AOAC SPIFAN ERP Meeting Book-March 16, 2016

VitK-01 w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Validation of a LC-MS/MS Method for Vitamin K Analysis

LOD 0.15 0.03 0.18 LOQ 0.50 0.10 0.60

The estimated LOQ of vitamin K1 method for trans, cis and total vitamin K1 is 0.50 µg/100g, 0.10 µg/100g and 0.60 µg/100g respectively ( in reconstituted samples and RF samples) and meets the SMPR requirement. The SMPR stipulates the LOQ of the method to be < 1 µg/100g of the reconstituted final product.

Analytical Range of the method: The analytical range of the method based on the LOQ and calibration curve is calculated to be 0.5-100 mcg/100g, 0.1-100 mcg/100g and 0.6 -100 µg/100g for trans vitamin k1, cis-vitamin K1 and total vitamin K1 respectively. The analytical range needs mentioned in the SMPR are 1.0 – 100 µg/100 g of the reconstituted final product. System suitability requirements: 1. The method blank processed like a sample in the method should not provide any peaks in the area where vitamin K1 (cis- or trans), vitamin K2-7 and K2-4 elutes when monitored for their specific transitions. The method blank is expected to provide a good internal standard of the expected magnitude in the method blank sample. 2. The method should be able to resolve accurately cis- and trans-phylloquinone from cis- phylloquinone. 3. The lowest calibrant ( 5 ng/mL) and the mid-point calibrant 50ng/mL should provide a good chromatography of the vitamin K compounds and expected response.

The reference material:

NIST SRM 1849a (a milk based and infant and adult nutrition formula) with a certified value of total (trans- + -cis) of 1.06 + 0.07 mg/Kg was used a reference material to establish accuracy of the method in the current studies.

Conclusions:

1. A LC-MS/MS method was developed and validated for analysis of trans-and cis- vitamin K1, vitamin K2-7 and K2-4. 2. HPLC conditions and mass spec parameters were optimized. 3. The extraction conditions were also optimized to provide best and consistent extraction of the targeted analytes. 4. The method accuracy was evaluated and established by the analysis of NIST SRM 1849a ( a milk based infant and adult nutrition formula) reference material. 5. The SPIFAN samples were analyzed by the method in replicates on multiple days.

Silliker Laboratories, Chemistry Research Center, Date: 1/30/15

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