Establishment of a Common Calibrator for Mass Establishment of a Common Calibrator for Mass Spectrometry Analysis of Serum Testosterone R.F. Greaves , C.S. Ho, K.E. Hoad, J Joseph, B. McWhinney, J.P. Gill, T. Koal, V. Vamathevan, E.J. Murby, C.Fouracre, Y-P. Iu, B. Cooke, C. Boyder, H.T Pham, L.M. Jolly Ronda.Greaves@rmit.edu.au AACB-APFCB Mass Spectrometry Harmonisation Group Biocrates Calibrator Set (nmol/L) Level Mean NMIA Result Biocrates Weighed- In Values 1 0.033 0.035 2 0.123 0.139 3 0.676 0.693 4 2.609 2.770 5 10.157 10.400 6 20.003 20.800 7 33.208 34.700 Comparison of Biocrates weighed in values compared to NMIA values assigned by RMP Intercept : 0.0006 [ -0.0224 to 0.0131 ] Slope : 1.043 [ 1.023 to 1.058 ] Passing-Bablok agreement test N = 7 Common calibrator package insert values compared to NMI target values 0 10 20 30 40 NMI 0 10 20 30 40 Biocrates Question Topic Responses Calibration Standard Material Source Sigma, NMIA, WEQAS, Lipomed, Steraloids. All purity>98%. Working/Secondary Calibrator Matrix Stripped serum, Calibrants in solvent, Biocrates matrix Lowest Calibrator (nmol/L) Median 0.14, Range 0 - 0.80 Highest Calibrator (nmol/L) Median 39, Range 10-50 Internal Standard Material Isotopically-labelled testosterone D2, D3, D5 Sample Preparation Volume Used 100uL, 200uL, 500uL, 1000uL Preparation Liquid/liquid extraction, Protein precipitation followed by liquid/liquid extraction, SPE Extraction Solvent MTBE, SPE, ethyl acetate LC-MSMS Analysis LC-MSMS Instrument Waters Quattro Premier XE, Waters Xevo TQD, Waters Quattro Micro, Agilent 6490 Jetstream, Thermo TSQ Vantage Mobile Phase in Various Gradient Methods Ammonium acetate / formic acid / methanol, Water / methanol / formic acid , Water / acetonitrile / formic acid, Acetonitrile / formic acid / methanol, Acetonitrile / methanol / water Run Time (min) Median 8, Range 3-32 MRM for Quantitation and Confirmation 289 > 97, 289 > 109 Method Validation Parameters LOQ (nmol/L) [CV=20%] Median 0.3, Range 0.06 - 0.5 LOQ (nmol/L) [S/N = 10] Median 0.1, Range 0.02 - 0.5 % Recovery Median 98.6%, Range 90-102% Internal QC BioRad - Liquichek Immunoassay Plus, BioRad - Lyphochek Immunoassay Plus, Biocrates QC, In-house, NIST SRM 971 & ERM DA346a Reporting concentration range (nmol/L) 0.3 - 40, <0.2 to 20, 0.2 - 50, 0.8 - 19.2, 0.03 - 34.6, 0.5 - 50 Separate epi-testosterone Yes, Unknown, No Post-analytical Reference female intervals (nmol/L) <1.7, <2.0, 0.3- 2.6, 0.3- 3.8 Reference male intervals (nmol/L) >8 (1),8 - 35 (1), 8.3 - 33 (1), 9.0 - 35 (1) Results The questionnaire demonstrated that most laboratories prepared samples by liquid-liquid extraction and one used solid phase extraction, with consistent MRM’s for the testosterone quantifier/qualifier ions. The source of the laboratory’s calibrator, number of calibration levels and deuterated sites on the internal standard differed. Recalculation of the human testosterone samples against the common calibrator demonstrated a significant change (p<0.05) in results for the male serum (NMIA assigned value 16.19 nmol/L) whereas the change for the female serum (NMIA assigned value 0.57 nmol/L) was not statistically significant for the group. Conclusions This is the first such report to fully characterise the LC-MSMS methods for serum testosterone in common use by clinical diagnostic laboratories. In addition, to characterising the LC-MSMS methods, this pilot study demonstrates the utility of the Biocrates material as a common calibrator and offers 16 initial recommendations to standardise testosterone LC-MS/MS methods. The assignment of reference values to the common calibrator by the reference measurement procedure provides a mechanism to establish traceability. Reference values for future calibrator batches will be made traceable to SI units. Whilst, the common calibrator has achieved a change in results this change is not consistent for all laboratories. Hence further harmonisation of the analytical method through, at least in part, the implementation of the proposed recommendations is supported. This is required before common reference intervals can be recommended for testosterone. Continued monitoring, will be achieved through collaboration and participation in the RCPAQAP Endocrine program. Materials and Methods All laboratories participated in the RCPA Quality Assurance Programs (QAP) for serum testosterone. In 2013 a detailed questionnaire was sent to participating laboratories in conjunction with a seven level common calibrator set (from Biocrates), external quality assurance set (from RCPAQAP) and two de-identified human serum samples. Samples were analysed by each laboratory’s (n=8) routine LC-MSMS method on two separate occasions. In addition, gas chromatography MS analysis was performed by one of these laboratories. Target values were assigned by the National Measurement Institute of Australia (NMIA). ANOVA was performed to determine if recalculation led to a change in results. Introduction In 2010 the Mass Spectrometry Harmonisation Working Group was established to progress the harmonisation and standardisation of mass spectrometry based clinical laboratory methods. The first project is to pursue the harmonisation of the measurement of serum steroids, with serum testosterone as the key analyte. Here we describe the outcome of the first two project goals: 1. provide detailed information on the serum testosterone liquid chromatography tandem mass spectrometry (LC-MS/MS) methods used in Asian-Pacific clinical biochemistry laboratories; and 2. standardise serum testosterone results through the use of a common calibrator. Summary of the questionnaire Reference Measurement Procedures (RMPs) RMPs for testosterone in serum were developed by NMIA using isotope dilution mass spectrometry (IDMS). Target values were determined using LC- MS/MS analysis with confirmation by gas chromatography/high resolution mass spectrometry (GC-HRMS) analysis of the same samples. The reference values are metrologically traceable to the International System of Units (SI) for mass (kg), volume (m 3 ) and amount of substance (mole) within their stated uncertainties. Mean difference : 0.39 [ -0.129 to 0.908 ] Difference plot N = 7 Common calibrator package insert values compared to NMI target values 0 10 20 30 40 Mean -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Difference Unknown serum samples (one male and one female): raw results (blue) compared to results obtained using the common calibrator (green) for each laboratory 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 5 6 7 8 Testosterone umol/L Laboratory Unknown Female Serum Run 1 Run 1 Run 2 Run 2 Corrected 1 Corrected 1 Corrected 2 Corrected 2 Linear (NMI) Linear (Desirable BIAS) Linear (Desirable BIAS) Linear (QAP ALP) Linear (QAP ALP) 12 13 14 15 16 17 18 19 20 0 1 2 3 4 5 6 7 8 Testosterone nmol/L Laboratory Unknown Male Serum Run 1 Run 1 Run 2 Run 2 Corrected 1 Corrected 1 Corrected 2 Corrected 2 Linear (NMI) Linear (Desirable Bias) Linear (Desirable Bias) Linear (QAP ALP) Linear (QAP ALP)