Isotope Dilution Ultra-performance Liquid Research Articles

Discrimination of overheated pasteurized milk using mass spectrometry-based proteomics

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

Simultaneous determination of typical toxic aldehydes formed during food frying and digestion using isotope dilution UHPLC-QqQ-MS/MS

An isotope dilution ultra-performance liquid chromatography–triple quadrupole mass spectrometry method was developed to simultaneously detect two typical kinds of α,β-unsaturated aldehydes, namely 4-hydroxy-2-hexenal (4-HHE) and 4-hydroxy-2-nonenal (4-HNE), in foods. The proposed method exhibited a linear range of 10–1000 ng/mL with a limit of detection of 0.1–2.0 ng/g and a limit of quantification of 0.3–5.0 ng/g. The recovery rates of these typical toxic aldehydes (i.e., 4-HHE, 4-HNE) and their d3-labeled analogues were 91.54%–105.12% with a low matrix effect. Furthermore, this proposed method was successfully applied to a real frying system and a simulated digestion system, wherein the contents of 4-HHE and 4-HNE were determined for both. Overall, the obtained results provide strong support for further research into the production of 4-HHE and 4-HNE resulting from foods during oil digestion and frying.

Association between Antibiotic Exposure and Type 2 Diabetes Mellitus in Middle-Aged and Older Adults

Although previous studies have shown an association between clinically used antibiotics and type 2 diabetes, the relationship between antibiotic exposure from food and drinking water and type 2 diabetes in middle-aged and older adults is unclear. This study was aimed at exploring the relationship between antibiotic exposures from different sources and type 2 diabetes in middle-aged and older people, through urinary antibiotic biomonitoring. A total of 525 adults who were 45-75 years of age were recruited from Xinjiang in 2019. The total urinary concentrations of 18 antibiotics in five classes (tetracyclines, fluoroquinolones, macrolides, sulfonamides and chloramphenicol) commonly used in daily life were measured via isotope dilution ultraperformance liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry. The antibiotics included four human antibiotics, four veterinary antibiotics and ten preferred veterinary antibiotics. The hazard quotient (HQ) of each antibiotic and the hazard index (HI) based on the mode of antibiotic use and effect endpoint classification were also calculated. Type 2 diabetes was defined on the basis of international levels. The overall detection rate of the 18 antibiotics in middle-aged and older adults was 51.0%. The concentration, daily exposure dose, HQ, and HI were relatively high in participants with type 2 diabetes. After model adjustment for covariates, participants with HI > 1 for microbial effects (OR = 3.442, 95%CI: 1.423-8.327), HI > 1 for preferred veterinary antibiotic use (OR = 3.348, 95%CI: 1.386-8.083), HQ > 1 for norfloxacin (OR = 10.511, 96%CI: 1.571-70.344) and HQ > 1 for ciprofloxacin (OR = 6.565, 95%CI: 1.676-25.715) had a higher risk of developing type 2 diabetes mellitus. Certain antibiotic exposures, mainly those from sources associated with food and drinking water, generate health risks and are associated with type 2 diabetes in middle-aged and older adults. Because of this study's cross-sectional design, additional prospective studies and experimental studies are needed to validate these findings.

The Characteristics and Potential Risks of Neonicotinoid Residues in Soils of Different Types of Land Use in Hangzhou

Due to the wide existence of neonicotinoid insecticides (neonics) and their potential impact on ecosystems and human health, they have received special attention in recent years. Soil is not only a sink of neonics but also a source of neonics, so it plays a key role in the ubiquity of neonics in the environment. The purpose of this research was to compare neonics residues in soils of different types of land use and estimate their exposure to different populations via ingestion. A total of 130 soil samples from six different types were collected. The concentrations of seven neonics in soil were simultaneous determined using isotope-dilution ultra-performance liquid chromatography–tandem mass spectrometry. The results showed that at least one neonic was analyzed in all samples. The highest average concentration was 3.42 ng/g (clothianidin), followed by 3.39 ng/g (thiamethoxam), 3.06 ng/g (acetamiprid), 2.84 ng/g (imidacloprid), 2.66 ng/g (nitenpyram), 2.43 ng/g (thiacloprid), and 1.89 ng/g (dinotefuran). IMI and ACE were the most commonly found neonics in soil. The neonic levels in different soils varied significantly. The integrated neonic residue in cropland was much higher than that in other types of land. The risk assessment revealed that the average daily dose (ADD) through ingestion contact with soil was acceptable to children and adults. With the increasing evidence that neonics could cause a variety of toxic effects on mammals and humans, ingestion exposure caused by neonics in soil should also receive continuous attention in future studies.

Determination of quinolone antibiotics in environmental water using automatic solid-phase extraction and isotope dilution ultra-performance liquid chromatography tandem mass spectrometry

The widespread use of quinolones in humans and animals has become a major threat to public health. In this study, a simple, rapid, sensitive, and high throughput method based on automatic solid-phase extraction and isotope dilution ultra-performance liquid chromatography tandem mass spectrometry was described for the determination of trace quinolones in environmental water. The proposed automated solid-phase extraction method was initially optimized, and the optimum experimental conditions found were 1 L water sample with 0.5 g/L Na2EDTA (pH 3) extracted and enriched by CNW Poly-Sery HLB cartridge at a flow rate of 50 mL/min and eluted by 8 mL of methanol. The linearity of the method ranged from 0.05 to 100 μg/L for 15 quinolones, with correlation coefficients ranging from 0.9993 to 0.9999. The limits of detection were in the low ng/L level, ranging from 0.005 to 0.051 ng/L. Finally, the optimized method was applied for determining trace levels of 15 quinolones in Wahaha pure water, tap water, river water, and seawater samples with good recoveries of 93 %–119 % and satisfactory relative standard deviations of 0.1 %–13.9 %. Fourteen quinolones were detected, and ofloxacin was the predominant congener in river water and seawater.

A novel single step solid-phase extraction combined with bromine derivatization method for rapid determination of acrylamide in coffee and its products by stable isotope dilution ultra-performance liquid chromatography tandem triple quadrupole electrospray ionization mass spectrometry

This work aimed to establish a novel determination method for acrylamide in coffee and its products by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acrylamide in samples were prepared by a single-step solid-phase extraction clean-up using mixed mode sorbents. The bromine derivatization efficiency of acrylamide and its internal standard were improved at an acidic condition. After derivation, the retention capability of acrylamide and its resistance to interference were significantly improved. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.2 and 4 μg/kg for roasted and instant coffees, while they were 0.24 and 0.8 μg/kg for ready-to-drink coffees. The average recoveries for acrylamide ranged from 99.3 to 102.2% in coffee and its products. All the results showed that the developed method was simple, quick, specific and suitable for screening and determination of acrylamide in batch samples of coffee and its products.

Determination of Parabens, Bisphenol A and Its Analogs, Triclosan, and Benzophenone-3 Levels in Human Urine by Isotope-Dilution-UPLC-MS/MS Method Followed by Supported Liquid Extraction.

The development of a rapid analytical approach for determining levels of antibacterial agents, plasticizers, and ultraviolet filters in biosamples is crucial for individual exposure assessment. We developed an analytical method to determine the levels of four parabens—bisphenols A (BPA) and its analogs, triclosan (TCS), triclocarban, and benzophenone-3 (BP-3)—in human urine. We further measured the levels of these chemicals in children and adolescents. We used a supported liquid extraction (SLE) technique coupled with an isotope-dilution ultraperformance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) method to assess the detection performance for these chemicals. Forty-one urine samples from 13 children and 28 adolescents were assessed to demonstrate the capability and feasibility of our method. An acceptable recovery (75.6–102.4%) and matrix effect (precision < 14.2%) in the three-level spiked artificial urine samples were achieved, and good performance of the validated ID-UPLC-MS/MS method regarding linearity, limits of detection, and quantitation was achieved. The within-run and between-run accuracy and precision also demonstrated the sensitivity and stability of this analytical method, applied after SLE. We concluded that the ID-UPLC-MS/MS method with SLE pretreatment is a valuable analytical method for the investigation of urinary antibacterial agents, plasticizers, and ultraviolet filters in humans, useful for human biomonitoring.

Quantification of major milk proteins using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry and its application in milk authenticity analysis

An authenticity-based method was developed to detect economically motivated adulteration (EMA) in milk. To achieve this goal, 46 peptides released from trypsin digested milk proteins were identified and marker peptides were further selected based on their digestibility and stability characteristics to allow further quantifications of the milk proteins in the original samples using an isotope-dilution ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQMS). Furthermore, to detect EMA in milk samples, 22 authentic milk powder samples were analyzed using the isotope-dilution UPLC-TQMS, and an authenticity threshold value --70.26 g/100 g as the content of all five major milk proteins to total protein—was established to evaluate milk samples with high adulteration risks. Finally, the threshold value was validated using soy protein adulterated milk samples. Results showed that the reported authenticity-based method was sensitive enough to detect the tampering of milk samples with as little as 10 % soy protein added. It is clear that this newly developed authenticity-based detection method is a powerful tool to detect unknown adulteration risks in milk samples. • Selected 5 marker peptides from 46 identified peptides based on their digestibility and stability characteristics. • Established and validated an isotope-dilution UPLC-TQMS method to quantitatively analyze major milk proteins. • Developed a milk authenticity threshold value to detect milk adulterations. • Validated the threshold value using artificially adulterated milk samples.

Method Improvement for Analysis of Enrofloxacin and Ciprofloxacin in Chicken Meat: Application of In-Sample Addition of Trace Ethylenediaminetetraacetic Acid to Isotope Dilution Ultra-Performance Liquid Chromatography–Mass Spectrometry

Method Improvement for Analysis of Enrofloxacin and Ciprofloxacin in Chicken Meat: Application of In-Sample Addition of Trace Ethylenediaminetetraacetic Acid to Isotope Dilution Ultra-Performance Liquid Chromatography–Mass Spectrometry

Plasma oxysterols: Altered level of plasma 24-hydroxycholesterol in patients with bipolar disorder

Cholesterol and its oxygenated metabolites, including oxysterols, are intensively investigated as potential players in the pathophysiology of brain disorders. Altered oxysterol levels have been described in patients with numerous neuropsychiatric disorders. Recent studies have shown that Bipolar disorder (BD) is associated with the disruption of cholesterol metabolism. The present study was aimed at investigating the profile of oxysterols in plasma, their ratio to total cholesterol and their association with clinical parameters in patients with BD. Thirty three men diagnosed with BD and forty healthy controls matched for age and sex were included in the study. Oxysterol levels were measured by isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry. Significantly higher levels were observed for cholestane-3β,5α,6β-triol, 27-hydroxycholesterol (27−OHC) and Cholestanol in patients with BD. The concentration of 24-hydroxycholesterol (24−OHC) was significantly lower in patients compared to controls. 24−OHC was also negatively correlated to MAS subscale score (r =-0.343; p = 0.049). In patients, 24−OHC was inversely correlated with age (r = -0.240; p = 0.045). Multivariate analysis found that BD acute decompensation was independently related to the rise in plasma 24−OHC (p = 0.002; OR = 0.966, 95 % CI [0.945 – 0.987]). However, the 24−OHC assay relevance as a biomarker of this disease deserves further investigation in other studies.

Characterization of volatile organic compound (VOC)exposures and their determinantsamong U.S. hairdressers serving an ethnic clientele

Background: Hairdressers are routinely exposed to volatile organic compounds (VOCs) linked to adverse health effects. Over 32% of U.S. hairdressers are women of color who may experience disproportionate VOC exposures from their occupational and personal use of products formulated for ethnic (Black/Latina) clientele. While these exposures may reflect potential health disparities, little is known about VOC exposures and their determinants in this worker population.Objective: To characterize concentrations of urinary VOC (UVOC) exposure biomarkers and their determinants among female hairdressers primarily serving an ethnic clientele.Methods: We recruited 23 minority female hairdressers predominantly serving an ethnic clientele, and 17 office workers in the Maryland/DC metro area. We quantified concentrations of 28 biomarkers, representing 20 VOC parent compounds in end-of-shift urine samples, using isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry. UVOC concentrations in hairdressers were compared to those among office workers and women in the U.S. general population (NHANES). We administered questionnaires to capture demographics and workplace behaviors.Results: The majority of hairdressers (65%) were black with a mean age of 40 years. Half of office workers were black with a mean age of 34 years. Geometric mean concentrations for 26 of the 28 UVOCs were higher in hairdressers than office workers, with statistically significant concentration differences observed for 10 biomarkers (p&lt;0.05). Also, geometric mean concentrations for 21 of 23 biomarkers quantified by both NHANES and our study, were between 1.5-3.7 times higher in hairdressers than women participating in NHANES. Salon services including semi-permanent hair coloring and hair extensions with glue, were associated with higher concentrations of toluene, xylene and acrylonitrile.Conclusions: Our work is among the first to characterize VOC exposures among minority female hairdressers serving an ethnic clientele. Larger studies are warranted to further examine determinants of VOC exposures and associated adverse health effects among this disparately exposed population.

8-Hydroxyguanosine as a possible RNA oxidative modification marker in urine from colorectal cancer patients: Evaluation by ultra performance liquid chromatography-tandem mass spectrometry

Oxidative RNA damage has been found to be associated with a variety of diseases, and 8-hydroxyguanosine (8-OHG) is a typical marker of oxidative modification of RNA. This guanosine modification is an emerging biomarker for disease detection and determination of 8-OHG in human urine is favored because it is noninvasive to patients. However, due to its poor ionization efficiency in mass spectrometry and trace amount in urine, accurate quantification of this modified nucleoside is still challenging. Herein, a rapid, accurate, sensitive and robust method using solid-phase extraction (SPE) combined with isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for detection of this oxidative RNA modification in human urine. The limit of detection can reach 1.5 fmol and the method exhibits good precision on intra-day (1.8–3.3%) and inter-day (0.6–1.2%) analyses. Satisfactory recovery (87.5–107.2%) at three spiked levels was achieved by using HLB cartridge for urine pretreatment. Using this method, we quantified 8-OHG in urine from 65 colorectal cancer (CRC) patients and 76 healthy volunteers. The measured level of urinary 8-OHG for CRC patients and healthy controls is 1.91 ± 0.63 nmol/mmol creatinine and 1.33 ± 0.35 nmol/mmol creatinine, respectively. We found the content of 8-OHG in urine was raised in CRC patients patients, implying this oxidative RNA modification marker could act as a potential noninvasive indicator for early screening of CRC. In addition, this study will make contributions to the investigations of the influences of oxidative stress on the formation and development of CRC.

Comparison of Aristolochic acid I derived DNA adduct levels in human renal toxicity models

Aristolochic acid (AA) dependent human nephropathy results either from environmental exposure to Aristolochiaceae plant subspecies or their use in traditional phytotherapy. The toxic components are structurally related nitrophenanthrene carboxylic acids, i.e. Aristolochic acid I (AAI) and II (AAII). AAI is considered to be the major cause of Aristolochic acid nephropathy, characterized by severe renal fibrosis and upper urothelial cancer. Following enzymatic activation in kidney and/or liver, AAI metabolites react with genomic DNA to form persistent DNA adducts with purines. To determine whether AAI can be activated in human renal cells to form DNA adducts, we exposed telomerase immortalized renal proximal tubular epithelial cells (RPTEC/TERT1), the human embryonic kidney (HEK293) cell line, as well as primary human kidney cells (pHKC) to AAI in vitro. We modified an isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry (ID-UPLC-MS/MS) based method for the quantification of dA-AAI adducts in genomic DNA. In addition, time dependent accumulation of adducts in renal cortex and bladder tissue from AAI/II treated Eker rats were used to validate the detection method. AAI-induced toxicity in human renal cells was determined by dA-AAI adduct quantification, the impact on cell viability, and NQO1 expression and activity. Our findings demonstrated adduct formation in all cell lines, although only pHKC and RPTEC/TERT1 expressed NQO1. The highest adduct formation was detected in pHKC despite low NQO1 expression, while we observed much lower adduct levels in NQO1-negative HEK293 cells. Adduct formation and decreased cell viability correlated only weakly. Therefore, our data suggested that i.) enzymes other than NQO1 could be at least equally important for AA bioactivation in human renal proximal tubule cells, and ii.) the suggested correlation between adduct levels and viability appears to be questionable.

Changes in markers of liver function in relation to changes in perfluoroalkyl substances - A longitudinal study

BackgroundWhile it is known that perfluoroalkyl substances (PFASs) induce liver toxicity in experimental studies, the evidence of an association in humans is inconsistent. ObjectiveThe main aim of the present study was to examine the association of PFAS concentrations and markers of liver function using panel data. MethodsWe investigated 1002 individuals from Sweden (50% women) at ages 70, 75 and 80 in 2001–2014. Eight PFASs were measured in plasma using isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). Bilirubin and hepatic enzymes alanine aminotransferase (ALT), alkaline phosphatase (ALP), and γ-glutamyltransferase (GGT) were determined in serum using an immunoassay methodology. Mixed-effects linear regression models were used to examine the relationship between the changes in markers of liver function and changes in PFAS levels. ResultsThe changes in majority of PFAS concentrations were positively associated with the changes in activity of ALT, ALP, and GGT and inversely associated with the changes in circulating bilirubin after adjustment for gender and the time-updated covariates LDL- and HDL-cholesterol, serum triglycerides, BMI, statin use, smoking, fasting glucose levels and correction for multiple testing. For example, changes in perfluorononanoic acid (PFNA) were associated with the changes liver function markers βBILIRUBIN = −1.56, 95% confidence interval (CI) −1.93 to −1.19, βALT = 0.04, 95% CI 0.03–0.06, and βALP = 0.11, 95% CI 0.06–0.15. ConclusionOur longitudinal assessment established associations between changes in markers of liver function and changes in plasma PFAS concentrations. These findings suggest a relationship between low-dose background PFAS exposure and altered liver function in the general population.

Sensitive analysis of steroid estrogens and bisphenol a in small volumes of water using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry.

An isotope-dilution ultra-performance liquid chromatography-electrospray tandem mass spectrometry method combined with dansylation was established to sensitively quantify four steroid estrogens (estrone, 17α-estradiol, 17β-estradiol and 17α-ethynylestradiol) and bisphenol A in sewage influent and effluent. A simple hexane extraction was performed from a small volume (10 mL), followed by dansyl chloride derivatization and purification with a silica cartridge. The method effectively reduced the matrix effects in sample extract and permitted the selective and sensitive determination of target compounds from complicated matrices. The detection limits of the method for steroid estrogens were 0.20-0.90 ng L-1 in influent and 0.10-0.20 ng L-1 in effluent samples. For bisphenol A, the limits detection of the method were 20 and 0.80 for influent and effluent samples, respectively. Recoveries of 85%-96% were observed in all matrices. The method was applied to analyze residual estrogens and bisphenol A in sewage influent and effluent samples from Beijing, China. The concentrations of bisphenol A (636-1200 ng L-1) were up to 250 times higher than those of steroid estrogens. Estrone was the dominant estrogen in influent and effluent samples, while similar concentrations of 17α-estradiol and 17β-estradiol were detected in all samples.

Quantification of glycocholic acid in human serum by stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry.

A rapid, accurate and sensitive stable isotope dilution ultra performance liquidchromatography electrospray ionization tandem mass spectrometry (ID-UPLC-ESI-MS/MS) method for the determination of glycocholic acid (GCA) in human serum was developed and validated. Serum samples were spiked with D5-glycocholic acid and then pretreated with protein precipitation. The analysis was performed on a Waters BEH C18 column (100 mm×2.1mm, 1.7μm), followed by ESI-MS/MS detection in negative ion mode under multiple reaction monitoring mode. The calibration curves covered a concentration range from 0.2 to 400ng/mL. The limit of detection and limit of quantification was 0.01ng/mL and 0.05ng/mL, respectively. The method showed satisfactory precision on intra-day (2.3-6.1%) and inter-day (2.4-4.6%) analyses and achieved good recovery at three spiked levels (103.7-114.3%). Moreover, this established method was successfully applied for quantification of GCA in serum samples from healthy volunteers, patients with hepatocellular carcinoma (HCC) and patients with other cancers. We demonstrated that the level of GCA in patients with HCC was significantly higher not only than that in healthy controls, but also than that in patients with other cancer, whereas no significant difference of GCA level was observed between healthy control group and other cancers group.

Simultaneous Determination of 19 Plant Growth Regulator Residues in Plant-originated Foods by QuEChERS and Stable Isotope Dilution-Ultra Performance Liquid Chromatography-Mass Spectrometry.

In this study, a highly sensitive and fast method of QuEChERS (acronym of quick, easy, cheap, effective, rugged, safe) combined with isotope dilution-ultra performance liquid chromatography coupled to tandem mass spectrometry was developed for the simultaneous determination of 19 plant growth regulators in plant-originated foods. The samples were initially extracted with acetonitrile containing 1% acetic acid and then the QuEChERS method was applied after the pH value was adjusted to 5.5 - 6.0, using 3-indolepropionic-d2 acid and forchlorfenuron-d5 as internal standard. The targeted 19 plant growth regulators were separated on an HSS T3 column using acetonitrile: 5 mmol/L ammonium acetate as the mobile phase. Quantitative results were based on multiple reaction monitoring mode after ionization in positive and negative electrospray ionization mode. Good linearity was achieved within a wide range and all the correlation coefficients were greater than 0.997. The limit of quantification was 0.060 - 6.0 μg/kg. Rate of recovery and relative standard deviation were 72.3 - 115.8 and 1.78 - 5.32%, respectively. The method was successfully applied to measure 19 plant growth regulator residues in 280 plant-originated commercial foods collected from local supermarkets in China. Twelve plant growth regulators were found in some of the analyzed samples.

Fully international system of units-traceable glycated hemoglobin quantification using two stages of isotope-dilution high-performance liquid chromatography–tandem mass spectrometry

Glycated hemoglobin (HbA1c), defined as hemoglobin (Hb) molecules having a stable adduct of glucose on the N-terminal of the β-chains, has been endorsed as a diagnostic tool for diabetes mellitus and a prediction indicator for the development of diabetes complications. Here we describe an accurate procedure using two stages of isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS) for the quantification of HbA1c that provides full traceability to International System of Units (SI). First, synthetic peptides representing specific markers of HbA1c (G-hexa) and hemoglobin A0 (Hexa) were certified by amino acid analysis via acid hydrolysis as reference materials (RMs) for the next step. For this peptide certification, three amino acids (proline, valine, and leucine) were determined by hydrolysis with 10M hydrochloric acid at 130°C for 48h followed by ID-LC–MS/MS. Then, HbA1c content in blood was quantified with the ratio of specific proteolytic peptides from HbA1c and HbA0 via enzyme digestion using ID-LC–MS/MS with the certified peptides as RMs and isotope-labeled peptides as internal standards. Results demonstrate complete traceability to SI-units throughout this procedure. Reliability was confirmed through comparative studies with commercially available RMs for HbA1c, and other routine HbA1c diagnostic methods as well. Following full method validation, we applied this procedure to the certification of candidate hemolysate-certified RMs for HbA1c content, as well as 52 real clinical samples. All of the results showed the suitability of this method to act as a primary reference measurement procedure for HbA1c in complex biological samples.

A reference measurement procedure for amino acids in blood using isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry.

We described a reference measurement procedure for amino acid (AA) quantification in blood samples based on deproteinization with 5-sulfosalicylic acid (SSA) and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (LC-MS) method. The serum was deproteinized with 15% v/v SSA and the supernatant was injected directly into the LC-MS system without further processing. Compared with the use of other precipitants and water as a control, five model AAs-valine, isoleucine, leucine, tyrosine, and phenylalanine-in the SSA-treated samples showed ionization enhancement as well as stable background signals without significant ion suppression effects. Five analytes were clearly separated within 3min using gradient elution and ion-pair chromatography of water and acetonitrile containing 0.1% v/v trifluoroacetic acid. The limit of detection range of this method was 2-52fmol, and the RSDs of accuracy and precision from intra- and inter-day assays were within 2.7%. The method was applied to various blood samples including serum, whole blood and plasma, with no reasonable measurement bias revealed. The quantification accuracy of this method was then assessed using commercially available plasma certified reference material (CRM) for AA, and the results agreed well within certified values. We finally applied this method to the determination of candidate serum CRM. The optimized protocol was found to be suitable for the accurate quantification of five AAs in serum, and may satisfactorily serve as a primary method for AA measurement in various blood matrices.

Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels

Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.