AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Table 17: Measured concentrations of standards through processing against solvent standard curve

5-CH3- THF

5-CHO- THF

10-CHO- FA

FA

THF

10-CH3-FA

Target Conc. (ng/mL) Meas. Conc. (ng/mL)

200 193 210 209 204

200 207 225 205 212

100

100

100

100

75.0 79.6 75.2 76.6 3.4%

41.9 56.4 46.0 48.1

75.1 82.0 81.3 79.5 4.8%

63.1 65.3 66.4 64.9 2.6%

Average (ng/mL)

%RSD

4.7%

5.2%

15.5% 48.1%

Accuracy

102.0%

106.2%

76.6%

79.5%

64.9%

Method Validation Results:

The Evaluation of Accuracy of the Method by Analysis of Reference Materials : The method accuracy was evaluated by analysis of a variety of Reference materials. The reference values for total folate or some of the selected folate vitamers is available for some of the Reference materials. The Results are presented in Table 24. Folic Acid value in NIST SRM 1849a matched closely with its Certfied value. 5-methyl-THF value obtained by our method was almost twice of reported by NIST. The NIST value for this vitamer is based on the NIST in house analysis by a LC-MS/MS method and it’s a reference value and not a certified value. It’s possible there method is based on older methodology and not using internal std. and other features required for accurate analysis of 5-methyl-THF. Total folate as folioc acid in this reference material was found to be close to that obtained using the microbiological analysis. Table 18: The analysis of folate vitamers in Reference Materials Reference Material Matrix Folate Vitamer Results Days of analysis Mean mcg/g % RSD n

Reference Value

mcg/g

Folic Acid 5-CH3-THF Total Folate* 5-CH3-THF Total Folate*

2.28 0.17 2.42 2.62 2.71

6.6

50 41 41 35 34

17 14 14 17 12

2.293+ 0.062 0.084 + 0.0031

Infant Formula Milk Based

12.1

NIST 1849a

2.31 (Micro value) 2.14 + 0.42 3.15 + 0.28

5.8 9.5

Dried Vegetable Powder

BCR 485

12.5

NIST 3280

Vitamin Tablet Total Folate*

383.1

1.6

3 n

1

394 + 22

Mean ng/g

% RSD

ng/g

NIST 1546**

Meat

Total Folate*

3

14.3 12* *Total Folate as Folic Acid; **NIST SRM 1546 Total Folate Value is an Information Value (no uncertainty provided). 5.1 15

5-methyl-THF in BCR 485 was estimated by the method close to the reported upper range and total folate as folic acid was about 96% of the lowest range specified for this reference material. Folic acid in NIST SRM 3280, a multivitamin and mineral dietary supplement tablet matrix, estimated by the method matched closely to the NIST value. Similarly total folate value as folic acid in the NIST SRM 1546, meat sample, was found to match with the reference value indicated by the NIST for this sample.

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