Determination of steroidal and nonsteroidal drugs in human body matrices by LC-MS/MS, GC-MS, HPLC and ELISA

There is an ever-increasing need to develop analytical methods for the detection of steroids, NSAIDs, fibrates, iothalamates and fatty acids for therapeutic, forensic, medical and pharmaceutical applications. Hence, the primary aim of the project was to develop new methods for detection of these classes of drugs in different human. body matrices using chromatographic techniques like enzyme-linked immunosorbent assay [ELISA], HPLC-UVIDADÆLSD, LC-MS/MS and GC-MS/MS. The new methods developed were capable of detecting the drugs: prednisolone, cortisol, prednisone, nandrolone, stanozolol, testosterone, diclofenac, fenofibric acid, iothalamic acid, monoolein, diolein, triolein, palmatic acid, stearic acid and linolenic acid. The methods were validated for LLOD, specificity, accuracy, precision, linearity and accuracy according to FDA and ICH guidelines in a cGLP-accredited lab. The assay was linear in the range 1 to 400 pg/mg for stanozolol, 0.5 to 400 pg/mg for testosterone and 3 to 400 pg/mg for nandrolone. The linear range for the routine assays were from 1 to 25 ng/mI with no interference from matrices. The calibration standards and quality controls were prepared in fresh human matrices. New internal standards were developed and added to all aliquots of samples. All methods showed good precision, accuracy, recovery and linearity. LLODs for non-steroidal anti inflammatory drugs were found to be in the range of 0.02 ng/ml to 10 ng/mI in plasma, urine and synovial fluid. Corticosteroids were analysed in the range of 5 ng/ml to 50 ng/mI. The anabolic steroids assays were capable of detecting 0.25 pg testosterone, 0.5 pg stanozolol and 2.5 pg nandrolone as a LLOD per mg of hair when approximately 20 mg of hair was processed. The assay was linear in the range 1 to 400 pg/mg for stanozolol, 0.5 to 400 pg/mg for testosterone and 3 to 400 pg/mg for nandrolone. lothalamates were found to be in the range of 0.25 ug/ml to 0.5 ug/ml, fibrates 5 to 25 ng/mI and diuretics in 1-5 ng/ml range. The extended lower limits of detection and lower limits of quantification would help in the detection of these drugs even at very low concentrations in matrices. Hair, urine, plasma, synovial fluids etc from subjects that were found positive for steroidal, non-steroidal drugs, corticosterides and others by ELISA screens were successfully quantified on HPLC, GC-MS and LC-MS/MS. In conclusion, the results obtained demonstrate the application of hair analysis to detect steroids in line with WADA requirements at lower concentrations. These novel methods have been developed and validated in human plasma, urine, synovial fluids, gastrodeudenal fluids and hair using improved extraction techniques. In addition, new solvents have been added to the samples as a deproteination agent, which increased the recovery of most drugs. Narrow bore column technology has been used to detect lower levels of drugs in matrices. These revised processes allowed clean extraction and near-quantitative recovery of analyte (>95%).