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Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS.

ABSTRACT: 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 3?l/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 0.5?l 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.

SUBMITTER: Broccardo CJ

PROVIDER: S-EPMC4391816 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS.

Broccardo Carolyn J CJ Schauer Kevin L KL Kohrt Wendy M WM Schwartz Robert S RS Murphy James P JP Prenni Jessica E JE

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 20130704

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, ...[more]

PMID: 23891914

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Project description:Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17β-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17β-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17β-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

Dataset Information

Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS.

Publications

Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS.

Similar Datasets

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