SAT-011 Quantification of Testosterone, Androstenedione and 17-Hydroxyprogesterone Collected Using Mitra®Micro Sampling Devices

Introduction Measurement of total plasma testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) typically requires a venous sample. A highly sensitive and specific assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after extraction from a Mitra® volumetric absorptive microsampling (VAMs) device. VAMs devices are small absorptive polymer tips which absorb a fixed amount of blood (1); they can be dried after collection of finger prick blood and stored prior to analysis. Differences in binding in whole blood compared to serum require the quantification of haematocrit using a published protocol (2), thus allowing an estimated serum result to be generated. Method Pre-mixed calibrator/sample (10 µL) was combined with deionised water and mixed internal standard, after vortexing MTBE was added and mixed by inversion for 1 minute. The supernatant was transferred to a clean 2 mL deep-well plate, dried down and re-constituted with 100 µL 50:50 methanol/water. Mitra® tips were prepared from anonymised whole blood samples in the laboratory and compared to the corresponding plasma using the same method. Results Mean recovery was 86% for A4, 102% for T and 97% for 17OHP, the lower limit of quantification was 1 nmol/L for A4, 1 nmol/L for testosterone and 4 nmol/L for 17OHP. Ion suppression was within acceptable criteria for all the analytes measured. For the samples prepared in the laboratory, comparisons for T, A4 and 17OHP the R-squared values were 0.95, 0.93 and 0.95 respectively with a minimal bias. Summary We have developed a robust assay for LC-MS/MS measurement of VAMs for T, A4, and 17OHP in a routine clinical laboratory. It is simple, reproducible and easy to perform and may have implications for monitoring of T therapy in hypogonadal males or screening/monitoring of patients with congenital adrenal hyperplasia. In-house comparisons of T, A4 and 17OHP compared well when haematocrit was used to adjust for the space occupying effect of red cells. References 1. Spooner N, Denniff P, Michielsen L, De Vries R, Ji Q.C, Arnold ME, Woods K, Woolf EJ, Xu Y, Boutet V, Zane P, Kushon S, Rudge JB (2015) A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated with blood hematocrit. Bioanalysis 7, 653-659. 2. Prediction of haematocrit in dried blood spots from the measurement of haemoglobin using commercially available sodium lauryl sulphate. G Richardson, D Marshall, BG Keevil, Ann Clin Biochem. 2018 May;55(3):363-367