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

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.

The role of the CCQM OAWG in providing SI traceable calibrators for organic chemical measurements.

Metrological traceability for organic chemical measurements is a documented unbroken chain of calibrations with stated uncertainties that ideally link the measurement result for a sample to a primary calibrator in appropriate SI units (e.g., mass fraction). A comprehensive chemical purity determination of the organic calibrator is required to ensure a true assessment of this result. We explore the evolution of chemical purity capabilities across metrology institute members of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology's Organic Analysis Working Group (OAWG). The OAWG work program has promoted the development of robust measurement capabilities, using indirect "mass balance" determinations via rigorous assessment of impurities and direct determination using quantitative nuclear magnetic resonance spectroscopy methods. A combination of mass balance and qNMR has been shown to provide a best practice approach. Awareness of the importance of the traceability of organic calibrators continues to grow across stakeholder groups, particularly in key areas such as clinical chemistry where activities related to the Joint Committee for Traceability in Laboratory Medicine have raised the profile of traceable calibrators.

Purity assessment of organic calibration standards using a combination of quantitative NMR and mass balance.

Quantitative NMR spectroscopy (qNMR) has been examined for purity assessment using a range of organic calibration standards of varying structural complexities, certified using the traditional mass balance approach. Demonstrated equivalence between the two independent purity values confirmed the accuracy of qNMR and highlighted the benefit of using both methods in tandem to minimise the potential for hidden bias, thereby conferring greater confidence in the overall purity assessment. A comprehensive approach to purity assessment is detailed, utilising, where appropriate, multiple peaks in the qNMR spectrum, chosen on the basis of scientific reason and statistical analysis. Two examples are presented in which differences between the purity assignment by qNMR and mass balance are addressed in different ways depending on the requirement of the end user, affording fit-for-purpose calibration standards in a cost-effective manner.

The development of an efficient mass balance approach for the purity assignment of organic calibration standards.

The purity determination of organic calibration standards using the traditional mass balance approach is described. Demonstrated examples highlight the potential for bias in each measurement and the need to implement an approach that provides a cross-check for each result, affording fit for purpose purity values in a timely and cost-effective manner. Chromatographic techniques such as gas chromatography with flame ionisation detection (GC-FID) and high-performance liquid chromatography with UV detection (HPLC-UV), combined with mass and NMR spectroscopy, provide a detailed impurity profile allowing an efficient conversion of chromatographic peak areas into relative mass fractions, generally avoiding the need to calibrate each impurity present. For samples analysed by GC-FID, a conservative measurement uncertainty budget is described, including a component to cover potential variations in the response of each unidentified impurity. An alternative approach is also detailed in which extensive purification eliminates the detector response factor issue, facilitating the certification of a super-pure calibration standard which can be used to quantify the main component in less-pure candidate materials. This latter approach is particularly useful when applying HPLC analysis with UV detection. Key to the success of this approach is the application of both qualitative and quantitative (1)H NMR spectroscopy.

Production of certified reference materials for the sports doping control of the REV-ERB agonist SR9009.

Two human metabolites of the REV-ERB agonist SR9009, identified by researchers with an interest in sports doping control, have been synthesized and assessed for purity. The synthesis employed was a modification of published procedures for the parent SR9009, careful attention to the purification of intermediates and the final product ensuring materials of the highest purity were available for certification. For each candidate material impurities of related structure were identified and quantified as a relative mass fraction using high performance liquid chromatography-ultraviolet (HPLC-UV) detection and proton nuclear magnetic resonance (1 H NMR) spectroscopy. The quantification of water, occluded solvent, and inorganic residue was assessed using Karl Fischer, 1 H NMR, and thermogravimetric analysis, thereby completing the assessment of all impurities typically characterized by the mass balance approach. Summation and subtraction from 1000 mg/g afforded the mass fraction of the main component, the associated uncertainty ensuring certified reference material status can be applied to the resulting pure substance calibration standards. The availability of these standards to the sports doping control community will facilitate delivery of metrological traceability to the SI unit for mass (kg) to routine testing results and aid method development for the detection and quantification of SR9009 abuse.