Collaborative study: Quantification of total folate in food using an efficient single-enzyme extraction combined with LC-MS/MS

Quantification of the specific folate vitamers to estimate total folate in foods is not standardized. A collaborative study, including eight European laboratories, was conducted in order to determine the repeatability and reproducibility of the method for folate quantification in foods using the plant-origin γ-glutamyl hydrolase as part of the extraction procedure. The seven food samples analyzed represent the food groups; fruits, vegetables, dairy products, legumes, offal, fish, and fortified infant formula. The homogenization step was included, and six folate vitamers were analyzed using LC-MS/MS. Total folate content, expressed as folic acid equivalent, was 17–490 μg/100 g in all samples. Horwitz ratio values were within the acceptable range (0.60–1.94), except for fish. The results for fortified infant formula, a certified reference material (NIST 1869), confirmed the trueness of the method. The collaborative study is part of a standardization project within the Nordic Committee on Food Analysis (NMKL).

Highlights: Specific LC-MS/MS method for folate analysis in food is inter-laboratory validated; Total folate results acceptable in six tested food samples; for salmon unacceptable; HorRat values for total folate and 5-CH3-H4folate were within the acceptable range; Acceptability of HorRat values for other studied vitamers depends on food matrix; For total folate: repeatability at 2.3%–15% and reproducibility at 8.7%–58%

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Many thanks to Dr. Andrew Hanson (University of Florida) and Dr. Rocío Díaz de la Garza (Technologíco de Monterrey) who generously gave us the permission to produce the GGH used in this study, as well as permission to establish a commercial production in the future. Authors are very grateful to Merck and Phenomenex for providing a significant discount for the internal standards and the SPE columns used, and to NMKL to be responsible for shipping cost. We would like to thank the following individuals for their contribution with the analyses: Nina Wollertsen, Tone Myhra, Susanna Petrova, and Torill Berg at IMR (Bergen, Norway); Sylvie Bénet at Nestlé Research (Lausanne, Switzerland); Brian Hermansen at Eurofins Vitamin Testing (Vejen, Denmark); Marlène Daminato at Laboratoire Aquanal (Pessac, France); Tiago Moreira at INSA (Lisbon, Portugal) and to Heidi Jahn at DTU Food (Denmark) for assisting with standards and packing.