Trienzyme Treatment Research Articles

Folate Content and Yolk Color of Hen Eggs from Different Farming Systems.

This study aimed to compare folate contents in hen eggs from four different farming systems, namely organic, free range, barn, and cage one. Folate retention during egg boiling was studied as well. The contents of individual folate vitamers were determined using the high-performance liquid chromatography method (HPLC), following trienzyme treatment. Folate content in eggs differed significantly (p < 0.05) due to the rearing system, with the highest mean content determined in the eggs from organic farming (113.8 µg/100 g). According to this study, one egg (60 g) may provide 40–86 µg of folates, which corresponds to 10–22% of the recommended daily intake for adults, 400 µg according to the Nutrition Standards for the Polish Population. The predominant folate form found in egg was 5-methyltetrahydrofolate, which showed considerably greater stability under boiling compared to 10-formylfolic acid present in a lower amount. In most eggs tested, the losses in total folate content did not exceed 15%. The color of yolk of the most folate-abundant organic eggs, had the highest value of lightness (L*) and the lowest value of redness (a*). This, however, does not correspond to consumer preferences of intense golden yolk color.

The contribution of minor folates to the total vitamin B9 content of Infant formula and clinical nutrition products

In the present study an optimization of trienzyme treatment combining α-amylase, protease and γ-carboxy peptidase allowing complete sample preparation within a working day for the analysis of vitamin B9 (folate) in infant formula and adult/pediatric nutritional products is presented. The optimized sample preparation was applied to a set of samples representing most of the products in the marketplace. Results on Standard Reference Material 1849a were well in agreement with certified values. The main contributor to total folate was folic acid, 5-methyl-tetrahydrofolate was the only minor contributor in milk-based products. Soy-based formulas contained polyglutamates of 5-formyl-tetrahydrofolate. The relative contribution of polyglutamates to the total folate content remained low in the types of product included in this study. The results suggest that a simple di-enzyme treatment could be enough for these products, nevertheless, this should be carefully evaluated prior to making a decision on the use of tri- or di-enzyme treatment.

Validation of Folate-Enriched Eggs as a Functional Food for Improving Folate Intake in Consumers.

Functional foods enriched with folate may be beneficial as a means of optimizing folate status in consumers. We recently developed novel eggs enriched with folate through folic acid supplementation of the hen’s feed, but their potential to influence consumer folate status is unknown because the natural folate forms incorporated into the eggs may not necessarily be retained during storage and cooking. This study aimed to determine the stability of natural folates in folate-enriched eggs under typical conditions of storage and cooking. Total folate was determined by microbiological assay following tri-enzyme treatment in folate-enriched eggs and un-enriched (barn and free-range) on the day they were laid, after storage (up to 27 days) and after using four typical cooking methods (boiling, poaching, frying, scrambling) for different durations. On the day of laying, the folate content of enriched eggs was found to be significantly higher than that of un-enriched barn or free-range eggs (mean ± SD; 123.2 ± 12.4 vs. 41.2 ± 2.8 vs. 65.6 ± 18.5 µg/100 g; p < 0.001). Storage at refrigerator and room temperature for periods up to the Best Before date resulted in no significant losses to the folate content of folate-enriched eggs. Furthermore, folate in enriched eggs remained stable when cooked by four typical methods for periods up to the maximum cooking time (e.g., 135 ± 22.5, 133.9 ± 23.0 and 132.5 ± 35.1; p = 0.73, for raw, scrambled for 50 s and scrambled for 2 min, respectively). Thus, natural folates in folate-enriched eggs remain highly stable with little or no losses following storage and cooking. These findings are important because they demonstrate the feasibility of introducing folate-enriched eggs into the diet of consumers as functional foods with enriched folate content. Further studies will confirm their effectiveness in optimizing the biomarker folate status of consumers.

DETERMINATION OF FOLATE RETENTION DURING TOFU PREPARATION USING TRIENZYME TREATMENT AND MICROBIOLOGICAL ASSAY

Megaloblastic anaemia, neural tube defects, coronary heart disease, and some forms of cancer have been linked to an inadequate dietary intake of folate. Legumes are good sources of folate, however, processing may affect folate availability in legume foods. This study investigated the folate content and retention during the preparation of tofu through the trienzyme treatment (protease, α-amylase, and human plasma) and microbiological assay using Lactobacillus casei. The results showed that the total folate content was considerably reduced during tofu preparation, from 273.4 µg 100 g-1 dwb in soybean seed to 108.4 µg 100 g-1 dwb in tofu, yielding a folate retention of 40%. Folate losses were mainly due to leaching into the water during soaking and pressing as well as heat destruction during milling and boiling. High folate loss (51%) was particularly found in the whey. A shorter soaking time (8 hours), optimum extraction, and pressing methods were suggested with respect to minimizing folate losses during tofu preparation. This study suggests that the consumption of 100 g of deep-fried tofu may satisfy 21% of the recommended dietary intake (RDI).

DETERMINATION OF FOLATE RETENTION DURING TOFU PREPARATION USING TRIENZYME TREATMENT AND MICROBIOLOGICAL ASSAY

Megaloblastic anaemia, neural tube defects, coronary heart disease, and some forms of cancer have been linked to an inadequate dietary intake of folate. Legumes are good sources of folate, however, processing may affect folate availability in legume foods. This study investigated the folate content and retention during the preparation of tofu through the trienzyme treatment (protease, α-amylase, and human plasma) and microbiological assay using Lactobacillus casei. The results showed that the total folate content was considerably reduced during tofu preparation, from 273.4 µg 100 g-1 dwb in soybean seed to 108.4 µg 100 g-1 dwb in tofu, yielding a folate retention of 40%. Folate losses were mainly due to leaching into the water during soaking and pressing as well as heat destruction during milling and boiling. High folate loss (51%) was particularly found in the whey. A shorter soaking time (8 hours), optimum extraction, and pressing methods were suggested with respect to minimizing folate losses during tofu preparation. This study suggests that the consumption of 100 g of deep-fried tofu may satisfy 21% of the recommended dietary intake (RDI).

Microbiological Assay of Folic Acid Content in Some Selected Bangladeshi Food Stuffs

Folic acid concentration in six common food samples of Bangladesh including lentil (Lens culinaris Medik) and bengal gram(Cicer arietinum), representatives of legumes; spinach (Spinacia oleracea) and basil (Ocimum basilicum), representatives of green leafy vegetables; milk, representative of a common animal source food and topa boro rice (Oryza sativa), a representative of cereal-grains were measured in the present study by microbiological assay using a new trienzyme folic acid extraction method. In this experiment, we estimated that, lentil, bengal gram, spinach, basil, milk and topa boro rice contained about 63 µg, 48 µg, 195 µg, 131 µg,10 µg and 42 µg of folic acid per 100 g of food sample respectively. For the assay of this water-soluble vitamin, Lactobacillus casei (ATCC 7469) was employed as test organism. Trienzyme treatment was performed to release bound folic acid using protease, and a-amylase with chicken pancreases as the conjugase. The highest folic acid content was recorded for spinach (Spinacia oleracea), the well-known richest source of folic acid, followed by basil (Ocimum basilicum). The lowest value was recorded for milk. Topa boro rice, a kind of parboiled rice was also found to be a fair source of folic acid. For these six types of Bangladeshi foods studied, the content of folic acid ranged from 10 to 195 µg/ 100 g, indicating a very wide range of folic acid content in foods.

국내 소비되는 봄나물의 Trienzyme 추출법을 적용한 엽산 함량 분석

본 연구에서는 국내 엽산 분석 데이터의 질적?양적 향상을 위해 첫째, AOAC법의 trienzyme 추출법과 식품공전의 암모니아 가수분해법을 비교 분석하고 둘째, 엽채류에 있어 trienzyme 추출법의 검증을 위해 국제표준인증물질을 분석하고 정밀성과 정확성을 측정하였다. 마지막으로 검증된 추출법을 적용하여 시판 봄나물(생것, 데친것)의 총 엽산 합량을 L. casei를 이용한 미생물학적 정량법을 적용하여 비교 분석하였다. 연구 결과 CRM 485(mixed vegetable), CRM 487(pig liver), CRM 121(wholemeal flour), 시금치, 돌미나리의 경우 암모니아 추출법보다 trienzyme 추출법을 적용할 경우 엽산 함량이 시료에 따라 1.2에서 5.8배까지 증가하였다. 따라서 나물 등 엽채류 식품에 함유된 천연 엽산 분석에는 trienzyme 추출법 적용이 필요한 것으로 생각된다. Trienzyme 추출법을 적용하여 분석한 봄나물(생것, 데친 것)의 엽산 함량은 습물 기준(wet weight basis) 나물 생시료의 경우 오가피(146.9 μg/100 g), 부지갱이(142.8 μg/100 g), 갯기름(140.4 μg/100 g)의 엽산 함량이 15개의 분석시료 중 높은 값을 나타내었다. 반면 건물 기준(dry weight basis)을 적용한 절대량 비교에서는 민들레(1,643.0 μg/g), 시금치(1,284.8 μg/g), 비름나물(1,222.5 μg/g) 순으로 높은 엽산 함량을 나타내었다. 가죽나물, 참두릅, 세발나물, 돌미나리, 참취나물 경우 100°C에서 30초간 데쳤을 때 엽산 함량은 차이가 없었으며(P>0.05) 갯기름나물을 제외한 나머지 9개 시료에서는 엽산 함량이 11∼63% 감소하였다(건물 기준 비교). 본 연구에 적용된 엽산 trienzyme 추출법은 상대적으로 부족한 국내 다소비 식품의 엽산 분석 연구에 활용될 것으로 생각되며 나아가 국가표준식품성분표 작성을 위한 엽산 데이터베이스 구축의 기초 자료가 될 것이다.

Determination of folate/folic acid level in milk by microbiological assay, immuno assay and high performance liquid chromatography

A concurrent determination of folate versus folic acid in milk by microbiological assay (MA) with Lactobacillus rhamnosus as the assay organism, Enzyme Linked Immuno Sorbent Assay (ELISA) by competitive binding rapid ELISA kit (RIDASCREEN®) and high-pressure-liquid chromatography (HPLC) was done for detection of the folate form and its level. MA gave total folate content as Lb. rhamnosus showed similar response to most folate isomers formed by the tri-enzyme treatment in comparison with the other two methods which specifically estimated the folic acid. In case of ELISA, specificity was apparently limited to folic acid and dihydro folic acid and thereby showed a lower response for other folate derivatives. Estimation by HPLC with UV detector was highly specific and hence only folic acid could be detected without any cross reactivity. Among the different methods HPLC was observed to be the most sensitive method for determination of folic acid and hence can efficiently determine the folic acid fortification level while MA remained highly efficient, sensitive and reproducible method for estimation of total folate indicating its potential use for dietary folate estimation.

Iron and Folate Contents of Tajik Legumes

Seven varieties of Tajik legumes and two Tajik snack type ready-to-eat (RTE) whole/split chickpeas were analyzed for iron in raw and cooked legumes and for naturally occurring folate content in cooked legumes. Iron was measured according to AACC method 40 - 41B. Folate contents were determined by microbiological (Lactobacillus casei subsp. Rhamnosus ATCC 7469) and high-performance liquid chromatography analysis utilizing a tri-enzyme treatment (pro-tease, α-amylase and conjugase). Folate derivatives of tetrahydrofolate, 5-formyl-tetrahydrofolate and 5-methyl- -tetrahydrofolate were identified and quantified. Iron content for Tajik legumes ranged from 5.52 to 13.27 mg/100 g for raw; 2.81 to 4.12 mg/100 g for cooked and 4.37 and 4.76 mg/100 g for RTE chickpeas. The total folate content of cooked legumes ranged from 53 to 81 µg/100 g for beans; 133 to 203 µg/100 g for peas, and from 39 to 22 µg/100 g for small and large lentils, respectively. The predominant form of folate in legumes was tetrahydrofolate, followed by 5-formyl-tetrahydrofolate and 5-methyl-tetrahydrofolate.

Testing of folate conjugase from chicken pancreas vs. commercial enzyme and studying the effect of cooking on folate retention in Thai foods

Crude enzyme from chicken pancreas as a source of folate conjugase was prepared in a lyophilised form. Homogeneity, stability and activities were checked against a commercial enzyme. Subsequently, the prepared crude enzyme was used to investigate the process of folate extraction in various food matrices and study the effect of cooking on folate retention in several Thai foods. The lyophilised enzyme was homogeneous and contained 4 μg endogenous folate per g of prepared lyophilised crude enzyme. The stability of the lyophilised enzyme and the diluted enzyme, kept at 4 °C, was at least 12 months and 3 days, respectively. The activity of the prepared lyophilised folate conjugase, used at the level of 20 mg/g sample, was evaluated by a microbiological assay of total folate in brown rice, egg, whole milk powder, soybeans and asparagus. It showed comparable results to that of the commercial enzyme. Single-enzyme treatment (folate conjugase alone) was sufficient for the determination of folate content in brown rice, soybeans and asparagus. However, for egg and whole milk powder, it was necessary to apply tri-enzyme treatment as this showed a significantly higher level (at P < 0.05) of total folate than that obtained using only single- or di-enzyme (protease + folate conjugase) treatments. Among the representative foods studied, the levels of total folate ranked in order of concentration, were soybeans > egg > asparagus > brown rice > steamed-mackerel (common form sold) (305, 117, 95, 41 and 24 μg/100 g wet weight basis, respectively). After cooking, the true retention of total folate in the cooked foods ranged from 60% in brown rice (cooked in an electric rice cooker) up to 82% in boiled asparagus. On a wet weight basis, the total folate content in boiled soybeans, boiled asparagus, boiled egg, fried mackerel and cooked brown rice was 100, 84, 72, 22 and 12 μg/100 g, respectively. One boiled egg (50 g) contributes 36 μg folate, which is equal to 18% of the Thai Recommended Daily Intake (RDI), and thus it can be considered as a good source of folate based on one serving size. A serving of boiled soybeans (70 g) or boiled asparagus (80 g) is an excellent source of folate, each contributing about 35% of the Thai RDI.

Determination of the Folate Content in Cladodes of Nopal (Opuntia ficus indica) by Microbiological Assay UtilizingLactobacillus casei(ATCC 7469) and Enzyme-Linked Immunosorbent Assay

Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

Folic Acid Content of Ready‐to‐Eat Cereals Determined by Liquid Chromatography‐Mass Spectrometry: Comparison to Product Label and to Values Determined by Microbiological Assay

ABSTRACTTwelve popular ready‐to‐eat breakfast cereals fortified with folic acid were sampled in the United States in 2006, and the data have been incorporated into the USDA National Nutrient Database for Standard Reference. Cereals were collected from three statistically selected retail outlets in each of four primary census regions, and four composites of each product were prepared using random groupings of three locations each. Folic acid was determined using a validated LC‐MS method, with 13C‐folic acid as an internal standard, after trienzyme treatment and solid phase extraction. A cereal reference material (AACC VMA399) was analyzed as a control. Selected samples were also assayed using the standard microbiological method, with and without trienzyme extraction, to generate an estimate of endogenous folate. On average, as shown on the label, folate content was underestimated. In seven cereals, folate was within 5% of the declared value; in four cereals, it was 5–20% higher; and in two cereals, it was &gt;20% greater, representing –75 to +69 μg/serving (mean 17) of the label value, equivalent to –19% to +17% of the 400 μg/daily value. The microbiologically determined folic acid was higher than LC‐MS by 10–67% (mean 40%). Therefore, use of label values might underestimate folate intake from some breakfast cereals.

Folate content in foods commonly consumed in Egypt

The folate content in some Egyptian foods was determined using RP-HPLC-FL. Trienzyme treatment was used for legumes, dienzyme treatment for cereals and starchy vegetables, and monoenzyme treatment for vegetables and fruits. The highest folate content (633 μg/100 g) was found in dried Jew’s mellow due to low water content, followed by legumes (e.g. 150 μg/100 g for chick peas) and leafy vegetables (100 μg/100 g). For other foods, folate content ranged from 10–90 μg/100 g. In all foods, the predominant folate form was 5-CH 3–H 4folate, except for dried Jew’s mellow, which contained more than 80% 10-HCO–PteGlu. Using folate data from our own analyses and food tables and food consumption data, the dietary folate intake per capita in Egypt was estimated. However, representative and validated food composition data for folate in Egyptian foods are needed for estimating and evaluating the adequacy of the population’s folate intake.

Optimisation and validation of a liquid chromatography–tandem mass spectrometry method for folates in rice

An LC–MS/MS method has been developed and validated for the analysis of six monoglutamate folates in rice. Accurate determination of folates in rice seeds required the optimisation of an extraction procedure encompassing homogenisation, heat treatment, tri-enzyme treatment (α-amylase, protease and deconjugase), centrifugation and ultrafiltration. The resulting extract was loaded onto a Polaris C 18-A column, followed by gradient elution and detection by tandem mass spectrometry. The validated method is sensitive, reproducible, accurate, and linear between 1 and 400 μg/100 g rice (dry weight). The limit of detection and limit of quantification in diluted rice matrix ranged between 0.2 and 1.2 μg/100 g and between 0.6 and 4 μg/100 g, respectively, depending on the compound. Intra- and inter-day precision varied, respectively, from 1.0 to 9.1% RSD, and from 3.4 to 13.5% RSD, except for one compound (19.1% at low concentrations). The accuracy ranged from 96.9 to 115.4%. Matrix effects were compensated for by use of isotopically labelled internal standards. Most folates in rice extracts were stable when kept for 2 weeks at −20 and −80 °C, but degraded after three freeze–thaw cycles or at 4 °C, even when stored protected from light. Our validated method offers a sensitive and versatile approach to determine the level of six different monoglutamate folates in a complex matrix as rice.

Folate contents of some selected Fijian foods using tri-enzyme extraction method

Folic acid and total folate contents of 18 common foods in the Fijian diet were assayed. Foods were purchased from the central and eastern parts of Viti Levu in Fiji. Tri-enzyme treatment was performed to release bound folates using protease and α-amylase, with chicken pancreas as the conjugase. The highest total folate content was recorded for egg yolk ( Gallus domesticus) at 256 μg/100 g, followed by long beans ( Vigna sesquipedalis) which contained 130 μg/100 g of total folate (fresh weight basis). The local leafy vegetable called Bele ( Abelmoschus manihot) and the Drumstick leaves ( Moringa oleifera) available in Fiji also had high total folate contents, above 100 μg/100 g (fresh weight basis). For the 18 foods studied, the content of folic acid ranged from 3 to 189 μg/100 g and the total folate content was in the range of 3–256 μg/100 g, indicating a very wide range of folate content in the foods studied.

Determination of Folate Concentrations in Diverse Potato Germplasm Using a Trienzyme Extraction and a Microbiological Assay

Folate deficiency is a leading cause of birth defects and is implicated in several other diseases. We are interested in how much folate concentrations vary among potato germplasm. We determined total folate concentrations of potato tubers from 67 cultivars, advanced breeding lines, or wild species. Folates were extracted by a tri-enzyme treatment and analyzed by using a Lactobacillus rhamnosus microbiological assay. Folate concentrations varied from 521 +/- 96 to 1373 +/- 230 ng/g dry weight and were genotype and location dependent. The highest folate concentrations were mostly found in color-fleshed potatoes. Variations of folate concentrations within either color- or white-fleshed tubers were similar ( approximately 2-fold). Skin contained approximately 30% higher folate concentrations than flesh. Storage of tubers for 7 months generally led to an increase in folate contents. Semiquantitative RT-PCR analyses showed that higher folate contents were correlated with lower mRNA expression of some folate genes.

Use of the affinity/HPLC method for quantitative estimation of folic acid in enriched cereal-grain products.

In 1998, the United States introduced mandatory fortification of enriched cereal-grain products with folic acid to reduce the incidence of neural tube defects. As a consequence, substantial amounts of folic acid, the synthetic form of folate, were added to the American diet, and the ability to assess folic acid intake took on greater importance. The purpose of the current study was to separate and quantify folic acid and 5-methyltetrahydrofolate, the most prominent naturally occurring folate in fortified foods, with a reliable and robust method. Folates were heat-extracted from food samples. A trienzyme treatment (alpha-amylase, rat plasma conjugase, and protease) was applied to the extracts followed by purification by affinity chromatography. Folic acid and 5-methyltetrahydrofolate were separated and quantified by reversed-phase HPLC with fluorescence and UV detection. A gradient elution with phosphate buffer and acetonitrile was used to separate the different forms of folates. The method gave a linear response in a range of 0.1-3 mumol/L and 0.0125-0.25 mumol/L for folic acid and 5-methyltetrahydrofolate, respectively. These ranges were similar to the expected levels in the samples. The CV of the peak areas of folic acid and 5-methyltetrahydrofolate for 5 commercial wheat flour samples extracted and run separately on the same day was 2.0 and 5.7% and, run over 5 consecutive days, was 7.2 and 7.3%, respectively. Total folate values in 45 samples of fortified food measured by HPLC and by the traditional microbiological assay demonstrated a high correlation (r(2) = 0.986).

Determination of folate in some cereals and commercial cereal-grain products consumed in Poland using trienzyme extraction and high-performance liquid chromatography methods

A trienzyme treatment (rat plasma conjugase, α-amylase, protease) followed by affinity chromatography (with folate-binding protein from bovine milk) and reversed-phase high-performance liquid chromatography with UV and fluorescence detections was performed for quantification of different folate derivatives in some commercial cereal-grain products and cereals cultivated in Poland. The method described allowed good separation of the main folate vitamins. Of the commercial cereal-grain products analyzed, buckwheat groats (30 μg/100 g) had the highest content of total folate, followed by rye flour (29 μg/100 g), barley groats (21 μg/100 g) and wheat flours (19–20 μg/100 g). Two of the ready-to eat breakfast products were found to be fortified with folic acid with the level slightly exceeding the label declaration. The total folate content of whole-grain wheat flours was more than half as much as in white flours (33–40 μg/100 g), and the highest folate content was found in rye whole grains (123–135 μg/100 g). 5-Formyltetrahydrofolate was the predominating folate form in the cereal products analyzed, above all in rye flour.

High-Performance Liquid Chromatographic Determination of Naturally Occurring Folates during Tempe Preparation

A trienzyme treatment (protease, alpha-amylase, and human plasma conjugase), followed by purification using SPE with SAX cartridges and reversed-phase HPLC with UV-PDA detection, was performed for determination of the distribution of various folate forms and content at various stages of tempe preparation. The major folate form in soybean identified was 5-formyl tetrahydrofolate (5-CHO-H4folate), followed by 10-formyl tetrahydrofolate (10-CHO-PGA), and 5-methyl tetrahydrofolate (5-CH3-H4folate), whereas folic acid was not detected and tetrahydrofolic acid (H4folate) was not detectable. The most predominant form in tempe was also 5-CHO-H4folate, followed by 10-CHO-PGA, whereas the quantities of 5-CH3-H4folate and folic acid were negligible. Quantities and retention of folate significantly decreased during the first boiling, dehulling, soaking, and second boiling procedures, yielding folate retention of 32%. A remarkable increase in folate content was found after fermentation, 5.2-fold higher than that of the boiled soybean. This may be due to de novo formation of folate by Rhizopus oligosporus, the principal mold in tempe fermentation. HPLC results were approximately 38-55% lower than the values obtained from the microbiological assay using Lactobacillus casei.

Stability of 5-methyltetrahydrofolate in frozen fresh fruits and vegetables

The stability of 5-methyltetrahydrofolate (5MTHF) in homogenized fresh fruits and vegetables representing samples for the USDA National Food and Nutrient Analysis Program was evaluated. Samples were homogenized in liquid nitrogen and 5MTHF was measured after 0, 2, 7, 30 days and then at 3-month intervals for a total of 12 months storage at −60 ± 5 °C, utilizing extraction by a tri-enzyme treatment, purification by strong anion-exchange solid-phase extraction, and quantification by reverse-phase HPLC. Method validation included analysis of a reference material and interlaboratory analysis of selected samples by HPLC and LC-MS. A canned spinach composite was assayed in each analytical batch to monitor inter-assay precision. No change in 5MTHF content was detected in any of the samples after 12 months. Concentrations ranged from <10 μg/100 g in bananas to >100μg/100 g in spinach. Relative standard deviations were generally <7% within assay and <11% between assays.