MSACL 2023 Abstract

Background:
Recent epidemiological studies have reported that vitamin D deficiency poses a risk of developing not only bone diseases but also various diseases such as cancer and autoimmune diseases. Serum concentration of 25(OH)D is used as a marker to reflect vitamin D sufficiency. Vitamin D metabolites are excreted in the urine in the form of glucuronidation. The relationship between urinary and blood vitamin D metabolites is not yet well understood.　Quantitative analysis of urinary vitamin D metabolites is important for understanding vitamin D metabolism. Vitamin D metabolites in urine are very low concentration, requiring sensitive measurements. In this study, we aimed to develop LC-MS/MS based method to accurately measure vitamin D metabolites in human urine.

Methods:
The JeoQuantTM Kit for LC-MS/MS analysis of vitamin D metabolites (JEOL, Japan) was slightly modified and used for the quantitative measurement of urinary vitamin D metabolites. Deglucronidation was done using β-Glucuronidase. Urine samples were collected from healthy volunteer subjects (22 subjects, collected on 4 separate days, total n= 88) and stored at −80 °C until their use. A urine sample (300 µL) was added with 50µL β-glucuronidase (from E. coli, Type IX-A, 5000 units) in water, then incubated at 37 °C for 1 h. After incubation, 350 μL of sample was mixed with 50 μL of IS solution, followed by solid liquid extraction (ISOLUTE SLE+: Biotage, Sweden) and derivatized as manufacturers instruction. The pretreated samples were analyzed using an AQUITY UPLC I-Class system coupled to a Xevo TQ-XS mass spectrometer (Waters, USA).

Results:
Glucuronic acid conjugated vitamin D metabolites in 24-hour urine collections were treated with β-glucuronidase and released under optimized reaction conditions. The lower limits of quantification (LOQ) is as follows; 25(OH)D3: 3.4pg/mL, 3-epi-25(OH)D3: 5.9pg/mL, 25(OH)D2: 1.4pg/mL, 24,25(OH)2D3: 5.9pg/mL. In 24-hour urine collections, 25 (OH) D3 and 24,25(OH)2D3 could be quantified, but 3-epi-25(OH) D3 and 25(OH)D2 were less than LOQ. The intra-assay CVs were 10.0% and 8.4% for the low level samples and 3.6% and 4.9% for the high level samples for 25(OH)D3 and 24,25(OH)2D3, respectively. Plasma 25(OH)D3 concentrations correlated very well with urinary 24,25(OH)2D3 concentrations (r= 0.63, n= 88).

Conclusions:
We developed the LC-MS/MS based method using JeoQuantTM for the simultaneous quantification of vitamin D metabolites in urine by the treatment with β-Glucuronidase. This method can be expected to be useful for understanding the urinary excretion of vitamin D metabolism and for assessing the clinical conditions caused by vitamin D deficiency.