Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

Use of Theoretical Subjects to Develop a Method for Assessing Equivalence of Dietary Vitamin A in a Mixed Diet.

BACKGROUND: Although the vitamin A (VA) equivalency of provitamin A carotenoids from single foods or capsules has been studied using several approaches, there is currently no reliable method to determine VA equivalency for mixed diets. OBJECTIVES: To reach the objective of identifying a method to determine the VA equivalency of provitamin A carotenoids in mixed diets, we tested a new approach using preformed VA as proxy for provitamin A. METHODS: We studied 6 theoretical subjects who were assigned physiologically plausible values for dietary VA intake, retinol kinetic parameters, plasma retinol pool size, and VA total body stores. Using features in the Simulation, Analysis and Modeling software, we specified that subjects ingested a tracer dose of stable isotope-labeled VA on day 0 followed by 0-µg supplemental VA or 200, 400, 800, 1200, 1600, and 2000 µg VA daily from day 14 to day 28; we assigned VA absorption to be 75%. For each supplement level, we simulated plasma retinol specific activity (SAp) over time and calculated the mean decrease in SAp relative to 0 µg. Group mean data were fitted to a regression equation to calculate predicted VA equivalency at each supplement level on day 28. RESULTS: For each subject, higher VA supplement loads resulted in lower SAp, with the magnitude of the decrease differing among subjects. The mean predicted amount of absorbed VA was within 25% of individual subjects' assigned amount for 4 of the 6 subjects, and the mean ratio of predicted to assigned amount of absorbed VA over all supplement loads ranged from 0.60 to 1.50, with an overall mean ratio of 1.0. CONCLUSIONS: Results for preformed VA suggest that this protocol may be useful for determining VA equivalency of provitamin A carotenoids in free-living subjects if mixed diets with known provitamin A content were substituted for the VA supplements.

Challenges in the determination of total vitamin B12 by cyanidation conversion: insights from stable isotope dilution assays.

Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations.

Development and Validation of a Stable Isotope Dilution Headspace-SPME-GC/MS Method for the Determination of Vanillin in Fragrant Vegetable Oils.

It has been reported that vanillin has been intentionally added to enhance the taste and flavor of low-quality vegetable oils. Therefore, it is crucial to investigate the accurate concentrations of vanillin in three types of fragrant vegetable oils commonly consumed in China. In this study, a method has been developed for the quantification of vanillin in commercial fragrant vegetable oils using the stable isotope dilution assay (SIDA) and headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography/mass spectrometry (GC/MS). The limit of detection (LOD) and limit of quantification (LOQ) of the analyte were determined to be 20 µg kg-1 and 50 µg kg-1, respectively. The validation study demonstrated that the recoveries ranged from 89% to 101%, with intra-day and inter-day precision being less than 7.46%. A survey of 80 commercially available fragrant vegetable oils was performed using the present method. Vanillin was found to be widely present in fragrant vegetable oils, with sesame oils showing the highest average content (842.6 µg kg-1), followed by rapeseed oils (262.1 µg kg-1) and peanut oils (115.0 µg kg-1). The results indicate that the proposed method is a simple, accurate, and eco-friendly approach for determining the presences of vanillin in fragrant vegetable oils.

The Combined Effect of Lactic Acid Bacteria and Galactomyces geotrichum Fermentation on the Aroma Composition of Sour Whey.

The increase in demand for food flavorings due to the shortening and simplification of food production technology also entails an increase in the demand for new technologies for their production. The biotechnological production of aromas is a solution characterized by a high efficiency, an independence from environmental factors and a relatively low cost. In this study, the influence of the implementation of lactic acid bacteria pre-fermentation into the production of aroma compounds by Galactomyces geotrichum on a sour whey medium on the intensity of the obtained aroma composition was analyzed. The monitoring of the culture in terms of biomass buildup, the concentration of selected compounds, and the pH resulted in the confirmation of interactions between the analyzed microorganisms. The post-fermentation product underwent a comprehensive sensomic analysis for the identification and quantification of the aroma-active compounds. The use of gas chromatography-olfactometry (GC-O) analysis and the calculation of odor activity values (OAVs) allowed 12 key odorants to be identified in the post-fermentation product. The highest OAV was found for phenylacetaldehyde with a honey odor (1815). The following compounds with the highest OAVs were 2,3-butanedione with a buttery aroma (233), phenylacetic acid with a honey aroma (197), 2,3-butanediol with a buttery aroma (103), 2-phenylethanol with a rosy aroma (39), ethyl octanoate with a fruity aroma (15), and ethyl hexanoate with a fruity aroma (14).

Creatinine standardization: a key consideration in evaluating whole blood creatinine monitoring systems for CKD screening.

Early detection of CKD using point of care creatinine and eGFR testing improves patient management outcomes. We undertook a field study to evaluate the use of a whole blood creatinine/eGFR device to screen a rural Nicaraguan population to determine the variability between creatinine methods and specimen types. All specimens including capillary and venous dried blood spots (DBS) were tested with an isotope dilution liquid chromatography mass spectrometry (ID-LCMS) gold standard method. This is to our knowledge the first time a capillary whole blood (POC) method has been directly compared to the gold standard IDMS method, through the novel approach of using dried blood spots. Capillary and venous whole blood specimens were obtained and tested directly with the BCMS method, and then, DBS samples were prepared. Venous plasma specimens were tested using three laboratory analyzer creatinine methods. DBS were sent to the site performing ID-LCMS. Control samples were also prepared to assess the stability of shipment and storage of DBS. The ID-LCMS method was aligned using primary and secondary standards. Sixty-six (66) patients participated in the study, and the CKD prevalence rate was 7.8%. While all creatinine methods showed a good correlation to ID-LCMS, there was a positive bias (mean absolute bias range: 0.21-0.63 mg/dL). All methods used were 100% sensitive, but specificity varied from 62.7 to 94.9% with PPV ranging from 25 to 62.5%. A correction factor was used to align the values from each method to ID-LCMS which improved the specificity of each method. This study used a unique DBS approach to align capillary whole blood creatinine to ID-LCMS. To ensure reliability of BCMS for identifying screened patients with CKD, it is important to establish IDMS traceability and alignment prior to undertaking CKD studies.

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17ß-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC-MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01-2 ng mL-1 for PREGS, DHEAS, and CRTS and of 0.05-10 ng mL-1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL-1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6-112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 µL saliva samples.

Breast Milk-Derived Retinol Is a Potential Surrogate for Serum in the 13C-Retinol Isotope Dilution Test in Zambian Lactating Women with Vitamin A Deficient and Adequate Status.

BACKGROUND: Vitamin A (VA) deficiency (VAD) affects ∼19 million pregnant women worldwide. The extent of VAD in Zambian women of reproductive age is unknown owing to lack of survey inclusion or the use of static serum retinol concentrations, a low-sensitivity biomarker. OBJECTIVES: This cross-sectional study employed isotopic techniques to determine VA status with serum and milk among women aged 18-49 y (n = 197) either lactating with infants aged 0-24 mo or nonlactating with or without infants. METHODS: Assistants were trained and piloted data collection. Demographic data, anthropometry, and relevant histories were obtained including malaria and anemia. For retinol isotope dilution (RID), baseline fasting blood and casual breast milk samples were collected before administration of 2.0 µmol 13C2-retinyl acetate and 24-h dietary recalls. On day 14, blood (n = 144) and milk (n = 66) were collected. Prevalence of total liver VA reserves (TLR) ≤0.10 µmol/g was defined as VAD with comparison to the DRI assumption of 0.07 µmol/g as minimally acceptable for North Americans. RESULTS: When a 20% adjustment for dose lost to milk was made in the RID equation for lactation, mean total body VA stores (TBS) for lactating women were 25% lower than for nonlactating women (P < 0.01), which was not the case without adjustment (P = 0.3). Mean ± SD TLR for all women were 0.15 ± 0.11 µmol/g liver. Using retinol purified from breast milk instead of serum for RID analysis yielded similar TBS and TLR, which were highly correlated between methods (P < 0.0001). Serum retinol ≤0.70 µmol/L had 0% sensitivity using either VAD liver cutoff and milk retinol ≤1.0 µmol/L had 42% sensitivity for VAD at 0.10 µmol/g. CONCLUSIONS: Determining accurate VA status among women of reproductive age, especially lactating women, forms a basis for extrapolation to the general population and informing policy development and program implementation.

Identification of Rotundone as an Important Contributor to the Flavor of Oak-Aged Spirits.

Experiments were conducted to identify a compound responsible for a spicy, woody, incense-like odor note in oak-aged spirits. The target compound was extracted from oak wood and various oak-aged spirits and analyzed by multidimensional (heart-cut) gas chromatography-mass spectrometry-olfactometry (MD-GC-MS-O), and was unambiguously identified as the sesquiterpene ketone, 5-isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexadydro-1(2H)-azulenone (rotundone). Quantitation of the trace-level target compound was done by stable isotope dilution analysis (SIDA) in a variety of oak-aged spirits, including bourbon, rye, Tennessee whiskey, scotch, rum, and tequila. The content of rotundone was found to increase as a function of years of barrel aging for 4-, 8-, and 12-year-old bourbons obtained from the same manufacturer, thus confirming its origin to be from oak. In addition, odor-activity values (OAVs) were compared for selected potent odorants, including rotundone, in the same 4-, 8-, and 12-year-old bourbons, which indicated the relative importance of rotundone in the overall flavor of oak-aged spirits.

Immunoaffinity Targeted Mass Spectrometry Analysis of Human Plasma Samples Reveals an Imbalance of Active and Inactive CXCL10 in Primary Sjögren's Syndrome Disease Patients.

One of the most important advantages of mass spectrometry is the ability to quantify proteins and their modifications in parallel to obtain a holistic picture of the protein of interest. Here, we present a hybrid immunoaffinity targeted mass spectrometry (MS) method that combines efficient pan-antibody enrichment of a specific protein from plasma with the selectivity of high-resolution targeted MS analysis to quantitate specific proteoforms of interest. We used this approach to quantify plasma levels of the chemokine CXCL10 that has been associated with many immunological disorders such as systemic lupus erythematosus and primary Sjögren's Syndrome (pSS). The hybrid approach enabled sensitive, specific, and simultaneous quantification of total, full-length (active) CXCL101-77 and DPP4-truncated (inactive) CXCL103-77 in human plasma down to the low pg/mL level, reaching ELISA sensitivities. Samples from 30 control subjects and 34 pSS patients (n = 64) were analyzed. The ratio of CXCL101-77 to truncated CXCL103-77 was significantly increased in patients with pSS and provided the highest correlation with pSS disease activity. Therefore, this CXCL10 proteoform ratio represents an interesting exploratory disease activity biomarker to further investigate. As this strategy can be readily adapted to other plasma proteins and proteoforms of interest, we are convinced that it will lead to a more detailed understanding of proteoforms in physiology and pathology yielding more relevant biomarkers and drug targets.

Development of a LC-MS/MS method using stable isotope dilution for the quantification of individual B6 vitamers in fruits, vegetables, and cereals.

Vitamin B6 comprises an important set of molecules tightly interwoven with the human amino acid, fatty acid, and carbohydrate metabolism. Analytical methods striving for the quantification of individual B6 vitamers so far mostly rely on methods based on HPLC in combination with fluorescence detection, but their application encounters multiple difficulties due to the chemical divergence of the single vitamers. The present study describes the development of a method based on LC-MS/MS and stable isotope dilution assay (SIDA) for the simultaneous quantification of five vitamers (PN, PL, PM, PMP, and PNG) of the B6 group in food samples. [13C3]-PN, [13C3]-PL, and [13C6]-PNG were applied as internal standards for the analysis of PN, PL, and PNG. PM and PMP were quantified via matrix-matched calibration referring to [13C3]-PN. The developed method was validated using starch matrix. The limits of detection and quantification ranged from 0.0028 to 0.02 mg/kg and from 0.0085 to 0.059 mg/kg, respectively, for all analytes. Calculated recoveries varied from 92 to 111%. Intra-injection precisions ranged from 0 to 9%, inter-day precisions from 4 to 10%, and intra-day precisions from 4 to 10%. A total of 14 plant-based food samples including fruits, vegetables, and cereals were examined for their content of vitamin B6 using the validated method. Furthermore, the first quantitation of PNG without enzymatic steps or divergent internal standards was undertaken utilizing LC-MS/MS and SIDA.

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry.

Mitogen-activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three-tiered kinase module-MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase-that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine-glutamic acid-tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal-regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)-selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID-SRM-MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono-phosphorylatable mutants ERKT202A and ERKY204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.

Stable isotope dilution assay for the accurate determination of tricaine in fish samples by HPLC-MS-MS.

Tricaine methanesulfonate is one of most commonly used anesthetics in fish during blood sampling, artificial propagation and long-distance transportation. In this study, an accurate method for the quantitative determination of tricaine in fish samples by a stable isotope dilution assay coupled with high-performance liquid chromatography-triple quadrupole mass spectrometry was developed. Tricaine-D5 was synthesized and used as an isotopically labeled internal standard for the determination of tricaine. The analytical performance of the method was validated for tricaine determination in marine fish and freshwater fish. The determination of tricaine was linear in the range of 2.0-200.0 µg L-1 . The limit of detection and limit of quantitation for fish muscle tissues were 1.0 and 4.0 µg kg-1 , respectively. Good recoveries were obtained in the range of 92.08-97.50%. The inter- and intra-assay relative standard deviations (RSD values) were investigated, and the values were 0.39-3.01 and 0.85-2.77%, respectively. The values of CCα and CCß were 10.21-10.43 and 10.42-10.87 µg kg-1 , respectively. The clearance of MS-222 from grass carp was further studied using our method. The results demonstrate that MS-222 could be well absorbed and rapidly eliminated after bath administration.

Quantification of Cl-PAHs and their parent compounds in fish by improved ASE method and stable isotope dilution GC-MS.

This study aimed at developing a simple and accurate method for determination of emerging chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) in fish by stable isotope dilution gas chromatography tandem mass spectrometry. Fish samples were extracted by improved accelerated solvent extraction (ASE) method. Matrix effects were observed, and matrix-matched calibration was verified with good intra-day and inter day precisions (lower than 16.1% and 15.1% respectively). Method detection limits were 0.10-5.62 ng g-1 (dry weight) with satisfactory linearity, and recoveries ranged from 50% to 150%, with relative standard deviation values less than 18.5% at different concentration levels. This improved ASE method was proved to be suitable for analyzing Cl-PAHs in fish samples, with good analytical selectivity, linearity, recovery and precision. Furthermore, the composition analysis revealed that chlorinated compounds of phenanthrene, pyrene and acenaphthene were dominated in Cl-PAHs contaminants. The correlationship between the pollution of Cl-PAHs and their corresponding parent structures in fish samples was also analyzed in detail.

Analysis of Potent Odour-Active Volatile Thiols in Foods and Beverages with a Focus on Wine.

Certain volatile thiols are some of the most potent odour-active molecules that are found in nature. Thiols play significant roles in the aroma qualities of a range of foods and beverages, including wine, with extremely low odour detection thresholds (nanogram per litre range). A fundamental understanding of their formation, fate, and impact essentially depends on the development of suitable analytical methods. The analysis of volatile thiols in foods and beverages is a challenging task when considering (1) the complexity of food and beverage matrices and (2) that thiols are highly reactive, low molecular-weight volatiles that are generally present at trace to ultra-trace concentrations. For the past three decades, the analytical evaluation of volatile thiols has been intensively performed in various foods and beverages, and many novel techniques related to derivatisation, isolation, separation, and detection have been developed, particularly by wine researchers. This review aims to provide an up-to-date overview of the major analytical methodologies that are proposed for potent volatile thiol analysis in wine, foods, and other beverages. The analytical challenges for thiol analysis in foods and beverages are outlined, and the main analytical methods and recent advances in methodology are summarised and evaluated for their strengths and limitations. The key analytical aspects reviewed include derivatisation and sample preparation techniques, chromatographic separation, mass spectrometric detection, matrix effects, and quantitative analysis. In addition, future perspectives on volatile thiol research are also suggested.

Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid.

Absolute protein quantification by selected reaction monitoring (SRM, also MRM) is an alternative to immunoassays, and the gold standard here is the addition of stable-isotope labeled (SIL) proteins (PSAQ). Cerebrospinal fluid (CSF) is the preferred source of biomarkers for neurological diseases, and recent improvements in mass spectrometry enable the quantification of disease-relevant proteins in CSF. We used alpha-synuclein SRM to investigate alternatives to the PSAQ approach in human CSF regarding precision and accuracy, including SIL peptides, winged SIL (WiSIL) peptides, and quantitative protein epitope signature tags (QPrESTs). All approaches yielded precise results in CSF with CV values <15% in several runs for all four measured peptides. PSAQ and QPrEST also showed good accuracy (deviation ≤15%), whereas SIL and WiSIL peptides yielded deviations up to 54% that greatly depended on the measured peptide. Total protein concentration in CSF did not affect precision and accuracy. Thus, our study indicates that all four approaches are suitable for relative quantification of alpha-synuclein in CSF. QPrESTs are a valuable alternative to PSAQ in terms of precision and accuracy, although SIL and WiSIL peptides can yield accurate results as well when peptides are selected consciously.

Headspace solid-phase microextraction and gas chromatographic analysis of low-molecular-weight sulfur volatiles with pulsed flame photometric detection and quantification by a stable isotope dilution assay.

Low-molecular-weight volatile sulfur compounds such as thiols, sulfides, disulfides as well as thioacetates cause a sulfidic off-flavor in wines even at low concentration levels. The proposed analytical method for quantification of these compounds in wine is based on headspace solid-phase microextraction, followed by gas chromatographic analysis with sulfur-specific detection using a pulsed flame photometric detector. Robust quantification was achieved via a stable isotope dilution assay using commercial and synthesized deuterated isotopic standards. The necessary chromatographic separation of analytes and isotopic standards benefits from the inverse isotope effect realized on an apolar polydimethylsiloxane stationary phase of increased film thickness. Interferences with sulfur-specific detection in wine caused by sulfur dioxide were minimized by addition of propanal. The method provides adequate validation data, with good repeatability and limits of detection and quantification. It suits the requirements of wine quality management, allowing the control of oenological treatments to counteract an eventual formation of excessively high concentration of such malodorous compounds.

Stable isotope labeling-assisted GC/MS/MS method for determination of methyleugenol in food samples.

BACKGROUND: Methyleugenol is a common phenylpropanoid compound found in many plants and has been widely used as a flavoring agent in people's daily life. In this study, a stable isotope dilution assay-coupled gas chromatography/triple quadrupole mass spectrometry (SIDA-GC/MS/MS) method was developed for the quantitative determination of methyleugenol in food samples. Methyleugenol-D3 was synthesized and used as an isotope internal standard for the determination of methyleugenol. The QuEChERS (quick, easy, cheap, effective, rugged and safe) method was applied to the clean-up of food sample extracts. Confirmation and quantification were carried out by GC/MS/MS. RESULTS: The analytical performance of the method was validated. The determination range of methyleugenol was linear from 4 to 500 µg L-1 . Method detection limits for solid food samples, semi-solid food samples and liquid beverages were 50, 50 and 1 µg kg-1 respectively. Satisfactory recoveries in the range 94.29-100.27% were obtained. Intra- and inter-day precision was also validated and the values were all lower than 9%. The method was successfully applied to quantify methyleugenol in different kinds of food samples. CONCLUSION: This article describes a new method for the accurate quantification of methyleugenol in food samples based on a stable isotope labeling-assisted GC/MS/MS method. Methyleugenol-D3 was synthesized and used as an isotope internal standard for the determination of methyleugenol. Excellent results were generated with the method, and the detection sensitivity and accuracy of the method were good. © 2018 Society of Chemical Industry.

Assessment of bioavailable B vitamin content in food using in vitro digestibility assay and LC-MS SIDA.

Standardized analytical methods, where each B vitamin is extracted from a given sample individually using separate procedures, typically ensure that the extraction conditions provide the maximum recovery of each vitamin. However, in the human gastrointestinal tract (GIT), the extraction conditions are the same for all vitamins. Here, we present an analytically feasible extraction protocol that simulates conditions in the GIT and provides a measure of the content of bioavailable vitamins using LC-MS stable isotope dilution assay. The results show that the activities of both human gastric and duodenal juices were insufficient to liberate absorbable vitamers (AV) from pure cofactors. The use of an intestinal brush border membrane (IBBM) fraction derived from the mucosal tissue of porcine small intestine ensured at least 70% AV recovery. The rate of AV liberation, however, was strongly dependent on the cofactor, e.g., in the case of NADH, it was magnitudes higher than in the case of thiamine diphosphate. For some vitamins in some food matrices, the use of the IBBM fraction assay resulted in lower values for the content of AV than conventional vitamin determination methods. Conventional methods likely overestimate the actual bioavailability of some vitamins in these cases. Graphical abstract Assessment of bioavailable B vitamin content in food.

Origins of the difference between food folate analysis results obtained by LC-MS/MS and microbiological assays.

To investigate the often reported disagreement in food folate quantitation between the microbiological assay and high-performance liquid chromatography methods, different foods were analyzed both by a microbiological assay and by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. For the LC-MS/MS analysis we emphasize the need for complete deconjugation of polyglutamic folate forms. Moreover, our results revealed no need for an additional enzyme treatment except in the deconjugation step. To check the efficiency of deconjugation without additional sample preparations, the amount of diglutamates was quantified and samples were screened for additional polyglutamates. A thorough investigation of a substance with a polyglutamate chain deconjugated like the folates revealed that it was an oxidation product of 5-methyltetrahydrofolate, a pyrazino-s-triazine called MeFox in previous reports. The latter is not microbiologically active and, therefore, does not contribute to the amount of total folates. But we found it is commonly present in foods, especially in those low in ascorbic acid. The microbiological assay showed different responses to the single vitamers. Therefore, it was necessary to perform calibration with the folate that had the highest portion in the folate distribution. The investigations showed that both methods can provide similar results when they both include a deconjugation step. This is particularly important for LC-MS/MS but probably also for the microbiological assay. Additionally, consideration of the folate distribution was found to be crucial for the accurate calibration of the microbiological assay.