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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 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.

Background: Mass spectrometry (MS)-based 17-hydroxyprogesterone (17OHP) methods show considerable variation in results in external quality assurance (EQA) programs. An understanding of the current status of MS-based serum/plasma 17OHP quantification is important to facilitate harmonization. Methods: A 50-item e-survey related to (1) laboratory characteristics, (2) pre-analytical considerations and (3) analysis of 17OHP was developed and circulated to clinical MS laboratories via professional associations in Asia Pacific, Europe and North America. Results: Forty-four laboratories from 17 countries completed the survey. Sample preparation varied between laboratories with protein precipitation and liquid-liquid extraction being the most common processes. Analyte separation was most commonly achieved by liquid chromatography (LC) using a C18 column and mobile phases of water, methanol and formic acid. The ions selected for quantification were 331>97 m/z or 331>109 m/z. Alternative transition ions were used as qualifiers. Twenty-seven of 44 respondents reported preparing their calibrators in-house and variations in material purity and matrix were evident. Nine of 44 laboratories did not participate in an EQA program, and half did not know if their method separated out isobars. The reference intervals, and also their partitioning, reported by the laboratories were highly discrepant, in some cases, by multiple folds. Conclusions: Although MS-based methods are similar in many facets, they are highly disparate. Five recommendations have been developed as an outcome of this survey to support the continued improvement of analysis of serum/plasma 17OHP by MS.

Background:Serum dihydrotestosterone (DHT) is an important analyte for the clinical assessment of disorders of sex development. It is also reportedly a difficult analyte to measure. Currently, there are significant gaps in the standardisation of this analyte, including no external quality assurance (EQA) program available worldwide to allow for peer review performance of DHT. We therefore proposed to establish a pilot EQA program for serum DHT.Methods:DHT was assessed in the 2015 Royal College of Pathologists of Australasia Quality Assurance Programs’ Endocrine program material. The material’s target (i.e. “true”) values were established using a measurement procedure based on isotope dilution gas chromatography (GC) tandem mass spectrometry (MS/MS). DHT calibrator values were based on weighed values of pure DHT material (>97.5% purity) from Sigma. The allowable limits of performance (ALP) were established as ±0.1 up to 0.5 nmol/L and ±15% for targets >0.5 nmol/L.Results:Target values for the six levels of RCPAQAP material for DHT ranged from 0.02 to 0.43 nmol/L (0.01–0.12 ng/mL). The material demonstrated linearity across the six levels. There were seven participating laboratories for this pilot study. Results of the liquid chromatography (LC) MS/MS methods were within the ALP; whereas the results from the immunoassay methods were consistently higher than the target values and outside the ALP.Conclusions:This report provides the first peer comparison of serum DHT measured by mass spectrometry (MS) and immunoassay laboratories. Establishment of this program provides one of the pillars to achieve method harmonisation. This supports accurate clinical decisions where DHT measurement is required.