Simultaneous determination of VD2, VD3, 25(OH) D2, and 25(OH) D3 in human plasma using electrospray LC–MS/MS as well as its application to evaluate VD plasma levels in depressive, schizophrenic patients and healthy individuals

Abstract

Vitamin D measurements in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been widely used but remain challenging at very low concentration levels. Rapid, high recovery, sensitive and reliable measurements of vitamin D, as well as its primary metabolites using LC-MS/MS are urgently needed for a routine clinical laboratory. Herein, we reported a novel electrospray LC-MS/MS method for determining vitamin D and its primary metabolites using the supported liquid extraction method to achieve higher recoveries, with optimized pH values to achieve optimal derivatization efficiency for higher sensitivity and selected chromatographic conditions to shorten the separation time. The method has been validated with respect to selectivity, recovery, matrix effects, accuracy and precision, stabilities, carryover and dilution effects. The method has been successfully applied to quantify the VD plasma concentrations of depressive, schizophrenic patients and healthy individuals. The result showed that there were significant differences in plasma VD levels between mental disorder patients with healthy individuals, and the total VD levels in mental disorder patients were much higher than healthy individuals, which might require larger clinical samples for validation. K E Y W O R D S LC-MS/MS, PTAD derivatization optimizing, supported liquid extraction, vitamin D 1 | INTRODUCTION Vitamin D not only plays an essential role in the control of calcium homeostasis and bone mineralization in the human body, but is also related to the occurrence of benign and malignant tumors, infectious diseases, autoimmune diseases, cardiovascular diseases and mental health (Howe & Dellavalle, 2007). The typical forms of vitamin D in the human body are vitamin D2 (VD2, ergocalciferol) and vitamin D3 (VD3, cholecalciferol), which derive from plants and animals (Zmijewski, 2019). The metabolites of these two types of vitamin D are both called 25(OH) D, which is a stable storage form and serum determination marker of vitamin D in vivo. Interestingly, VD3 converts Haiyan Lyu and Suping Wang contributed equally to this work.

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis

“…The stability of vitamin D 3 metabolites in biological samples has been thoroughly investigated, mostly stabilities in sample matrices prior to sample preparation, in particular freeze-thaw stability [11][12][13][14][15], bench-top stability [15], long-term stability [15,16] and stability regarding differently preserved blood samples (lithium heparin plasma, EDTA plasma) [13] and exposure to light and temperature [14]. Some studies also describe the stability of vitamin D 3 metabolite derivatization products.…”

“…The authors noticed no significant differences in peak areas [ 19 ]. For 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatives, Lyu et al observed no significant analyte loss when the extract was left for 24 h at room temperature [ 15 ]. Similarly, He et al described stable extracts when left in the autosampler for 96 h at 4 °C [ 11 ].…”

Association of serum 25-hydroxyvitamin D (25(OH)D) levels with the gut microbiota and metabolites in postmenopausal women in China

“…10 Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China. 11 School of Basic Medical Science, Central South University, Changsha, China.…”

“…In the kidneys, the enzyme 1α-hydroxylase (CYP27B1) forms an active form of VD, namely, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) [10]. Eventually, 1,25(OH) 2 D 3 is metabolized by the renal CYP24A1 enzyme in the kidney [11]. Clinically, serum 25-hydroxyvitamin D (25(OH)D) levels ≥ 20 ng/ml are considered adequate, while serum 25(OH)D levels < 20 ng/mL are defined as VD deficiency [12].…”

Association of serum 25-hydroxyvitamin D (25(OH)D) levels with the gut microbiota and metabolites in postmenopausal women in China

“…In the kidneys, the enzyme 1α-hydroxylase (CYP27B1) forms an active form of VD, namely, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) [8]. Eventually, 1,25(OH) 2 D 3 is metabolized by the renal CYP24A1 enzyme in the kidney [9]. Clinically, serum 25-hydroxyvitamin D (25(OH)D) levels ≥ 20 ng/ml are considered adequate, while serum 25(OH)D levels < 20 ng/mL are de ned as VD de ciency [10].…”