UPLC-MS/MS method for determination of sofosbuvir in human plasma.

INTRODUCTION: A sensitive and rapid method for quantitation of Sofosbuvir in human plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). MATERIALS AND METHODS: Sofosbuvir d3 was used as an internal standard. Sofosbuvir and internal standard in plasma sample were extracted using ethyl acetate (liquid liquid extraction). A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid: methanol (30:70, v/v). The reconstituted samples were injected into a Gemini C18 (50×4.6mm, 5µm) column. RESULTS: Using MS/MS in the multiple reaction monitoring mode, Sofosbuvir and Sofosbuvir d3 were detected without severe interferences from human plasma matrix. Sofosbuvir produced a protonated precursor ion ([M+H]+) at m/z 428.35 and a corresponding product ion at m/z 279.26. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 431.38 and a corresponding product ion at m/z 282.37. The calibration curves for the analyte was linear (R2≥0.9956, n=4) over the concentration range of 4.063-8000.010ng/mL. Stability studies revealed that Sofosbuvir was stable in plasma during bench top (7h at room temperature), in injector (20h), at the end of five successive freeze and thaw cycles and long term at -70°C±15°C for 15 days. CONCLUSION: The developed method was validated as per the guidelines of USFDA and the obtained results were found to be within the limits and could be successfully employed for the determination of Sofosbuvir in human plasma for regular and pharmacokinetic studies.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM anti-idiotypes directed against anti-HBs molecules.

A simple and specific enzyme-linked immunosorbent assay (ELISA) has been developed to detect circulating IgG and IgM anti-idiotypic antibodies directed against anti-HBs molecules using 96-well polyvinyl microtitre plates as the solid phase and HRPO-labelled goat anti-HBs as conjugate. Anti-idiotype reactions were observed in the supernatant portion after precipitation of immune complexes from sera with polyethylene glycol 6000 (PEG). Both IgG and IgM with anti-idiotype activity were detected concurrently in HBsAg-positive sera from HBV-infected patients and asymptomatic HBV carriers. Anti-idiotype activity was absent in HBsAg-negative sera from healthy persons, and in patients with non-A, non-B hepatitis and viral hepatitis A. However, such antibodies could be demonstrated in the sera of two out of eight HBsAg vaccine recipients negative for anti-HBs but in none of 11 recipients positive for anti-HBs after receiving a booster immunising dose of HBsAg vaccine. Those sera showing positive anti-idiotype reactions were free from rheumatoid factor and HBsAg/IgM or HBsAg/IgG complex activity. An analysis of anti-idiotype positive sera for anti-HBs, HBeAg and HBV-specific DNA-polymerase activity demonstrated these markers in 20%, 30% and 60% of cases, respectively. The presence of anti-idiotypic antibodies was presumed to permit a more active multiplication of hepatitis B virus.

Amebic liver abscess and circulating immune complexes of Entamoeba histolytica proteins.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect amebic antigen in polyethylene-glycol-precipitable circulating immune complexes. Seventy-nine percent of 191 patients with amebic liver abscesses and 46% of 26 Entamoeba histolytica cyst passers had positive tests. None of the samples from 43 apparently healthy controls, 8 patients with past amebic liver abscesses or 31 patients with Giardia lamblia infections were positive. One patient out of 32 with viral hepatitis and 1 patient out of 27 with intestinal tuberculosis had positive tests.

Studies on HBsAg binding with polymerised human serum albumin by ELISA.

A simple and sensitive ELISA was developed to characterize the interaction between polymerised human serum albumin (pHSA) and HBsAg, using pHSA-coated polyvinylmicrotitre plates as solid phase and anti-HBs-coupled HRPO as the conjugate. The interaction was found to be specific and dependent on the size of albumin polymer. pHSA-binding activity (pHSA-BA) was studied in both HBsAg-negative and HBsAg-positive sera from various liver diseases including acute viral hepatitis, fulminant hepatitis, cirrhosis of liver, chronic active hepatitis, and healthy HBsAg carriers. pHSA-BA was detected only in HBsAg-positive sera. Analysis of HBsAg-positive sera indicated pHSA-BA in high proportions of patients sera as compared to sera from healthy HBsAg carriers. pHSA-BA was detected both in the presence and absence of HBe markers, though the mean BA was relatively high in presence of HBeAg. The effect of human serum immunoglobulins (IgG, IgA, and IgM) on the BA was investigated and a correlation between pHSA-BA and HBsAg-IgM complex positivity in sera was established. Finally, the probable role of human serum IgM in facilitating the binding process was discussed.

A dot immunobinding assay (DIA) on nitrocellulose membrane for the serological detection of antibodies to Entamoeba histolytica.

A rapid, cheap, simple and specific serological test of adequate sensitivity for detecting IgG antibodies against Entamoeba histolytica antigen is described. Axenically cultured amoebic antigen was used to precoat the nitrocellulose membrane. The strips were incubated with test samples and later with horseradish peroxidase (HRPO) labelled protein-A conjugate. A dark blue spot was obtained by treatment with peroxidase substrate, 4-chloro-1-naphthol, in positive samples. Serum samples from 32 healthy controls, 45 patients with acute amoebic liver abscess and 10 asymptomatic E. histolytica cyst passers were tested. This test was positive in 93% of cases of amoebic liver abscess, 3% of healthy controls and none of the cyst passers; its sensitivity (97%) and specificity (93%) were as good as that of the ELISA test and, because it is simple, quick and cheap it is recommended as a serological test of choice for the diagnosis of invasive amoebiasis.

Enzyme linked protein-A: an ELISA for detection of amoebic antibody.

Enzyme linked protein-A was used to develop an enzyme linked immunosorbent assay (ELISA) system for the detection of circulating antibodies to amoebic antigen. The specificity of protein-A to bind IgG only through Fc receptors, makes the test more specific for the detection of IgG antibodies to amoebic antigen. The ELISA system was used to detect amoebic antibody in control subjects (56), patients with amoebic liver abscess (79) and Entamoeba histolytica cyst-passers (10) and the results compared with those of indirect haemagglutination assay (IHA). The ELISA was more sensitive and detected 74.7% of cases with amoebic antibody in amoebic liver abscess compared with 66.7% detected by IHA. The test was more specific, sensitive and easy to perform and is recommended as a test of choice for the serological diagnosis of amoebic liver abscess.

Entamoeba histolytica: elevated nitroblue tetrazolium reduction activity in polymorphs during amoebic liver abscess.

Peripheral blood polymorphonuclear leucocytes (PMN) from patients with invasive amoebiasis, i.e. amoebic liver abscess (ALA) and acute amoebic dysentery, showed marked elevation of nitroblue tetrazolium dye (NBT) reduction. This dramatic change was not observed in PMN from patients with non-invasive amoebiasis, i.e. non-suppurative hepatic amoebiasis, or in asymptomatic Entamoeba histolytica cyst passers. A small number (12%) of patients with viral hepatitis displayed increased NBT reduction. 10 to 12 days after recovery following treatment, the majority (75%) of ALA patients failed to show increased NBT reduction. Our results suggest that the PMN-NBT reduction test could be useful as an aid to the diagnosis of ALA.

Lipids changes in alcoholic & non-alcoholic cirrhotics.

Fasting plasma samples from 29 patients of cirrhosis were analysed for cholesterol and triglycerides and their lipo-protein fractions. The patients included 11 alcoholic cirrhotics consuming over 130 g/day of absolute ethanol and 18 non-alcoholic cirrhotics. The difference in lipid values between the two patient groups was not significant except that VLDL cholesterol was raised in alcoholic cirrhotics (P less than 0.05). However, in comparison to normal healthy controls, the values were significantly altered. The dietary intake, in the two groups showed no difference, except that non-alcoholic cirrhotics consumed more animal proteins. Low intake of exogenous fat and reduced synthesis of endogenous cholesterol in cirrhotic patients seemed to influence the total lipid values.

A simple spot-test for circulating Entamoeba histolytica antigen-antibody complexes in patients with amoebic liver abscess.

An enzyme linked immunosorbent assay (ELISA) is described for the detection of E. histolytica immune complexes in serum. Antiamoebic antibody purified by affinity chromatography was used both to precoat strips of nitrocellulose membrane and as an enzyme carrier. These strips were incubated with samples of concentrated test serum and the enzyme conjugate. Following treatment with the peroxidase substrate 3-amino-9-ethylcarbazole the presence of E. histolytica antigens was visualized as red spots. Blocking of positive test sera with excess antibodies inhibited this reaction. Serum samples from 47 patients with amoebic liver abscess, 43 patients with other enteric diseases and 35 healthy controls were investigated. The spot test was positive in 75 per cent of patients with amoebic liver abscess, and in 12 per cent diseased controls whereas all the healthy controls were negative. The spot test is simple and sensitive and does not require prior separation of the immune complexes. The test is recommended as an aid to the diagnosis in patients suspected to have amoebic liver abscess.

Role of immune serum globulin in post transfusion virus B infection.

Fifty four blood recipients were administered prophylactic immune serum globulin (31) or hepatitis B immune globulin (23) and followed up for six months. None of the patients developed either acute hepatitis B or HBsAg carrier state. However, 7 (14%) had anicteric self limiting non-B post-transfusion hepatitis. Twenty (40%) of the blood recipients developed anti-HBs during the follow up period suggesting either HBV exposure by subdetectable levels of HBsAg present either in blood or immunoglobulin preparation or due to passive transfer by administration of immunoglobulins.

Plasma lipid profile in gallstone patients from North India.

One hundred and thirty five patients with gallstones along with eighty nine matched controls were studied ultrasonographically to look for any association with hyperlipidemias. Plasma cholesterol and triglycerides were estimated by colorimetric methods and lipoproteins were classified according to Beaumont's classification. Male to female ratio in gallstone patients was 1:3. Mean plasma cholesterol and triglyceride values were higher in male gallstones patients as compared to controls (166.40 +/- 54.21 vs 40.26 +/- 32.80 mg/dl, p <0.01 and 182.65 +/- 84.49 vs 133.18 +/- 52.37 mg/dl, p <0.01 respectively). In female gallstone patients, on the other hand, only plasma triglyceride levels were raised as compared to control (182.65 +/- 84.49 vs 133.18 +/- 52.32 mg/dl, p <0.01). Prevalence of type IIb and type IV was 24.32% and 29.72% in male gallstone patients and 13.2 and 39.70% respectively in female gallstone patients. Thus, more than half of our gallstone patients had hyperlipidemia, the commonest types amongst them being type IIb and type IV.

Serological pattern of hepatitis B virus markers (HBsAg, anti-HBs, IgM anti-HBc and HBV specific DNA polymerase) in leprosy patients.

Sera of 134 lepromatous (LL/BL) and 57 tuberculoid (TT/BT) leprosy patients were analysed for four HBV markers. HBsAg was detected in 6.71% of lepromatous and 3.5% of tuberculoid sera. The per cent positivity of lepromatous and tuberculoid sera for anti-HBs antibodies was 30.59% and 35.08%, respectively. The positivity of normal sera for HBsAg and anti-HBs was 3.60% and 21.69%, respectively. The difference in the positivity of three groups of sera (lepromatous, tuberculoid and normal) for HBsAg or anti-HBs was not statistically significant. Anti-HBc (IgM) antibodies were detected in 6% of lepromatous sera. HBV-specific DNA-polymerase activity was found in 22.22% of HBsAg positive (but anti-HBc negative) sera, and 66.66% of anti-HBc positive (but HBsAg negative) sera. The pattern of acute HBV infection in leprosy patients followed the typical pattern prevalent in the normal population.

Relationship of examination stress to serum lipid profile.

Although mental stress as well as hypercholesterolaemia have been individually linked with atherosclerosis, the relationship between mental stress and hypercholesterolaemia is poorly understood. Serum lipid profile was studied in eight male medical student volunteers before, near and after examinations. Identical observations were also made on seven well-matched control volunteers. As compared to pre-exam levels, total serum cholesterol (T-C) increased significantly (P less than 0.05) near exams, and so did low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C). The HDL-C/T-C and HDL-C/LDL-C ratios remained essentially constant throughout the study. Control subjects did not show any significant change in serum lipid profile. Further serial measurement in five of the subjects revealed that examination-related changes were transient. Moreover, a second examination after about 40 days did not evoke any change in the lipid profile. The response to examination stress may be related to the enhanced utilisation of cholesterol in the adrenal cortex for steroidogenesis.

Effect of sieved buckwheat (Fagopyrum esculentum) flour supplementation on lipid profile and glucose tolerance.

Buckwheat (Fagopyrum esculentum) was picked up for a study of its effects on lipid profile and glucose tolerance in view of its relatively high fibre content. In earlier studies, we demonstrated that supplementing the daily diet with 100 g whole buckwheat flour raised the high density lipoprotein cholesterol (HDL-C)/cholesterol ratio and improved glucose tolerance. In the present study, 12 human volunteers replaced part of their cereal intake at lunch by a preparation made from 100 g sieved buckwheat flour for a period of 4 weeks. At the end of 4 wk, there was a significant rise in HDL-C from 42.8 +/- 11.4 mg/100 ml to 55.2 +/- 15.3 mg/100 ml (P less than 0.05), and in HDL-C/cholesterol ratio from 26.7 +/- 7.0% to 33.8 +/- 10.2% (P less than 0.02): The other changes in lipid profile were not significant. There was also no significant change in fasting blood glucose or oral glucose tolerance in the 5 subjects on whom the test was done. Comparing the results with the observations made earlier on whole buckwheat, it is still not possible to say to what extent the effects of buckwheat on lipids may be attributed to its fibre content. The effects on glucose tolerance, on the other hand, seem to be more directly related to the fibre content.