ESPE Abstracts (2019) 92 P1-158

Influence of Internal Standards Choice on Quantification of 17α-hydroxyprogesterone (17OHP) Using Mass Spectrometric Based Methods

Ronda Greaves1,2, Michaela F. Hartmann3, Rosita Zakaria2, Chug Shun Ho4, Yolanda B. de Rijke5, Sjoerd van den Berg5, Brian Cooke6, Kirsten Hoad6, Peter Graham7, Stephen Davies8, Lindsay Mackay8, Tze Ping Loh9, Stefan A. Wudy3


1Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Victoria, Australia. 2School of Health and Biomedical Sciences, RMIT University, Victoria, Australia. 3Steroid Research & Mass Spectrometry Unit, Laboratory for Translational Hormone Analytics in Pediatric Endocrinology, Justus Liebig University, Giessen, Germany. 4Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. 5Erasmus MC, University Medical Centre, Rotterdam, Netherlands. 6PathWest, Fiona Stanley Hospital, Perth, Australia. 7RCPA Quality Assurance Programs, Chemical Pathology, Sydney, Australia. 8National Measurement Institute of Australia, Sydney, Australia. 9Department of Laboratory Medicine, National University Hospital, Singapore, Singapore


Objective: This project aims to evaluate the effect of two isotopically labelled internal standards on the quantification of 17OHP by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) as an orthogonal technique.

Methods: Three LC-MS/MS and one GC-MS/MS laboratories, spanning four countries worldwide, who routinely measure serum17OHP, compared two internal standards as part of their patient runs. The only change to the respective laboratory standard operating procedure was the substitution of their internal standard (if different to in-house method) with: 1) IsoSciences carbon-13 labelled 17OHP-[2,3,4-13C3]; and 2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-D8]. Statistical interpretation of the data is based on the slope from the Passing Bablok regression, difference from the Bland Altman plots and the Student two tailed paired t-test, with confidence intervals (CI) of 95% and level of significance P<0.05 applied.

Results: The three LC-MS/MS and one GC-MS/MS laboratories successfully evaluated the two internal standards against altogether 232 patient samples. Analysis of the 13C- and D-labelled internal standard results from the individual laboratories, along with the combined all laboratory data, demonstrated agreement: the Passing-Bablok regression slope to include one in the CI; and Bland Altman difference to include zero in the CI. The all laboratory data t-test demonstrated a p>0.05

Conclusions: Overall, the comparison between the results of 13C- and D-labelled internal standards for 17OHP showed not influence by the internal standard used.