Measurements of serum cortisol and urine normetanephrine by LC–MS/MS
SEPARATION, POSITIVE
Serum cortisol and urine normetanephrine were measured using Liquid chromatography tandem mass spectrometry (LC–MS/MS) methods by a Waters Acquity I-class UPLC system coupled to the Xevo TQ-XS tandem mass spectrometer (Waters Corp., Milford, MA, USA) operated in positive electrospray mode. Serum cortisol was extracted using the Extrahera™ automation system (Biotage, Uppsala, Sweden) under positive pressure supplied by a nitrogen generator. In a 96-position 2 mL deep well plate, 200 μL of calibration standards (Chromsystems, München, Germany NIST SRM971 traceable), quality control materials, and serum samples were added to each well with 50 μL internal standard solution containing cortisol-d4 (IsoSciences, King of Prussia, PA, USA). 200 μL of isopropanol:water 50:50 (v/v) was then added to dissociate the binding proteins. After mixing, the samples were loaded onto ISOLUTE® supported liquid extraction (SLE +) 400 μL plate (Biotage). Elution was carried out by adding two cycles of 750 µL of methyl tertiary butyl ether (MTBE). The eluents were collected into a corresponding deep well plate. Positive pressure was applied at each stage to remove residual solvent. Samples were then dried to completeness under a gentle stream of nitrogen gas heated to 60 °C, then reconstituted with 100 μL of 50:50 methanol/water before being vortexed and sealed. 10 μL of the extract was injected into the LC–MS/MS. Chromatographic separation was achieved using a CORTECS™ core–shell C18 50 × 2.1 mm, 2.7 µm, reversed-phase (Waters Corp., Milford, MA, USA) column heated at 30˚C. Mobile phases used were (A) water and (B) methanol in 0.1% formic acid, pumped at the flow rate of 0.4 mL/min in 70:30% (A:B), gradually increased to 100% (B) then returned to the starting gradient at 4 min. Tandem mass spectrometry detection was based on the mass-to-charge (m/z) precursor to product ion transitions specific to each compound: cortisol (363.3 > 121.2) and cortisol-d4 (367.3 > 121.2) as an internal standard. The inter-assay CV for serum cortisol was < 6.0% across the assay range of 0.3–806 nmol/L.Urine normetanephrine was extracted using solid phase extraction method (Chromsystems Biogenic amino #80,600, München, Germany) as per the manufacturer’s instruction. The assay was calibrated using human urine-based calibration standards and controls (Chromsystems) that are traceable to certified reference materials. m/z transitions were: normetanephrine (166.1 > 106.1) and normetanephrine-d3 (169.1 > 109.1) as internal standard. The inter-assay CV for normetanephrine was 3.4–6.2% across the assay range of 20.5–10,311 nmol/L. Urine results obtained from LC–MS/MS analysis were adjusted for variations in renal function by dividing by urine creatinine. Urine creatinine was analysed using Roche 2nd generation kinetic colorimetric assay based on the Jaffé method performed on the COBAS® C501 analyser (Roche, Burgess Hill, UK). The inter-assay CV was < 2.1%. The final results are expressed as nmol/L per mmol/L creatinine.