Single-laboratory validation of a method for the determination of vitamin D3 in dietary supplements by liquid chromatography with tandem mass spectrometry (LC-MS/MS).
A single-laboratory validation (SLV) for the analysis of vitamin D3 was performed in four types of dietary supplements (capsules, soft gels, syrups, and tablets) using LC-MS/MS. Samples were treated by alkaline saponification for oil-based soft gels and utilized EDTA solution for capsules, syrups, and tablets prior to n-hexane extraction. Vitamin D3 in sample extracts was separated on a reversed-phase C18 column (100 x 2.1 mm, 2.7 pm) using a mobile phase of a 95 + 5 (v/v) mixture of 5 mM ammonium formate in methanol containing 0.1% formic acid and 5 mM ammonium formate in 0.1% formic acid running at a flow rate of 0.2 mL/min. Vitamin D3 was confirmed by the presence of three fragment ions at m/z 107, 159, and 259 within a defined retention time window from the precursor ion at m/z 385. Quantitation was based on the peak area at m/z 367 to that of the internal standard (d3-vitamin D3) at m/z 370 with reference to the respective response ratios of the calibration standards. The linear response of vitamin D3 ranged from 0.10 to 6.29 mg/L and the correlation coefficient (r) of the six-point calibration curves was > 0.999. Accuracy, in terms of the spiked recoveries from blank syrup and starch powder at three different concentration levels, was 101-103%. Precision, determined by two different analysts over a period of 5 weeks, ranged from 2.7 to 7.0% for the four preparations. The SLV demonstrates the present LC-MS/MS method is reliable and robust for the determination of vitamin D3 in the studied dietary supplements. Considering the attainment of satisfactory SLV results, further validation through intra-laboratory collaborative study is recommended.
Victor K M
VK
Lam
Ray C T
RC
Hung
Ella L M
EL
Wong
Johnny Y W
JY
Fok
Yiu-Chung
YC
Wong
D050071:Bone Density Conservation Agents / Q000737:chemistry*; D002762:Cholecalciferol / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D019587:Dietary Supplements / Q000032:analysis*; D015203:Reproducibility of Results; D013607:Tablets; D053719:Tandem Mass Spectrometry / Q000379:methods*
D050071:Bone Density Conservation Agents; D013607:Tablets; D002762:Cholecalciferol