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

Sameh A Ahmed; Hani M J Khojah; Sultan S Al-Thagfan; Yaser M Alahmadi; Yasser A Mohammed

doi:10.1016/j.talanta.2022.123497

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

Talanta. 2022 Aug 15:246:123497. doi: 10.1016/j.talanta.2022.123497. Epub 2022 Apr 22.

Authors

Sameh A Ahmed¹, Hani M J Khojah², Sultan S Al-Thagfan², Yaser M Alahmadi², Yasser A Mohammed³

Affiliations

¹ Pharmacognosy and Pharmaceutical Chemistry Department, College of Pharmacy Taibah University, AlMadinah AlMunawarah, 30001, Saudi Arabia. Electronic address: shassan@taibahu.edu.sa.
² Clinical and Hospital Pharmacy Department, College of Pharmacy, Taibah University, AlMadinah AlMunawarah, 30001, Saudi Arabia.
³ Ohud Hospital, Al Madinah AlMunawarah, Saudi Arabia.

Abstract

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 °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 × 2.1 mm, 1.8 μm) 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^-1 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.

Keywords: 1,25-dihydroxyvitamin D3; 2-nitrosopyridine; 25-hydroxyvitamin D3; CMDR; COVID-19; UHPLC-ESI-MS/MS; Vitamin D3.

MeSH terms

COVID-19* / diagnosis
Cholecalciferol
Chromatography, High Pressure Liquid / methods
Humans
Microwaves
Reproducibility of Results
Tandem Mass Spectrometry* / methods

Substances

Cholecalciferol