Development and Validation of Facile RP-HPLC Method for Simultaneous Determination of Timolol Maleate, Moxifloxacin Hydrochloride, Diclofenac Sodium and Dexamethasone in Plasma, Aqueous Humor and Pharmaceutical Products.

The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 µL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32-1.57 and 1.29-3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

Effects of chelating agents on heavy metals in Hepatitis C Virus (HCV) patients.

Heavy metals are released into the environment through both human and natural sources, may have a direct hepatic toxicity and are involved in chronic liver diseases. Modification in the regulation of heavy metals metabolism enhanced hepatitis c virus (HCV) replication which ultimately reduced outcomes of anti-viral therapy in chronic HCV patients. Chelation therapy with new drugs seems to eradicate HCV and may prevent liver complications. The present study was planned to explore the effects of MiADMSA (lipophilic chelating agent) for achieving maximum heavy metals elimination in hepatitis c virus patients with minimum side effects. For this purpose concentration of heavy metal was determined in HCV patients and established correlation of heavy metals between healthy persons and HCV patients. Atomic absorption spectrophotometer (AAS) was used to explore them. Concentrations of heavy metal in different samples (blood serum, nails and hair) of patients and healthy individuals. Result revealed that heavy metals (Lead, Cobalt, Cadmium, Manganese, Iron and Cooper) concentration were significantly higher in blood of HCV patients as compared to normal persons, but some metals like Ni and Zn were present in normal concentration and in low concentration respectively. After chelation with monoisoamyl DMSA (MiADMSA) a significant amount of heavy metals was excreted in the urine in a dose dependent manner. It was generally observed from the results that TDS is a better treatment option than BD for chelation of heavy metals in hepatitis c virus patients. This chelation therapy will be helpful to reverse the HCV related health problems.

Analysis of toll-like receptors-9 (TLR9) gene polymorphism (rs5743836) in Pakistani patients with HCV.

Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response during HCV infections. The goal of this study was to evaluate the association of TLR9 gene polymorphism (rs5743836) in Pakistani patients infected with genotype 3a of HCV. Total 500 subjects were recruited, 400 HCV patients and 100 healthy individuals. Genotyping of TLR9 (-1237T/C, rs5743836) was carried out in 400 HCV patients (323 interferon responders and 77 interferon non responder) and control group by applying High resolution melting (HRM) curve assay. No remarkable differences in distribution of genotype between HCV (p<0.0001; OR= 3.21, 95% CI= (2.514.12) and control groups (p<0.0001; OR=0.092, 95%CI= (0.0580.14) were observed. In conclusion TLR9-1237T/C gene polymorphism may not be considered as a molecular risk for patients with HCV in Pakistan.