Influence of isotopically labeled internal standards on quantification of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography mass spectrometry

Tze Ping Loh 1 , Chung Shun Ho 2 , Michaela F. Hartmann 5 , Rosita Zakaria 3 , 4 , Clara Wai Shan Lo 2 , Sjoerd van den Berg 12 , Yolanda B. de Rijke PhD 6 , Brian R. Cooke 7 , Kirsten Hoad 7 , Peter Graham 8 , Stephen R. Davies 9 , Lindsey G. Mackay 9 , Stefan A. Wudy 5 , and Ronda F. Greaves 4 , 10 , 11
  • 1 Department of Laboratory Medicine, National University Hospital, Singapore, Singapore
  • 2 Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
  • 3 Murdoch Children’s Research Institute, Parkville, Australia
  • 4 School of Health and Biomedical Sciences, RMIT University, Melbourne, Australia
  • 5 Division of Pediatric Endocrinology & Neonatology, Steroid Research & Mass Spectrometry Unit, Justus Liebig University, Giessen, Germany
  • 6 Clinical Chemistry Erasmus MC, University Medical Center, Rotterdam, the Netherlands
  • 7 PathWest, Fiona Stanley Hospital, Perth, Australia
  • 8 Royal College of Pathologists of Australasia Quality Assurance Programs, Sydney, Australia
  • 9 National Measurement Institute Australia, Sydney, Australia
  • 10 Department of Paediatrics, University of Melbourne, Melbourne, Australia
  • 11 Biochemical Genetics, Parkville, Australia
  • 12 Dept. Clinical Chemistry and Dept. Internal Medicine, Erasmus MC, Rotterdam, the Netherlands
Tze Ping Loh
  • Department of Laboratory Medicine, National University Hospital, Singapore, Singapore
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, Chung Shun Ho
  • Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
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, Michaela F. Hartmann
  • Division of Pediatric Endocrinology & Neonatology, Steroid Research & Mass Spectrometry Unit, Justus Liebig University, Giessen, Germany
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, Rosita Zakaria
  • Murdoch Children’s Research Institute, Parkville, VIC, Australia
  • School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia
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, Clara Wai Shan Lo
  • Biomedical Mass Spectrometry Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
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, Sjoerd van den Berg
  • Dept. Clinical Chemistry and Dept. Internal Medicine, Erasmus MC, Rotterdam, the Netherlands
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, Yolanda B. de Rijke
  • Clinical Chemistry Erasmus MC, University Medical Center, Rotterdam, the Netherlands
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, Brian R. Cooke, Kirsten Hoad, Peter Graham
  • Royal College of Pathologists of Australasia Quality Assurance Programs, Sydney, Australia
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, Stephen R. Davies, Lindsey G. Mackay, Stefan A. Wudy
  • Division of Pediatric Endocrinology & Neonatology, Steroid Research & Mass Spectrometry Unit, Justus Liebig University, Giessen, Germany
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and Ronda F. GreavesORCID iD: https://orcid.org/0000-0001-7823-8797
  • Corresponding author
  • School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia
  • Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia
  • Biochemical Genetics, Parkville, VIC, Australia
  • orcid.org/0000-0001-7823-8797
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Abstract

Objectives

Our recent survey of 44 mass spectrometry laboratories across 17 countries identified variation in internal standard (IS) choice for the measurement of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The choice of IS may contribute to inter-method variations. This study evaluated the effect of two common isotopically labeled IS on the quantification of 17OHP by LC-MS/MS.

Methods

Three collaborating LC-MS/MS laboratories from Asia, Europe and Australia, who routinely measure serum 17OHP, compared two IS, (1) IsoSciences carbon-13 labeled 17OHP-[2,3,4-13C3], and (2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-2H]. This was performed as part of their routine patient runs using their respective laboratory standard operating procedure.

Results

The three laboratories measured 99, 89, 95 independent samples, respectively (up to 100 nmol/L) using the 13C- and 2H-labeled IS. The slopes of the Passing-Bablok regression ranged 0.98–1.00 (all 95% confidence interval [CI] estimates included the line of identity), and intercept of <0.1 nmol/L. Average percentage differences of −0.04% to −5.4% were observed between the two IS materials, which were less than the optimal bias specification of 7% determined by biological variation, indicating no clinically significant difference. The results of 12 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) proficiency samples (1–40 nmol/L) measured by the laboratories were all within the RCPAQAP analytical performance specifications for both IS.

Conclusions

Overall, the comparison between the results of 13C- and 2H-labeled IS for 17OHP showed good agreement, and show no clinically significant bias when incorporated into the LC-MS/MS methods employed in the collaborating laboratories.

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