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Data Supplement Development and validation of DBS steroid assay Calibrators, controls and internal standards: Calibrators: Standards for androstenedione, 17-hydroxyprogesterone (17-OHP), and testosterone were obtained in certified concentrations of 1 mg/mL. Dehydroepiandrosterone sulfate (DHEAS) was weighed as a solid and diluted in methanol to a concentration of 1 mg/mL. A master steroid stock solution in methanol was prepared containing final concentrations of 16,000 µmol/L DHEAS, 12,500 nmol/L Andro, 16,000 nmol/L Testo and 37,500 nmol/L 17-OHP. Steroid-free whole blood was used to make dilutions from the steroid stock solution in order to give a six-point standard curve for each compound, spanning the target ranges of 0.02 – 20 µmol/L for DHEAS, 0.04 – 40 nmol/L for androstenedione, 0.3 – 300 nmol/L for 17OHP and 0.1 – 70 nmol/L for testosterone. The resulting six whole blood calibrators were each spotted onto a set of Whatman filter papers and allowed to dry. DBS calibrators were stored at -20°C until use. After spotting the filter paper, the remaining whole blood calibrators were centrifuged and the plasma was retrieved for analysis using the regular steroid assay being used clinically in the lab. The concentration of the steroids measured in the plasma samples was determined by replicate analysis. The values obtained for the calibrators from the plasma analysis were then used to set the calibrator concentrations of the dried blood spot calibrators. Controls Controls were created in the same manner as the calibrators. Aliquots from the master steroid stock solution were pipetted into whole blood pools created using washed red blood cells mixed with steroid free serum. Two control levels were created with target concentrations of 0.3 and 16 µmol/L for DHEAS, 1.2 and 35 nmol/L for androstenedione, 15 and 275 nmol/L for 17-OHP and 5 and 65 nmol/L for testosterone. The spiked pools were mixed gently, spotted onto filter paper, allowed to dry and then stored at -20 C until use. Internals standards: All deuterated steroids were diluted to final concentrations of 17.35 nmol/L [d3]-testosterone, 150 nmol/L [d8]-17-hydroxyprogesterone, 17.45 nmol/L [d7]-androstenedione and 1.35 µmol/L [d2]-DHEAS. Aliquots were stored at -80 C until use. Assay Procedure and Validation Methanol alone was found to extract the four steroid hormones together with the greatest overall efficiency. Steroid recoveries are reported in Table S1. All reagents and samples were allowed to reach room temperature prior to use. Six calibrators, a blank and two levels of controls were analyzed with each run. Chromatography and mass spectrometry parameters for the un-derivatized assays are presented in Table S2. Parameters for the derivatized assay are presented in Table S3. LLOQ was defined and determined as the lowest measurable concentration of each steroid hormone with acceptable imprecision (<20%). Recovery was determined as the average percent of the target concentration recovered from DBS calibrators run as patient samples. Five samples were assayed at each of three concentrations across the linear range for the recovery studies. Imprecision studies were performed using the DBS controls, with 10 controls at each level assayed within a single run for with-in run precision, and values at each control level from assays performed over 20 different days used for between-run precision. Matrix effects and interferences had been previously studied with the validation of the serum steroid assay and included assaying unextracted native steroids, extracted native steroids and native steroids spiked into bovine serum albumin, serum, hemolyzed serum, lipemic serum and icteric serum and then extracted. These studies showed no more than 15% loss or gain of concentration in any of the samples assayed. In this DBS study, calibrators, controls and patient samples were all dried blood spots with the same matrices. Thus for matrix effects, only different hemoglobin concentrations in the DBS were studied. In the serum assay, ion suppression was noted to be a problem affecting concentration when the Internal Standard abundance of the patient samples was less than 60% of the average Internal Standard abundance of the calibrators. For this reason, ion suppression was monitored throughout the DBS work by determining that the IS abundance in DBS patient samples was always at least 80% of the average IS abundance of the DBS calibrators. DBS vs plasma Paired plasma and DBS samples were prepared and analyzed as described in the main manuscript. 60 paired samples were analyzed for each steroid except DHEAS which only had 20 paired samples analyzed. Samples with results below the LLOQ for the DBS or plasma steroid hormones were not used to determine the relationship between DBS and plasma concentrations in patient samples. RESULTS The ions used to identify and quantify the steroid hormones are shown in Tables S2 and S3. Hormone assays were linear from 0.3 - 40 nmol/L for androstenedione, 0.3 – 70 nmol/L for testosterone, 0.3 – 300 nmol/L for 17-OHP and 0.1 – 20 mol/L for DHEAS. The lower limit of quantification was defined as the lowest value that could be measured with a coefficient of variation < 20%. Analytical measurement range and LLOQ are shown in Table S4. The imprecision data for the assay are shown in Table S5 and show that CVs ranged from 3.1 – 23.4% across the various steroid hormones and concentrations. Table S1. Steroid recovery from DBS. Analyte 17-OH-progesterone Androstenedione Testosterone DHEAS Percent recovery across linear range 98 – 112 99 – 113 99 – 107 92 – 101 Table S2. Instrument parameters for performing un-derivatized analysis Time (min) Buffer A (%) 0 100 1 100 6.1 10 6.2 0 8 0 8.1 100 9 100 Buffer A – 50% Methanol: 50% water Buffer B – 100% Methanol Column temperature = 35°C Injection volume 20 μL Flow rate – 0.4 mL/min Compound Androstenedione Androstenedione [d7]-Androstenedione Testosterone Testosterone [d3]-Testosterone 17-hydroxyprogesterone 17-hydroxyprogesterone [d8]-17hydroxyprogesterone Dwell = 0.1 sec MS1 (m/z) 287 287 294 289 289 293 331 331 339 Buffer B (%) 0 0 90 100 100 0 0 MS3 (m/z) 97 109 100 97 109 97 97 109 100 Retention time (min) 3.16 Transition Primary Secondary 3.99 Primary Secondary 4.44 Primary Secondary Cone voltage 36 V 36 V 40 V 40 V 40 V 40 V 40 V 40 V 40 V Collision energy 24 eV 24 eV 24 eV 24 eV 24 eV 24 eV 24 eV 24 eV 24 eV Table S3. Instrument parameters for performing un-derivatized analysis Time (min) Buffer A (%) 0 78 1 78 6 50 7 0 8 78 10 78 Buffer A – 0.1% formic acid in water Buffer B – 0.1% formic acid in acetonitrile Column temperature = 35°C Injection volume 20 μL Flow rate – 0.2 mL/min Compound DHEAS DHEAS [d2]-DHEA Dwell = 0.1 sec MS1 (m/z) 460 460 382 Buffer B (%) 22 22 50 100 22 22 MS3 (m/z) 362 95 96 Retention time (min) 3.58 Transition Primary Secondary Cone voltage 48 V 48 V 50 V Collision energy 25 eV 35 eV 30 eV Table S4. Linear range and LLOQ Analyte linear range 17-OH-progesterone Androstenedione Testosterone DHEAS LLOQ 0.30 – 300 nM 0.30 – 40 nM 0.30 – 70 nM 0.1 – 20 µM concentration CV (%) 0.3 nM 0.3 nM 0.3 nM 0.1 µM 15.7 13.8 12.9 16.6 Table S5. Imprecision studies. Analyte 17-OH-progesterone Androstenedione Testosterone DHEAS Within run CVs (%) Conc CV Conc 18 nM 4.2 260 nM 1.5 nM 13.3 32 nM 4.5 nM 12.3 64 nM 0.3 µM 13.3 16 µM CV 4.9 8.0 8.1 3.1 Between run CVs (%) conc CV conc 18 nM 16.9 260 nM 1.5 nM 23.2 32 nM 4.5 nM 23.4 64 nM 0.3 µM 21.1 16 µM CV 6.2 10.4 9.4 10.4 Optimization of Blood Spot Extraction for Amino Acid Analysis Preliminary experiments were performed to assess the utility of using multiple punches from one blood spot because these spots are known to be heterogeneous from the center to the annulus. Three whole blood samples were spotted onto standard Whatman 903 paper and residual sample centrifuged to generate plasma. Spot concentrations were expressed as a percentage of the determined plasma value to account for differences in endogenous amino acid concentrations. Figure S1 below shows spot concentrations six representative amino acids determined using a one, three, or five punches from the larger spot. Variability in spot concentrations using 3 punches was improved over a single spot with 5 punches yielding no additional benefit. FIGURE S1. Amino acid extraction efficiency. A pool of EDTA anticoagulated whole blood was spotted onto Whatman 903 paper. An aliquot of the same pool was supplemented with amino acid calibrator solutions to increase the concentration of each amino acid by 75 µmol/L. Three punches from the native and supplemented pool from 10 spots each were subject to extraction with methanol and recovery of added amino acid was calculated. Mean percent recovery ± SD of the amino acids detectable in blood spots are presented below. Table S6. Amino Acid Phenylalanine Tyrosine Isoleucine Leucine Valine Threonine Serine Glycine Methionine Glutamine Glutamate Citrulline Arginine Ornithine Homocitrulline Alanine Hydroxyproline Proline Lysine β-aminoisobutyric β-alanine Sarcosine γ-aminobutyrate Histidine α-amino-n-butyrate Recovery, % (mean ±SD) 80±6 75±8 70±4 72±9 77±10 98±11 93±10 100±11 78±7 114±21 82±9 84±7 97±5 51±4 97±7 85±11 90±6 83±11 60±8 78±4 60±4 110±8 67±5 73±8 94±7