生物分析および生物医学ジャーナル

A sensitive and reproducible high performance liquid chromatography (HPLC) method has been developed and validated for the quantification of prochlorperazine sus- tained release tablets in human plasma, after solid phase extraction (SPE). Best chromatographic resolution was achieved on a reverse-phase Phenomenex C 18 column us- ing the mobile phase consisted of a mixture of 20 mM disodium hydrogen ortho phosphate–acetonitrile (95:5) in an isocratic elution with a total run time of 12 min. Linear plot was obtained in the concentration range of 15–300 ng/ml ( r 2 = 0.99). Lower limit of quantification (LLOQ) was found to be 15 ng/ml. Average recovery of the analyte was found to range from 98.25 to 99.13% in plasma at the concentrations of 45, 150 and 270 ng/ml. The intra and inter-day relative standard deviations of low quality con- trol (LQC), medium quality control (MQC) and high qual- ity control (HQC) of prochlorperazine were found to be 2.63, 3.25, 2.83 and 3.57, 5.88 and 3.78 respectively. The present method was successfully applied in the pharma- cokinetic study of prochlorperazine in human plasma.

The aim of study was to assess the pharmacokinetics of ethionamide in the local population of healthy huma n sub- jects. Serum samples were taken from each of the selected subject at different time intervals. These samples were analyzed by using High Performance Liquid Chromatog - raphy consisting of reverse phase C18 column, UV de tec- tor set at 291nm. The mobile phase was consisted of 0.02 M disodium hydrogen phosphate and acetonitrile (75: 25) and delivered at a rate of 1.5ml/min. The value of C max was found to be 1.941 ± 1.487 μ g/ml (mean ± SEM) and T max was 1.75 ± 1.487 hours (mean ± SEM). The area un- der the curve (AUC) was 8.745 ± 0.536 (mean ± SEM). The elimination half life (t ½ ) was found out as 1.995 ± 1.157 hours (mean ± SEM). The total body clearance (Cl) was determined as 32.591 ± 0.298 ml/hr/kg (mean ± S EM). It was concluded that ethionamide (Ethomid ® Schazoo- Lahore, Pakistan) found in consistent with the valu es re- ported in the available literature. The study will be ben- eficial and valuable in designing dosage regimen fo r the patients on ethionamide therapy and can be utilized as guideline in accessing the bioavailability and phar maco- kinetics parameters in clinical situations.

T he phar macokinetics of the antiepileptic a gent lamotrigine (CAS 84057-84-1) was investigated after single oral doses in 14 healthy volunteers. After t he ad- ministration of single oral doses of 2x100 mg lamot rigine chewable/soluble tablets to healthy volunteers, blo od samples were collected for the next 96 h. The pharm aco- kinetic modelling of lamotrigine showed that the dr ug exhibited two compartment open model with regard to the goodness of fits, Residual Sum of Squares (RSS), Ak aike’s Information Criteria (AIC), Schwartz Criteria (SC), stan- dard deviation of the regression (Sr), and determin ation coefficient (r 2 ). The time-concentration curves showed a mean time to reach peak plasma concentration, C max (t max ) of 2.0 h. The pharmacokinetic parameters were calcu lated based on the plasma curves. Area under the curve of con- centration versus time from zero to infinity ( 0 AUC → ∞ ), sys- temic clearance (Cl), apparent volume of distributi on (V darea ), apparent volume of distribution at steady state (V dss ), apparent volume of distribution for I.V. (V dext ), and mean residence time (MRT) were found to be 128±31 μ g.h/ mL, 1.63±0.39 L/h, 88.5±28.6 L, 83.2±23.6 L, 93.2±3 5.6 L, and 62.6±13.7 h (mean±SD), respectively. Compart - mental analysis demonstrated that oral lamotrigine tab- lets obey two compartment open model with rapid abs orp- tion and a relatively long half life.

A sensitive and reproducible high performance liqui d chromatography (HPLC) method has been developed and validated for the quantification of metolazone in h uman plasma, after solid phase extraction (SPE). A Good reso- lution was achieved on a reverse-phase LichroCART Purospher ® C 18 column using the mobile phase acetoni- trile–0.5% triethylamine (35:65) in isocratic eluti on with a total run time of 15 min. The analyte, metolazone , was detected by using high performance liquid chromatog ra- phy with the support of photo diode array detector. Limit of detection and Lower limit of quantification was found to be 1 and 2.5 ng/mL. The present method was succe ss- fully applied in the pharmacokinetic study of metol azone in human plasma.

Purpose: To study the absorption of main flavonoids in Hypericum japonicum extract (HJE) with liver protective property; Method: HPLC-ESI-MS was introduced to identify and evaluate the flavonoids in HJE; Caco-2 cell monolayer model was established and validated, and the compounds in HJE, including quercetin (Q), quercetin-3-Orhamnoside (Q-3-R), quercetin-7-O-rhamnoside (Q-7-R) and quercetin-3-O-glucoside (Q-3-G) were administrated in individual, paired or mixed form of the compounds to the monolayer to evaluate their apparent permeability coefficients (Papp value). The transport of HJE was also investigated, mixture of pure components and HJE Inhibitor was added to investigate the transport mechanism of the compound mixture. The absorption of the four main ingredients in HJE was then investigated in vivo. Result: transportation of Q, Q-3-R Q-3-G but not Q-7-R through Caco-2 monolayer was observed when they were administrated individually. Increase of the transport of Q-3-G and Q-7-R and decrease in Q were observed when the four compounds were given in paired form; when the four flavonoids were given as a whole (either in mixture of pure compounds or in HJE), mass permeability of Q-3-R, Q-7- R and Q-3-G was found. In vivo study identified the in vitro investigation that the major active components of HJE could be absorbed after orally administrated to mice. Conclusion: The increased transport of mixed active components in HJE gives rise to the enhanced hepatoprotetive effect of HJE, and therefore supports the use of botanical drugs.

A simple, rapid and selective method was developed for esti- mation of simvastatin from human plasma. The method involves simple protein precipitation techniques using etofy lline as inter- nal standard. Chromatographic separation was carrie d out on a reversed phase C 18 column using mixture of methanol: 2mM ammonium acetate and 500 μl of 0.5% formic acid (80 :20, v/v) at a flow rate of 1.0 ml/min with UV-VIS detection at 418.35 nm. The retention time of simvastatin and internal standard were 5.41 and 1.086 min, respectively. The method was va lidated and found to be linear in the range of 1.0-10.0 ng/mL. An open, ran- domized, two-treatment, two period, single dose cro ssover, bioequivalence study in 12 fasting, healthy, male, volunteers was conducted. After dosing, serial blood samples were collected for the period of 24 . 0 h . Various pharmacokinetic parameters including AUC 0– t , AUC 0– ∞ , C max , T max , T ½ , and elimination rate constant ( K el ) were determined from plasma concentration of both formulations. Log transformed values were comp ared by nalysis of variance (ANOVA) followed by classical 90% con- fidence interval for C , AUC 0– t and AUC 0– ∞ and was found to be within the range. These results indicated that t he analytical method was linear, precise nd accurate. Test and r eference for- mulation were found to be bioequivalent.

A simple, economic, reproducible, robust and precis e HPLC method for estimation of swertiamarin in both 60 % methanolic extract of Enicostemma littorale and mar- keted formulations was developed and validated in p resent investigation. The mobile phase composed of methano l and water (90:10 % v/v), gave a sharp and well-defi ned peak of swertiamarin at the retention time of 10.15 ± 1.52 min. The limit of detection (LOD) and limit of quan tifica- tion (LOQ) were 17.25 and 56.92 μ g/ml respectively. The proposed method with high degree of precision and a ccu- racy was employed for the estimation of swertiamari n in methanolic extract and in marketed formulation.

Aim

To compare the bioavailability of two ramipril tabl et for- mulations – 10 mg Prohytens ® tablets as test formulation and 10 mg Triatec ® tablets as reference formulation.

Methods

A single-dosed, open-label, randomized two-way cros s- over design under fasting period with two weeks was h out period was evaluated in 24 subjects. For the an alysis of pharmacokinetic properties, the blood samples we re drawn taken up to 72 hours after dosing. Plasma con cen- tration of ramipril and ramiprilat were determined using liquid chromatography – tandem mass spectrometry method with TurboIonSpray mode. Pharmacokinetic pa- r a meter s AUC 0 -t, AUC 0- ∞ a nd C max wer e tes ted for bioequivalence after log-transformation of data and ratios of t max were evaluated non-parametrically.

Results

The point estimates and 90% confidence intervals (C I) for AUC 0-t, AUC 0- ∞ and C max for ramipril were 93.21% (85.67-101.41%), 93.45% (85.88-101.69%), 94.02% (80.09-110.38%) and for ramiprilat were 92.26% (87. 76- 96.99%), 94.59% (89.71-99.73%) and 91.55% (84.88- 98.74%).

Conclusion

These results indicated that the two formulations o f ramipril were bioequivalent and thus may be prescri bed interchangeably.

The purpose of this study is to investigate the effect of using replicate design on the intra/inter subject variability and bioequivalence of drugs in healthy volunteers. Model drugs used for analysis were amoxicillin/clavulanic acid combination. 24 healthy subjects participated in this study using 4-phase replicate cross over design. Individual disposition kinetic parameters of areas under plasma concentrations (AUC0-t) and maximum concentration (Cmax) were calculated by non-compartmental analysis using Kinetic program V 4.2 using all phases. The 90 % confidence intervals for log-transformed AUC0-t and Cmax were calculated for phases I & II; then for phases I, II and III; and for phases I, II, III and IV respectively. The intra and inter-subject variability values did not show a trend to decrease by the increase in phases included in analysis in both drugs and for both parameters. In addition, the 90 % confidence intervals for log-transformed AUC0-t and Cmax passed the 80-125 % limit range in both drugs for all phase combinations, even though Cmax variability was shown high for clavulanic acid. However, individual bioequivalence was shown for AUC and not shown for Cmax of both drugs. These results suggest not using replicate design as an approach to show the high inter/intra subject variability of highly variable drugs and hence justify wider acceptance limits of 75-133 % as recommended by the draft EMEA guideline. Literature information about drug high variability should be adequate to justify using wider acceptance limits of 75-133%.

A specific, fast and sensitive LC–MS/MS assay was d e- veloped for the determination of cimetidine in huma n plasma using nizatidine as the internal standard (I S). The limit of quantification was 5.0 ng/ml and the metho d was linear in the range of 5.0 to 5000 ng/ml. The cimet idine and IS retention times were 1.35±0.3 and 1.40±0.03 min, respectively. Method intra-batch precision and accu racy ranged from 2.0 to 5.4%, and 92.1 to 103.7%, respec tively. Inter-batch precision ranged from 4.2 to 6.3%, whil e In- ter-batch accuracy ranged from 97.0 to 106.6%.

The analytical method was applied to evaluate the p har- macokinetic and relative bioavailability of two dif ferent pharmaceutical formulations containing 400 mg of cimetidine containing. This study evaluated 29 volu nteers in a randomized, 2-period crossover study with 14 d ays washout period between doses. The geometric mean an d respective 90% CI of cimetidine test/reference perc ent ratios were 95.73% (87.76 - 104.43%) for C max, 100.80% (95.98 - 105.96%) for AUC 0-t and 100.90 (96.06 - 105.88) for AUC 0-inf . Based on the 90% confidence interval of the individual ratios (test formulation/reference formu lation) for C max and AUC 0-inf , it was concluded that the test formu- lation is bioequivalent to the reference one with r espect to the rate and extent of absorption of cimetidine. In addi- tion, using the Kruskal-Wallis Test no statistical differ- ences of Tmax and the Cmax were observed related to the sex of the volunteer.

Staurosporine has been shown to possess an array of important biological properties such as anti-fungal, anti-bacterial, antihypertensive and anti-cancer. Methods available to screen any plant or bacterial extract for this compound are lengthy and laborious. We present here a simple HPLC-MS-MS method for the highly selective identification of Staurosporine in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate Staurosporine from the matrix and positive ion electrospray mass spectrometry was used for detection and confirmation. Presence of Staurosporine in bacterial extracts was confirmed by matching retention times and parent-daughter ion mass spectra (using Multiple Reaction Monitoring, MRM mode) of the standard Staurosporine with those of the bacterial extracts. Detection of Staurosporine was achieved down to 11 ng/mL bacterial extract. This method can be easily adapted to screen any plant extract for Staurosporine. The simplicity and the speed of this method make it possible for the analyst to screen a large number of extracts before embarking on lengthy and costly isolation and purification of Staurosporine in a selected few.