Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair

•Quantitative steroid analysis via online SPE LC–MS/MS within 4 min total run time.•Uniform procedure increases convenience in sample preparation regardless the matrix.•In complex matrices quantitation by MS3 proves to be superior to MS2.•Hair cortisol analysis suffers from massive pre-analytical issues, often unconsidered.

Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC–MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS3 (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100 mL of human plasma/serum, saliva, urine and 10–20 mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC–MS/MS or LC–MS3. Steroids can be analyzed in 4 min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS3 detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37 pmol/L (estradiol) up to 3.1 nmol/L (DHEAS). General accuracy was 89–107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (−71% mean), aldosterone (−40% mean) and plasma aldosterone (−45% mean). The comparison of MS2 and MS3 quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS3 was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS3 can be superior to MS2 regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC–MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair.