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Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
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Table 17: Measured concentrations of standards through processing against solvent standard curve
FA
5-CH3-
THF
5-CHO-
THF
THF
10-CH3-FA
10-CHO-
FA
Target Conc.
(ng/mL)
200
200
100
100
100
100
Meas. Conc.
(ng/mL)
193
207
75.0
41.9
75.1
63.1
210
225
79.6
56.4
82.0
65.3
209
205
75.2
46.0
81.3
66.4
Average (ng/mL)
204
212
76.6
48.1
79.5
64.9
%RSD
4.7%
5.2%
3.4%
15.5%
4.8%
2.6%
Accuracy
102.0%
106.2%
76.6%
48.1%
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
Reference Value
Mean mcg/g
% RSD
n
mcg/g
NIST 1849a
Infant Formula
Milk Based
Folic Acid
2.28
6.6
50
17
2.293+ 0.062
5-CH3-THF
0.17
12.1
41
14
0.084 + 0.0031
Total Folate*
2.42
5.8
41
14
2.31 (Micro
value)
BCR 485
Dried
Vegetable
Powder
5-CH3-THF
2.62
9.5
35
17
2.14 + 0.42
Total Folate*
2.71
12.5
34
12
3.15 + 0.28
NIST 3280
Vitamin Tablet Total Folate*
383.1
1.6
3
1
394 + 22
NIST 1546**
Meat
Total Folate*
Mean ng/g
% RSD
n
3
ng/g
14.3
5.1
15
12*
*Total Folate as Folic Acid; **NIST SRM 1546 Total Folate Value is an Information Value (no uncertainty provided).
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.
2011.06 (Fol-22) w/SLV
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