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

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

Formula Weight

Precursors [M+H] +

Product Ions

Collision Energy

Folate Vitamers

FA

441 459 473 445 456 455 469

442 460 474 446 457 456 470

295 313 327 299 412 309 295

20 22 20 20 27 20 35

5-CH3-THF 5-CHO-THF 5 ,6,7,8-THF 10-CH3-FA 10-CHO-FA Total Folate

5,10-CH=CH-THF

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 ( 13 C-Folic acid) is used as the internal standard.

Chemicals:

1.) Folic Acid (Standard) Schircks Laboratories 2.) (6R,S)-5-Methyl-5,6,7,8- Schircks Laboratories Tetrahydrofolic acid, calcium salt 3.) (6S)-5,6,7,8- Tetrahydrofolic Schircks Laboratories

Cat. No. 16.203

Cat. No. 16.235

Cat. No. 16.207

acid 4.) 7,8-Dihydrofolic acid 5.) (6S)-5-Formyl-5,6,7,8-

Schircks Laboratories

Cat. No. 16.206

Schircks Laboratories

Cat. No. 16.221

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