The setup and validation of an LC-MS/MS assay of Androstenedione & Testosterone a labs experience Keith Mulready Specialist Senior Medical Scientist
Why measure Testosterone? Hypogonadism in Males Cancer monitoring Associated with various disease states Delayed or precocious puberty in kids Ambiguous genitalia in infants Androgen excess in females Polycystic ovarian syndrome (PCOS) All predicated upon accurate measurement of circulating T levels in both sexes 2
The Rotterdam criteria for PCOS Oligo- and/or anovulation Clinical or biochemical signs of hyperandrogenism Polycystic ovaries 3
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UKNEQAS for Steroid Hormones 5
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Why measure Androstenedione? T most commonly ordered measurement for investigation of female hyperandrogenemia A used as secondary test with elevated T A not routinely available on chemistry analysers Spironolactone interferes with Siemens CAC assay To save money and resources now flawed Measuring A only when T raised would miss the high A-normal T PCOS phenotype IA gives results >twice MS for A (UKNEQAS) 7
PCOS patients had higher levels of serum androgens and urinary androgen metabolites than controls (all P<.001). All subjects with T above the normal reference range [high T (HT)] also had high A (HA/HT group, n 56). However, the remaining 30 patients had normal T levels, either in the presence of HA(HA/NT; n20) or NA(NA/NT; n10)....these data endorse the hypothesis that increasing androgen burden is associated with an adverse metabolic phenotype and that concurrent measurement of both A and T highlights a PCOS cohort that appears to be at increased metabolic risk. We also believe on the basis of these data that serum A is a more sensitive indicator of PCOS-related androgen excess than serum total T concentrations. Clin Endocrinol Metab, March 2014, 99(3):1027 1036. 8
Androstenedione EQA performance 9
Sample Preparation MAX (Mixed-Mode Anion exchange) Two step extraction Zinc Sulphate Precipitation 200µL Serum Internal Standard in 50/50 MeOH/H2O 2% Ammonia (aq) 0.2M Zinc Sulphate Vortex for 3 minutes MeOH Vortex for 2 minutes H 2 O Centrifuge @3000rpm for 4 minutes MAX µelution Condition and Equilibrate with MeOH and H 2 O, respectively Load 600µL supernatant to the plate Wash with 0.1% Ammonia in 20% MeOH (aq) Elute with MeOH and H 2 O 10
ACQUITY UPLC system UPLC Conditions Injection volume: 20µL Column: HSS C18 SB 2.1 x 50mm, 1.8µm Run time: 3.8 mins (injection to injection 4.5 mins) Flow rate: 0.6mL/min Mobile Phase A: Water 2mM Amm Ace + 0.1% formic acid Mobile Phase B: Methanol 2mM Amm Ace + 0.1% formic acid Column Temperature: 45 o C Gradient : TIME %A %B CURVE Initial 50 50 Initial 1.50 50 50 6 2.75 40 60 6 2.76 2 98 11 3.30 50 50 11 11
MS conditions Xevo TQ-MS Mass Spectrometer Ion mode: ESI+ Capillary Voltage: 0.40kV Desolvation Temperature: 450 C Source Temperature: 150 C MRM parameters are shown in the table below: Compound Parent (m/z) Androstendione 287.2 97.1 (Quan) 109.1 (Qual) Daughter (m/z) Cone (v) CE (ev) 30 22 (Quan) 24(Qual) Androstenedione D7 294.2 100.1 30 22 Androstenedione 13C3 290.2 100.1 30 22 Testosterone 289.2 97.1 (Quan) 109.1 (Qual) 30 22 (Quan) 24(Qual) Testosterone 13C3 292.2 100.2 30 22 12
Metrological Traceability Need to fulfil the requirements of the IVD directive and ISO15189 MMUH utilises WEQAS Testosterone Calibrators which were prepared using a certified reference material and value assigned by a JCTLM listed Reference Measurement Lab using a validated ID-GCMS method. The traceability chain is maintained. Cerilliant CRM for calibration 13
Testosterone Targeted Calibrators Sample ID (lot 101308) WEQAS Target Conctraceable to SI units (nmol/l) Measured in-house Cal Concs (nmol/l) Expected Working Cal Concs (nmol/l) Level 0 0 0.15 0 Level 1 0.50 0.33 0.17 Level 2 1.04 0.63 0.52 Level 3 2.88 1.57 1.74 Level 4 7.63 3.37 3.47 Level 5 15.10 8.27 8.68 Level 6 23.48 23.48 26.03 Level 7 38.32 49.48 52.05 14
Importance of Internal Standard selection a tough lesson learned!! Pre-Ad7 issue Something not right??? 15
Digging deeper!! Dilute Ad7 1:10 Extraction minus Ad7.endogenous 16
Guidance for Industry Bioanalytical Method Validation published by the FDA for method validation 17
Parameter Validation Passing&Bablok equation Table: Androstendione and female and male Testosterone comparisons Proportional Bias Constant Bias Andro: Ext Lab v MMUH MS y = 1.0 x 7.1 e -016 N N Andro: EQA MM v MMUH MS y = 1.0 x 0.04 N N Female T: Ext Lab v MMUH MS y = 1.0 x + 1.7e -016 N N Female T: EQA MM v MMUH MS y = 0.93 x + 0.04 Y N Male T: IA v MMUH MS y = 1.32 x 0.95 Y N Male T: EQA MM v MMUH MS y = 0.97 x + 0.08 N N Compound L 1 L 2 L 3 L 4 L 5 Androstendione 5.2 4.1 3.7 4.7 4.4 Testosterone 3.1 3.7 3.7 4.6 4.7 Compound L 1 L 2 L 3 L 4 L 5 Androstendione 6.8 7.2 6.1 7.4 7.5 Testosterone 5.9 6.4 7.2 8.0 8.1 Table: Intra-assay precision (CV%) Table: Inter-assay precision (CV%) Reason NEQAS has NOT introduced Restricted ALTM for Male Testosterone WEQAS QCRM Pool 620 Target = 28.93nmo/L MMUH=33.48, 27.73, 29.70 (av bias of +4%) 18
NEQAS Testosterone examples 19
Validation LOQ A = 0.13 nmol/l, T = 0.15 nmol/l (<20%CV and 80-120% accuracy). LOD A = 0.08 nmol/l, T = 0.10 nmol/l (S:N >3:1) Extract stability: on-board stability @ 10 o C for up to 48hrs (CV s <0.8%) Carryover Numerous stability studies Av injection precision (CV=1%) Interference: Branching ratio s Linearity: linear up to 70nmol/L, R 2 >0.995 Recovery: Mean recovery A=105%, T=97% Isobars/epimers.DHEA and Epi-testosterone clearly resolved Where is epi peak in authentic patient samples?????? 20
Ion suppression Post-column infusion Andro Post-column infusion Testo 21
Conclusion We have developed a sensitive, robust, accurate and precise LC- MS/MS method for simultaneous determination of A & T. Sole laboratory providing this esoteric test by LC-MS/MS in the Republic of Ireland. 4 in 1 method to include 17-OHP + DHEAS being validated!!! 22
Acknowledgments MMUH Dr Marguerite McMahon Rachel Cullen Dr Maria Fitzgibbon Waters Rob Ennis Dom Foley Lisa Calton 23
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