A new liquid chromatographic method for determination of some statin drugs and activity HMGCR level by different methods.

Abstract

This thesis concerns the separation and determination of the three statin drugs (Rosuvastatin, Atorvastatin and Simvastatin) in pharmaceutical dosage forms which play an essential role in treating with hypercholesterolemia diseases and measurement the HMGCR enzyme activity by two methods. The study starts with an overview of the chromatography technique and how this technique is being introduced firstly as a separation technique. The most famous types of statin drugs have been taken in this chapter to analyze pharmaceuticals using the HPLC technique and how these drugs affect the hypercholesterolemia disease. Methodology and experimental part have been clarified and reviewed which includes chemicals, tools and devices, and the general procedure for the preparation of standard solutions. The study also covers effect of the ACN content, the buffer concentration and the pH effect on the retention of the three pharmaceuticals. Moreover, serum lipid profile assay, Enzyme-Linked Immunosorbent Assay kit of HMGCR enzyme, the primer of the enzyme are used in this study and the RT-PCR instrument. The research findings can be interpreted as follows:  HILIC stationary phases (ZIC-1 and ZIC-4) are investigated for chromatographic Rosuvastatin separation. The eluent's retention behavior in various sodium buffer containing acetonitrile and pH of the Rosuvastatin was reported. The separation mechanism leads to a mode for Rosuvastatin according to hydrophobic interaction. The calibrating graphs were produced for two exchangers, and linear range (0.1-7µgmL-1 ), RSD% (0.34± 0.22 and 0.56± 0.08), LOD (0.0094 and 0.0081µg mL-1 ), LOQ (0.0284 and 0.0245µg mL-1 ), respectively. For pharmaceutical samples, the methods proposed have been effective. Additionally, the proposed methods' findings are compared to the standard method, demonstrating that their precision and accuracy are equivalent.  The chromatographic separation and quantification of Atorvastatin, zwitterionic stationary phases with a high capacity were prepared using zwitter-molecules connected to a PS / DVB particle. The retention activity of atorvastatin was determined using eluent at varied vii pH values, mobile phase concentrations, and ACN percentages. Separation strategies are based on separating Atorvastatin from hydrophobic interactions. With direct calibration curves, a linearity of 0.1-7 µg mL-1 for two columns was obtained, as well as RSD percent (0.705 ± 0.075 and 0.6 ± 0.05), LOD (0.0020 and 0.0013µg mL-1 ), and LOQ (0.0060 and 0. 0039μg.mL-1). Additionally, the results of the proposed methods are compared to those of the standard method, proving that their precision and accuracy are comparable.  The chromatographic Simvastatin separation, hydrophilic stationary phases (ZIC-1 and ZIC-4) have been investigated. The retention activity of the eluent in different acetonitrile containing sodium acetate buffer amounts and pH was investigated for Simvastatin. According to hydrophobic interaction, the separation mechanism leads to a mixed mode for the Simvastatin. The calibrating graphs were produced for two exchangers, and linear range (0.05-4µgmL-1 ), RSD percent (0.495 ± 0.055 and 0.39 ± 0.03), LOD (0.023 and 0.015µgmL-1), LOQ (0.069 and 0.045µgmL-1 ), respectively. For pharmaceutical samples, the methods proposed have been effective. And the results of the proposed methods are compared with the comparison method, and their precision and accuracy are comparable.  The study aslo comprises fifty subjects divided into two groups; controls group which involves 20 persons (12 males and 8 females), and the patients` group applies 30 patients (14 males and 16 females) before and after treatment. The blood samples have been collected from patients in Baghdad Teaching Hospital during the period from February to June 2021, including the ages from 33-78-years.  This study shows a significant difference between the control group and the patients `group after statin drugs intake in lipid profile. The mean of T. Cho (mg/dL) has appeared to be high significantly increased (P= <0.001) in patients` group (226.166 ± 53.9) compared with control group (149.4 ± 21.84). The mean of Tg (mg/dL) has appeared to be significantly increased (P = 0.011) in patients` group (212.633 ± 119.57) compared with control group (141.45 ± 8.75). The mean of VLDL (mg/dL) has appeared to be significantly increased (P = 0.011) in patients` group (42.52 ± 23.91) compared with control group (28.29 ± 1.75), viii The mean of LDL (mg/dL) has appeared to be significantly increased (P= 0.001) in patients` group (130.46 ± 54.73) compared with control group (67.26 ± 22.99), while the mean of HDL (mg/dL) was appeared to be no significant (P = 0.85) in patients group (53.17 ± 13.98) compared with control group (53.84 ± 8.89).  Serum levels of HMGCR activity are being estimated by the enzyme-linked immunosorbent assay (ELISA) technique. And whole blood levels of HMGCR activity are evaluated by Real-Time PCR. The first detection method (ELISA kit) in this study is the atorvastatin and rosuvastatin drugs, as shown, inhibited HMG-CoA reductase activity in a concentration-dependent manner which finds a significant decrease in HMG-CoA reductase activity in patients with hypercholesterolemia after statin treatment than before statins treatment (15.7± 7.5; 26.3 ± 11.1) respectively. The Real-Time PCR detection method in this study has inhibited HMG-CoA reductase activity through a copy number gene of this enzyme which conducts a significant decrease in HMG-CoA reductase activity in patients, with hypercholesterolemia, after statin treatment than before statins treatment 348442.4 ± 485652.53; 110.7 ± 20.424) respectively. There were significant differences between RT-PCR and designed ELISA depending on hyperlipidemia patients before and after statin treatment.