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Abstract INTRODUCTION: Endogenous steroids are essential to the regulation of several metabolic pathways including energy metabolism, stress, and fertility. Dried blood spots (DBS) offer an alternative to conventional venipuncture blood collection by allowing less invasive sample collection at home. A challenge for the lab is the small sample size collected from the dried blood spot resulting in low concentrations of endogenous analytes. To assist with accurate quantitation of select steroids in this biological matrix, dispersive solid-phase (dSPE) extraction was utilized for increased sensitivity and selectivity by reducing matrix interference. Triple quadrupole mass spectrometry with its sensitive quantitation capability was used to analyze endogenous steroids. Optimized source conditions and MRM transitions on the mass spectrometer further improved limit of detection in matrix.
OBJECTIVES: The primary objective of this study is to compare quantitative results from serum and plasma separator cards with immunoassay methods.
METHODS: Four steroids (cortisol, testosterone, DHEA-S, and progesterone) were evaluated. Calibration curves and quality control samples were prepared by spiking certified reference standards into charcoal striped serum. Correlation samples were prepared by spiking analytes into serum and adding two drops to the collection card and drying for 15min. The collection disk was removed and reconstituted with isotope-labeled internal standard solution, incubated, and digested. The samples were then taken through an automated dSPE extraction protocol. A triple quadrupole mass spectrometer (LCMS-8060NX) coupled to a HPLC (Nexera X2) was used. Separation occurred over 3 minutes using a Velox SP-C18 column (100mm x 3.0mm I.D., 2.7µm). The spiked serum samples were additionally analyzed on immunoassay and the results were compared.
RESULTS: A triple quadrupole mass spectrometer with polarity switching was utilized to allow for positive and negative ionization in a single method (quantitation MRM: cortisol (ESI+): 363.05>105.10, testosterone (ESI+): 289.20>109.10, progesterone (ESI+): 315.20>109.05, and DHEA-S (ESI-): 367.05>97.05). Accuracy and precision for quality control inter-day (n=6) and intra-day (n=20, 3days) were less than 10% for all analytes. Calibrations were linear with correlation coefficients (r2) greater than 0.99 over the concentration range 5-1,250 ng/mL for cortisol, 80-10,000 ng/mL for DHEA-S, 0.1-25 ng/mL for testosterone, and 0.1-25 pg/mL for progesterone. Correlation between serum aliquots and reconstituted serum collection cards ranged between -4.0% -30% differences and correlation between plasma collection cards are currently in evaluation. dSPE is completed using Hydrophilic-Lipophilic Balanced (HLB) absorbent, developed to cleanup both polar and non-polar analytes from aqueous samples to achieve these low reference ranges. The steps for the sample extraction are automated using hands-free automated electronic pipettes. Chromatography for all analytes have been evaluated on a SP-C18 (2.7um 2.1x100mm) column with an injection-to-injection time of 6 min (Cortisol, 2.1min, DHEAs: 2.4min, testosterone: 3.1min, and progesterone: 3.8min).
CONCLUSION: A highly sensitive and accurate LC-MS/MS method was developed for the quantification of four steroids on DBS cards. The approach to utilizing DBS rather than traditional immunoassay has several advantages such as non-invasive collection and smaller sample volume. Automated sample extraction increases sample throughput by analyzing all analytes on a single LC-MS/MS method.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
