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

Blank Milk Form * Samples were spiked with each targeted folate compound at three different levels. Total folic calculated as sum of levels of spiked/analyzed folate compounds as folic acid. Values are averages of replicates of spike recovery irrespective of the spike level. Each value is average of 9 independent analyses.

The SPIFAN samples were spiked with each targeted folate compound (at equal level of each of seven targeted compound) and total spike level as folic acid calculated as sum of concentration of all folate compounds as folic acid. Results in the above Table. % spike recovery of folic acid ranged from 92 to 104%. All the spiked samples provided spike recovery within the recovery range 90-110% stipulated by the SMPR. Folate recovery analysis was satisfactory in all of the studied SPIFAN matrices. The repeatability of % spike recovery analysis in multiple replicate was evaluated as % RSD value. % RSD of % spike recovery analysis of folic acid in replicates analyzed in SPIFAN matrices ranged from 1.7 % to 6.7 %. The repeatability of folate recovery analysis was found to be satisfactory in all of the studied SPIFAN matrices.

LOD and LOQ of the Method:

The LOD and LOQ of the method were calculated based on replicate analysis of one of the SPIFAN samples. The folate results in the sample were calculated as mcg/g (as is). The LOD was calculated as 3 X of the standard deviation and LOQ as 10X of the standard deviation of the replicates of the sample.

Sample Folate Average (mcg/100g, as is) (n=3) = 1.108 Sample Folate Std Dev (mcg/100g, as is) (n=9) = 0.03 LOD of the method (mcg/g reconstituted) = 0.09 LOQ of the method (mcg/g reconstituted) = 0.3

Distribution of Minor Folates in SPIFAN Matrices : There was a discussion in last Folate ERP about distribution of minor folate isomers in SPIFAN matrices. Clear answer at that time was not available. ERP made a suggestion to the members and observers to look for answer to the question. We reviewed folate data of SPIFAN matrices and calculated distribution of minor folate compounds in the samples. Minor folate compounds Includes = 5- CHO-THF + THF + 10-CH3-FA + 10-CHO-FA + Methenyl-THF . The data are presented in Table 30.

Table 30: Distribution of minor folate compounds in SPIFAN matrices

% Minor* Folates of total

Sample

Matrix

Lot #/ID

REC'D AS

n

LCS

Break Fast Cereal

9 9

14.18

1

SRM 1849A

CLC10-b

POWDER

3.39

39