+/-0.76% and was stable over the 7-h study period. CONCLUSION: The in vitro and in vivo performance of the microdialysis technique was established for the study of sinomenine. It would prove to be a useful and reliable tool to study the pharmacokinetics and pharmacodynamics of sinomenine. The data obtained in our study highlight the importance of a systematic examination of microdialysis linear probe validation.

Pharmacokinetic study of free-form sinomenine in rat skin by microdialysis coupled with liquid chromatography-electrospray mass spectrometry.

Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one) is a pure alkaloid extracted from the Chinese medical plant. In this report a liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method with in vivo microdialysis for the pharmacokinetic study of free-form sinomenine in rat skin has been developed. A microdialysis probe was surgically implanted into the subcutaneous tissue of the rats and an isotonic phosphate buffer (PBS) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC-ESI-MS. The chromatographic separation was achieved within 4.2 min by using a narrow-bore Xterra C(18) column (2.1 x 150 mm, 5 microm) with acetonitrile-(10 mmol/L ammonium acetate buffer, 0.1% acetic acid) (15:85, v/v). Ion signal m/z 330.1 for sinomenine was measured in the positive mode. Linearity was established for the range of concentrations of 2.0-10000.0 ng/mL with a coefficient of determination (r) of 0.9989. The intra- and inter-day reproducibility of the present method was better than 6%. The lower limit of quantification (LLOQ) was 1.0 ng/mL. The proposed method described provides more authentic information on pharmacokinetics and metabolism at the site of action by using the coupling of microdialysis to LC-ESI-MS technique than the traditional sampling methods.