Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant.

AIM: To investigate if any mutations in hepatitis C virus (HCV) internal ribosome entry site (IRES) can inhibit the translation of viral polyprotein. MATERIALS AND METHODS: A 26-year-old male patient infected with HCV 10 years ago was followed up. After 9 years of chronic infection. The patient had managed to resolve the infection for a period of 9 months, after which the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. The IRES structures of the viral strains that disappeared were comparable with those that are currently active using structural mutational analysis. RESULTS: A novo mutational position 254 combined with a rarely observed mutation at position 253 in the stem of the IIId subdomain were observed and the new conformation had an octa-apical loop (AGUGUUGG) and a shift in the 3 ` GU from the loop to the stem. CONCLUSIONS: These mutations were found to be highly deleterious, and they affected the direct binding of the IIId loop to the 40S ribosomal subunit with a subsequent inhibition of translation of viral polyprotein and clearance of the virus.

Validated liquid chromatographic-fluorescence method for the quantitation of darifenacin in mice plasma and its application to a pharmacokinetic study.

A highly selective, sensitive, and rapid high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of darifenacin in mouse plasma. Bisoprolol was used as an internal standard (IS). Darifenacin and the IS were extracted using the deproteinisation technique, followed by injection of an aliquot of the supernatant into the chromatographic system. The chromatographic separation was achieved on a reversed phase C18 column with a mobile phase of acetonitrile: 0.1% diethyl amine (pH 3.5) (60:40, v/v) pumped at a flow rate of 1.0 mL min(-1). The analytes were detected at 210 and 314 nm for excitation and emission, respectively. The assay exhibited a linear range of 100-3000 ng mL(-1), with a lower detection limit of 35 ng mL(-1). The method was statistically validated for linearity, accuracy, precision, selectivity and stability according to the FDA guidelines. The intra- and inter-assay coefficients of variation did not exceed 13.5% from the nominal concentration. The accuracy for darifenacin was within ±15% of the theoretical value. The assay was successfully applied in a pharmacokinetic study.

Spectrofluorometric determination of some beta-blockers in tablets and human plasma using 9,10-dimethoxyanthracene-2-sodium sulfonate.

A simple and sensitive spectrofluorometric method was developed for the quantitative determination of some beta-blockers, namely arotinolol, atenolol and labetalol as hydrochloride salts. The method is based on the reaction of these drugs as n-electron donors with the fluorogenic reagent 9,10-dimethoxy-2-anthracene sulfonate (DMAS) as pi-acceptor in acidic medium. The obtained ion-pairs were extracted into chloroform and measured spectrofluorometrically at 452 nm after excitation at 385 nm. The fluorescence intensity-concentration plots are rectilinear over the ranges of 0.5-5 microg x ml(1), 1.0-11.0 microg x ml(1) and 0.6-6.4 microg x ml(1) for labetalol, atenolol and arotinolol, respectively. The different parameters affecting the reaction pathway were thoroughly studied and optimized. No interference was observed from the common pharmaceutical excipients. The proposed method was successfully applied to the analysis of tablets and the results were statistically compared with those obtained by reference methods. The method was further extended to the in vitro determination of the drugs in spiked human plasma, the % recoveries (n = 3) ranged from 96.98 +/- 1.55 to 98.28 +/- 2.19. A proposal of the reaction pathway was postulated.

Stereoselective determination of pyridoglutethimide enantiomers in serum with a chiral cellulose-based high-performance liquid chromatographic column using solid phase extraction and UV detection.

A sensitive method for the separation and determination of R(+)- and S(-) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for R(+)- and S(-)-pyridoglutethimide enantiomers were in the range 86-91% at 300-900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9-3.9 and 1.5-4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9-3.3 and 1.5-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100-1500 ng/ml for each enantiomer show correlation coefficient (r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (S/N = 2).

Analysis of certain tranquilizers in biological fluids.

A review with 282 references is presented that deals with the reported methods of analysis of phenothiazines, thioxanthenes, and benzodiazepine derivatives of pharmaceutical interest. The review includes the methods adapted in biological fluids.

Analysis of pharmaceutically-important thioxanthene derivatives.

A review with 92 references is presented that deals with the reported methods of analysis of the thioxanthene derivatives of pharmaceutical interest. The review includes the methods adopted in dosage forms and biological fluids. A brief discussion of the metabolism and pharmacokinetics of this class of compounds is also reported.

A selective fluorimetric method for the determination of some 1,4-benzodiazepine drugs containing a hydroxyl group at C-3.

A highly selective and sensitive fluorimetric method was developed for the determination of four 1,4-benzodiazepine drugs containing a hydroxyl group at carbon 3, namely oxazepam, lorazepam, cinolazepam and temazepam. The method is highly specific because other benzodiazepinee lacking the hydroxyl group at C-3 do not react similarly and hence do not interfere. The proposed method involves reduction of the target compound using Zno/HCl at room temperature with the formation of a highly fluorescent derivative within 15 min. The different experimental parameters were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is applicable over the concentration range of 0.1-1.2 micrograms ml-1 for both temazepam and cinolazepam, and 0.2-2.5 and 1-8 micrograms ml-1 for oxazepam and lorazepam respectively. The recoveries of the title compounds from spiked urine ranged from 90.0 to 92.0% and for serum from 94.1 to 95.4% with a limit of detection (S/N = 2) of 4 ng ml-1 for all drugs. The mechanism of the fluorimetric reaction is discussed.