Development and validation of an UHPLC–MS/MS method for extended serum steroid profiling in female populations

Bioanalysis ◽  
2020 ◽  
Author(s):  
Olivier Salamin ◽  
Federico Ponzetto ◽  
Michel Cauderay ◽  
Julien Boccard ◽  
Serge Rudaz ◽  
...  
Keyword(s):  
Serum Samples ◽  
Complementary Approach ◽  
Steroid Profiling ◽  
The World ◽  
Combined Uncertainty ◽  
Different Populations ◽  
Development And Validation ◽  
Endogenous Steroid ◽  
Conjugated Steroids

Aim: Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC–MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. Results: The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20 %). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. Conclusion: The UHPLC–MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.

Steroidomics for highlighting novel serum biomarkers of testosterone doping

Bioanalysis ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1169-1185 ◽  
Author(s):  
Federico Ponzetto ◽  
Julien Boccard ◽  
Raul Nicoli ◽  
Tiia Kuuranne ◽  
Martial Saugy ◽  
...  
Keyword(s):  
High Performance ◽  
Biomarker Discovery ◽  
High Resolution Mass Spectrometry ◽  
Serum Markers ◽  
Serum Biomarkers ◽  
Serum Samples ◽  
Steroid Profiling ◽  
Efficient Alternative ◽  
Detection Capabilities ◽  
Endogenous Steroid

Aim: Quantification of testosterone (T) and 5α-dihydrotestosterone serum concentrations proved to be an efficient alternative to urinary steroid profiling for the detection of T doping. In this context, additional serum markers could be discovered by exploratory untargeted steroidomics studies. Results: Endogenous steroid metabolites were monitored by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry in serum samples collected during a T administration clinical trial. A three-step workflow for accurate review of annotation was used and multifactorial data analysis allowed highlighting promising serum biomarkers. Longitudinal monitoring of selected compounds was performed to assess T abuse detection capabilities. Conclusion: Application of serum steroidomics showed high potential for biomarker discovery of T doping, suggesting longitudinal monitoring of steroid hormones in serum as a significant improvement in detection of endogenous steroids abuse.

Development and Validation of a Biolayer Interferometry Method for Determination of Human Anti-PD-1 Monoclonal Antibody Concentration in Cynomolgus Serum

2020 ◽  
Vol 20 (4) ◽  
pp. 257-267
Author(s):  
K. V. Ulyanova ◽  
A. A. Kazarov ◽  
M. S. Pantyushenko ◽  
A. A. Olenev ◽  
I. V. Lyagoskin ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Test System ◽  
Test Method ◽  
Antibody Concentration ◽  
Malignant Tumours ◽  
Serum Samples ◽  
Biolayer Interferometry ◽  
Validation Parameters ◽  
Development And Validation

Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer. Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients. The development of monoclonal antibodies (mAbs) specific for PD-L1 or PD-1 is a promising area of immunotherapy of malignant tumours. However, before a therapeutic antibody-based product enters the market, it is necessary to ensure its safety and efficacy, i.e. perform a full scope of preclinical and clinical studies.The aim of the study was to develop and validate a bioanalytical method that does not require additional labeling and that could be used for determination of mAbs specific for human PD-L1 in the blood serum of a biological test system during preclinical studies.Materials and methods: an antigen in the form of a dimer of PD-1 extra-cellular domain covalently bonded to the Fc-fragment of human IgG (R&D Systems, USA) was used in the study. The antigen was immobilised on Dip and Read™ Protein A biosensors (Fortebio, USA). The therapeutic anti-PD-1 antibody GNR-051 was developed and produced by IBC “Generium” (Russia). The healthy cynomolgus monkey serum samples used as matrix were obtained from the Research Institute of Medical Primatology (Sochi, Russia). The assessment of binding was performed using Octet® QKe interferometer (Fortebio, USA) by real-time analysis of the dose-dependent rate of the antigen-antibody complex formation.Results: the paper presents experimental data on the development and validation of the test method for determination of the therapeutic PD-1-binding mAb concentration in cynomolgus monkey serum in the antibody concentration range from 2 to 2500 µg/mL. The authors assessed the calibration curve reliability, between-run and within-run precision and accuracy, dilution linearity, specificity and selectivity of the test method.Conclusions: the authors developed and validated the biolayer interferometry-based method for determination of therapeutic mAbs concentration. The method was shown to comply with the Eurasian Economic Union’s regulatory requirements in terms of the main validation parameters: analytical range, accuracy, precision, and selectivity. 

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