Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization.

In current clinical practice, a thorough understanding of vitamin D metabolism is in high demand both for patients with various diseases and for healthy individuals. Analytical techniques that provide simultaneous measurement of multiple metabolites are preferred. Herein, the development of an HPLC-DMS-MS/MS method for the quantitation of vitamin D compounds (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3, 24,25(OH)2D3, and D3) in serum is described. The selected sample preparation procedure based on the combination of liquid-liquid and solid-phase extraction, which excluded a lengthy derivatization step, was compared with other common approaches. Sensitivity was increased through the implementation of differential ion mobility separation. The proposed assay allowed us to determine the low abundant 1,25(OH)2D3 with the detection limit of 10 pg/mL. The validation study showed good linearity (r2 > 0.99), a wide analytical range (2.5-75 ng/mL for 25(OH)D3), and acceptable precision (<7%) for all metabolites. The recovery ranged from 71% to 93% and the matrix effect from 0.80 to 0.95 depending on the metabolite; accuracy determination was performed using DEQAS controls.

Dynamic Evaluation of Vitamin D Metabolism in Post-Bariatric Patients.

BACKGROUND: findings from the previously conducted studies indicate altered regulatory mechanisms of calcium and vitamin D metabolism in obese patients and a role for bariatric surgery in regulating vitamin D metabolism; however, the available data is controversial and does not provide an adequate understanding of the subject. METHODS: we evaluated serum parameters of vitamin D and mineral metabolism (vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3, and 24,25(OH)2D3), vitamin D-binding protein (DBP), free 25(OH)D, fibroblast growth factor 23 (FGF-23), parathyroid hormone (PTH), total calcium, albumin, phosphorus, creatinine, magnesium) in 30 patients referred for bariatric surgery in comparison with 30 healthy volunteers of similar age, sex and baseline 25(OH)D3. Patients were also followed up with repeated laboratory assessments 3 months and 6 months after surgery. During the first 3 months, patients were prescribed high-dose cholecalciferol therapy (50,000 IU per week), with subsequent correction based on the results of the 3-month visit examination. RESULTS: Preoperatively, patients with morbid obesity were characterized by a high prevalence of vitamin D deficiency (median 25(OH)D3 level 11.9 (6.8; 22.2) ng/mL), significantly lower levels of active vitamin D metabolite 1,25(OH)2D3 (20 (10; 37) vs. 39 (33; 50) pg/mL, p < 0.001), lower serum albumin-adjusted calcium levels (2.24 (2.20; 2.32) vs. 2.31 (2.25; 2.35) mmol/L, p = 0.009) and magnesium levels (0.79 (0.72; 0.82) vs. 0.82 (0.78; 0.85) mmol/L, p = 0.043) with simultaneous similar PTH levels (p = 0.912), and higher DBP levels (328 (288; 401) vs. 248 (217; 284) mg/L, p < 0.001). The 25(OH)D3 levels remained suboptimal (24.5 (14.7; 29.5) ng/mL at the 3-month visit and 17.9 (12.4; 21.0) ng/mL at the 6-month visit, p = 0.052) despite recommended high-dose cholecalciferol supplementation. Patients also demonstrated an increase in 1,25(OH)2D3 levels (38 (31; 52) pg/mL at the 3-month visit and 49 (29; 59) pg/mL at the 6-month visit, p < 0.001) without a change in PTH or calcium levels during the follow-up. CONCLUSION: our results of a comprehensive laboratory evaluation of vitamin D status and mineral metabolism in patients undergoing bariatric surgery highlight the importance of improving current clinical guidelines, as well as careful monitoring and education of patients.