A nutritional supplement during preconception and pregnancy increases human milk vitamin D but not B-vitamin concentrations.

BACKGROUND & AIMS: Optimal maternal vitamin status during pregnancy and lactation is essential to support maternal and infant health. For instance, vitamin D3 is involved in infant bone development, and B-vitamins are involved in various metabolic processes, including energy production. Through a double-blind randomised controlled trial, we investigated the effects of maternal supplementation from preconception throughout pregnancy until birth on human milk (HM) concentrations of vitamin D3 and B-vitamins. In addition, we aimed to characterise longitudinal changes in milk concentrations of these vitamins. METHODS: Both control and intervention supplements contained calcium, iodine, iron, ß-carotene, and folic acid, while the intervention also contained zinc, vitamins B2, B6, B12, and D3, probiotics, and myo-inositol. HM samples were collected across 4 time points from 1 week to 3 months post-delivery from 158 mothers in Singapore, and 7 time points from 1 week to 12 months from 180 mothers in New Zealand. HM vitamin D was quantified using supercritical fluid chromatography and B-vitamins with mass spectrometry. Potential intervention effects on HM vitamins D3, B2, B6, and B9, as well as other B-vitamin (B1 and B3) concentrations were assessed using linear mixed models with a repeated measures design. RESULTS: Over the first 3 months of lactation, HM 25-hydroxyvitamin D3 concentrations were 20% (95% CI 8%, 33%, P = 0.001) higher in the intervention group, with more marked effects in New Zealand. There were no observed intervention effects on HM concentrations of vitamins B1, B2, B3, B6, and B9. In New Zealand mothers, longitudinally, vitamin D3 concentrations gradually increased from early lactation up to 12 months, while vitamins B1 and B2 peaked at 6 weeks, B3 at 3 weeks, and B6 and B9 at 3 months. CONCLUSIONS: Maternal supplementation during preconception and pregnancy increased HM vitamin D, but not B-vitamin concentrations in lactation. Further studies are required to examine the discrete benefits of vitamin D supplementation starting preconception vs during pregnancy, and to further characterise the effects of supplementation on later offspring health outcomes. CLINICAL TRIAL REGISTRATION: Registered at ClinicalTrials.gov on the 16 July 2015 (identifier NCT02509988); Universal Trial Number U1111-1171-8056. This study was academic-led by the EpiGen Global Research Consortium.

Circulating Structurally Related (-)-Epicatechin Metabolite Species and Levels after Sustained Intake of a Cocoa Powder High in Polyphenols Are Comparable to Those Achieved after a Single Dose.

BACKGROUND: While the bioavailability of cocoa polyphenols, particularly of the monomer (-)-epicatechin, has been investigated after a single-dose intake, the effect of sustained cocoa consumption on the metabolic profile of the structurally related (-)-epicatechin metabolites (SREMs) has not been investigated. METHODS: A randomized, controlled crossover clinical trial in healthy young adults (18-40 year) was conducted to evaluate SREMs after consumption of a single-dose and after daily consumption of 1.3 g of polyphenol-rich cocoa powder for 28 days. The circulating SREMs were measured by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). RESULTS: Twenty subjects (eleven males and nine females) were enrolled. The SREMs concentrations increased to 1741 ± 337 nM after a single-dose and to 1445 ± 270 nM after sustained supplementation. Sulfate conjugates showed higher levels in females (p < 0.05). The epicatechin-3'-glucuronide (E3'G) and epicatechin-3'-sulfate (E3'S) were the most abundant metabolites in all subjects. A high intra-individual correlation (r = 0.72, p < 0.001) between SREMs concentrations after single-dose and sustained supplementation was observed. The antioxidant capacity of plasma did not change in response to the intervention and was not correlated with any of the SREMs. CONCLUSION: The individual SREMs profile and concentrations after a 28-day supplementation are comparable to those after a single dose.

Recommendations for Oil Extraction and Refining Process to Prevent the Formation of Monochloropropane-diol Esters in Sunflower Oil.

The aim of this study was to identify the chlorine source during sunflower oil production and propose mitigation strategies in order to prevent monochloropropane-diol ester (MCPDE) formation. Whole sunflower seeds, the separated kernel, hulls, and pressed cake were studied to pinpoint the location of chlorine donors originating from the crop. Acid-water-based degumming, bleaching, cooling, and heat treatment were performed to mimic the current refining process practices. Various oil extraction and refining scenarios were tested. MCPDE and total monochloropropane-diol (MCPD) content of the heat-treated samples were determined by liquid chromatography-HRMS and by an AOCS Official method. The results show that the oil produced from crop hulls and the bleaching clay used are the strongest chlorine sources boosting the MCPDE formation. Using a mixture of pressed and solvent extracted cake oil as model, total 3-MCPD decreased by a factor of 2 when applying static cooling in combination with a washed bleaching clay.

Biomarker-based validity of a food frequency questionnaire estimating intake in Brazilian children and adolescents.

This study evaluated the validity of nutrient and food group intakes estimated by an FFQ against biomarkers. A 71-item semiquantitative FFQ was administered to 210 Brazilian children and adolescents aged 9-13 years. Intakes were correlated with biomarkers in plasma and red blood cells. Correlations between nutrients and their biomarkers were presented for animal protein, myristic acid (C14:0), EPA, DHA, ß-carotene, folate, and vitamins B3, B5 and B6. Food groups and biomarkers were correlated as follows: fish products with EPA and DHA; milk and dairy with C14:0, pyridoxal 5'-phosphate and vitamin B12; total vegetables and dark green and orange vegetables with ß-carotene; 5-methyltetrahydrofolate with green vegetables; and flour products with para-aminobenzoylglutamic acid. This FFQ is a valid tool for ranking Brazilian children and adolescents according to their intake of several nutrients and food groups.

Vitamins and carotenoids in human milk delivering preterm and term infants: Implications for preterm nutrient requirements and human milk fortification strategies.

Differences in vitamin and carotenoids content of human milk (HM) produced for infants born at term and preterm is poorly understood. In this study, HM was collected weekly for four and two months post-partum for preterm and term groups, respectively. Nutrients of interest, from single full breast expressions were measured by liquid chromatography coupled with mass spectrometry. Microbiological assay was employed for vitamin B12. When compared at equivalent post-partum age, vitamins B1, B2, B6, and B9 were significantly higher in preterm than in term HM, but only during the first two weeks. No significant differences were observed for A, E, B3 and B12 between groups. Lycopene was the only carotenoid exhibiting a significant higher concentration in term than in preterm HM between weeks 1 and 4 post-partum. When compared at equivalent post-menstrual age, preterm milk was significantly higher for vitamins B1, B2, B3, B6 and B9 and lower levels of vitamins A, E, ß-carotene, ß-cryptoxanthin, lutein, zeaxanthin and lycopene compared to their term counterparts. These results suggest that preterm breastfed infants at term equivalent age may receive lower amounts of these micronutrients than breast-fed term neonates, possibly highlighting the need to supplement or fortify their nutritional intake with vitamins and carotenoids. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT #02052245.

Mitigating the formation of monochloropropanediol diesters in vegetable oils by removing their residual sediments.

This study investigates whether the formation of monochloropropane diol fatty acid esters (MCPDE) can be mitigated by removing the residual sediments from vegetable oils. Settling and centrifugation were conducted in crude sunflower and palm oil and the purified oils and their sediment-rich fractions were heated and analyzed for their MCPDE content. Increased MCPDE levels by factors of x2 to x6 were found in the sediment-rich fractions of settled sunflower oils compared to the sediment-free oil. The sediment-containing fraction could be however purified by ultracentrifugation resulting in the mitigation of MCPDE levels by a factor of 10. The effect of residual sediment on the MCPDE formation was also confirmed in the case of palm oil showing x2 to x10 more MCPDE formation in the sediment containing fractions compared to the purified oil. These results confirm that the mechanical removal of the trace sediments from crude vegetable oils results in reduced MCPDE levels.

Removal of organochlorines from vegetable oils and its benefits in preventing formation of monochloropropanediol diesters.

This paper reports the first results on depleting certain organochlorines from vegetable oils without the use of any solvent in order to mitigate monochloropropanediol diesters (MCDPE). The concept is based on separating the organochlorines from the bulk oil by using trapping agents (e.g. monoacylglycerols) that can be easily separated from the oil. The process starts by mixing and homogenizing crude vegetable oils with the trapping agent and subsequently separating the trapping agent from the oil bulk via crystallization. The proof-of-concept of the approach is demonstrated on a spiked sunflower model system, solvent extracted crude sunflower oil, industrially produced crude soybean and corn oils. The depletion of organochlorines in the crude oils and its beneficial effect on the MCPDE content in the heat treated samples is measured by LC-MS. The depletion efficacy of the monitored organochlorines was estimated to be in the 60-95 % range. Both the melting point and polarity of the trapping agents affected the depletion efficacy of the organochlorines. Trapping agents with higher melting point and polarity, such as monostearin were more effective in comparison to high melting point but less polar agents such as palm stearin or agents rich in polar but low melting point monolinolein/monoolein. The effect of organochlorine depletion on the subsequent MPCDE levels in heat treated oil was in the range of 60-90 % reduction depending on the type of the studied oil.

Quantification of Vitamins A, E, and K and Carotenoids in Submilliliter Volumes of Human Milk.

Background: Human milk is the optimal nutrition for all newborns in the first 6 months of life. In order to assess the nutritional needs of the breastfed infant, human milk is often characterized for multiple nutrients. Objective: To ensure that we minimize the volume of milk dedicated for research and optimize the number of nutrients characterized, we developed analytical methodologies for the determination of vitamins A (retinol), E (alpha and gamma tocopherol), K (phylloquinone and menaquinone-4), and five carotenoids (ß-carotene, lycopene, ß-cryptoxanthin, lutein, and zeaxanthin) using <1 mL human milk. Method: Vitamins E and K and carotenoids are simultaneously isolated from 750 µL milk by liquid-liquid extraction (LLE). Tocopherols and carotenoids are determined by normal-phase LC with fluorescence and ultraviolet detection respectively. Vitamin K is analyzed on the same extracts after resuspension and clean-up by reversed phase liquid chromatography coupled to tandem MS. The analysis of vitamin A involves saponification of 200 µL milk followed by LLE and determination by normal-phase LC with UV detection. Results: Full single-laboratory validation at four different concentration levels is presented. Recovery rates were within 90-105% in all except one case (retinol at 1.9 µg/mL, 88% recovery), with RSDs of repeatability and intermediate reproducibility below 10 and 15%, respectively for all the compounds. Conclusions and Highlights: To the best of best knowledge, this is the first report that allows for the characterization and quantification of vitamins A, E, and K and five carotenoids in <1 mL human milk.

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.

Clinical and Vitamin Response to a Short-Term Multi-Micronutrient Intervention in Brazilian Children and Teens: From Population Data to Interindividual Responses.

SCOPE: Micronutrients are in small amounts in foods, act in concert, and require variable amounts of time to see changes in health and risk for disease. These first principles are incorporated into an intervention study designed to develop new experimental strategies for setting target recommendations for food bioactives for populations and individuals. METHODS AND RESULTS: A 6-week multivitamin/mineral intervention is conducted in 9-13 year olds. Participants (136) are (i) their own control (n-of-1); (ii) monitored for compliance; (iii) measured for 36 circulating vitamin forms, 30 clinical, anthropometric, and food intake parameters at baseline, post intervention, and following a 6-week washout; and (iv) had their ancestry accounted for as modifier of vitamin baseline or response. The same intervention is repeated the following year (135 participants). Most vitamins respond positively and many clinical parameters change in directions consistent with improved metabolic health to the intervention. Baseline levels of any metabolite predict its own response to the intervention. Elastic net penalized regression models are identified, and significantly predict response to intervention on the basis of multiple vitamin/clinical baseline measures. CONCLUSIONS: The study design, computational methods, and results are a step toward developing recommendations for optimizing vitamin levels and health parameters for individuals.

Concentrations of Carotenoids and Tocopherols in Breast Milk from Urban Chinese Mothers and Their Associations with Maternal Characteristics: A Cross-Sectional Study.

Milk composition remains the best estimate of infant requirements. The aims of this study were to quantify carotenoids and tocopherols in human milk from healthy Chinese mothers, and to explore their associations with lactation stage, region, socio-economic and obstetric characteristics, and dietary intake. Human milk was obtained from 509 healthy mothers, and concentrations of carotenoids and tocopherols were analyzed by Ultra High Performance Liquid Chromatography. The mothers' socio-economic and obstetric characteristics and dietary intake through a single 24-h dietary recall were evaluated. The median concentrations (µg/100 mL) of each component of 0-4 days, 5-11 days, 12-30 days, 31-60 days, 61-120 days, and 121-240 days postpartum were respectively as follows: ß-carotene 8.0, 2.8, 2.1, 1.7, 1.9, 1.8; ß-cryptoxanthin 6.2, 3.4, 2.4, 1.7, 1.8, 2.1; lutein 5.7, 7.0, 2.2, 2.9, 2.8, 3.7; lycopene 6.3, 2.5, 1.8, 1.4, 1.4, 1.5; zeaxanthin 1.0, 1.4, 0.8, 0.8, 1.0, 1.1; α-tocopherol 645, 382, 239, 206, 212, 211; γ-tocopherol 68, 63, 70, 73, 68, 88. The levels of those components varied significantly among different lactation stages and presented regional differences. Associations of carotenoid contents with maternal education, delivery mode, and present body mass index were found in multivariate analyses. These results suggested that lactation stage, region, and socio-economic and obstetric factors were associated with human milk concentrations of carotenoids and tocopherols in healthy Chinese mothers.

Regional, socioeconomic, and dietary factors influencing B-vitamins in human milk of urban Chinese lactating women at different lactation stages.

BACKGROUND: Adequate B-vitamins concentrations in human milk are considered to be a prerequisite for healthy development of infants in early life. This study aims to determine the concentrations of B-vitamins in human milk from Chinese women and the relationships between their concentrations and different geographical origin, lactation stages, socioeconomic characteristics, and dietary intake. METHODS: Human milk was obtained from 443 healthy lactating women from Beijing (n = 150), Suzhou (n = 146), and Guangzhou (n = 147) cities. Thiamine, riboflavin, vitamin B3 (nicotinamide and nicotinic acid), and vitamin B6 (pyridoxal, pyridoxine, and pyridoxamine) in human milk were analyzed by high performance liquid chromatography-tandem mass spectrometry. Pantothenic acid, biotin, and folates in human milk were analyzed by microbiological assay. The information from one 24-h dietary recall and socioeconomic characteristics were collected by interview and structured questionnaire, respectively. RESULTS: B-vitamins concentrations in human milk varied greatly among individuals. The median concentrations of B-vitamins of postpartum 5-11 d, 12-30 d, 31-60 d, 61-120 d, and 121-240 d were respectively as follows: thiamine 3.13, 5.07, 4.28, 5.65, 6.28 (µg/100 g); riboflavin 20.8, 20.2, 11.9, 13.6, 15.6 (µg/100 g); vitamin B3 194.0, 300.0, 261.0, 212.5, 218.0 (µg/100 g); pantothenic acid 236.5, 291.0, 254.0, 179.0, 189.0 (µg/100 g); vitamin B6 6.34, 7.58, 8.60, 9.34, 10.20 (µg/100 g); biotin 0.462, 0.834, 0.606, 0.523, 0.464 (µg/100 g); folates 0.730, 2.390, 2.440, 2.420, 2.330 (µg/100 g). The levels of B-vitamins presented regional differences and varied significantly among different lactation stages. The inversely associations of thiamine, vitamin B6, and folates with maternal BMI were found in multivariate analyses (p < 0.05), as well as higher pantothenic acid, folates, and biotin concentrations in lactating women with supplement intake when compared with those without (p < 0.05). Riboflavin concentrations associated with regular exercise was found in multivariate analyses (p < 0.05). CONCLUSIONS: The present study indicated regional and socioeconomic factors, lactation stage, and supplement intake may influence B-vitamins concentrations of human milk in healthy Chinese mothers. Further studies on accurate and complete analysis of all vitamin forms are crucial for giving a more comprehensive understanding of vitamin status in human milk. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01971671. Registered 13 October 2013.

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans.

Determination of folate in infant formula and adult/pediatric nutritional formula by ultra-high performance liquid chromatography-tandem mass spectrometry: First Action 2013.13.

A UHPLC-MS/MS method for the determination of folate (vitamin B9) in infant formula and adult/pediatric nutritional formula was assessed for compliance with standard method performance requirements set forth by the AOAC INTERNATIONAL Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN). A single-laboratory validation (SLV) study was conducted as the first step in the process to validate the method. In the study, 12 matrixes, representing the range of infant and adult nutritional products, were evaluated for folate [the sum of supplemental folic acid plus 5-methyl tetrahydrofolic acid (5-Me THF)]. Method response was linear in the range of 1.0-900 ng/mL, corresponding to 0.33-300 microg/l100 g in reconstituted sample. LOD for folic acid and 5-Me THF, expressed in reconstituted product, were 0.10 microg/100 g and 0.05 microg/100 g, respectively, and LOQ were 0.33 microg/100 g and 0.10 microg/100 g, respectively. Repeatability was <5.3% and intermediate precision was <5.5%. Recovery rates of spiking at 50 and 100% of target values in nonfortified products were within 90-110%. Evaluation of trueness was performed on Certified Reference Material (SRM 1849 Infant/Adult Nutritional Formula) and gave 96.4% of theoretical value. Based on the results of the SLV, the method meets the SPIFAN requirements for AOAC First Action status for the determination of folates in infant formula and adult/pediatric nutritional formula.

Vitamin C in infant formula and adult/pediatric nutritional Formula by ultra-performance liquid chromatography with Ultraviolet detection: first action 2012.22.

During the AOAC Annual Meeting held in Las Vegas, NV from September 30 to October 3, 2012, the Stakeholder Panel on Infant Formula and Adult Nutritionals convened to review single-laboratory validation data submitted for the method, Vitamin C in Adult/Pediatric Formula by Ultra-Performance Liquid Chromatography with Ultraviolet Detection. This method is a modified version of the method "HPLC-UV Determination of Total Vitamin C in a Wide Range of Fortified Food Products" previously published in Food Chem., 94, 626-631 (2006). The SLV data from the modified method were reviewed and compared to the standard method performance requirements (SMPR 2012.012), and it was concluded that the method meets the requirements. The method was approved as AOAC Official First Action. The method is based on the acidic extraction of ascorbic acid in the presence of Tris[2-carboxyethyl] phosphine (TCEP) as a reducing agent. Separation was achieved on a C18 column with a sodium acetate eluent (pH 5.4) combined with TCEP and decylamine as an ion-pairing agent. Accuracy rates were between 90 and 100%. Repeatability RSD (RSD,) ranged from 1.4 to 2.5%, and intermediate reproducibility RSD (RSDiR) ranged from 1.3 to 7.5%.

Identification of cholesteryl ester of ferulic acid in human plasma by mass spectrometry.

Epidemiological data suggests that regular consumption polyphenol rich foods and beverages is associated with a reduced risk of certain pathological conditions. While the in vivo "per se" antioxidant benefit of polyphenols still has not been clearly demonstrated, it has been suggested that phenolic acids can be incorporated into low-density lipoproteins (LDL). In the present study, we hypothesized that esterification of phenolic acids - such as ferulic acid - with lipophilic substances such as cholesterol can occur in vivo. To prove this hypothesis, we have synthesized pure cholesteryl-ferulate standard and used gas- and liquid chromatography coupled with mass spectrometry to confirm the presence of endogenous form in human plasma. The detection and identification of cholesteryl ferulate was based on: (1) matching gas- and liquid chromatographic retention time with the reference standard; (2) accurate mass of the molecular ion; (3) matching electron ionization mass spectrum and (4) matching electrospray product ion spectrum. The identified cholesteryl ferulate demonstrated an in vitro antioxidant capacity in various assays. The present study confirmed that phenolic acid can be found in human plasma as lipophilic conjugates which exert antioxidant capacity. These molecules can potentially be involved in the protection of lipoproteins against oxidative damages.

Specific dietary preferences are linked to differing gut microbial metabolic activity in response to dark chocolate intake.

Systems biology approaches are providing novel insights into the role of nutrition for the management of health and disease. In the present study, we investigated if dietary preference for dark chocolate in healthy subjects may lead to different metabolic response to daily chocolate consumption. Using NMR- and MS-based metabolic profiling of blood plasma and urine, we monitored the metabolic response of 10 participants stratified as chocolate desiring and eating regularly dark chocolate (CD) and 10 participants stratified as chocolate indifferent and eating rarely dark chocolate (CI) to a daily consumption of 50 g of dark chocolate as part of a standardized diet over a one week period. We demonstrated that preference for chocolate leads to different metabolic response to chocolate consumption. Daily intake of dark chocolate significantly increased HDL cholesterol by 6% and decreased polyunsaturated acyl ether phospholipids. Dark chocolate intake could also induce an improvement in the metabolism of long chain fatty acid, as noted by a compositional change in plasma fatty acyl carnitines. Moreover, a relationship between regular long-term dietary exposure to a small amount of dark chocolate, gut microbiota, and phenolics was highlighted, providing novel insights into biological processes associated with cocoa bioactives.

Absorption of dimethoxycinnamic acid derivatives in vitro and pharmacokinetic profile in human plasma following coffee consumption.

SCOPE: This study reports the 24 h human plasma pharmacokinetics of 3,4-dimethoxycinnamic acid (dimethoxycinnamic acid) after consumption of coffee, and the membrane transport characteristics of certain dimethoxycinnamic acid derivatives, as present in coffee. METHODS AND RESULTS: Eight healthy human volunteers consumed a low-polyphenol diet for 24 h before drinking 400 mL of commercially available coffee. Plasma samples were collected over 24 h and analyzed by HPLC-MS(2) . Investigation of the mechanism of absorption and metabolism was performed using an intestinal Caco-2 cell model. For the first time, we show that dimethoxycinnamic acid appears in plasma as the free aglycone. The time to reach the C(max) value of approximately 0.5 µM was rapid, T(max) = 30 min, and showed an additional peak at 2-4 h for several subjects. In contrast, smaller amounts of dimethoxy-dihydrocinnamic acid (C(max) ∼ 0.1 µM) peaked between 8 and 12 h after coffee intake. In the cell model, dimethoxycinnamic acid was preferentially transported in the free form by passive diffusion, and a small amount of dimethoxycinnamoylquinic acid hydrolysis was observed. CONCLUSION: These findings show that dimethoxycinnamic acid, previously identified in plasma after coffee consumption, was rapidly absorbed in the free form most likely by passive diffusion in the upper gastrointestinal tract.

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.