AOAC OMA# 2011.06 Final Action Review-OMB

1552  S zpylka et al . : J ournal of AOAC I nternational V ol . 95, N o . 6, 2012

References:  J. AOAC Int . 95 , 1547(2012); AOAC SMPR 2011.006, J. AOAC Int . 95 , 294(2012)

respectively. % Ca = formula weight (FW) Ca ÷ FW Ca folate salt × 100. ( b ) Determine the concentration (μg/100 g) of folate vitamer in the sample as follows (concentrations are calculated for each daughter ion using corresponding response factors):

Results and Discussion

System Linearity Study Standards of folates expressed as FA with known amounts of internal standard were analyzed by the method. The standards were not taken through the extraction process, only the UPLC-MS/MS procedure was performed to determine the linear range of the instrument’s response. The following plots illustrate the instrument’s response with increased analyte concentration. A plot of the peak area ratios (PA-Folates/PA-IS) versus the concentration showed a linear relationship between 0.01 and 100 μg/mL (ppm) with R 2 of 0.9941 (Figure 1). A further plot of the response factors versus folates concentration showed linearity at 95% confidence level between 0.025 and 95.5 μg/mL. Express as μg/100 g to 19 100 μg/100 g. The LOD of total folates by this method defined as three times the SD of the blank is 0.01 μg.mL. This would correspond to 2 μg/100 g of total folates for a sample weight of 1 g with 2 μL injection on the analytical column. But the linear response of the instrument to concentration is between 10.0 μg/100 g and 191 000 μg/100 g for a sample weight of 1 g with 2 μL injection onto the analytical column. Therefore, the method’s detection limit is 10 μg/100 g for a 1 g sample weight with 2 μL injection volume on the analytical column. The detection limit can be reduced to 5 μg/100 g when the sample weight is increased to 2 g.

where C = concentration in μg/100 g folate vitamer in the sample; PA 2 = peak area of folate vitamer in the sample chromatogram; PA 1 = peak area of the IS in the sample chromatogram; W 1 = weight, in μg, of IS added to the sample before extraction; W 2 = weight, in grams, of the sample; F = RF of folate vitamers using corresponding daughter ions from Table 2011.06E ; and 100 = conversion of concentration of folate vitamers to μg/100 g. ( c ) Determine the concentration ( m g/100 g) of folate vitamer in the sample expressed as folic acid:

100

FW folic acid FW folate vitamer acid form

C PA W fa 2

1

PA W F 1 2

where C fa

= concentration in µ g/100 g folate vitamer in sample

expressed as folic acid; PA 2 sample chromatogram; PA 1 sample chromatogram; W 1

= peak area of folate vitamer in = peak area of internal standard in = weight, in µ g, of internal standard

added to sample before extraction; W 2 = weight, in grams, of sample; F = RF of folate vitamers from calculation, G ( a ); 100 = conversion of concentration of folate vitamers to µ g/100 g. Folate salt conversion to free acid = (FW folate salt vitamer – FW salt) + 2 = FW folate vitamer acid form See Table 2011.06F . ( d ) Report total concentration of folate present in assay as a summation of folate vitamers, G ( b ), or folate vitamers expressed as folic acid, G ( c ), with respect to the method blank: C T = [(5-CH 3 -THF) + (5-CHO-THF) + (THF) + (FA) + (10-CHO-FA) + (10-CH 3 -FA)] – B where B = total folates present in method blank. ( e ) For the conjugase efficiency test, calculate the percent conversion of polyglutamates to monoglutamates by rat plasma conjugase as follows:

Accuracy

Accuracy estimation was obtained by running a study using four spiked matrixes. The samples used were NIST 1846 (infant formula), NIST 3244 (protein powder containing ephedra), multigrain cereal, and milk-based yogurt beverage. These

where PA (FA) reaction; PA (IS) reaction; μg (IS)

= peak area of folic acid peak after conversion = peak area of IS peak after conversion

= μg IS added; F = RF for folic acid from G ( a ); 441.3 = formula weight of folic acid in μg/μmole used to convert μg of recovered folic acid to μmoles for comparison; 0.0042882 = μmoles of pteroyltri-γ-glutamate (FA-glu 3 ) obtained from converting the μg of “rat plasma test solution” to μmoles for comparison, i.e., 0.15 mL × 20 μg/mL/699.2 μg/μmoles; 699.2 μg/μmoles is the formula weight of FA-glu 3 ; 100 = conversion to percent.

Figure 1. Plot of the peak area ratios versus concentration shows a linear relationship between 0.01 and 100 μg/mL (ppm) with R2 of 0.9941