Isotope-dilution Ultraperformance Liquid Chromatography Research Articles

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.

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.

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

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.

Urinary Antibiotics of Pregnant Women in Eastern China and Cumulative Health Risk Assessment.

Exposure to antibiotics during pregnancy can pose a systematic effect on human health. A few biomonitoring studies have demonstrated an extensive exposure of children to antibiotics, but there is still a lack of data for pregnant women. To assess the exposure of pregnant women to antibiotics and potential health risk, we investigated 536 pregnant women aged 16-42 years from two geographically different study sites in Eastern China in 2015. We measured 21 antibiotics of five categories (seven fluoroquinolones, three phenicols, four tetracyclines, three macrolides, and four sulfonamides) in urine using the isotope dilution ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The hazard index (HI) was calculated on the basis of estimated daily exposure dose and acceptable daily intakes. A total of 16 antibiotics were found in urine, with detection frequencies between 0.2 and 16.0%. Antibiotics were overall detected in 41.6% of urine, and two or more antibiotics were detected in 13.1% of urine. Ciprofloxacin, ofloxacin, and trimethoprim were most frequently detected in urine, with detection frequencies between 10 and 20%. The majority of the antibiotics tested had an estimated daily exposure dose less than 1 μg/kg/day, and 4.3% of pregnant women had a HI value of more than 1. These findings indicated that pregnant women were frequently exposed to antibiotics and some individuals were in the potential risk of adverse microbiological effects induced by antibiotics.

Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol

Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N 2-((furan-2-yl)methyl)-2′-deoxyguanosine (N 2-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC–MS/MS). Surprisingly, low levels of adducts that may represent N 2-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N 2-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine.

Distribution of DNA adducts and corresponding tissue damage of Sprague-Dawley rats with percutaneous exposure to sulfur mustard.

Sulfur mustard (SM) is a highly reactive alkylation vesicant and cytotoxic agent that has been recognized as an animal and human carcinogen. Although the exact mechanism of toxicology is vague, DNA alkylation seems to be responsible for the triggering of apoptosis. In this study, after male adult Sprague-Dawley rats were cutaneous exposed to a low concentration of SM at parts-per-million levels, their lungs, livers, pancreases, spleens, marrow, and brains were collected at 11 different time points and analyzed. N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N7-HETEG), N3-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N3-HETEA), and bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) as the biomarkers for DNA damage were measured in the vital tissues by isotope dilution ultraperformance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS). At the same time, general variations and pathological changes were monitored and detected to evaluate the tissue damage. Time- and dose-dependent data showed that SM had strong permeability and reactivity and that three SM-DNA adducts were detected in all investigated tissues only after 10 min after exposure. Obvious dose-dependency was observed except in the brain and pancreas. Most times to peak (tmax) of all three adducts were less than 3 h, while half-lifetimes (t1/2) were less than 24 h. We also suggested that the lipophilic SM can easily pass through the blood-brain barrier and can be stored in the fatty organs. To the best of our knowledge, the abundant adducts in marrow were found and reported for the first time. The surveillance of N7-HETEG in vivo, which was the most abundant adduct, may be the most efficient indicator to validate SM exposure even without any symptoms. Bis-G can be regarded as a biomarker of effect, which is directly related to the extent of damage. The most abundant Bis-G was found in the most sensitive tissues, marrow, spleen, and lung, which is in good accordance with histopathologic results. General variations and pathological changes were evaluated as well. After cutaneous exposure to SM, the body weights of rats heavily decreased in the first 4 days and were inversely proportional to the applied doses, and then recovered at the last experimental day except for those of the rats at the highest dosing level, in which the relative weights of rat spleens were obviously lost. Moreover, we found remarkable histological changes of the lung and skin, such as encephalemia, at the very beginning of the sampling procedure, and plentiful mononuclear cells in marrow appeared 6 h after exposure. The micronucleus test of marrow cells showed that the micronucleus rate had a positively dose-dependent effect.

Fast and sample cleanup-free measurement of nicotine and cotinine by stable isotope dilution ultra-performance liquid chromatography–tandem mass spectrometry

We developed a stable isotope dilution ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-D₄ and cotinine-D₄ as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5-35,000 μg/L (r>0.999). Limits of Detection were 0.7 μg/L for nicotine and 0.4 μg/L for cotinine, and Lower Limits of Quantification were 1.7 μg/L for nicotine and 1.1 μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were <4% and <2%, respectively, the interassay CVs were <6% for nicotine and <4% for cotinine. The inaccuracy was <6% for both substances. The mean recovery was 103.2% (range 96.8-105.1%) for nicotine and 97.4% (range 94.3-99.2%) for cotinine. A method comparison showed that the values of nicotine metabolites in human urine samples (n=98) measured by a commercially available chemiluminescent immunoassay tested on analyzer IMMULITE 2000 were much higher than the cotinine concentration in the same urine samples measured by our UPLC-MS/MS assay. The Passing-Bablok regression line was: immunoassay=4.62 (UPLC-MS/MS)+3.64 [μg/L]; r=0.75. This robust, sensitive and interference-free UPLC-MS/MS assay permits rapid and accurate determination of nicotine and cotinine in human urine.

Simultaneous determination of albendazole and its metabolites in fish muscle tissue by stable isotope dilution ultra-performance liquid chromatography tandem mass spectrometry

A rapid, specific, and sensitive method utilizing ultra-performance liquid chromatography tandem mass spectrometry was developed and validated to determine albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole 2-aminosulfone in fish muscle tissue. The fish samples were extracted with ethyl acetate, then the organic phase was evaporated to dryness, and the residue was reconstituted in methanol-water solution and cleaned up by n-hexane. Reversed-phase separation of target compounds was achieved using a BEH C18 column and a gradient consisting of 0.2% (v/v) formic acid and methanol. Tandem mass spectrometry analyses were performed on a triple-quadrupole tandem mass spectrometer. In the whole procedure, the isotope-labeled internal standards were used to correct the matrix effect and variations associated with the analysis. The method was validated with respect to linearity, specificity, accuracy, and precision. The method exhibited a linear response from 0.1 to 20 ng mL(-1) (r(2) > 0.9985). The limit of quantitation for albendazole (ABZ), albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO(2)), and albendazole 2-aminosulfone (ABZ-2-NH(2)SO(2)) was 0.1, 0.1, 0.1, and 0.2 ng g(-1), respectively. The mean recoveries of ABZ, ABZSO, ABZSO(2), and ABZ-2-NH(2)SO(2) spiked at a level of 0.2-5.0 ng g(-1) were 95.3-113.7%, and the relative standard deviations of intra- and inter-day measurements were less than 6.38%. The method was later successfully applied to the determination of albendazole and its three metabolites in 60 fish samples collected from local markets.

Simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultraperformance liquid chromatography–tandem mass spectrometry

A simple and sensitive analytical method was developed for the simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Samples were directly purified through HLB cartridge. The organic phase was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by UPLC–MS/MS on an Acquity UPLC ® BEH C 18 column with gradient elution. The samples were quantified using clenbuterol-D 9, chloramphenicol-D 5 and diethylstilbestrol-D 8 as internal standards. The proposed method was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total time required for the analysis of one sample was about 50 min.