Phenols such as bisphenols, parabens, and triclosan are common environmental endocrine disruptors. Previous epidemiological studies have suggested that phenols may affect semen quality, but the results were inconsistent. In addition, most existing studies have been limited to the effects of a single chemical compound, ignoring the health effects of mixed exposure to multiple chemicals. Thus, we aimed to explore the associations between individual and mixed exposure to phenols and various semen quality parameters. In this study, a rapid and sensitive method was used to determine 18 phenolic compounds in urine samples of 799 volunteers who donated sperm samples to the Shanghai Human Sperm Bank. A spot urine sample was collected from each subject on the day of their clinic visit and stored at -20 ℃ until testing. Urine samples (200 μL) were extracted and added with 20 μL of an internal standard and 50 μL of β-glucuronidase solution. The mixtures were then incubated for 12 h at 37 ℃. After hydrolysis, the samples were extracted twice using ethyl acetate (500 μL). The concentrations of the 18 phenolic compounds were measured using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Semen quality parameters were analyzed using a computer-aided semen analyzer. Multiple linear regressions were used to detect the associations between individual phenol exposure and semen quality parameters. In addition, weighted quantile sum (WQS) models were used to explore the associations between mixed-phenol exposure and semen quality parameters. After adjusting for potential covariates, the results of multiple linear regressions showed that exposure to ethyl paraben (EtP) was significantly negatively associated with sperm concentration and total sperm count (P<0.05). In addition, exposure to mixed phenols was significantly associated with decreased sperm concentration; methyl paraben (MeP) and EtP were identified as the main contributors to this decrease. Thus, phenol exposure may be associated with decreased semen quality in young males, particularly with respect to sperm concentration and total sperm count.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) have been extensively used as synthetic fluorine-containing compounds in various consumer products, including surfactants, cookware, lubricants, clothing, and food packaging, since the 1950s. Evidence has shown that PFASs cross the placental barrier and interfere with fetal thyroid hormone homeostasis, which is crucial for fetal growth and neurobehavioral development in children aged 2-9 years. However, no epidemiological data on the association between prenatal PFAS exposure and neonatal neurobehavioral development are available. In this study, we explored the association between prenatal PFAS exposure and neonatal neurobehavioral development based on the Ezhou cohort study. Blood samples (10 mL) were collected during the third trimester of pregnancy (28-36 weeks) at the Ezhou maternal and child health hospital. The blood specimens were centrifuged at 4000 r/min for 15 min immediately after collection, separated, stored at -80 ℃. The samples were analyzed for seven PFASs, namely, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroheptanesulfonic acid (PFHpS), and perfluorooctane sulfonamide (PFOSA). The PFASs were separated using a C18 column (100 mm×2.1 mm, 1.7 μm) at an oven temperature of 40 ℃, injection volume of 10 μL, and flow rate of 0.4 mL/min via gradient elution with methanol and ammonium acetate aqueous solution. The instrument was operated in negative electrospray ionization mode with multiple reaction monitoring. The correlation coefficients (r2), limits of detection (LODs) and quantification (LOQs), and spiked recoveries of the seven PFASs were 0.993-0.999, 0.006-0.020 ng/mL, 0.020-0.066 ng/mL, and 84.6%-116.8%, respectively. Neonatal behavioral neurological assessment (NBNA) was used to evaluate newborn cognitive development 72 h after birth; this tool consisted of five clusters, including behavior (six items), passive muscle tone (four items), active muscle tone (four items), primitive reflexes (three items), and general assessment (three items). Each item was rated on a three-point scale (0, 1, or 2), with the 20 items having a maximum score of 40. A total of 379 mother-newborn pairs were included in the analysis. The PFASs with the highest exposure levels was PFOA, with median levels of 19.4 ng/mL. Linear regression models were used to test the effects of ln-converted PFAS levels in newborns. After adjusting for confounding factors, the linear regression model showed that PFOS exposure during pregnancy was associated with decreased active muscle tone(β(95% CI): 0.36(-0.64, 0.08)) and general assessment(β(95% CI): 0.34(-0.61, 0.07)) in all newborns. Furthermore, PFNA exposure was associated with decreased passive muscle tone(β(95% CI): 0.38(-0.74, 0.01)) and total NBNA(β(95% CI): 0.37(-0.68, 0.06)). PFDA exposure was associated with decreased behavior(β(95% CI): 0.28(-0.54, 0.01)), while PFHxS exposure was associated with elevated total NBNA(β(95% CI): 0.27(0.05-0.48)). Gender stratification analysis showed that PFOS exposure during pregnancy was associated with decreased active muscle tone(β(95% CI): 0.54(-0.73, 0.35)) and general assessment(β(95% CI): 0.50(-0.88, 0.13)), PFNA exposure during pregnancy was associated with decreased passive muscle tone(β(95% CI): 0.67(-1.2, 0.14)) and total NBNA(β(95% CI): 0.45(-0.91, 0.01)), PFDA exposure during pregnancy was associated with decreased behavior(β(95% CI): 0.44(-0.71, 0.17)), PFHxS exposure was associated with elevated total NBNA(β(95% CI): 0.41(0.02-0.80)) in male newborns, and PFOA exposure was associated with decreased general assessment(β(95% CI): -0.27(-0.51, 0.02)), and PFDA exposure was associated with elevated behavior(β(95% CI): 0.46(0.40-0.52)) in female newborns. The proposed method separates and detects various PFASs without the need for cumbersome pretreatment processes, and has the advantages of low LODs, satisfactory recoveries, and accurate precision. Thus, it allows for the simultaneous analysis of trace PFASs in microserum samples from pregnant women. Our results also showed that prenatal PFAS exposure can lead to neurobehavioral disorders in offspring, with male newborns showing greater sensitivity than female newborns.

The discovery and identification of mushroom toxins has long been an important area in the fields of toxicology and food safety. Mushrooms are widely favored for their culinary and medicinal value; however, the presence of potentially lethal toxins in some species poses a substantial challenge in ensuring their safe consumption. Therefore, the development of a robust and sensitive analytical method is necessary for accurately identifying the risks associated with mushroom consumption. The study of mushroom toxins, which are characterized by their diversity and substantial variations in chemical structures, present a considerable challenge for achieving precise and high-throughput analysis. To address this issue, the present study employed a robust approach combining a solid-phase extraction (SPE) purification technique with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to establish an analytical method for the detection and quantification of five amatoxins and two tryptamines (psilocybin and bufotenine) present in some mushrooms. Several optimization procedures were undertaken, including optimizing the chromatographic conditions, mass spectrometric parameters, and sample extraction and purification. The procedure involved the extraction of dry mushroom powder with methanol containing 0.3% formic acid, followed by purification using a strong cation exchange cartridge (SCX). The analytes were separated on a T3 chromatographic column (100 mm×2.1 mm, 1.8 μm) using mobile phases of acetonitrile and 5 mmol/L ammonium acetate solution containing 0.1% formic acid. The multiple reaction monitoring (MRM) mode was employed for data acquisition. Amatoxins were quantified using matrix-matched standard calibration curves, whereas isotopic internal standards were used to quantify tryptamine. The results showed that all seven toxins exhibited good linearities (r2>0.99) within the optimized concentration range. The limits of detection (LODs) for bufotenine, psilocybin, and amatoxins were determined as 2.0, 5.0, and 10 μg/kg, respectively, while the limits of quantification (LOQs) were determined as 5.0, 10, and 20 μg/kg, respectively. The LOD and LOQ values further underscore the ability of the method to detect minute quantities of toxins, making it particularly well suited for screening food samples for potential contamination. Using dried shiitake mushroom powder as the matrix, the recoveries of the two tryptamines ranged from 80.6% to 117%, with relative standard deviations (RSDs) ranging from 1.73% to 5.98%, while the recoveries of amatoxins ranged from 71.8% to 115%, with RSDs varying from 2.14% to 9.92% at the three concentration levels. The consistent and satisfactory recoveries of amatoxins and tryptamines demonstrated the ability of this method to accurately quantify the target analytes even in a complex matrix. Comparison with the results of supplementary test method recognized by State Administration for Market Regulation for food (BJS 202008) demonstrated comparable results, indicating no significant differences (p>0.05) in amatoxin contents. The newly developed method is rapid, accurate, precise, meets the required standards, and is suitable for the detection of seven toxins in wild mushrooms. As part of the application of this method, a comprehensive investigation of the distribution of toxins in wild mushrooms from Fujian Province was undertaken. In this study, 59 wild mushroom samples from nine cities were collected in the Fujian province. Species identification was conducted using rDNA-internal transcribed space (rDNA-ITS) molecular barcode technology, which revealed the presence of toxins in the two samples. Notably, one specimen named Amanita fuligineoides contained α-amanitin, β-amanitin, and phalloidin in quantities of 607, 377, and 69.0 mg/kg, respectively. Additionally, another sample, identified as Tricholomataceae, had a psilocybin concentration of 12.6 mg/kg.

The widespread and frequent use of antibiotics to treat diseases or encourage animal growth has resulted in their persistence and accumulation in water, soil, and sediments. As a typical emerging pollutant in the environment, antibiotics have become an important research focus in recent years. Antibiotics are commonly found at trace levels in water environments. Unfortunately, the determination of various types of antibiotics, all of which exhibit different physicochemical properties, remains a challenging endeavor. Thus, developing pretreatment and analytical techniques to achieve the rapid, sensitive, and accurate analysis of these emerging contaminants in various water samples is an essential undertaking.In this paper, a solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for the simultaneous determination of 22 antibiotics including 4 penicillins, 12 quinolones and 6 macrolides in environmental water samples was developed. Based on the characteristics of the screened antibiotics and sample matrix, the pretreatment method was optimized, focusing on the SPE column, pH of the water sample, and amount of ethylene diamine tetra-acetic acid disodium (Na2EDTA) added to the water sample. Prior to extraction, a 200 mL water sample was added with 0.5 g of Na2EDTA and pH-adjusted to 3 using sulfuric acid or sodium hydroxide solution. Water sample enrichment and purification were achieved using an HLB column. HPLC separation was carried out on a C18 column (100 mm×2.1 mm, 3.5 μm) via gradient elution with a mobile phase composed of acetonitrile and 0.15% (v/v) formic acid aqueous solution. Qualitative and quantitative analyses were performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode using an electrospray ionization source. The results showed correlation coefficients greater than 0.995, indicating good linear relationships. The method detection limits (MDLs) and limits of quantification (LOQs) were in the ranges of 2.3-10.7 ng/L and 9.2-42.8 ng/L, respectively. The recoveries of target compounds in surface water at three spiked levels ranged from 61.2% to 157%, with relative standard deviations (RSDs) of 1.0%-21.9%. The recoveries of target compounds in wastewater at three spiked levels were 50.1%-129%, with RSDs of 1.2%-16.9%. The method was successfully applied to the simultaneous determination of antibiotics in reservoir water, surface water, sewage treatment plant outfall, and livestock wastewater. Most of the antibiotics were detected in watershed and livestock wastewater. Lincomycin was detected in 10 surface water samples, with a detection frequency of 90%, and ofloxacin showed the highest contents (127 ng/L) in livestock wastewater. Therefore, the present method exhibits excellent performance in terms of MDLs and recoveries compared with previously reported methods. The developed method presents the advantages of small water sample volumes, wide applicability, and fast analysis times; thus, it can be considered a rapid, efficient, and sensitive analytical method with excellent potential for monitoring emergency environmental pollution. The method could also provide a reliable reference for formulating antibiotic residue standards. The results provide strong support for and an improved understanding of the environmental occurrence, treatment, and control of emerging pollutants.

Glyphosate (GLY) and glufosinate (GLUF) are non-selective translocated herbicides that are used in agricultural and non-agricultural land worldwide. The extensive use of GLY and GLUF may lead to their accumulation in soil, which causes soil pollution and affects the soil micro-ecological environment; the accumulated GLY and GLUF also migrate to groundwater via leaching. However, GLY, GLUF, and their metabolites are highly water-soluble and lack chromogenic and fluorescent groups, making them difficult to analyze. Currently, derivatization methods are mostly used to detect GLY, GLUF, and their metabolites. However, these methods also have some drawbacks, such as complex operation, long time consumption, and poor stability. In addition, these compounds are easily passivated and made inactive in soil; they also react with organic matter, humic acid, metal oxides, and heavy metal ions, making their extraction from soil difficult. To date, the method for the determination of GLY, GLUF, and their metabolites in soil is limited. Therefore, it is necessary to establish a quick and sensitive method to determine the residues of GLY, GLUF, and their metabolites in soil. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of GLY, GLUF, and their metabolites in soil. Parameters like extraction solvent, extraction temperature, extraction time, and adsorbents, which affected the extraction efficiencies, were optimized. Finally, the soil samples were extracted with 0.5 mol/L ammonia solution in a bath shaker at 50 ℃, and then centrifuged at 10000 r/min for 5 min. The supernatant was filtered through 0.2-μm syringe filters and then determined by HPLC-MS/MS. A Dikma Polyamino HILIC column (150 mm×2.0 mm, 5 μm) was used for chromatographic separation with good peak shape and high response of the target compounds. Ammonium acetate (0.2 mmol/L) with 0.07% ammonia aqueous solution and acetonitrile were used as the mobile phase. The flow rate of the elute was 0.25 mL/min. MS/MS was conducted under multiple reaction monitoring (MRM) mode using an electrospray ionization (ESI) source, and was quantified by the external standard method using matrix-matched calibration curves. All the target compounds were ionized in the negative ionization mode. The linear ranges of GLY and its metabolites were between 5.0 and 500 μg/L, and those of GLUF and its metabolites were between 2.0 and 500 μg/L. Linear correlation coefficients were greater than 0.99. The limit of detection (LOD) and limit of quantification (LOQ) were assessed using signal-to-noise (S/N) ratios of 3 and 10, respectively. The LOD and LOQ values of both GLY and (aminomethyl)phosphonic acid (AMPA) were 4.0 and 13.3 μg/kg, respectively. The LOD and LOQ values of GLUF, MPP, and N-acetyl glufosinate (NAG) were 2.0 and 6.7 μg/kg, respectively. Method accuracy was acquired by recovery test at three spiked levels (0.02, 0.05, 0.2 mg/kg). The average recoveries of five targets spiked in soil with low organic matter content were 74.2%-101%, and the relative standard deviation (RSD) was 0.93%-6.8%; the average recoveries of the five targets spiked in soil with high organic matter content were 90.8%-116%, and the RSD was 0.40%-7.1%. The established method was used to determine 20 soil samples in peach orchard, and the detection rates of AMPA, GLY, MPP, GLUF and NAG were 45%, 25%, 10%, 5% and 5%, respectively. The maximum residues were 147, 35.2, 154, 21.6 and 11.0 μg/kg, respectively. This method is simple, rapid, green, inexpensive, allows pretreatment without organic reagents, and affords high accuracy, high sensitivity, and good reproducibility. The method is suitable for testing a large number of soil samples with different organic matter contents. It can provide reliable technical support for the study of residue status and environmental behavior in soil.

Carbamate pesticides are a class of synthetic pesticides having wide antimicrobial spectrum, good insecticidal efficacy, and a short residual period. These pesticides are used in agriculture, forestry, and animal husbandry. Their widespread use in the last two decades has led to the existence of drug residues in the environment, which are transferred to food, thereby raising concerns regarding the potential threat to human health. Rapid and accurate detection of carbamate pesticide residues in food is of great significance for food safety, and this requires pretreatment to purify the target components and maximize the accuracy and precision of the analysis. A rapid and accurate analytical method based on online solid phase extraction/purification-high performance liquid chromatography-tandem mass spectrometry (online SPE-HPLC-MS/MS) was established for the determination of eight carbamate pesticides in tomato, rice, and cabbage. About 5.0 g of tomato (without water), 2.0 g of cabbage, and 2.0 g of rice (mixed with 3 mL of water) were vortexed at 1000 r/min for 1 min. After adding 2 g of sodium chloride and 10 mL of acetonitrile containing 0.5% (v/v) formic acid, the samples were extracted and centrifuged. The supernatants were combined after the samples were extracted again. The reconstituted solutions were then purified on a CAPCELL PAK C18 column (50 mm×2.0 mm, 15 μm). When the volume ratio of 0.1% (v/v) formic acid aqueous solution and acetonitrile (used as the mobile phases) was 90∶10 and 35∶65, the eight carbamate pesticides could be completely adsorbed and eluted. The carbamate pesticides were separated on an ACQUITY UPLC CSH C18 column (100 mm×2.1 mm, 1.7 μm) under gradient elution and analyzed in the multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+). Under the optimum conditions, the calibration curves of the eight carbamate pesticide residues showed good linearity (r>0.995) within their respective linear ranges. The limits of quantification (LOQs) and limits of detection (LODs) were in the range of 0.05-1.0 ng/mL (S/N=10) and 0.01-0.3 ng/mL (S/N=3). The recoveries were in the range of 73.76%-112.32% at three spiked levels (2, 10, and 20 ng/mL), with relative standard deviations of 1.28%-13.14% (n=6). The online purification method showed better enrichment and purification ability for the target substances than did the offline purification method and greatly improved the pretreatment efficiency. The loading and purification could be completed within 12 min. The developed method has the advantages of high recovery rate, good reproducibility, accuracy, rapidness, sensitivity, and environment friendliness. It can be used for the determination of the eight carbamate pesticides in plant foods, such as tomato, rice, and cabbage.

Histone post-translational modifications (HPTMs) have been believed to play crucial roles in the regulation of gene transcription. Thus, aberrant modification of histone can contribute to the occurrence and development of diseases such as tumors. To date, formalin fixed paraffin-embedded (FFPE) clinical tissues are believed to be one of the most valuable sample resources in the study of human diseases. Therefore, it is of great significance to reveal the molecular mechanism of cancer and discover the markers of tumor. Proteomics, based on high performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS), has become a powerful tool for HPTM identification. However, HPTM analysis of FFPE samples is yet to be explored; it has not been reported in China to our best knowledge. In this study, a new method based on HPLC-MS/MS was developed for the extraction and separation of histone proteins and analysis and quantification of HPTMs in FFPE tissues. First, the strategy for the extraction and separation of histone proteins from FFPE samples were optimized. After comparing the extraction efficiency of two different methods, which include the acid extraction of histone and extraction of total protein, a novel method was developed for histone extraction, separation, and HPTMs analysis by integrating dewaxed hydration treatment of FFPE tissues with protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Furthermore, the effects of operation parameters on histone extraction and HPTM identification were investigated, including number of paraffin embedded sections and chemical derivation of histone proteins. Thereafter, the identification and quantification of HPTMs were performed using reversed-phase HPLC-MS/MS in data independent acquisition (DIA) mode. Finally, the optimized methods were applied to quantitative analysis of HPTMs in FFPE tissues. A variety of HPTMs were identified; they included lysine methylation, acetylation, crotonylation, etc. Therefore, the spectrum of HPTMs on global level was set for human breast cancer and paracancerous tissues from two cases of FFPE clinical tissues. The sites holding differential HPTM level in cancer and paracancerous tissues were further obtained by quantifying the individual HPTMs. Thus, the relationship between HPTM level and tumor was discussed. Abnormal HPTM sites were characterized using cluster analysis, thus their similar tendency was found. For example, abnormal HPTM sites such as H3K9me3, H3K9ac, and H3K27me3 in cancers are associated with epigenetic regulation. It indicated that different epigenetic events might occur in cancer and paracancerous tissues. Interestingly, we found that the level of H4K20me3 were both significantly down-regulated in the two cancer tissues. HPTM had been thought to be a potential biomarker in breast cancer; therefore, these positive results indicated that our method is effective for HPTMs of clinical FFPE samples. Our study provides a useful tool for the isolation and analysis of HPTMs in clinical FFPE samples, showing the potential for the detection of epigenetic biomarker in cancer.

Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 μm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 μm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R2) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1-108%) except for AZA1 (65.8-68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L-1; DTX1 783 pg L-1; YTX 86.6 pg L-1; desMeC 15.6 pg L-1; PTX2 1.11 × 103 pg L-1) appeared in June 2014. Based on toxins present in phytoplankton samples, it is implied that seafood in the Bohai Sea is more likely to be contaminated by OA group and PTX group toxins, and spring is the high-risk season for toxin contamination.

In this paper an analytical method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the determination of coumarin and its derivatives in tobacco products was developed. The MS/MS fragmentation pathways of the eight coumarins were elucidated. The new analytical method was defined based on two main axes, an extraction procedure with acetonitrile and analyte detection performed by HPLC-MS/MS in electron impact mode. The excellent selectivity and sensitivity achieved in multiple reaction monitoring (MRM) mode allowed satisfactory confirmation and quantitation for the coumarin flavor additives. Under the optimized gradient elution conditions, it took only 4.5 min to separate all eight coumarins. Good linearity for all the analytes were confirmed by the correlation coefficient r², ranging from 0.9987 to 0.9996. The limits of detection (LODs) and limits of quantitation (LOQs) of these compounds were in the range of 0.5-1.7 μg/kg and 1.7-5.2 μg/kg, respectively. The average recoveries at three spiked levels (LOQ, 1.5LOQ, 2LOQ) were all in the range of 69.6%-95.1% with RSDs (n = 6) lower than 5.3%. The method of HPLC-MS/MS developed in this study was initially applied to the research of coumarin flavor additives in tobacco products collected from the located market in Beijing from China and proved to be accurate, sensitive, convenient and practical.