4-octyl itaconate inhibits high glucose induced renal tubular epithelial cell fibrosis through TGF-ß-ROS pathway.

Diabetic kidney disease (DKD) is one of the most serious complications of diabetes and has become the leading cause of end-stage kidney disease, causing serious health damage and a huge economic burden. Tubulointerstitial fibrosis play important role in the development of DKD. Itaconate, a macrophage-specific metabolite, has been reported to have anti-oxidant, anti-inflammatory effects. However, it is unknown whether it perform anti-fibrotic effect in renal tubular epithelial cells. In this current study, we observed that in human renal tubular epithelial cells (HK2), high glucose induced an increase in transforming growth factor ß (TGF-ß) production, and upregulated the expressions of fibronectin and collagen I through the TGF-ß receptor as verified by administration of TGF-ß receptor blocker LY2109761. Treatment with 4-octyl itaconate (4-OI), a derivant of itaconic acid, reduced the TGF-ß production induced by high glucose and inhibited the pro-fibrotic effect of TGF-ß in a dose-dependent manner. In addition, we found that 4-OI exerted its anti-fibrotic effect by inhibiting the excessive production of ROS induced by high glucose and TGF-ß. In summary, 4-OI could ameliorate high glucose-induced pro-fibrotic effect in HK2 cell, and blocking the expression of TGF-ß and reducing the excessive ROS production may be involved in its anti-fibrotic effect.

[Study on determination methods and analysis of micronutrients in take-away meals].

OBJECTIVE: To comprehensively analyze the trace nutrient contents in take-away meals, the simultaneous detection method of common vitamins in take-away meals were explored based on the samples' matrix, and the content of trace nutrients in take-away meals was analyzed combined with inductively coupled plasma-mass spectrometry(ICP-MS) detection of common elements. METHODS: Fifty-seven take-away meals were collected randomly and analyzed. Vitamins were determined by high performance liquid chromatography-ultraviolet detector tandem fluorescence detector after pretreatment of samples including enzymatic digestion, hydrolysis and extraction. The separation was performed on a C_(18) column(250 mm×4.6 mm, 5 µm) with ion-pair acid reagents as the mobile phase for water-soluble vitamins and methanol for fat-soluble vitamins. Vitamin B_1, vitamin B_2, nicotinic acid, nicotinamide and vitamin A were detected by ultraviolet detector(UVD), while vitamin B_6 and E by fluorescence detector(FLD). Elemental analysis of calcium, magnesium, sodium, potassium, zinc, selenium and copper in the take-away meals was carried out according to GB 5009.268-2016 by ICP-MS to comprehensively evaluate the contents of micronutrients. RESULTS: Through optimization of chromatography and sample pretreatment conditions, the sensitivity of the established detection method can meet the needs of micronutrient evaluation with the detection limits and quantification limits of vitamins in the range of 0.002-0.098 mg/100 g and 0.007-0.327 mg/100 g, respectively. Good precision was obtained(<10%). The spiked recovery rates were 80.5%-103.8%(n=6). The result showed that the contents of micronutrients in take-away meals were generally low. The detection rates of vitamins ranged from 21.1% to 98.2%. CONCLUSION: The proposed method is simple and sensitive, and the contents of vitamins and elements determined were low in the collected take-away meals.

Establishment and application of a screening method for 354 organic toxicants in blood and urine with high-performance liquid chromatography-high-resolution orbitrap mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-high-resolution orbitrap mass spectrometry method was developed for the simultaneous screening of 354 organic poisons and metabolites in blood and urine, including drugs, medications, pesticides, rodenticides, veterinary drugs, alkaloids, and mycotoxins with a multi-toxicant chromatography-mass spectrometry information library. The method and library showed good prospects in clinical poisoning screening and forensic toxicological identification. Blood and urine samples were extracted successively with ethyl acetate in acidic and alkaline conditions; then, the extract was blown to nearly dry by nitrogen gas and redissolved with methanol-aqueous solution (v:v, 50:50), and the dissolved solution was analyzed by LC-MS/MS after filtering. Precursor ions' m/z was set for identification, retention time, fragment ions, and isotopic pattern which were used for confirmation. No interference peaks were found in the blank samples, showing good specificity. The LODs of toxicants in urine and blood were 1.00×10-3-50.0 ng/mL and 2.07×10-3-50.0 ng/mL, respectively, while the LOQs were 3.30×10-3-1.67×102 ng/mL and 6.91×10-3-1.67×102 ng/mL. The intra-day precision and inter-day precision of urine samples were 2.31-9.13% and 4.75-12.3%, respectively, which were 1.92-10.8% and 2.01-12.1% in blood samples. The established method was applied to analyze 9 cases of clinical poisoning patients, and bromadiolone, carbofuran, and amanitins were detected, respectively. A total of 382 biospecimens from drug abusers were analyzed with the proposed method, which indicated that some drugs were detected in 62 cases, mainly including methamphetamine, heroin, and MDMA. The results were consistent with the information from traditional liquid chromatography-triple quadrupole mass spectrometry.

Association between nickel exposure and body compositions in the United States: a population-based cross-sectional study.

BACKGROUND: Increasing body fat or decreasing muscle and bone mass were associated with worse health outcomes in the adult population. The effects of nickel exposure on body composition are not known. The aim of the current study was to investigate the relationship between urinary nickel levels and body compositions. MATERIALS AND METHODS: Two thousand seven hundred sixty-two participants were included in the analysis from the National Health and Nutrition Examination Surveys of 2017-2018 after excluding participants who have missing data on urinary nickel and those with missing all body mass component data. We used weighted generalized linear models to explore the relationship between urinary nickel and body mass components under interpolating missing covariable values. Simultaneously, sensitivity analyses and subgroup analysis were conducted to verify stability of analysis result. Curve fitting and saturation effect analysis were used to explore the possible nonlinear relationship between urine nickel and body compositions. RESULTS: Among the 2,762 participants, the average urinary nickel level was 1.58 ug/L. The weighted generalized linear models, the sensitivity analyses and subgroup analyses found no significant linear relationship between urinary nickel and body compositions. For body weight, BMI, TLM, ALM, TRF, TOF and BMC, the urine nickel saturation effect values were 0.76, 0.74, 0.5, 0.67, 0.64, 0.48, and 0.45 ug/L, respectively. For each 1 ug/L rise in urinary nickel levels at levels below the turning point, body weight increases (ß = 9.06, 95% CI = 2.75, 15.36, p = 0.01), BMI increases (ß = 3.20, 95% CI = 1.36, 5.05, p = < 0.001), TLM decreases (ß = -47.39, 95% CI = -97.38, 2.59, p = 0.06), ALM decreases (ß = -37.25, 95% CI = -63.25, -11.24, p = 0.01), TRF increases (ß = 20.68, 95% CI = 1.50, 39.86, p = 0.03), TOF increases (ß = 57.92, 95% CI = -0.12, 115.95, p = 0.05), and BMC decreases (ß = -6.84, 95% CI = -12.64, -1.04, p = 0.02). CONCLUSIONS: In summary, our study demonstrated that a dose-response relationship exists between urinary nickel and body compositions, with a low inflection point level of urinary nickel for the saturation effect.

A simplified D3 -creatine dilution method for skeletal muscle mass determination with dynamic correction of creatinine and D3 -creatinine using ultra-performance liquid chromatography-tandem mass spectrometry.

This study developed a simple method for muscle mass determination based on D3 -creatine dilution by removing the matrix effects of ultra-performance liquid chromatography-tandem mass spectrometry analysis through mutual correction of creatinine and D3 -creatinine. Rats were administered an oral tracer dose of D3 -creatine at age 6 weeks. Creatinine and D3 -creatinine in urine were detected using ultra-performance liquid chromatography-tandem mass spectrometry after diluting 20 times to obtain D3 -creatinine enrichment factor (mole percent excess). The mole percent excess obtained from peak area could be used to calculate muscle mass using the improved formula. The limit of detection was 0.500 ng/mL for D3 -creatinine. Creatinine and D3 -creatinine could be mutually corrected because of the same matrix effect, and D3 -creatine spillage was negligible within 0.22%. Isotopic steady time was consistent with that obtained using conventional methods. Bland-Altman plots demonstrated the satisfying consistency between the proposed method and magnetic resonance imaging. This is a simple and rapid measuring method of muscle mass based on D3 -creatine dilution that requires no accurate quantification of creatinine and D3 -creatinine concentrations and no urine sample collection to obtain D3 -creatine spillage.

Research progress in human biological monitoring of aromatic hydrocarbon with emphasis on the analytical technology of biomarkers.

Aromatic hydrocarbons are unsaturated compounds containing carbon and hydrogen that form single aromatic ring, or double, triple, or multiple fused rings. This review focuses on the research progress of aromatic hydrocarbons represented by polycyclic aromatic hydrocarbons (including halogenated polycyclic aromatic hydrocarbons), benzene and its derivatives including toluene, ethylbenzene, xylenes (o-, m- and p-), styrene, nitrobenzene, and aniline. Due to the toxicity, widespread coexistence, and persistence of aromatic hydrocarbons in the environment, accurate assessment of exposure to aromatic hydrocarbons is essential to protect human health. The effects of aromatic hydrocarbons on human health are mainly derived from three aspects: different routes of exposure, the duration and relative toxicity of aromatic hydrocarbons, and the concentration of aromatic hydrocarbons which should be below the biological exposure limit. Therefore, this review discusses the primary exposure routes, toxic effects on humans, and key populations, in particular. This review briefly summarizes the different biomarker indicators of main aromatic hydrocarbons in urine, since most aromatic hydrocarbon metabolites are excreted via urine, which is more feasible, convenient, and non-invasive. In this review, the pretreatment and analytical techniques are compiled systematically for the qualitative and quantitative assessments of aromatic hydrocarbons metabolites such as gas chromatography and high-performance liquid chromatography with multiple detectors. This review aims to identify and monitor the co-exposure of aromatic hydrocarbons that provides a basis for the formulation of corresponding health risk control measures and guide the adjustment of the exposure dose of pollutants to the population.

Associations between urinary concentrations of bisphenols and serum concentrations of sex hormones among US. Males.

BACKGROUND: Bisphenol A (BPA) exposure and its structural analogs (BPS and BPF) might cause endocrine alterations and adverse physiological effects. Few studies to date have directly explored the association between its structural analogs (BPS, BPF) and sex hormones in adult male participants. Therefore, we aimed to assess the associations between BPA, BPS, BPF, and sex hormones in American adult men. METHODS: We used data from the U.S. National Health and Nutrition Examination Survey 2011-2016. We excluded participants without data available on sex hormones and urinary bisphenols. Furthermore, participants consuming sex hormone medications were excluded. Multivariable regression models were performed to assess the association between bisphenols and sex hormones. RESULTS: In this study, 2367 participants were included. Of 2367, in 1575 participants, the data on BPS and BPF were available. We found that a per unit increase in BPF was associated with 0.575 ng/dL higher total testosterone (TT) (Model 2: 95% CI: 0.047, 1.103, P = 0.033). However, there was no significant association between BPA or BPS and TT. Furthermore, increased BPA and BPS levels were associated with higher levels of sex hormone-binding globulin (SHBG) (Model 2: ß = 0.364, 95% CI: 0.158, 0.571; ß = 0.25, 95% CI: 0.071, 0.429, respectively). Additionally, participants in the highest BPA exposure quartile (quartile 4) had 4.072 nmol/L higher levels of SHBG than those in quartile 1 (Model 2: 95% CI: 0.746, 7.397, P = 0.017; P for trend =0.005). Both BPA and BPS were negatively associated with free testosterone (FT, nmol/L) after full adjustment (Model 2, ß = - 0.01%, P = 0.0211, P = 0.0211; Model 2, ß = - 0.01%, P = 0.0258, respectively). However, BPF was positively associated with FT (Model 2, ß = 0.0029%, P = 0.0028). CONCLUSION: Our study indicated that exposure to both BPA and its substitutions could alter sex hormone levels. This finding supports the possibility that human exposure to bisphenols at environmental levels might affect the endogenous hormone balance.

Association between testosterone and serum soluble α-klotho in U.S. males: a cross-sectional study.

PURPOSE: Testosterone plays a crucial role in males, and the deficiency of testosterone leads to multiple adverse health conditions. Klotho is a recently discovered protein encoded by antiaging gene klotho. Both the levels of testosterone and klotho change with aging, so the relationship between them is worth exploring. The purpose of this study was to investigate whether total testosterone is associated with serum klotho levels in U.S. males aged 40-79 years. METHODS: Included in this study were 3750 male participants from the 2011 to 2016 National Health and Nutrition Examination Survey, aged 40-79 years with included information on klotho and sex hormones. The sex steroid hormone levels and klotho concentrations were assayed in laboratories using the recommended methods according to Nutrition Examination Survey guidelines. The association between sex hormones and klotho was calculated using multivariate linear regression models after adjustment for several possible confounding variables. RESULTS: Among the 3750 participants, the total testosterone concentration was 399.048 ± 184.780 ng/dL, and the testosterone deficiency prevalence was 1160 (30.942%). The geometric mean of serum klotho levels was 791.000 pg/mL. In the adjusted models, klotho increased 0.165 pg/mL for every 1 ng/dL increase of total testosterone (p = 0.004). In addition, estradiol (ß 2.232; 95% CI 0.588-3.876; p = 0.032) and sex hormone-binding globulin (ß 2.013; 95% CI 1.173-2.583; p = 0.002) were also positively associated with klotho concentrations. CONCLUSION: This study reported a significant association between klotho and sex hormones in the U.S. male population. The levels of klotho in men increased with total testosterone, estradiol and sex hormone-binding globulin levels, which may have implications for future research and clinical practice.

Changes in Gel Characteristics, Rheological Properties, and Water Migration of PSE Meat Myofibrillar Proteins with Different Amounts of Sodium Bicarbonate.

The changes in the gel and rheological properties and water-holding capacity of PSE meat myofibrillar proteins with different amounts of sodium bicarbonate (SC, 0−0.6/100 g) were studied. Compared to the PSE meat myofibrillar proteins with 0/100 g SC, the texture properties and cooking yield significantly increased (p < 0.05) with increasing SC; meanwhile, adding SC caused the gel color to darken. All samples had similar curves with three phases, and the storage modulus (G') values significantly increased with the increasing SC. The thermal stability of the PSE meat myofibrillar proteins was enhanced, and the G' value at 80 °C increased with the increasing SC. Because water was bound more tightly to the protein matrix, the initial relaxation times of T21 and T22 shortened, the peak ratio of P21 significantly increased (p < 0.05), and the P22 significantly decreased (p < 0.05), which implied that the mobility of the water was reduced. Overall, SC could improve the thermal stability of the PSE meat myofibrillar proteins and increase the water-holding capacity and textural properties of the cooked PSE meat myofibrillar protein gels.

[Determination of Haloacetic Acids, Disinfection Byproducts, in Tap Water with Reversed-Phase Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry].

Objective: To establish a method for quantitative analysis of haloacetic acids (HAAs), disinfection byproducts, in tap water with reversed-phase ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry. Methods: Tap water samples were collected and 0.70 g/L ascorbic acid was added to eliminate residual chlorine. Then, the water samples were directly injected into the instrument for analysis after filtration. After separation on a pentafluorobenzene (PFP) column with an inner diameter of 1.0 mm at a higher linear velocity and a lower volume flow rate compared with those of a narrow-bore column, nine HAAs, namely, monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), trichloroacetic acid (TCAA), bromodichloroacetic acid（BDCAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were examined by negative electrospray ionization and full MS/dd-MS 2 acquisition mode. In order to adjust for the matrix effect, matrix matching calibration curves were used to quantitate the nine HAAs. Results: Good linearity was obtained for each of the nine HAAs within their respective linear ranges. The detection limits and quantification limits of the method were 0.020-1.0 µg/L and 0.060-3.0 µg/L. The recoveries were 69.8%-119%. Conclusion: The proposed method showed strengths in separation speed and qualitative accuracy. It did not require for complicated pretreatment procedures and can meet the need of tap water sample analysis.

[The Screening Approaches for 34 Common Drugs and Metabolites in Biological Samples by Liquid Chromatography Orbital Trap Mass Spectrometry].

Objective: To establish a high-performance liquid chromatography orbital trap mass spectrometry (HPLC-Obitrap MS) method for screening 34 common drugs and metabolites in biological samples. Methods: The target analytes in urine and blood samples were extracted with ethyl acetate, concentrated by nitrogen blowing and redissolved. The hair samples were washed with water and acetone, dried and cut into bits of about 1 mm, and then crushed in a freezing grinder. The analytes were extracted with methanol, and after filtration, the filtrate was used for instrumental analysis. Hypersil Gold PFP (2.1 mm×100 mm, 3 µm) column was used for chromatographic separation. Methanol and 5 mmol/L ammonium acetate solution were used as mobile phase with gradient elution at a flow rate of 400 µL/min. Mass spectrometry was done by electrospray positive and negative ion alternation mode. The data were collected using Full MS and Full MS/dd-MS2 mode. Xcalibur 4.0 software was used to control instruments and to collect data, and TraceFinder 3.3 was used for screening and identification. Results: The method's detection limits for 34 drugs and their metabolites in blood, urine and hair samples were 3.30-10700 ng/L, 4.43-5440 ng/L, 0.0350-4.21 µg/kg, respectively. The intra-day and inter-day precisions of the spiked samples at the levels of 5.0, 10, and 20 µg/L were 3.50%-6.00% and 4.18%-9.90%, respectively. A total of 1125 biological samples of urine, blood and hair were collected and screened. The results showed that 96.7% of the drug users were taking a single drug, while 3.3% were mixed drug users. The main types of drug of abuse were methamphetamine (75.8%), heroin (18.5%), ketamine (2.4%) and other drugs (3.3%), and 87.9% of the positive samples were from male users. Compared with the results of high-performance liquid chromatography triple quadrupole mass spectrometry, this method can be used to identify more types of drugs in one run and to conduct retrospective analysis. Conclusion: The method established in the study is simple and sensitive and is well suited for the screening of common drugs and metabolites in biological samples.

[Determination of halogenated carboxylic acids in drinking water by ion chromatography-orbitrap mass spectrometry method].

OBJECTIVE: To establish a method for determination of the common and emerging halogenated carboxylic acids(HCAs) in drinking water by ion chromatography(IC) and quadrupole-orbitrap(Q-Orbitrap) high resolution mass spectrometry(HRMS) combined the traditional quantitative detection with semi-target analysis. METHODS: Effects on the type of chromatographic column, the composition of mobile phase, the flow rate of acetonitrile added post column, the column temperature, and the injection volume were studied in detail for IC-HRMS method, also for HRMS conditions. Drinking water sample was directly injected into IC-HRMS for analysis after filtration. The chromatography separation was performed on an AS21 anion exchange chromatography column(2 mm×250 mm) with the gradient elution using 800 mmol/L methylamine-water as mobile phase, and acetonitrile was added after column. The detection was conducted on HRMS with the electrospray ionization negative mode. And the quantitative analysis of 8 haloacetic acids(HAAs) and semi-target screening of 19 HCAs were achieved by full MS/dd-MS~2 mode. RESULTS: Good linearity(r>0.996) was obtained for each of 8 HAAs for IC-HRMS. The method detection limits(MDLs) and method quantification limits(MQLs) were in the range of 0.50-2.5 µg/L and 1.7-8.3 µg/L, respectively. Intra-and inter-day relative standard deviations(RSDs) were in the range of 1.50%-11.0% and 4.58%-10.9%, respectively. The recoveries were in the range of 61.3%-117%(n=6). The proposed method was applied to analyze 39 drinking water samples, and dichloroacetic acid and trichloroacetic acid were detected and quantified, with concentrations ranging from 1.35 to 48.0 µg/L. Besides, five HCAs(difluoroacetic acid, trifluoroacetic acid, bromochloroacetic acid, monochloropropionic acid and dichloropropionic acid) were preliminary identified with semi-target screening method. CONCLUSION: The developed method was simple, rapid, no sample preparation except filtration and low reagent cosumption, which could meet the need of drinking water monitoring and achieve comprehensive screening of HCAs in drinking water. In addition, full MS/dd-MS~2 data acquisition mode could provide retrospective analysis of existing data by adding the emerging or interesting HCAs into the screening compound database.

[Study on the Analytical Method of Solid Phase Extraction-Ultra Performance Liquid Chromatography Tandem Quadrupole Linear Ion Trap Mass Spectrometry for 12 Perfluorinated Compounds in Human Urine].

OBJECTIVE: To establish a method for simultaneous determination of 12 kinds of perfluorinated compounds (PFCs) in human urine based on ultra performance liquid chromatography tandem quadrupole linear ion trap mass spectrometry (UPLC-QTtrap-MS). METHODS: After pH adjustment with 2% formic acid, the urine samples were loaded on a WAX solid phase extraction (SPE) cartridge for extraction, purification and concentration. The eluates were collected, concentrated to dryness under nitrogen, and reconstituted with 10 mmol/L ammonium acetate aqueous solution-methanol ( V water∶ V methanol = 70∶30) before injection. UPLC was performed on a C 18 cartridge, and methanol and 10 mmol/L ammonium acetate aqueous solution was used as mobile phases with gradient elution. QTtrap-MS was operated in multiple reaction monitoring (MRM) mode, and the internal standard calibration curves were applied for quantitative analysis. RESULTS: Good linearity was obtained in the linear range, with the method detection limits and method quantification limits being 0.032 ng/L-6.5 ng/L and 0.10 ng/L-21 ng/L, respectively, for the 12 kinds of PFCs. The spiked recoveries of the 12 kinds of PFCs were 91.5%-114%, with the intra-day precision and the inter-day precision being 0.57%-16.0% and 1.88%-20.1%, respectively. The established method was applied to the determination of 12 kinds of PFCs in the urine samples of primary school students collected in one area. Nine kinds of PFCs were detected in the urine samples in this area. Among the PFCs detected, perfluorobutanesulfonic acid (PFBS) and perfluorooctanoic acid (PFOA) were the main PFCs found in the student urine samples. CONCLUSION: The method established in this study could be used to simultaneously examine 12 kinds of PFCs in urine. The method combined SPE with isotope internal standard correction and achieved good sensitivity and accuracy.

Takeaway food in Chengdu, Sichuan province, China: Composition and nutritional value.

BACKGROUND AND OBJECTIVES: The popularity of takeaway has caused health problems. To analyse the basic nutrients and composition of popular takeaway meals in Chengdu, China. METHODS AND STUDY DESIGN: We randomly collected 105 takeaway meals from takeaway platforms. The quality of ingredients such as grains, vegetables, and meat were assessed and weighed. The samples were then homogenised, and the nutrients were detected following the AOAC Official Methods of Analysis. RESULTS: Compared with Chinese and US dietary reference intakes, the average energy, protein, salt, fat, vitamin, and available carbohydrate contents exceeded dietary recommendations for one takeaway meal. By contrast, the whole grain, vegetable, fruit, dairy product, egg, mineral, and dietary fibre contents were insufficient. Food compositions and basic nutrients differed among takeaway meals prepared with various cooking methods and meats. Fried rice had the lowest nutritional value. The fried dish set meal had high energy density. The nutrient content of poultry takeaway meals was more balanced compared with other meals assessed, and salt and fat were excessive in mixed meat meals. In addition, meatless takeaway meals tended to have high fat content because of excess vegetable oil added for better taste. CONCLUSIONS: Takeaway meals should have lower contents of energy, fat, carbohydrate, and salt and higher contents of whole grains, vegetables, fruits, dairy products, and eggs. Attention should be paid to the high energy density of the fried dish set meal to prevent resultant health problems such as obesity. Consumers, takeaway outlets, and government agencies need to work together to address the health problems.

[Determination of 8-oxo-2'-deoxyguanosine and Cotinine in Urine by Hydrophilic Chromatography-tandem Mass Spectrometry with Isotope Dilution].

OBJECTIVE: To develop an assay for determination of 8-oxo-2'-deoxyguanosine and cotinine in human urine by hydrophilic chromatography tandem mass spectrometry (HILIC-MS/MS) with isotope dilution. METHODS: The urine supernatant was 1∶5 diluted with 3 mmol/L ammonium formate aqueous solution containing 15N 5-8-OHdG and D 3-cotinine as internal standard. After being filtered through a 0.22 µm water filter, the sample solution was injected into ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Separation was performed on ACQUITY UPLC® BEH HILIC column (50 mm×3.0 mm, 1.7 µm) with isocratic elution (A∶B=10∶90) at 40 ℃. The mobile phase was composed with acetonitrile (B) and 3 mmol/L ammonium formate water soulution (A). The flow rate was 0.3 mL/min. Positive ion scan-multiple reaction monitoring (MRM) mode were used for monitoring and internal standard curves were applied for quantification. RESULTS: Good linearity was obtained under the optimal conditions. Detection limits for 8-OHdG and cotinine were 0.064 µg/L and 0.035 µg/L respectively, the quantitation limits were 0.21 µg/L and 0.12 µg/L respectively, and the recoveries of the spiked urine samples were 92.6%-102% and 102%-106% respectively. Statistical analysis of 40 urine sample determination results obtained by using the above assay showed that there were significant differences in tobacco smoke exposure and tobacco-specific nitrosamine intake between active and passive smoker ( P<0.05). The concentration of NNAL and cotinine were higher in urine samples of active smoker. Tobacco smoke exposure was positively correlated with tobacco specific nitrosamine intake in both active and passive smokers (the correlation coefficients were 0.487 and 0.786 respectively, P<0.05). CONCLUSION: We successfully established a simple and fast assay for simultaneously detecting 8-oxo-2'-deoxyguanosine and cotinine in human urine. It was sensitive and accurate for quntification via the calibration by the isotope internal standards, and can meet the needs of batch analysis.

[Determination of Aromatic Compounds Metabolites in Human Urine by Solid Phase Extraction-liquid Chromatography-Tandem Mass Spectrometry].

OBJECTIVE: To establish the method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with solid phase extraction (SPE) for simultaneous determination of the biological metabolites of aromatic compounds, including N-acetyl-S-phenyl-L-cysteine (SPMA), N-acetyl-S-benzyl-cysteine (SBMA), p-nitrophenol (PNP), methylhippuric acids (MHA), p-Aminophenol (PAP), mandelic acid (MA), phenylglyoxylic acid (PGA) and 1-hydroxypyrene (1-OHP) in urine. METHODS: After adding 20 µL of ß-glucuronidase and 1 mL ammonium acetate buffer solution in 1 mL of urine, the sample was digested in a 37 ℃ incubator for 20 h. After digestion, the enzymatic hydrolysate was purified by PRIME HLB solid phase extraction column. The target compounds were eluted with 4 mL of acetonitrile and blown to dryness with nitrogen, reconstituted with 0.20 mL of methanol. Injected the sample solution into LC-MS/MS system for analysis after filtering with 0.22 µm filter membrane. LC separation was carried out on a reversed-phase C18 column (2.1 mm×150 mm, 3.5 µm); gradient eluting was performed at a flow rate of 0.2 mL/min. The water containing 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The mass spectrometry was performed with multiple reaction monitoring (MRM) mode, using alternating positive and negative ions, and internal standard curves were used for quantification. RESULTS: The eight metabolites showed good linearity within the range of 1-100 ng/mL, with a correlation coefficients greater than 0.995, and the relative precision deviation (RSDs) was 0.050%-9.95%. The method detection limits (MDLs) of the eight target metabolites were 0.041-0.12 ng/mL. The proposed method was used for urine sample analysis and the spiked recoveries were 80.1%-114.0%. CONCLUSION: The established method is quick, sensitive and accurate; it meets the requirementof the biological monitoring of aromatic compounds for the general population and occupational population.

[Detecting Nicotine and Cortinine in Hair by Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry].

OBJECTIVE: To develop a method for detecting nicotine and cotinine in hair by hydrophilic interaction chromatography tandem mass spectrometry. METHODS: Hair samples were hydrolyzed in sodium hydroxide solution before extraction with dichloromethane. The samples were blown to dry with nitrogen and dissolved with mobile phase. The filtrate of the samples was injected into a chromatographic-mass spectrometry system for analysis. The separation was performed by a hydrophilic column, with which methanol-0.1% ammonia was used as the mobile phase. The quantitative detection of Nicotine and Cortinine was carried out with electron spray ionization-triple quadrupole mass spectrometry. The established method was used for detecting nicotine and cotinine in 602 hair samples of pregnant women and 31 hair and urine samples of volunteers. RESULTS: A standard curve was drawn for the established method of hydrophilic liquid chromatography tandem mass spectrometry. Good linearity was obtained for detecting nicotine and cotinine in the range of 0.030-100.000 µg/L, with a detection limit (MDL) of 0.007 6 µg/g and 0.004 4 µg/g, respectively. The inter-day and intra-day precisions reached a level of less than 10%. The recoveries of the spiked samples ranged from 81.0% to 102.0%. About 0.020-0.260 µg/g nicotine and 0.004 8-0.069 0 µg/g cotinine were detected in the pregnant women without exposure to secondhand smoking (SHS), compared with 0.029-0.350 µg/g nicotine and 0.005 6-0.085 0 µg/g cotinine in those exposed to SHS. Nicotine and cotinine were also found in the hair and urine samples of volunteers, which were correlated with smoking (P < 0.05). A dose-response relationship were found between smoking and hair nicotine. CONCLUSIONS: The proposed method is accurate and sensitive for detecting nicotine and cotinine in hair samples. Hair nicotine can be a specific biomarker for assessing exposure to tobacco smoking.

Bypass of an Abasic Site via the A-Rule by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

The abasic site is one the most common DNA lesions formed in cells; it induces a severe blockage of DNA replication and is highly mutagenic. We continue to use Gp90 exo-, the sole DNA polymerase from Pseudomonas aeruginosa phage PaP1, to study DNA replication upon encountering an abasic site lesion. Gp90 exo- can incorporate dNTPs opposite the abasic site, but extension past this site is extremely slow. Among the four dNTPs, dATP is preferentially incorporated opposite the abasic site, consistent with the A-rule. The incorporation is independent of the identity of the nucleotide 5' of the abasic site. The incorporation of dATP opposite the abasic site occurs by direct incorporation of dNTP opposite the abasic site without a -1 frameshift deletion. Extension from an A:abasic site pair by Gp90 exo- is slightly unfavorable relative to those from other abasic site pairs. Incorporation of dATP opposite the abasic site is preferential and shows a biphasic shape, indicating that this incorporation is much faster than the subsequent dissociation of the polymerase from DNA. The template sequence does not affect the dATP incorporation priority, burst amplitude, burst rate, or dATP dissociation constant. Surface plasmon resonance shows that the presence of an abasic site in the template weakens the binding affinity of Gp90 exo- to DNA in a binary or ternary complex in the presence of any one kind of dNTP. This study reveals that Gp90 exo- preferentially inserts A opposite an abasic site via the A-rule, like other DNA polymerases (e.g., Pol θ, KlenTaq, KF exo-, Pols α, δ/PCNA, and Thermococcus litoralis Pol Vent (exo-)), providing further insight into DNA replication mediated by P. aeruginosa phage PaP1 upon encountering an abasic site lesion.

Rapid and sensitive determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in human urine samples using UHPLC-MS/MS.

Bisphenol analogues, amphenicol antibiotics, and phthalate have widely aroused public concerns due to their adverse effects on human health. In this study, a rapid and sensitive method for determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in the urine based on ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated. The sample pretreatment condition on the base of mixed-mode anion-exchange (Oasis MAX) SPE was optimized to separate bisphenol analogues and amphenicol antibiotics from phthalate metabolites: the former were detected with a mobile phase of 0.1% ammonium water solution/methanol containing 0.1% ammonium water solution in negative mode, whereas the latter were determined with a mobile phase of 0.1% acetic acid solution/acetonitrile containing 0.1% acetic acid in negative mode. The limits of detection were less than 0.26 ng/mL for bisphenol analogues, 0.12 ng/mL for amphenicol antibiotics, and 0.14 ng/mL for phathalate metabolites. The recoveries of all target analytes in three fortification levels ranged from 72.02 to 117.64% with the relative standard deviations of no larger than 14.51%. The matrix effect was adjusted by isotopically labeled internal standards. This proposed method was successfully applied to analyze 40 actual urines and 13 out of 18 studied compounds were detected. Graphical abstract Simultaneous determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in human urine samples.