Development and validation of a novel HPLC method for the simultaneous analysis of dexamethasone 21- phosphate disodium and dexamethasone in plasma and skin dialysate: Application to pharmacokinetics.

摘要

It has been said and observed that dexamethasone which is a synthetic glucocorticoid has dermatological effect. But there is no such evidence of dexamethasone being a promising drug for skin disease. The main purpose of my thesis is to check whether the dexamethasone concentration can reach to minimum effective concentration in the dermis after giving an iv-infusion which is required to be effective for any pharmacological action. If the concentration in the skin reaches to MEC then there are chances that drug may have any further dermatological action. Dexamethasone has a lipophilic nature. Dexamethasone 21-phosphate sodium is the ester pro-drug which is an available hydrophilic pro-drug of dexamethasone. It readily converts in to active drug dexamethasone in vivo with the help of phosphatase enzyme.;For simultaneous determine of dexamethasone and dexamethasone 21-phosphate sodium (ester pro-drug) in microdialysis and plasma samples a new High Performance Liquid Chromatographic (HPLC) method has been developed. A reverse phase C18 column was used and mobile phase consist of methanol: 10 mM TBA (pH 3) (53: 47% v/v), flow rate of mobile phase was kept 1 ml/min and a UV detector was used with detection wavelength of 240 nm. The calibration curves for microdialysis were linear (R2 correlation coefficient was always greater than 0.99) for the range of 50-10,000 ng/ml and 100-10,000 ng/ml for DXM and DSP respectively. The calibration curves for plasma were linear (R 2 correlation coefficient was always greater than 0.99) for the range of 3000-50,000 ng/ml and 500-50,000 ng/ml for DXM and DSP respectively. As we know only the unbound drug concentration at the site of action is pharmacologically active, and we need needed the concentration of dexamethasone at a particular site of action that is skin. There are several techniques by which we can monitor the concentration on site of animal or human such as biopsies, saliva and skin blister fluid sampling, imaging techniques, microdialysis. But with thesis techniques only a limited number of time points samples were taken where as with Microdialysis MD sequential sampling over a long period of time was possible which could lead to better results. Moreover Microdialysis is semi invasive method.;Female pathogen-free New Zealand albino rabbit was selected to check the kinetics of dexamethasone and dexamethasone 21-phosphate sodium in vivo. The rabbit were tranquilized using appropriate dose of acetopromazine (intramuscular), microdialysis probes were implanted into the skin of rabbit. Retrodialysis was performed first in which the probe were perfused with dexamethasone-dexamethasone 21-phsphate sodium solution to assess probe recovery which was further used to correct the dialysate concentration to reflect the actual interstitial fluid concentration. For recovery the probes were perfused with lactated ringer's solution. Short intra venous infusions of two different doses of dexamethasone 21-phosphate sodium were administered to rabbit. The two separate doses were 1 and 2 mg/kg. Blood and microdialysis samples were collected at predetermined time intervals.;About 100% of plasma dexamethasone 21-phosphate sodium gets converted in to dexamethasone in 20 min after IV- Infusion. Plasma dexamethasone gets highest concentration in 20 min after infusion. All in vivo microdialysis recovery study (for both two doses of IV- Infusions) shows that skin only detects dexamethasone and gets equilibrated within approximately 90 min. After the peak concentrations of analytes, skin (dexamethasone) and plasma (dexamethasone 21-phosphate sodium and dexamethasone) decline differently.