A RAPID ISOCRATIC HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC-UV) METHOD FOR THE QUANTIFICATION OF RITONAVIR IN HUMAN PLASMA

Objective: An accurate, simple, and rapid HPLC-UV based method for the quantitative determination of ritonavir (RTV) in human plasma is developed.Methods: The method involved deproteinization of the sample with 0.125N NaOH and methyl tertiary butyl ether and evaporated to dryness. The residue was reconstituted with mobile phase (20 mM Sodium Acetate and Acetonitrile–55:45 v/v). The analysis was done using C8 column (250 x 4.6 mm ID) and detection at a wavelength of 212 nmResults: The method range was linear over the range 0.5–10.0 µg/ml as derived using calibration curve method. Mean intra-and inter-day variations over the ranges of the standard curves were less than 10 % and mean extraction recoveries from human plasma ranged from 96 to 110 %.Conclusion: A rapid and accurate method for quantitation of RTV in plasma was validated. The assay spans the concentration range of clinical interest. The easy sample preparation and small sample size makes this assay highly suitable for pharmacokinetic studies of RTV in HIV-infected patients with TB.Keywords: Ritonavir, Pharmacokinetics, HPLC, Plasma extraction, HI

Psycho-social dysfunction: Perceived and enacted stigma among tuberculosis patients registered under Revised National Tuberculosis Control Programme.

Objective: To examine the perceived and enacted stigma experienced by TB patients and the community. Methods: We interviewed 276 TB patients registered for treatment during January-March 2004 in government health facilities of two Tuberculosis Units of south India. Data on perceived and enacted stigma were collected after two months of starting treatment, using a semi-structured interview schedule. In addition, four Focus Group Discussions were conducted among Directly Observed Treatment (DOT) providers and community members. Narrative summaries were also taken down to collect additional qualitative information. Results: Of the 276 patients, 190 (69%) were males. There was no significant difference between the genders in relation to social stigma. Perceived stigma was higher than enacted stigma in both genders and significantly higher among males (Low self esteem p<0.05), change of behavior of community (p<0.05), ashamed to cough in front of others (p<0.05). Conclusion: Considering the social and emotional impact of the disease, it is essential to adopt support strategies to enhance acceptance and for a successful health programme

Association of metabolic dysregulation with volumetric brain magnetic resonance imaging and cognitive markers of subclinical brain aging in middle-aged adults: the Framingham Offspring Study.

ObjectiveDiabetic and prediabtic states, including insulin resistance, fasting hyperglycemia, and hyperinsulinemia, are associated with metabolic dysregulation. These components have been individually linked to increased risks of cognitive decline and Alzheimer's disease. We aimed to comprehensively relate all of the components of metabolic dysregulation to cognitive function and brain magnetic resonance imaging (MRI) in middle-aged adults.Research design and methodsFramingham Offspring participants who underwent volumetric MRI and detailed cognitive testing and were free of clinical stroke and dementia during examination 7 (1998-2001) constituted our study sample (n = 2,439; 1,311 women; age 61 ± 9 years). We related diabetes, homeostasis model assessment of insulin resistance (HOMA-IR), fasting insulin, and glycohemoglobin levels to cross-sectional MRI measures of total cerebral brain volume (TCBV) and hippocampal volume and to verbal and visuospatial memory and executive function. We serially adjusted for age, sex, and education alone (model A), additionally for other vascular risk factors (model B), and finally, with the inclusion of apolipoprotein E-ε4, plasma homocysteine, C-reactive protein, and interleukin-6 (model C).ResultsWe observed an inverse association between all indices of metabolic dysfunction and TCBV in all models (P &lt; 0.030). The observed difference in TCBV between participants with and without diabetes was equivalent to approximately 6 years of chronologic aging. Diabetes and elevated glycohemoglobin, HOMA-IR, and fasting insulin were related to poorer executive function scores (P &lt; 0.038), whereas only HOMA-IR and fasting insulin were inversely related to visuospatial memory (P &lt; 0.007).ConclusionsMetabolic dysregulation, especially insulin resistance, was associated with lower brain volumes and executive function in a large, relatively healthy, middle-aged, community-based cohort

UFLC/MS method for the estimation of ethambutol in human plasma and its application in tuberculosis patients

Ultra Fast liquid chromatography – mass spectrometry method (UFLC-MS), for detection and quantification of the anti-tuberculosis drug, ethambutol (EMB), in human plasma is described here. The method uses a single Quadrapole mass spectrometer equipped with positive electrospray ionization detector, linked with Shimadzu 20AD ultrafast LC system. Ethambutol was extracted from human plasma (100 μl) through a single step, simple and straight forward acetonitrile deproteinization method, achieving 98 - 102 % analyte recovery. Short C18 column (50 mm x 2.1 mm) was used with isocratic mobile phase system consisting of 80% aqueous acetonitrile and 0.5% formic acid as additive. Selective ion monitoring (SIM) mode was selected with EMB m/z 205.3 in positive ion mode (MH+). This method achieved wide range of detection from microgram to nanograms of EMB (Linearity R value = 0.9999), with great precision and accuracy. The limits of quantification and detection were achieved upto 19.18 ng/ml and 5.78 ng/ml respectively. This method was applied successfully to determine EMB in plasma samples from TB patients who were receiving EMB along with other anti-TB drugs

Using Family-Based Imputation in Genome-Wide Association Studies with Large Complex Pedigrees: The Framingham Heart Study

Imputation has been widely used in genome-wide association studies (GWAS) to infer genotypes of un-genotyped variants based on the linkage disequilibrium in external reference panels such as the HapMap and 1000 Genomes. However, imputation has only rarely been performed based on family relationships to infer genotypes of un-genotyped individuals. Using 8998 Framingham Heart Study (FHS) participants genotyped with Affymetrix 550K SNPs, we imputed genotypes of same set of SNPs for additional 3121 participants, most of whom were never genotyped due to lack of DNA sample. Prior to imputation, 122 pedigrees were too large to be handled by the imputation software Merlin. Therefore, we developed a novel pedigree splitting algorithm that can maximize the number of genotyped relatives for imputing each un-genotyped individual, while keeping new sub-pedigrees under a pre-specified size. In GWAS of four phenotypes available in FHS (Alzheimer disease, circulating levels of fibrinogen, high-density lipoprotein cholesterol, and uric acid), we compared results using genotyped individuals only with results using both genotyped and imputed individuals. We studied the impact of applying different imputation quality filtering thresholds on the association results and did not found a universal threshold that always resulted in a more significant p-value for previously identified loci. However most of these loci had a lower p-value when we only included imputed genotypes with with ≥60% SNP- and ≥50% person-specific imputation certainty. In summary, we developed a novel algorithm for splitting large pedigrees for imputation and found a plausible imputation quality filtering threshold based on FHS. Further examination may be required to generalize this threshold to other studies