Isotope Dilution Research Articles

Stable isotope dilution assay and HS-SPME-GCMS quantification of key aroma volatiles of Australian pineapple (Ananas comosus) cultivars

Pineapple aroma is one of the most important sensory quality traits that influences consumer purchasing patterns. Reported in this paper is a high throughput method to quantify in a single analysis the key volatile organic compounds that contribute to the aroma of pineapple cultivars grown in Australia. The method constituted stable isotope dilution analysis in conjunction with headspace solid-phase microextraction coupled with gas-chromatography mass spectrometry. Deuterium labelled analogues of the target analytes purchased commercially were used as internal standards. Twenty-six volatile organic compounds were targeted for quantification and the resulting calibration functions of the matrix -matched validated method had determination coefficients (R2) ranging from 0.9772 to 0.9999. The method was applied to identify the key aroma volatile compounds produced by popular pineapple cultivars such as ‘Aus Carnival’, ‘Aus Festival’, ‘Aus Jubilee’, ‘Aus Smooth (Smooth Cayenne)’ and ‘Aussie Gold (73-50)’, grown in Queensland, Australia. Pineapple cultivars varied in its content and composition of free volatile components, which were predominantly comprised of esters, followed by terpenes, alcohols, aldehydes, and ketones.

Chickens' eggs and the livers of farm animals as sources of perfluoroalkyl substances.

This study focuses on perfluoroalkyl substance (PFAS) content in chickens' eggs and the livers of farm animals. Chickens' eggs (n = 25) and the livers of cows (n = 10), chickens (n = 7) and horses (n = 3) were collected from various regions of Poland. Samples were analysed using the isotope dilution technique with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The mean lower bound (LB) sum of four PFAS (∑4 PFAS) concentrations (perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorohexanesulfonic acid (PFHxS)) were the highest in cows' livers (0.52 μg/kg) and much lower in chickens' (0.17 μg/kg) and horses' livers (0.13 μg/kg) and chickens' eggs (0.096 μg/kg). The ratio of ∑4 PFASs to the limits set by Commission Regulation (EU) 2023/915 was <7% for liver and <6% for eggs. Linear PFOS was the compound with the highest detection frequency (8% in eggs and 48% in all livers). In cows' livers it was detected in 80% of samples. The estimated exposure to LB ∑4 PFASs via consumption of liver tissue from farm animals (assuming 50 g and 100 g portions) was <52% of the tolerable weekly intake (TWI) for children and <17% of the TWI for adults. Dietary intake via the average portion of three eggs led to low exposure of <15% for children and <5% for adults. Neither eggs nor the livers of chickens or horses as analysed in this study are significant sources of PFASs, while cows' livers might contribute significantly to a child's overall dietary intake. Further investigation of PFOS in farm animal livers should be conducted.

Dosimetry of human exposure to furan and 2-methylfuran by monitoring urinary biomarkers

Furan and 2-methylfuran (2-MF) can form during food processing and accumulate in foods at various concentrations depending on processing technology and beverage/meal preparation methods applied prior to consumption. Here, we report a controlled dosimetry study with 20 volunteers (10 male, 10 female) to monitor dietary furan/2-MF exposure. The volunteers followed an eleven-day furan/2-MF-restricted diet in which they consumed freshly prepared coffee brew containing known amounts of furan and 2-MF on two separate occasions (250 mL and 500 mL on days 4 and 8, respectively). Urine was collected over the whole study period and analyzed for key metabolites derived from the primary oxidative furan metabolite cis-2-butene-1,4-dial (BDA) (i.e., Lys-BDA, AcLys-BDA and cyclic GSH-BDA) and the primary 2-MF metabolite acetylacrolein (AcA, 4-oxo-pent-2-enal) (i.e., Lys-AcA and AcLys-AcA). A previously established stable isotope dilution analysis (SIDA) method was utilized. Excretion kinetics revealed two peaks (at 0–2 and 24–36 h) for AcLys-BDA, Lys-BDA, AcLysAcA and LysAcA, whereas GSH-BDA showed a single peak. Notably, women on average excreted the metabolite GSH-BDA slightly faster than men, indicating gender differences. Overall, the study provided further insights into the spectrum of possible biomarkers of furan and 2-methyfuran metabolites occurring in the urine of volunteers after coffee consumption.

PFAS fate using lysimeters during degraded soil reclamation using biosolids.

Carbon- and nutrient-rich biosolids are used in agriculture and land reclamation. However, per- and polyfluoroalkyl substances (PFAS) typically present in biosolids raise concerns of PFAS leaching to groundwater and plant uptake. Here, we investigated PFAS persistence and leaching from biosolids applied to a site constructed artificially to mimic degraded soils. Treatments included biosolids and biosolids blended with mulch applied at different rates to attain either one and five times the agronomic N rate for vegetable crops and a control treatment with synthetic urea and triple superphosphate fertilizer. Leachates were collected for a 2-year period from 15-cm depth zero-tension drainage lysimeters. Soils were analyzed post biosolids application. PFAS were quantified using isotope-dilution, solid-phase extraction and liquid chromatography tandem mass spectrometry. Leachate profiles exemplified an initial high total PFAS concentration, followed by a sharp decline and subsequent small fluctuations attributed to pre-existing soil conditions and rainfall patterns. Quantifiable PFAS in leachate were proportional to biosolids application rates. Short-chain perfluoroalkyl acids (CF2<6) were dominant in leachate, while the percentage of longer chains homologues was higher in soils. A 43% biosolids blend with mulch resulted in 21% lower PFAS leachate concentrations even with the blend application rate being 1.5 times higher than biosolids due to the blend's lower N-content. The blending effect was more pronounced for long-chain perfluoroalkyl sulfonic acids that have a greater retention by soils and the air-water interface. Biosolids blending as a pragmatic strategy for reducing PFAS leachate concentrations may aid in the sustainable beneficial reuse of biosolids.

High-Coverage UHPLC-MS/MS Analysis of 67 Mycotoxins in Plasma for Male Infertility Exposure Studies.

Mycotoxins are a class of exogenous metabolites that are major contributors to foodborne diseases and pose a potential threat to human health. However, little attention has been paid to trace mycotoxin co-exposure situations in vivo. To address this, we devised a novel analytical strategy, both highly sensitive and comprehensive, for quantifying 67 mycotoxins in human plasma samples. This method employs isotope dilution mass spectrometry (IDMS) for approximately 40% of the analytes and utilizes internal standard quantification for the rest. The mycotoxins were classified into three categories according to their physicochemical properties, facilitating the optimization of extraction and detection parameters to improve analytical performance. The lowest limits of detection and quantitation were 0.001-0.5 μg/L and 0.002-1 μg/L, respectively, the intra-day precision ranged from 1.8% to 11.9% RSD, and the intra-day trueness ranged from 82.7-116.6% for all mycotoxins except Ecl, DH-LYS, PCA, and EnA (66.4-129.8%), showing good analytical performance of the method for biomonitoring. A total of 40 mycotoxins (including 24 emerging mycotoxins) were detected in 184 plasma samples (89 from infertile males and 95 from healthy males) using the proposed method, emphasizing the widespread exposure of humans to both traditional and emerging mycotoxins. The most frequently detected mycotoxins were ochratoxin A, ochratoxin B, enniatin B, and citrinin. The incidence of exposure to multiple mycotoxins was significantly higher in infertile males than in healthy subjects, particularly levels of ochratoxin A, ochratoxin B, and citrinin, which were significantly increased. It is necessary to carry out more extensive biological monitoring to provide data support for further study of the relationship between mycotoxins and male infertility.

Comparison of polyfunctional thiol, element, and total essential oil contents in 32 hop varieties from different countries

Volatile thiol 3-mercaptohexan-1-ol (3MH) and particularly 4-mercapto-4-methylpentan-2-one (4MMP) are highly potent flavour compounds in hops. For the determination, a simple and robust stable isotope dilution LC–MS/MS method was developed and applied to 32 hop varieties worldwide from harvest years 2019 and 2020. Limit of detection, precision, and recovery were 0.15 μg/kg, 10%, and 97–108%, respectively. Levels of 3MH and 4MMP ranged from 1.9 to 79.2 μg/kg and from undetectable to 37.1 μg/kg, respectively. Citra, Mosaic, and Strata were rich in both thiols. ICP analyses revealed, that variation of potassium content between the two harvest years was inversely correlated with that of manganese and rubidium (|r| ≥ 0.89) among 12 US varieties excluding Citra and Mosaic. Total essential oil content (0.34–2.7 mL/100 g) was inversely correlated with calcium content (|r| ≥ 0.65). Greatly varying thiol levels depending on variety, region and harvest year might lead to differing flavour results in beer.

Hair product use and urinary biomarker concentrations of non-persistent endocrine disrupting chemicals among reproductive-aged Black women

BackgroundStudies have shown an association between hair product use and adverse health outcomes. Scientists have hypothesized that exposure to endocrine-disrupting chemicals (EDCs) drives these associations, but few studies have directly evaluated associations between hair product use and biomarkers of EDCs. Even more limited are studies of Black women, who frequently use EDC-containing products (e.g., hair relaxers). ObjectiveWe estimated associations between hair product use and EDC biomarker concentrations. MethodsWe leveraged cross-sectional data from the Study of Environment, Lifestyle, and Fibroids, a cohort of females aged 23–34 years who self-identified as Black/African American from the Detroit-metropolitan area (USA; n = 425). On structured questionnaires, participants reported their past 24-h and past 12-month use of hair products, including relaxers/straighteners/perms, styling products, moisturizers, oils, and hair food. We quantified urinary concentrations of 19 phthalate/phthalate alternative metabolites, 7 phenols, and 4 parabens using high performance liquid chromatography isotope dilution tandem mass spectrometry. EDC biomarker concentrations were creatinine-adjusted and natural log-transformed. We used multivariable linear regression to estimate mean percent differences in EDC biomarker concentrations and 95% confidence intervals (CIs) associated with hair product use, adjusting for sociodemographic confounders. ResultsHair product use was associated with greater concentrations of multiple EDC biomarkers. Notably, use of hair products in the previous 24 h (compared with non-use) was associated with 16.2% (95% CI = 0.7%, 35.9%), 35.0% (95% CI = 2.6%, 77.6%), and 32.3% (95% CI = 8.8%, 92.0%) higher concentrations of mono-isobutyl phthalate, methyl paraben, and ethyl paraben, respectively. Use of hair relaxers/straighteners/perms, styling products, moisturizers, oils, and hair food in the past 12 months was also associated with higher concentrations of multiple phthalate, phenol, and paraben biomarkers. ConclusionHair product use was associated with higher biomarker concentrations of multiple phthalates, phenols, and parabens. These findings suggest that hair products are potentially important exposure sources for hormonally-active chemicals among Black women.

Evaluating reference materials and common-Pb corrections for high-resolution apatite U[sbnd]Pb geochronology

We report isotope dilution thermal ionization mass spectrometry (ID-TIMS) and laser ablation split stream inductively coupled plasma mass spectrometry (LASS) UPb data for a suite of widely available reference apatites: Fish Canyon Tuff, Mount Dromedary, TEMORA 2, and Duluth Complex anorthosite. We apply different common-Pb correction strategies to the UPb data sets: (1) anchoring to a Stacey and Kramers (1975) model Pb composition; (2) unanchored 2-D 238U/206Pb-207Pb/206Pb isochron regressions; and (3) unanchored 3-D 238U/206Pb-207Pb/206Pb-204Pb/206Pb isochron regressions. The different common-Pb corrections yield consistent dates within each ID-TIMS and LASS data set, with 3-D regression method producing the highest precision isochrons. FCT apatite produces an ID-TIMS 3-D isochron age of 28.8 ± 3.7 Ma with (207Pb/206Pb)i = 0.851 ± 0.021. Mount Dromedary apatite yields an ID-TIMS 3-D isochron age of 98.4 ± 0.5 Ma with (207Pb/206Pb)i = 0.839 ± 0.003. TEMORA 2 apatite has an ID-TIMS 3-D isochron age of 402 ± 7 Ma and (207Pb/206Pb)i = 0.839 ± 0.008. Duluth Complex anorthosite apatite yields an ID-TIMS 3-D isochron age of 1077 ± 9 Ma with (207Pb/206Pb)i = 0.849 ± 0.046. The MSWDs associated with isochrons calculated from both the ID-TIMS and LASS data sets are larger than expected for a single age population, revealing complexities that are otherwise not captured by 2-D isochron methods. In the case of FCT apatite, the ID-TIMS data indicate significant heterogeneity in the initial Pb ratio ((207Pb/206Pb)i = 0.845–0.856), invalidating this sample as a viable reference apatite for high-precision geochronology. Additionally, the common-Pb compositions of TEMORA 2 and Duluth Complex anorthosite apatites calculated using the ID-TIMS data deviate from bulk Earth Pb evolution models beyond 2σ uncertainty. The data emphasize the utility of unanchored age regressions in generating the highest fidelity apatite UPb dates. Further, TEMORA 2 and Duluth Complex apatite ages are both younger than their corresponding zircon UPb ages, highlighting the need to independently verify the ages of prospective reference apatites.

Development and performance of an analytical method for polychlorinated naphthalenes and other persistent organic pollutants in dried blood spots

BackgroundDried blood spots (DBSs) collected and archived in newborn screening programs (NSP) represent a potentially valuable resource for assessing exposure to a range of organic and inorganic chemicals in newborns. This study develops and optimizes a method to measure polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides (OCPs) in DBS using the isotope dilution technique, ultrasonic-assisted liquid-liquid extraction, simple cleanup, triple quadrupole GC-MS/MS analysis, and background correction. ResultsWe minimize the number of extraction repetitions and the volume of solvent, which helps increase throughput while minimizing the potential for contamination. We obtained high recovery and precision for most compounds, and method detection limits (MDLs) were sufficiently low to detect the more prevalent compounds based on representative sample of the US population. MDLs averaged 0.020 ng/mL (recovery: 107 %, precision: 4 %) for PCNs, 0.021 ng/mL (recovery: 97 %, precision: 4 %) for PCBs, 0.021 ng/mL (recovery: 117 %, precision: 2 %) for OCPs, and 0.021 ng/mL (recovery: 96 %, precision: 3 %) for PBDEs. Significance and noveltyTo our knowledge, this is the first study presenting an analytical method and for PCNs in DBS, and one of the few studies providing an assessment of method performance for persistent organic pollutants in DBS. The optimized method can be applied to a wide range of applications, including exposure assessment, environmental epidemiology, forensics, environmental surveillance, and ecological monitoring.

Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS.

Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy

A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Study of a new matrix certified reference material for simultaneous measurement of two plant growth regulators residues in tomato

A new matrix certified reference material (CRM) was developed for analysis of chlormequat chloride (CCC) and 2,4-dichlorophenoxyacetic acid (2,4-D) residues in tomato pulp. A tomato pulp CRM candidate with the same matrix composition and similar residues concentration level to the real sample was prepared by means of adding drugs. Liquid chromatography tandem isotope dilution mass spectrometry (ID-LC-MS/MS) was established to accurately determinate CCC and 2,4-D residues in tomato pulp CRM candidates. The characterization of mass fractions of CCC and 2,4-D in tomato pulp were conducted by 8 laboratories. The certified values for CCC and 2,4-D were assigned to be 4.1 mg/kg and 1.9 mg/kg, with expanded uncertainties of 0.4 mg/kg and 0.3 mg/kg at a confidence level of 95 % (coverage factor k = 2), respectively. The results of homogeneity testing show that the CRM was homogeneous for CCC and 2,4-D between and within bottles. The stability monitoring results indicated that the CRM could be stable for 14 months at −20 °C storage temperature and for 9 days under 30 °C transportation condition. The developed CRM has been authorized and numbered as GBW(E)100933. It would be conductive to method validation in the residues monitoring of CCC and 2,4-D in fruits and vegetable products.

Detection of soybean allergens based on isotope dilution mass spectrometry: Development, validation, and applications in complex food matrices

An effective and versatile method based on isotope dilution mass spectrometry (IDMS) has been developed for the detection of soybean allergens in complex food matrices. The sample treatment method and MRM conditions in MS have been optimized to achieve the highest extraction efficiency and maximum intensity. The signature peptides VLIVPQNFVVAAR from Gly m 6 G1 subunit, TISSEDEPFNLR from Gly m 5 beta subunit, QQQEEQPLEVR and LFEITPEKNPQLR from Gly m 5 alpha’ subunit were selected, and the corresponding isotopically labeled peptides were served as the internal standards to minimize the matrix effects. The standard solutions showed a good linearity ranging from 1 to 200 ng/mL. The recoveries were in the range of 84.61 %–115.63 % and relative standard deviations were in the range of 0.66 % to 12.77 %. The method detection limits (MDLs) and quantitation limits (MQLs) of Gly m 6 G1 subunit, Gly m 5 beta subunit, and Gly m 5 alpha’ subunit in four matrices were less than 2.5 μg/g and 10 μg/g. By utilizing the developed method, three soybean allergens were determined in soy milk and ham sausage which contained soybean. The butter cookies that were not made of soy were also detected to contain soybean allergens, possibly due to manufacturing contamination. Surprisingly, soybean allergens were detected in the sesame paste labeled as soybean free, showing the importance to re-evaluate the soybean allergens after production. Our work laid the groundwork for the development of a versatile and sensitive method to evaluate soybean allergens in food products.

Implementation and experience of using methods for analyzing the isotopic composition of Sm and Nd, Rb and Sr in rock samples at the Geoanalitik Center for Collective Use

Research subject. AGV-2 and BHVO-2 geochemical reference materials for studying the Sm, Nd and Rb, Sr isotope systems along with various rocks and mineral samples. Materials and Methods. An analysis of the isotopic composition of Sm, Nd and Rb, Sr was carried out using Neptune Plus and Triton Plus mass spectrometers. Aim. To implement analytical techniques for the isotopic composition of Sm and Nd, Rb, and Sr in various rock and mineral samples using two types of multicollector mass spectrometers – inductively coupled plasma NeptunePlus and thermal ionization TritonPlus (Thermo-Fisher), as well as a description of the procedure of processing experimental data and the experience in using techniques at the “Geoanalitik” shared research facilities of the IGG UB RAS for the period 2015-2023. Results. The analytical techniques implemented included (1) column chromatography using various ion-exchange resins, optimised for the ratio of labour costs/quality of analytical results; (2) the measurement of isotope ratios using two types of mass spectrometers; (3) the correction of mass bias of isotope ratios and the determination of Sm and Nd, Rb, and Sr concentrations by the isotope dilution method using 149Sm+150Nd and 85Rb+84Sr spikes. Testing of the techniques was carried out using the AGV-2 and BHVO-2 geochemical reference materials; their metrological characteristics were presented. When using TritonPlus, the reproducibility (BHVO-2, n=60) of measurements of 143Nd/144Nd, 147Sm/144Nd isotope ratios and Sm and Nd concentrations are ±0.000020, ±0.0004, ±1.3 and ±0.4, respectively; indicators of correctness of determining the ratios 143Nd/144Nd and 147Sm/144Nd – 0.001 and 0.25% and concentrations of Sm and Nd – 2%; the reproducibility (BHVO-2, n=63) of measurements of 87Sr/86Sr, 87Rb/86Sr isotope ratios and concentrations (Rb and Sr) are ±0.0025%, ±1.5%, ±2%, respectively. The uncertainty of a single measurement of the 143Nd/144Nd and 87Sr/86Sr isotope ratio, represented by the standard error of the average single measurement in the sample, does not exceed 0.0025%. Conclusions: the results obtained for the geochemical reference materials are in satisfactory agreement with those provided in the GeoReM database, as well as with the certified values provided by the United States Geological Survey (USGS). The described analytical techniques are used at the “Geoanalitik” shared research facilities of the IGG UB RAS to analyse various rock and mineral samples. The work presents a number of experimental results obtained, which are subsequently used in geochronological applications.

Formation of Key Aroma Compounds During 30 Weeks of Ripening in Gouda-Type Cheese Produced from Pasteurized and Raw Milk.

Gouda-type cheeses were produced on a pilot-scale from raw milk (RM-G) and pasteurized milk (PM-G). Sixteen key aroma compounds previously characterized by the sensomics approach were quantitated in the unripened cheeses and at five different ripening stages (4, 7, 11, 19, and 30 weeks) by means of stable isotope dilution assays. Different trends were observed in the formation of the key aroma compounds. Short-chain free fatty acids and ethyl butanoate as well as ethyl hexanoate continuously increased during ripening but to a greater extent in RM-G. Branched-chain fatty acids such as 3-methylbutanoic acid were also continuously formed and reached a 60-fold concentration after 30 weeks, in particular in PM-G. 3-Methylbutanal and butane-2,3-dione reached a maximum concentration after 7 weeks and decreased with longer ripening. Lactones were high in the unripened cheeses and increased only slightly during ripening. Recent results have shown that free amino acids were released during ripening. The aroma compounds 3-methylbutanal, 3-methyl-1-butanol, and 3-methylbutanoic acid are suggested to be formed by microbial enzymes degrading the amino acid l-leucine following the Ehrlich pathway. To gain insight into the quantitative formation of each of the three aroma compounds, the conversion of the labeled precursors (13C6)-l-leucine and (2H3)-2-keto-4-methylpentanoic acid into the isotopically labeled aroma compounds was studied. By applying the CAMOLA approach (defined mixture of labeled and unlabeled precursor), l-leucine was confirmed as the only precursor of the three aroma compounds in the cheese with the preferential formation of 3-methylbutanoic acid.

Influence of Milk Pasteurization on the Key Aroma Compounds in a 30 Weeks Ripened Pilot-Scale Gouda Cheese Elucidated by the Sensomics Approach.

Gouda cheese was produced from pasteurized milk and ripened for 30 weeks (PM-G). By application of gas chromatography/olfactometry and an aroma extract dilution analysis on the volatiles isolated by extraction/SAFE distillation, 25 odor-active compounds in the flavor dilution (FD) factor range from 16 to 4096 were identified. Butanoic acid, 2- and 3-methylbutanoic acid, and acetic acid showed the highest FD factors, and 2-phenylethanol, δ-decalactone, and δ-dodecalactone were most odor-active in the neutral-basic fraction. Quantitations by stable isotope dilution assays followed by a calculation of odor activity values (OAVs) revealed acetic acid, 3-methylbutanoic acid, butanoic acid, and butane-2,3-dione with the highest OAVs. Finally, an aroma recombinate prepared based on the quantitative data well agreed with the aroma profile of the PM-G. In Gouda cheese produced from raw (nonpasteurized) milk (RM-G), qualitatively the same set of odor-active compounds was identified. However, higher OAVs of butanoic acid, hexanoic acid, and their corresponding ethyl esters were found. On the other hand, in the PM-G, higher OAVs for 3-methylbutanoic acid, 3-methylbutanol, 3-methylbutanal, and butane-2,3-dione were determined. The different rankings of these key aroma compounds clearly reflect the aroma differences of the two Gouda-type cheeses. A higher activity of lipase in the RM-G and higher amounts of free l-leucine in PM-G on the other side were responsible for the differences in the concentrations of some key aroma compounds.

Abstract 128: Blood TMAO Predicts Abdominal Aortic Aneurysm Growth Rate And Need For Surgical Intervention

Background: Patients known to have abdominal aortic aneurysms (AAAs) undergo surveillance imaging to predict both the risk for acute aortic syndromes including aortic dissection and aortic rupture, and the need for surgical intervention. Aortic rupture is a feared consequence of rapidly expanding AAAs. Aims: A blood biomarker that predicts risk for AAA and distinguishes slow from fast AAA growth does not exist but could be a valuable tool for risk stratification. We recently reported systemic levels of the gut microbe-generated metabolite trimethylamine N-oxide (TMAO) are associated with the presence of AAA in humans, and directly contribute to AAA progression in animal models. Methods: We leveraged access 805 patients in two cohorts from Europe (n=237) and the United States (n=658) undergoing serial aortic imaging surveillance. Blood TMAO concentration was measured by stable isotope dilution mass spectrometry. Results: TMAO was positively associated with larger baseline and follow-up abdominal aortic diameter beyond traditional factors (Adjusted OR 3.87, 95% CI, 2.70-5.58, P&lt;0.001). In addition, TMAO predicted rapid AAA growth (HR 1.81, 95%CI, 1.23-2.67, P=0.0029). When TMAO was added to a predictive model of traditional risk factors for AAA, we saw a significant improvement in the net reclassification index (NRI, 0.43, P &lt; 0.001; C statistic, 0.77 (0.72–0.81) with TMAO vs. NRI 0.73 (0.69–0.78) without TMAO, P =0.005). Finally, including TMAO in a predictive model for assessing need for surgical intervention among AAA patients significantly improved the prognostic performance of the model (NRI, 0.44, P &lt; 0.001; C statistic, 0.78 (0.73–0.82) with TMAO vs. 0.75 (0.70–0.79) without TMAO, P =0.004) (Fig. 1). Conclusions: Elevated TMAO levels predict increased risk of AAA, and identify patients with fast-growing AAA and those who may benefit from early surgical intervention to prevent acute aortic syndromes.

Determination of the Nanoscale Silica Mass Fraction by AF4/ICP-MS with Isotope Dilution Analysis Using 29Si-Enriched Silica Nanoparticles.

A methodology based on the use of asymmetrical flow field-flow fractionation (AF4) coupled to ICP-MS with size fraction-targeted isotope dilution analysis (IDA) has been developed, validated, and applied for the first time to determine the mass fraction of nanoscale silica (SiO2). For this purpose, 29Si-enriched SiO2 nanoparticles, to be used as an IDA spike/internal standard, were synthesized and characterized in-house. Double IDA was used to quantify an aqueous suspension of Stöber silica particles of similar characteristics to those of the 29SiO2 nanoparticle (NP) spike using a representative test material of natural Si isotopic composition as the calibrant. For fumed SiO2 NP in a highly complex food matrix, a methodology based on single IDA with AF4/ICP-MS using the same 29SiO2 NP spike was developed and validated. Relative expanded measurement uncertainties (k = 2) of 4% (double IDA) and 8% (single IDA) were achieved for nanoscale silica mass fractions of 5143 and 107 mg kg-1 in water suspension and food matrix, respectively. To assess the accuracy of AF4/ICP-IDMS for the characterization of SiO2 NP in a food matrix, standard addition measurements on samples spiked with Aerosil AF200, also in-house characterized for Si mass fraction, were undertaken, with an average recovery of 95.6 ± 4.1% (RSD, n = 3) obtained. The particle-specific IDA data obtained for both SiO2 NP-containing samples were also compared with that of post-AF4 channel external calibration using inorganic Si standards. The mass fractions obtained by IDA agreed well with those obtained by external calibration within their associated measurement uncertainties.

Simultaneous determination of picomolar level of dissolved silver with other key trace metals in seawater samples using solid phase extraction and isotope dilution methods

Simultaneous determination of dissolved silver (dAg) with other key GEOTRACES trace metals is difficult because dAg in seawater tends to form negatively charged chloride species that result in only 75% recovery efficiency with commonly used NOBIAS PA-1 chelating resins. In this study, we developed a method using solid phase extraction coupled with isotope dilution that enables full quantification (97.9 ± 2.1%) of dAg along with other major trace metals including cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni), and lead (Pb) (recovery efficiency = 100% to 102%) in seawater samples. Seawater samples were first spiked with Ag-109 and allowed to reach isotopic equilibrium before extraction using NOBIAS PA-1 chelating resin. Then, dAg isotope ratios (Ag-109/Ag-107) before and after solid phase extraction were determined and used to quantify dAg. Determination of dAg with dissolved Cd, Cu. Mn, Ni, and Pb in reference seawater material CASS-6 resulted in deviations of between 1.0% and 8.8% from the consensus values, which are well within the standard error of measurement. We then successfully determined the concentrations of dissolved Cd (0.05–0.2 nM), Cu (0.5–13 nM), Mn (10–140 nM), Ni (2–12 nM), Pb (5–110 nM), and Ag (10–40 pM) in Otsuchi Bay, Japan and its surrounding rivers. Evaluation of the behavior of dAg under a salinity gradient using estuarine samples collected from Samunsam River, Malaysia shows increasing dAg concentration with salinity (R2 = 0.68), which suggests release of sedimental Ag under high ambient chloride concentrations. Our new method enables rapid and simultaneous measurements of dAg with other key GEOTRACES trace metals in a single analysis, which is expected to expedite analysis and increase availability of oceanic dAg data globally.

Effect of absolute blood volume measurement-guided fluid management on the incidence of intradialytic hypotension-associated events: a randomised controlled trial.

Ultrafiltration to target weight during haemodialysis is complicated by intradialytic hypotension-associated adverse events (IHAAEs) in 10-30% of dialysis treatments. IHAAEs are caused by critical reductions in absolute blood volume (ABV), due to the interaction of ultrafiltration, refill and compensatory mechanisms. Non-randomised studies have suggested that ABV-guided treatment, using an indicator dilution technique employing the blood volume monitor on the dialysis machine, could reduce the incidence of IHAAEs. We performed an open-label randomised controlled trial. Patients were randomly assigned to adjustment of target weight guided by ABV measurements or standard care. The primary outcome was the change in the incidence of IHAAEs from baseline, defined as the percentage of treatment episodes in a 4-week period where the patient had a systolic blood pressure <90mmHg or symptoms of impending hypotension. ABV measurements were compared with anthropomorphometric estimation and the gold standard using isotope dilution. A total of 56 patients were randomised, of whom 29 were allocated to ABV-guided treatment and 27 to standard care. Overall baseline incidence of IHAAEs was 26.0%. ABV-guided treatment significantly reduced the incidence of IHAAEs compared with standard care, with a mean change from baseline of -9.6% [95% confidence interval (CI) -17.3 to -1.8) versus 2.4% (95% CI -2.3-7.2). The adjusted difference between the groups was 10.5% (95% CI 1.3-19.8; P=.026). ABV measurement had moderate agreement with other methods to estimate blood volume. The sensitivity for the previously suggested threshold of a post-dialysis normalised blood volume of 65ml/kg was observed to be 74% in this study. ABV-guided volume management significantly reduced IHAAEs compared with standard care. The clinical relevance of the previously suggested threshold of 65ml/kg cannot be firmly concluded on the basis of our results. If confirmed in a larger trial, this intervention could potentially change dialysis practice and impact patient care in a clinically meaningful way.