N.K. Nair

Determination of artemether and dihydroartemisinin in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A selective reproducible high-performance liquid chromatographic assay for the simultaneous quantitative determination of the antimalarial compound artemether (ARM), dihydroartemisinin (DQHS) and artemisinin (QHS), as internal standard, is described. After extraction from plasma, ARM and DQHS were analysed using a Lichrocart/Lichrosphere 100 CN stainless-steel column and a mobile phase of acetonitrile-0.05 M acetic acid (15:85, v/v) adjusted to pH 5.0, and electrochemical detection in the reductive mode. The mean recovery of ARM and DQHS over a concentration range of 30-120 ng/ml was 81.6% and 93.4%, respectively. The within-day coefficients of variation were 0.89-7.01% for ARM and 3.45-8.11% for DQHS. The day-to-day coefficients of variation were 2.06-8.43% and 3.22-6.33%, respectively. The minimum detectable concentration for ARM and DQHS in plasma was 2.5 and 1.25 ng/ml for both compounds. The method was found to be suitable for use in clinical pharmacological studies.

In vitro and in vivo evaluation of self-microemulsifying drug delivery system of buparvaquone.

Aim: The aim of this study was to prepare a lipid-based self-microemulsifying drug delivery system (SMEDDS) to increase the solubility and oral bioavailability of a poorly water-soluble compound, buparvaquone (BPQ). Methods: The solubility of BPQ was determined in various vehicles, and pseudo-ternary phase diagrams were constructed to determine the microemulsion region. A series of formulations with different compositions were selected in the microemulsion region for assessment of self-emulsification time and droplet size. The optimized SMEDDS formulation was used for in vitro dissolution and pharmacokinetic studies in rabbits. Results: The optimum formulation of SMEDDS consisted of Capryol 90 (9.82%), Cremophor EL (70.72%), Labrasol (17.68%), and BPQ (1.78%). Emulsification time and the mean droplet size were found to be 1 minute and 18.0 ± 0.25 nm, respectively, for the optimum formulation. The cumulative percentage of drug released in 90 minutes was 100% in both SGF and SIF. The calculated absolute oral bioavailability for BPQ was found to be 40.10%. Conclusions: The optimum SMEDDS formulation was increased the rate and extent of absorption of BPQ. The formulation is suitable for oral administration of BPQ. It would be useful to conduct efficacy studies of BPQ in diseased animal models and subsequently for toxicokinetics studies.

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C18 Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50-400 ng/ml was 96.8 +/- 5.2%. The within-day and day-to-day coefficients of variation were 1.8-4.0 and 1.8-4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.

Typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: An evaluation by the bioactivity guided approach

AIM OF THE STUDY Typhonium flagelliforme (Lodd.) Blume (Araceae) is a Malaysian plant used locally to combat cancer. In order to evaluate its antiproliferative activity in vitro and to possibly identify the active chemical constituents, a bioactivity guided study was conducted on the extracts of this plant. MATERIALS AND METHODS The active extracts of Typhonium flagelliforme were fractionated by flash column chromatography and each fraction was evaluated for antiproliferative activity using MTT assay. The apoptotic effect of the active fraction was determined microscopically and by using TUNEL colorimetric assay. GC-MS and NMR were used to determine the chemical constituents of this active fraction. RESULTS Several fractions of the hexane and dichloromethane extracts were found to inhibit the growth of NCI-H23 non-small cell lung carcinoma cell line significantly, with IC(50)<15 microg/ml. However, most of these active fractions were also found to inhibit the growth of non-tumorigenic BALB/c 3T3 mouse fibroblast cell line except for fraction 21 of the dichloromethane extract (D/F21). This particular fraction was not only less cytotoxic to the non-tumorigenic cells, where the IC(50) was 48.6 microg/ml compared to IC(50) 7.5 microg/ml for NCI-H23, but it was also found to induce apoptosis in the cancer cell line. GC-MS analysis revealed that D/F21 contains hexadecanoic acid, 1-hexadecene, phytol and a derivative of phytol. The presence of non-saturated fatty acids in this fraction was confirmed by nuclear magnetic resonance spectroscopy. CONCLUSIONS D/F21 was found to be the active and cancer cell line specific fraction of Typhonium flagelliforme. Its major chemical constituents had been determined spectroscopically.

Validation of high performance liquid chromatography-electrochemical detection methods with simultaneous extraction procedure for the determination of artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine in human plasma for application in clinical pharmacological studies of artesunate-amodiaquine drug combination.

With the expanded use of the combination of artesunate (AS) and amodiaquine (AQ) for the treatment of falciparum malaria and the abundance of products on the market, comes the need for rapid and reliable bioanalytical methods for the determination of the parent compounds and their metabolites. While the existing methods were developed for the determination of either AS or AQ in biological fluids, the current validated method allows simultaneous extraction and determination of AS and AQ in human plasma. Extraction is carried out on Supelclean LC-18 extraction cartridges where AS, its metabolite dihydroartemisinin (DHA) and the internal standard artemisinin (QHS) are separated from AQ, its metabolite desethylamodiaquine (DeAQ) and the internal standard, an isobutyl analogue of desethylamodiaquine (IB-DeAQ). AS, DHA and QHS are then analysed using Hypersil C4 column with acetonitrile-acetic acid (0.05M adjusted to pH 5.2 with 1.00M NaOH) (42:58, v/v) as mobile phase at flow rate 1.50ml/min. The analytes are detected with an electrochemical detector operating in the reductive mode. Chromatography of AQ, DeAQ and IB-DeAQ is carried out on an Inertsil C4 column with acetonitrile-KH(2)PO(4) (pH 4.0, 0.05M) (11:89, v/v) as mobile phase at flow rate 1.00ml/min. The analytes are detected by an electrochemical detector operating in the oxidative mode. The recoveries of AS, DHA, AQ and DeAQ vary between 79.1% and 104.0% over the concentration range of 50-1400ng/ml plasma. The accuracies of the determination of all the analytes are 96.8-103.9%, while the variation for within-day and day-to-day analysis are <15%. The lower limit of quantification for all the analytes is 20ng/ml and limit of detection is 8ng/ml. The method is sensitive, selective, accurate, reproducible and suited particularly for pharmacokinetic study of AS-AQ drug combination and can also be used to compare the bioavailability of different formulations, including a fixed-dose AS-AQ co-formulation.

Optimization and validation of RP‐HPLC‐UV method with solid‐phase extraction for determination of buparvaquone in human and rabbit plasma: application to pharmacokinetic study

A simple, sensitive and specific reversed-phase high-performance liquid chromatographic method with UV detection at 251 nm was developed for quantitation of buparvaquone (BPQ) in human and rabbit plasma. The method utilizes 250 microL of plasma and sample preparation involves protein precipitation followed by solid-phase extraction. The method was validated on a C18 column with mobile phase consisting of ammonium acetate buffer (0.02 m, pH 3.0) and acetonitrile in the ratio of 18:82 (v/v) at a flow rate of 1.1 mL/min. The calibration curves were linear (correlation coefficient>or=0.998) in the selected range. The method is specific and sensitive with limit of quantitation of 50 ng/mL for BPQ. The validated method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions and BPQ was found to be stable. Partial validation studies were carried out using rabbit plasma and intra- and inter-day precision and accuracy were within 7%. This method is simple, reliable and can be routinely used for preclinical pharmacokinetic studies for BPQ.

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C18 column (Nucleosil, 250 x 4.6 mm I.D., 5 microns particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (lambda ex = 267 nm, lambda em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries of pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.

Determination of the antifilarial drug UMF-078 and its metabolites UMF-060 and flubendazole in whole blood using high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic (HPLC) method for the simultaneous determination of the antifilarial drug UMF-078 (I) and its metabolites UMF-060 (II) and flubendazole (III) is described. After a simple extraction from whole blood, the compounds were determined by HPLC using a C18 Inertsil ODS-2 reversed-phase column with methanol-0.05M ammonium acetate (pH 4.0) as the mobile phase and ultraviolet detection at 291 nm. The average recoveries of I, II and III over the concentration range 20-500 ng ml-1 were 69.9 +/- 4.7, 85.6 +/- 4.4 and 85.1 +/- 6.0%, respectively. The minimum detectable concentrations in whole blood for I, II and III were 10, 7 and 7 ng ml-1, respectively. This method was found to be suitable for pharmacokinetic studies.

Determination of a new antifilarial drug, UMF-058, and mebendazole in whole blood by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for simultaneous quantitative determination of a new antifilarial drug (UMF-058, I) and mebendazole (MBZ) is described. After a simple extraction from whole blood, both compounds were analysed using a C18 Nova Pak reversed-phase column and a mobile phase of methanol-0.05 M ammonium dihydrogenphosphate (50:50, v/v) adjusted to pH 4.0, with ultraviolet detection at 291 nm. The average recoveries of I and MBZ over a concentration range of 25-250 ng/ml were 92.0 +/- 7.7 and 84.4 +/- 4.4%, respectively. The minimum detectable concentrations in whole blood for I and MBZ were 7 and 6 ng/ml, respectively. This method was found to be suitable for pharmacokinetic studies.