A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC-MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3μl/min with overall method run times comparable to standard flow LC-MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5μl of sample, corresponding to a 100-400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample preparation and large injection volumes. The LOQs for cortisol (1.9ng/mL), cortisone (0.3ng/mL), and dihydrotestosterone (1.4ng/mL) were all within a biologically relevant range. A comparison of clinical serum samples was performed for the analysis of testosterone using this microfluidic LC-MS/MS assay and the Beckman Access II automated antibody-based measurement system. The immunoassay results were systematically higher due to matrix interference which was easily resolved with the increased chromatographic resolution obtained in the microflow LC-MS/MS assay.
Keywords: CLIA; ELISA; ESI; Immunoassay; LC–MS/MS; LLE; LOD; LOQ; MRM; Microflow; QC; RIA; RSD; SPE; Serum; Steroid; T; chemiluminescent immunoassay; electrospray ionization; enzyme-linked immunosorbent assay; epiT; epitestosterone; limit of detection; limit of quantitation; liquid chromatography with tandem mass spectrometry; liquid–liquid extraction; multiple reaction monitoring; quality control; radioimmunoassay; relative standard deviation; solid phase extraction; testosterone.
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