Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability
-
James M. Hawley
,
Joanne E. Adaway
Abstract
Background
Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability.
Methods
An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis.
Results
The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively.
Conclusions
We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.
Acknowledgments
The authors would like to thank Lee Williams (Biotage) for the provision of SLE plates and all volunteers who provided blood for the stability study.
Author contributions: BGK conceived the study. JMH, JEA, LJO and BGK developed the method. JMH validated the method and prepared the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Guarantor: BGK
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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©2020 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
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- Editorial
- Advancements in mass spectrometry as a tool for clinical analysis: Part I
- Drug adherence, testing and therapeutic monitoring
- Hyphenated mass spectrometry techniques for assessing medication adherence: advantages, challenges, clinical applications and future perspectives
- Method development for quantitative determination of seven statins including four active metabolites by means of high-resolution tandem mass spectrometry applicable for adherence testing and therapeutic drug monitoring
- Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath
- THC and CBD concentrations in blood, oral fluid and urine following a single and repeated administration of “light cannabis”
- Identification of metabolites of peptide-derived drugs using an isotope-labeled reporter ion screening strategy
- Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma
- Enhanced specificity due to method specific limits for relative ion intensities in a high-performance liquid chromatography – tandem mass spectrometry method for iohexol in human serum
- Small molecule biomarkers
- Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease
- Trimethylamine-N-oxide (TMAO) determined by LC-MS/MS: distribution and correlates in the population-based PopGen cohort
- Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability
- Short-term stability of free metanephrines in plasma and whole blood
- Validation of a rapid, comprehensive and clinically relevant amino acid profile by underivatised liquid chromatography tandem mass spectrometry
- UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects
- Independent association of plasma xanthine oxidoreductase activity with serum uric acid level based on stable isotope-labeled xanthine and liquid chromatography/triple quadrupole mass spectrometry: MedCity21 health examination registry
- Serum bile acids profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application on pediatric liver and intestinal diseases
- LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population
- Dried blood spots and alternative sample mediums
- Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and β-thalassemias in dried blood spots
- Candidate reference method for determination of vitamin D from dried blood spot samples
- Therapeutic drug monitoring of anti-epileptic drugs – a clinical verification of volumetric absorptive micro sampling
- Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry
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Articles in the same Issue
- Frontmatter
- Editorial
- Advancements in mass spectrometry as a tool for clinical analysis: Part I
- Drug adherence, testing and therapeutic monitoring
- Hyphenated mass spectrometry techniques for assessing medication adherence: advantages, challenges, clinical applications and future perspectives
- Method development for quantitative determination of seven statins including four active metabolites by means of high-resolution tandem mass spectrometry applicable for adherence testing and therapeutic drug monitoring
- Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath
- THC and CBD concentrations in blood, oral fluid and urine following a single and repeated administration of “light cannabis”
- Identification of metabolites of peptide-derived drugs using an isotope-labeled reporter ion screening strategy
- Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma
- Enhanced specificity due to method specific limits for relative ion intensities in a high-performance liquid chromatography – tandem mass spectrometry method for iohexol in human serum
- Small molecule biomarkers
- Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease
- Trimethylamine-N-oxide (TMAO) determined by LC-MS/MS: distribution and correlates in the population-based PopGen cohort
- Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability
- Short-term stability of free metanephrines in plasma and whole blood
- Validation of a rapid, comprehensive and clinically relevant amino acid profile by underivatised liquid chromatography tandem mass spectrometry
- UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects
- Independent association of plasma xanthine oxidoreductase activity with serum uric acid level based on stable isotope-labeled xanthine and liquid chromatography/triple quadrupole mass spectrometry: MedCity21 health examination registry
- Serum bile acids profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application on pediatric liver and intestinal diseases
- LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population
- Dried blood spots and alternative sample mediums
- Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and β-thalassemias in dried blood spots
- Candidate reference method for determination of vitamin D from dried blood spot samples
- Therapeutic drug monitoring of anti-epileptic drugs – a clinical verification of volumetric absorptive micro sampling
- Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry
- Obtaining information from the brain in a non-invasive way: determination of iron in nasal exudate to differentiate hemorrhagic and ischemic strokes
