| Immunoassays |
High sensitivities (as low as in the pg·mL−1 range) Use of small plasma or serum volumes Faster and cheaper compared to LC-MS/MS when measuring a single hormone No need to develop methods, as commercial assays for most hormones are already available
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Require specific high-affinity antibodies, measuring a single hormone per assay The amplification technologies that give immunoassays high sensitivities are often less reliable than direct quantification methods Antibody cross-reactivity Limited dynamic range Matrix interferences Lack of internal standards to calculate recovery limits Reliance on external calibration
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| LC-MS/MS |
Provides a robust platform with sufficient sensitivity and specificity for steroid hormone determination Able to determine cortisol in samples collected through non-invasive methods No cross-reactivity Can be used in the analytical validation of ELISAs and RIAs Allows for the simultaneous determination of several hormones Use of small sample volumes Fast analysis time
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Detection of several hormones in a low volume of serum or plasma can be challenging Similarity of chemical structures and fragmentation patterns demands sufficient chromatographic separation, especially for isomers High instrument costs Greater technical complexity, speed, and turnaround of analysis Greater complexity in sample preparation is required to avoid blockages and sample matrix effects
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| GC-MS |
Fast and precise Excellent chromatographic resolution Multi-class profiling potential Intensive fragmentation patterns leading to better structure elucidation
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More labor-intensive, requiring derivatization Reproducibility issues due to incomplete compound derivatization
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