The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in Guideline on Validation of Bioanalytical Methods was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method's application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis.

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Conjugated norandrosterone is the main urinary metabolite of anabolic steroids like nandrolone, norandrostenedione and norandrostenediol. Nandrolone traces of endogenous origin have been identified in human follicular fluid, and further investigations revealed urinary excretion of norandrosterone in pregnant and non-pregnant females and even males. A threshold level for the norandrosterone concentration in urine has been established when controlling the administration of prohibited nandrolone or its precursors in human doping control. This level has been set to 2 ng/mL for males and females. To investigate the excretion of conjugated norandrosterone in females more systematically, we collected daily urine samples from 12 female volunteers during a whole menstrual cycle. These samples were analysed for norandrosterone down to a limit of quantification and identification of 0.05 ng/mL (180 pmol/L). The results clearly show that all the volunteers excreted norandrosterone glucuronide in a characteristic pattern during one menstrual cycle. Concentrations in urine were considerably lower at the beginning of the follicular and the end of the luteal phases than midcyclic. Peak concentrations up to 0.8 ng/mL (2.9 nmol/L) were recorded and they were three to four times higher than the values at the beginning and end of the cycle. The time of the peak concentration was clearly related to the increased excretion of luteinizing hormone. These results strongly support the possibility of endogenous nandrolone production as a side reaction to enzymatic aromatisation. However, a threshold value of 2 ng/mL for reporting adversed findings in doping control of females was never reached in any of the samples.

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Whereas numerous immunoassays have been developed to ensure detection of the entire spectrum of isoforms displayed by the human chorionic gonadotropin (hCG) molecule, significant variation has been demonstrated in how these isoforms are recognized by the antibodies in different immunoassays. The aim of this study was to establish a method using the dual selectivity of the immunoextraction and the mass spectrometry detection for the differentiation between various hCG isoforms in clinically relevant samples. Immunoextraction of endogenous hCG isoforms using a monoclonal antibody (E27) on a 96-well microtitier plate, followed by in-well tryptic digestion, and SIM monitoring of the selected signature peptides, resulted in the qualitative differentiation between several hCG isoforms in serum or urine. We conclude that the orthogonal selectivity conferred by the combination of immunoaffinity extraction and LC-MS analysis offers valuable complementary information to the conventional immunoassays.