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2019, Cilt 17, Sayı 2, Sayfa(lar) 085-094
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A Simple, Sensitive and Rapid HPLC-UV Method for the Determination of 25(OH)D and Comparison of the İmmunoassay Method
Özgür Baykan
Balıkesir Üniversitesi Tıp Fakültesi Biyokimya Anabilim Dalı
Keywords: Methods; Chromatography; Vitamin D

Aim: Vitamin D deficiency is a common public health problem in our country. It is possible to find data reaching 90% in studies about determining deficiency prevalence. In recent years, this deficiency has been attracted the attention of the press and has directed people to a search for blood level monitoring. For these reasons, there are also serious increases in the number of vitamin D levels analyses in laboratories. Due to the high measurement costs per patient, it is also one of the most important laboratory expenses. It is aimed to develop an simple, sensitive and rapid ‘in-house’ analysis method in order to reduce laboratory costs and to determine 25(OH)D2 and D3 forms separately.

Material and Methods: Ultimate 3000HPLC-UV (Dionex, Germany) was used in the development of the method. Architect 25(OH)D kit (Abbott Diagnostic, USA) were used for method comparison study. Validation studies were performed using CLSI guidelines(EP5-A2,EP6-A,EP9-A2). Accuracy was evaluated using Certified Reference Material (UME CRM 1308). For chromatographic separation, reverse phase 150mmX4.6mm,(3.0 μm) ODS-1 (Thermo Scientific,USA) analytical column was used.

Results: Within run, between run and between day precisions (%CV) for 18.9ng/mL 25(OH)D3 were 2.6%, 2.7%, 6.3% and for 57.4ng/mL were 1.8%, 1.8% and 4.8% respectively. For 25(OH)D3, the LOD was 0.7ng/mL and the LOQ was 2.1ng/mL. Correlation between HPLC-UV and Abbott Architect systems was evaluated by Passing Bablok regression analysis (y=0,89x+1,81).

Conclusion: The compatibility between HPLC-UV and Abbott Architect suggests that both methods can be preferred. However,each laboratory should decide according to the their conditions in which the method is selected for the determination of 25(OH)D. Because in such cases where test performances are very similar. The number of patients, experienced staff presence and cost-determining parameters will be more prominent in the selection of methods,


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