An ultrasensitive UHPLC-ESI-MS/MS method augmented with a controlled microwave derivatization reaction for quantitation of vitamin D3 and its major metabolites in COVID-19 patients

It is established that vitamin D deficiency is correlated with the disease severity in COVID-19 patients. However, the reliable and sensitive quantitation of vitamin D3 (D3) and its metabolites remains a difficult challenge. Herein, a novel ultrasensitive and reliable UHPLC-ESI-MS/MS method was developed and validated for the quantitation of D3 and its major metabolites in COVID-19 patients. The mass spectral sensitivity was augmented via controlled microwave-assisted derivatization reaction (CMDR) with 2-nitrosopyridine (Pyr-NO) at 65 degrees C for 2 min. CMDR hyphenation with UHPLC-MS/MS improves detection sensitivity while shortening separation and derivatization reaction times. The precursor to product ion transitions for D3, 25-hydroxy D3 (25(OH)D3), 1,25-dihydroxy D3 (1,25-(OH)2D3) and calcipotriol (CPT) as an internal standard were m/z 493.4 -> 231.3, m/z 509.4 -> 231.3, m/z 525.4 -> 247.3, and m/z 521.4 -> 247.3; respectively. The separation of the formed derivatives was conducted using a gradient elution mode with mobile phase A: formic acid (0.1%) in water and mobile phase B: formic acid (0.1%) in acetonitrile. The elution started with 40% (v/v) of B for 0.3 min then increased linearly to 90% (v/v) at 2 min on an Agilent EclipsePlus C18 (50 x 2.1 mm, 1.8 mu m) column at a flow rate of 0.3 mL min(-1). The method was validated using FDA standards for bioanalytical method validation over a concentration range of 0.02-50 ng mL(-1) with correlation coefficient >= 0.9987 and the lower limit of quantitation (LLOQ) were 0.02-0.05 ng mL(-) in human plasma. The developed method has demonstrated excellent comparability to a well-established chemiluminescent immunoassay (CLIA) method for the analysis of D3 metabolites in human samples. The developed UHPLC-ESI-MS/MS method was implemented for routine and reliable quantitation of D3 and its major metabolites in COVID-19 patients.