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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
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Detailed Method:
Folate Analysis in Food, dietary supplements, feed and pet food samples
Definition:
The described method is
an UPLC-MS/MS method for determining total folates, and important
folate vitamers in various food, dietary supplement, feed and pet food matrices after trienzyme digestion. Rat
plasma conjugase is used in deconjugating various folate polyglutamates to folate monoglutamates after protease
and α-amylase digestion of the food matrix in the trienzyme digestion process.
Scope:
This method is applicable to all various food, dietary supplement, feed and pet food matrices.
Analyte Scope:
Table 1: Analyte Scope of the Method
Range:
The method is linear in the range of 10 μg/100g to 19100 μg/100g total folates. With appropriate adjustment
of sample weight and SPE clean up loading volume (Table 1), the range can be extended from 5 μg/100g to
2,000,000 μg/100g total folates in the food matrices and vitamin concentrates described above.
Principle:
An aliquot of a thoroughly blended mixed sample is accurately is taken for the analysis.
Appropriate amount of internal standard and phosphate buffer, pH 6 is added. The sample is extracted a pH
6 buffer containing different folate stabilizers and
labeled folic acid and methyl –THF as internal
standards by triple enzyme digestion.
Rat plasma conjugase is used to de-conjugate the polyglutamate
forms of folates to mono-glutamates. The clear extract is then subjected to a weak anion exchange (WAX)
SPE . The folates are eluted from SPE and analyzed by UPLC-MS/MS. Seven folate vitamers are quantified
and the total folates determined and expressed as folic acid. Isotopically labeled folic acid (
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C-Folic acid)
is used as the internal standard.
Chemicals:
1.) Folic Acid (Standard) Schircks Laboratories
Cat. No. 16.203
2.) (6R,S)-5-Methyl-5,6,7,8- Schircks Laboratories
Cat. No. 16.235
Tetrahydrofolic acid, calcium salt
3.) (6S)-5,6,7,8- Tetrahydrofolic Schircks Laboratories
Cat. No. 16.207
acid
4.) 7,8-Dihydrofolic acid
Schircks Laboratories
Cat. No. 16.206
5.) (6S)-5-Formyl-5,6,7,8-
Schircks Laboratories
Cat. No. 16.221
Folate Vitamers
Formula
Weight
Precursors
[M+H]
+
Product
Ions
Collision
Energy
FA
441
442
295
20
5-CH3-THF
459
460
313
22
5-CHO-THF
473
474
327
20
5 ,6,7,8-THF
445
446
299
20
5,10-CH=CH-THF
456
457
412
27
10-CH3-FA
455
456
309
20
10-CHO-FA
469
470
295
35
Total Folate
2011.06 (Fol-22) w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE