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A significant bias was found when using the Siemens ADVIA Centaur XP system for measurement of 25-hydroxyvitamin D (25OHD) with VACUETTE® tubes with Serum Clot Activator and Gel. Here, we examined whether other commonly used tubes or temperatures affected 25OHD results obtained with the Siemens ADVIA Centaur XP system. Serum was collected into five types of vacuum blood collection tubes from three manufacturers, and 25OHD was analyzed using the Siemens ADVIA Centaur XP system and liquid chromatography tandem mass spectrometry (LC-MS/MS) immediately or after storage at 4°C or -80°C for 48 h. Significantly higher 25OHD values were found when using the Siemens ADVIA Centaur XP system with VACUETTE® tubes with serum clot activator and gel and VACUETTE® tubes with clot activator but no gel compared with VACUETTE® tubes with no additives. The 25OHD values in all of these tubes were not significantly different from those obtained by LC-MS/MS. Moreover, after storage at -80°C for 48 h, the values obtained in IMPROVEVACUTER® tubes with serum clot activator and gel significantly increased, with a mean bias of 74.6% compared with the values before storage, on analysis with the Siemens ADVIA Centaur XP system. VACUETTE® tubes containing additives significantly affect the accuracy of 25OHD results obtained using the Siemens ADVIA Centaur XP system. Additionally, the composition of serum collected in IMPROVEVACUTER® tubes was affected by freezing, resulting in different measurements when using the Siemens 25OHD assay platform., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To develop a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method with ability to separate 3-epi 25OHD3 (EPI-LC-MS/MS) from 25OHD3, and evaluate the effects of 3-epi 25OHD3 on routine LC-MS/MS that cannot separate 3-epi 25OHD3 (NEPI-LC-MS/MS)., Design and Methods: Performance of the newly built EPI-LC-MS/MS was validated, and 982 samples were analyzed and compared by the two methods., Results: Both methods showed a linearity coefficient correlation exceeding 0.999 in the 6.25-500nmol/L range for 25OHD2 and 25OHD3. Moreover, they showed a between run coefficient variation (CV) and total CV of < 5% for 25OHD2 and 25OHD3. The results of the accuracy test showed that the bias was below 6.19% in the absence of 3-epi 25OHD3. Comparison of the 25OHD results obtained by the two methods for 982 patients (age 1-100years) revealed excellent clinical agreement (Cohen's kappa=0.875) and correlation (R2=0.973). Among the 982patients, only 73patients had 3-epi 25OHD3 (>6.25nmol/L); out of these 73patients, the 3-epi 25OHD3 level in 58patients was between 6.25 and 12.5nmol/L. In patients with <375nmol/L 25OHD (25OHD2+25OHD3), only 8 had 3-epi 25OHD3 levels exceeding 12.5nmol/L (range: 13.3 - 27.5nmol/L). Among samples containing 3-epi 25OHD3, only three were separated into different 25OHD-deficiency groups using the above methods., Conclusion: A rapid and precise EPI-LC-MS/MS method for measuring 25OHD with efficient separation of 3-epi 25OHD3 was developed. Our results showed that 3-epi 25OHD3 had little effect on the routinely used NEPI-LC-MS/MS., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Vitamin D deficiency, which is usually detected by using immunoassays or the more reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) methods, has recently been considered a public health problem worldwide. However, the vitamin D status in Chinese populations, as measured using the LC-MS/MS method, is not available. The objective of this multicenter study was to determine the vitamin D status and prevalence of vitamin D deficiency by using a reliable method in 5 large cities in China. From May 1 to September 31, 2013, we conducted a multicenter study on 2173 apparently healthy adults who were recruited from 5 Chinese cities. The 25-hydroxyvitamin D 25OHD2 and 25OHD3 levels were measured using the LC-MS/MS method. Intact parathyroid hormone (iPTH), calcium, phosphorus, and alkaline phosphate levels were also measured using an automatic analyzer. The mean 25OHD level of all participants was 19.4 ± 6.4 ng/mL (2.5-97.5%: 7.9-32.6 ng/mL), and only 109 (5.0%) participants had a 25OHD2 level >2.5 ng/mL (maximum, 22.4 ng/mL). In this study, the prevalence of severe vitamin D deficiency (<10 ng/mL), vitamin D deficiency (10-20 ng/mL), vitamin D insufficiency (20-30 ng/mL), and vitamin D sufficiency (>30 ng/mL) was 5.9%, 50.0%, 38.7%, and 5.4%, respectively. Women had a significant higher rate of deficiency than men (66.3% vs 45.3%, P < 0.01). Participants aged 18 to 39 years had a lower 25OHD level than elderly individuals (>59 years). Lifestyle may influence the 25OHD level more than the latitude, with participants in Dalian having the highest 25OHD level and the lowest deficiency rate. The serum iPTH level showed a significant negative correlation with the 25OHD level (r = -0.23, P < 0.01) after correcting for age and sex. In conclusion, the present study evaluated the vitamin D status using a reliable method, and our results indicate that vitamin D deficiency is prevalent among all age groups in China, especially among younger adults. We also observed significant differences in the 25OHD levels according to sex, age, and region among apparently healthy individuals.

Variations in vitamin D quantification methods are large, and influences of vitamin D analogues and blood collection methods have not been systematically examined. We evaluated the effects of vitamin D analogues 25OHD2 and 3-epi 25OHD3 and blood collection methods on vitamin D measurement, using five immunoassay systems and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum samples (332) were selected from routine vitamin D assay requests, including samples with or without 25OHD2 or 3-epi 25OHD3, and analysed using various immunoassay systems. In samples with no 25OHD2 or 3-epi 25OHD3, all immunoassays correlated well with LC-MS/MS. However, the Siemens system produced a large positive mean bias of 12.5 ng/mL and a poor Kappa value when using tubes with clot activator and gel separator. When 25OHD2 or 3-epi 25OHD3 was present, correlations and clinical agreement decreased for all immunoassays. Serum 25OHD in VACUETTE tubes with gel and clot activator, as measured by the Siemens system, produced significantly higher values than did samples collected in VACUETTE tubes with no additives. Bias decreased and clinical agreement improved significantly when using tubes with no additives. In conclusion, most automated immunoassays showed acceptable correlation and agreement with LC-MS/MS; however, 25OHD analogues and blood collection tubes dramatically affected accuracy. [ABSTRACT FROM AUTHOR]

Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH) 2 D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) is considered to be the “gold standard” for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC–MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D 3 (1α,25(OH) 2 D 3 ) and 1α,25-dihydroxy vitamin D 2 (1α,25(OH) 2 D 2 ). The analytes were extracted from the matrix by liquid–liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH) 2 D. The assay was sensitive with a low limit of quantitation of 10.0 pg/mL for both 1α,25(OH) 2 D 3 and 1α,25(OH) 2 D 2 using a 0.5 mL sample aliquot. Linearity was obtained over the range of 10.0 pg/mL to 500 pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100 ± 5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH) 2 D 3 and 1α,25(OH) 2 D 2 concentrations. [ABSTRACT FROM AUTHOR]