Hua-fen Liu1, Theresa Lee1, Susan Leonard1, Michael Nova2, Robert L. Fitzgerald3 and David A. Herold3
1 AB SCIEX, Foster City, CA, 94404;
2 Pathway Genomics, Inc., San Diego, CA 92130;
3VA Healthcare System San Diego, CA 92161 and University of California-San Diego, La Jolla, CA 92037
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Introduction:
Steroids are important biomarkers for a number of clinical conditions. Most clinical assays for steroids are based on immunoassays which have problems quantifying steroids due to cross-reactivity problems. A comprehensive steroid panel is challenging due to the need to separate structurally related compounds in a rapid manner that is capable of quantifying low concentrations present in complex biological specimens. , Most clinical assays are based on serum or plasma, however; dried blood spot (DBS) samples are conveniently obtained from a prick of the finger, or heel and spotted onto filter paper. The goal of this project is to develop a simple extraction method (without derivatization) that is capable of measuring a variety of clinically important steroids from DBS using LC-MS/MS.
Method:
Cortisol, 17-alpha hydroxyprogesterone (17-HP), progesterone, testosterone and 25-OH vitamin D3 are extracted from plasma or DBS using liquid-liquid extraction, prior to analysis with LC-MS/MS. Steroids were identified using multiple reaction monitoring and were quantified vs an internal standard. Concentrations of steroids in serum and DBS from 20 apparently healthy volunteers were compared. Method development included evaluation of: different dry blood spot sample preparation procedures, standard curve construction, interferences, separation and reproducibility of the assay. The dried blood spot calibration curves were constructed and spotted onto fresh filter paper in 60 µL aliquots, left to dry at room temperature for 24 hours, and stored at -20 degrees C until needed. The optimal sample preparation procedure entailed cutting out the dry blood spot, soaking it in solvent for 60 min, and then performing liquid-liquid extraction. The organic supernatants were then transferred to glass tubes and evaporated to dryness with nitrogen. Extracts were reconstituted with a solution of 50:50 methanol: water before loading onto the LC-MS/MS system. An QTRAP® 5500 was employed using a C18 reverse phase column.
Results:
Preliminary data demonstrated that the lower limit of quantitation for cortisol, 17-alpha hydroxyl progesterone, progesterone, testosterone, and 25-OH vitamin D3, respectively are: 5 ng/mL, 100 pg/mL, 100 pg/mL, 50 pg/mL, and 10 ng/mL. The reproducibility of the assay is around 15% in patient specimens. Good correlation was observed between dry blood spot and serum sample for testosterone (r2=0.96), cortisol (r2=0.96) and 25OH-vitamin D3 (r2=0.90). The serum to DBS ratio appeared to be related to lipophilicity with cortisol < testosterone < 17HP < progesterone < 25-OH Vitamin D3.
Conclusion:
Initial validation experiments have demonstrated that it is possible to quantify steroids from DBS specimens and that there is a good relationship between DBS concentrations and those measured in plasma.
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