AOAC 2018 Fat Soluble Vitamin Analysis by LCMSMS

Purpose

Fat-soluble vitamins (FSV) are required for a wide variety of physiological functions. Deficiencies in FSV have been associated with increased risk of cancer, type II diabetes mellitus, and a number of immune system disorders. Therefore, quantitative FSV assays are critical for addressing these issues. Currently, 59 different quantitative FSV assays have been published by AOAC and USP. Most of these assays employ liquid-liquid extraction (LLE) coupled with saponification, which is both time- and cost-consuming. We present a non-LLE assay for the analysis of FSV (see Table 1), and compare this non-LLE method with the LLE assay using samples with different matrices.

Method

Standard Preparation:

The standard is prepared in DMSO:water: isopropanol with a concentration range of 5.00-500 ng/mL. Approximately 2.00 grams of sample were extracted using a mixture of DMSO, water, and isopropanol. The extracted sample was filtered through a 0.20 μm filter before analysis.

UPLC-MS Conditions:

UPLC system: Nexera UPLC system including SIL-30AC auto-sampler, controller, column heater, and binary pump (SHIMADZU)

Column: 100×2.1 mm, 2.6 μm Kinetex F5 (Phenomenex)

Mobile Phase A: Formic acid and water

Mobile Phase B: Acetonitrile

Flow rate: 1.00 mL/min

Pump Gradient Cycle time: 10.0 minutes

MS detector: Triple Quadrupole 5500 MS (AB Sciex)

Results and Discussion

During the method development, different columns and mobile phases were investigated to separate D2 from D3 and K2-MQ4 from K2-MQ7—the Kinetex F5 column with acidic mobile phases provided sufficient resolution for these FSV. The method was successfully developed over the range of 5.00-500 ng/mL. The specificity experiment showed that there was no significant contribution between analytes/IS and no visible interference peaks showed in blank diluent at the expected retention time ( Fig. 2). The LLOQ has sufficient sensitivity S/N> 10) Fig. 2). System suitability consisted of six replicate injections of the middle standard solution. It was injected before sample analysis, and RSD was ≤7.55% ( Table 2). The response linearity study revealed that quadratic regression with 1/X weighing factor provides the best fit, and the correlation coefficient r2 is ≥0.995 Fig. 3 and Table 3). To develop a non-LLE extraction method, various extraction solvents were investigated, and the results indicated that a 1:1:1 mixture of DMSO, water, and IPA provided the highest recovery. Comparison to the LLE method revealed that most matrices can be efficiently extracted by a non-LLE method Fig. 4). However, several matrices, like some vitamin mineral tablets and protein shake,e still need a LLE/saponification to minimize potential interferences ( Fig. 5). Dyad Labs analyzes samples with a non-LLE method unless matrix interference is observed, in which the LLE extraction will be performed.

Conclusion

A specific, fast, and high-throughput non-LLE LC/MS/MS assay for quantifying FSV has been successfully developed and applied to various matrices.