Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS

Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89–107% at three separate fortification levels including the LOQ. Precision for replicates was ≤7.4% relative standard deviation (RSD) for milk and ≤11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.     

Determination of chloramphenicol, enrofloxacin and 29 pesticides residues in bovine milk by liquid chromatography-tandem mass spectrometry

A sensitive method for determination of chloramphenicol, enrofloxacin and 29 pesticides residues in bovine milk by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. Residues of the targets were extracted from milk with acetonitrile, cleaned up by C₁₈-SPE cartridge, and then determined by HPLC-MS/MS. The MS detection was operated in positive or negative ionization mode, depending on the compounds. For confirmation of each target compound, two precursor ion > product ion transitions were selected by multi-reaction monitoring mode (MRM). The method showed good linearity for all the tested compounds over the studied concentration range with correlation coefficient higher than 0.9910. Recoveries for the studied compounds at three spiked levels (0.05, 0.10, 0.19 mg kg⁻¹) in bovine milk were in the range of 71-107% with RSDs not larger than 13.7%, except that recoveries of trifluralin ranged between 62% and 70% at the spiked levels. Limits of quantitation for the analytes were estimated to range between 0.03 × 10⁻³ and 14.5 × 10⁻³ mg kg⁻¹. The proposed method was applied for the determination of the analytes residues in real samples. The found levels of the analytes in milk samples were lower than maximum residues levels (MRL).     

An LC-MS/MS method for creatine and creatinine analysis in paraquat-intoxicated patients

Abstract

A sudden increase in serum creatinine after paraquat intoxication has been reported in several clinical studies. However, this dramatic change of creatinine may be possibly due to an interconversion of creatine-creatinine in relation to paraquat toxicity. In order to investigate the creatine-creatinine relationship, a liquid chromatography tandem mass spectrometry in combination with electrospray ionization was developed and validated for simultaneous determination of creatine and creatinine in the serum. The chromatographic separation was achieved on a Gemini® C₆-Phenyl column with a gradient elution consisting of 0.1% formic acid in ultrapure water and methanol as the mobile phase. The method yielded suitable levels of specificity and selectivity, and calibration curves of creatine and creatinine in serum were linear over the concentration range of 0.5–200?µg mL^?1. The limit of quantification of both compounds was 0.5?µg mL^?1, and the method was accurate within the recovery range of 96.23–102.75%, indicating the robustness of the method. The method was successfully applied to toxicological samples from paraquat-intoxicated patients, and the concentrations of creatine and creatinine were quantified. High creatine concentrations in serum samples were observed which may lead to high serum creatinine despite normal kidney function as creatine is converted to creatinine in proportion to its concentration.     

Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%–102% and the limits of detection were in the range of 1.5–6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%–106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.     

用固相萃取-高效液相色谱-三重四极杆串联质谱法测定海洋沉积物中13种氟喹诺酮类抗生素

为进一步探索海洋沉积物中氟喹诺酮类抗生素的污染情况,基于固相萃取-高效液相色谱-三重四极杆串联质谱(SPE-LC-MS/MS)技术,建立了海洋沉积物中 13 种FQs的测定方法;采用高效液相色谱-三重四极杆串联质谱多反应监测离子模式(MRM)对FQs进行分离检测。结果表明：在优化实验条件下,13种FQs的质量浓度为0.50~100 μg·L⁻¹,目标化合物峰面积与内标物质峰面积之比与质量浓度的线性关系良好(R²>0.99),方法检出限为0.003~0.03 μg·kg⁻¹;在加标量为1 μg·kg⁻¹和10 μg·kg⁻¹时,空白加标的平均回收率为73.5%~124.6%和67.5%~118.5%,相对标准偏差(RSD)为1.0%~9.7%(n=7);以海洋沉积物为基质,13种目标物的加标回收率为67.7%~142.4%,RSD小于10.2%(n=6);使用该方法对广州某湾区海洋沉积物中 13 种 FQs 的残留量进行了实地检测,培氟沙星质量分数最高,为1.6 μg·kg⁻¹,氧氟沙星、环丙沙星和恩诺沙星质量分数次之,为0.7 μg·kg⁻¹。该方法实现了对海洋沉积物中 13种 FQs 的同时检测,具有快速、准确等优点,适用于海洋沉积物中13种FQs的测定。本研究成果可为海洋生态环境保护提供数据基础及技术支撑。     

固相萃取-液相色谱-三重四极杆串联质谱测定养殖废水中17种氟喹诺酮类抗生素

采用固相萃取-液相色谱-三重四极杆串联质谱(SPE-LC-MS/MS)技术,建立了养殖废水中17种氟喹诺酮类抗生素(FQs)的测定方法。水样在采用固相萃取法富集前,先用 0.45 μm 的聚四氟乙烯滤膜过滤,而后加入5%甲醇(体积比),用盐酸溶液将水样pH调节至2.0±0.5,经固相萃取柱富集,最后用9 mL 0.1%甲酸甲醇洗脱。以C18柱为分离柱,0.1%甲酸−5 mmol·L⁻¹ 甲酸铵水溶液和甲醇为流动相,采用液相色谱－三重四极杆串联质谱多反应监测离子模式(MRM)对目标化合物进行了检测和分析。在优化实验条件下,17种FQs的线性范围为0.50 -100 μg·L⁻¹时,目标化合物峰面积与内标物质峰面积之比与质量浓度的线性关系良好(R²>0.99),方法检出限为0.08-0.3 ng·L⁻¹。在加标量为0.01 μg·L⁻¹和0.09 μg·L⁻¹时,空白加标的平均回收率为58.6%-104.2% 和65.3%-91.0%,相对标准偏差(RSD)在2.1%-19.3%(n=6)。以养殖废水为基质,17种FQs的加标回收率在47.8%-118.7%,RSD小于20%(n=6)。应用该方法测定了广州某水产养殖场的养殖废水。结果表明,氧氟沙星检出浓度最高(9.36 ng·L⁻¹),达氟沙星次之(5.96 ng·L⁻¹)。该方法快速、准确,可适用于养殖废水中17种FQs的测定。     

SPE和LC-MS／MS联用同时检测水体中的15种抗生素

采用（SPE）固相萃取柱进行分离富集,使用LC—MS／MS联用仪进行检测,建立了一种高效、低成本的可同时检测水体中多种抗生素的方法,并在河北省子牙河流域汪洋沟水体抗生素污染现状调查中得到应用。实验结果表明,（1）最优实验条件的选择为：水样体积为1L,洗脱剂用量为10mL,pH为3．0;（2）回收率达到69．2％～86．3％,相对标准偏差在1．8％～8．4％之间,表明检测结果准确度和灵敏度均较高;（3）在所检测的15种抗生素中,头孢西丁、土霉素、氯唑西林和布洛芬4种抗生素是该河流的主要抗生素污染物,其含量比重（中位数）高达73．5％,检测浓度最高（中位数）的是磺胺类抗生素,浓度为100．4ng／L;（4）各抗生素的含量从上游到下游大体呈逐渐衰减的趋势,这主要归因于下游支流的稀释作用,但水体中抗生素浓度仍远高于已有报道的地区;（5）污染源解析表明,水体中抗生素主要来源于制药企业的废水,部分来源于附近居民的生活污水。     

Optimization of the ionization conditions for the trace analysis of PCDD/PCDF with ion trap MS/MS

Commercial ion trap mass spectrometer provides easy-to-operate MS/MS analysis for the determination of PCDD/PCDF. The limit of quantification is appropriately low (0.2 pg for 23478-P5CDF) because all the stages are performed in the trap and sample losses associated with the ion transportation are minimized. However, if excessive ions are injected into the trap, its electrical fields are distorted and an overall reduction in performance arises. Ionization condition is an important parameter as it affects the amount of the total ions produced. If the amount of interfering compounds are negligible, such as standard solution or cleaned-up sample, lower ionization condition (e.g. electron energy: 30 eV, emission current (EC): 150 μA) is preferable. On the contrary, in case excessive interfering ions are coexisting with PCDD/PCDF, such as crude extract or semi-cleaned-up sample, the ionization condition should be high (e.g. electron energy: 90 eV, EC: 350 μA) for the reproductive quantification.     

在线固相萃取液质联用法测定饮用水源中的痕量联苯胺

建立了在线固相萃取液相色谱串联质谱法测定水中痕量联苯胺的方法。样品经自动进样器注入在线固相萃取小柱后,用富集泵流动相实现对样品的富集洗脱,然后用分析泵流动相将样品从富集柱冲洗至色谱柱后用串联质谱仪进行检测。该方法可在11 min内自动完成对样品的富集、净化、进样和检测过程。当进样量为5 mL时,联苯胺在0.010~2.0 μg·L-1范围内具有良好的线性关系,相关系数R为0.999,检出限为0.8 ng·L-1,实际水样的回收率为85%~104%。该方法具有简单、快速、重现性好、灵敏高等特点,可用于环境水体中痕量联苯胺的检测。     

Application of an ELISA to the quantification of carbamazepine in ground, surface and wastewaters and validation with LC-MS/MS

Carbamazepine is a psychiatric pharmaceutical widely detected in aquatic environments. Due to its generalized occurrence and environmental persistence it might be considered as an anthropogenic pollution indicator. In this research, a previously developed enzyme-linked immunosorbent assay (ELISA), based on a commercial monoclonal antibody, was applied to the quantification of carbamazepine in ground, surface and wastewaters and results were validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS).The performance of the applied ELISA methodology was tested in the presence of high concentrations of sodium chloride and dissolved organic matter. The method was not significantly affected by matrix effects, being adequate for the quantification of carbamazepine in environmental samples, even without sample pre-treatment. This method allows the quantification of carbamazepine in the range of 0.03-10 μg L⁻¹, with a relative error lower than 30%. Due to a pH dependent cross-reactivity with cetirizine, an antihistaminic drug, the assay also enabled the quantification of cetirizine in the samples.The application of the developed method to the quantification of carbamazepine was performed by using environmental samples with very different matrices, collected in the geographical area of Ria de Aveiro, an estuarine system located in the North of Portugal. Carbamazepine was detected in all analyzed wastewater samples and in one surface water with concentrations between 0.1 and 0.7 μg L⁻¹. Validation with LC-MS/MS revealed that results obtained by ELISA are 2-28% overestimated, which was considered highly satisfactory due to the absence of sample pre-treatments.     

The application of HPLC ESI MS in the investigation of the flavonoids and flavonoid glycosides of a Caribbean Lamiaceae plant with potential for bioaccumulation

As part of an exchange technology program between the government of Barbados and Environment Canada, methanolic and aqueous extracts from the flavonoid-rich Lamiaceae family were characterized using negative-ion electrospray mass spectrometry. The species investigated is part of the Caribbean Pharmacopoeia, and is used for a variety of health issues, including colds, flu, diabetes, and hypertension. The extracts were investigated for structural elucidation of phenolics, identification of chemical taxonomic profile, and evidence of bio-accumulator potential. The methanolic and aqueous leaf extracts of Plectranthus amboinicus yielded rosmarinic acid, ladanein, cirsimaritin, and other methoxylated flavonoids. This genus also shows a tendency to form conjugates with monosaccharides, including glucose, galactose, and rhamnose. The aqueous extract yielded four isomeric rhamnosides. The formation of conjugates by Plectranthus amboinicus is thus evidence of high bioaccumulator significance.     

Assessing washing methods for reduction of pesticide residues in Capia pepper with LC-MS/MS

Abstract

The effects of washing treatments on removal rates of some pesticides residues (acetamiprid, chlorpyrifos and formetanate hydrochloride) on pepper were investigated. Method verification was conducted through spiking pepper samples at 0.1, 1.0 and 10.0 × MRL. QuEChERS method produced average recovery of 104.91% with relative standard deviation (RSD) of 13.41%. LOQ values of acetamiprid, chlorpyrifos and formetanate hydrochloride were estimated as 2, 10 and 5?µg/kg, respectively. Capia peppers grown in open fields were sprayed three times with pesticides. Peppers were harvested after 1st, 2nd and 3rd day of the treatments. Then the peppers were subjected to tap water, acetic acid and citric acid washing and ultrasonic cleaning treatments (for 2 and 5?min). Based on three different harvest times and two different washing durations, processing factors (PFs) and reduction rates were calculated for each washing treatment. The residues gradually decreased during washing treatments with increasing process duration. Similarly, a gradual reduction was noted with the progress of harvest times. This in turn corresponded to an increase in PF. Ultrasonic cleaning and citric acid (9%) washing were more effective than the others. Non-systemic pesticides (chlorpyrifos) were more readily removed than the systemic ones (acetamiprid). Similarly, highly soluble pesticides exhibited higher reduction.     

Development of a sensitive,competitive, indirect ELISA for the detection of fumonisin B1 in corn originating from Anhui province,China

Fumonisin B₁ (FB₁) is a secondary metabolite produced by Fusarium verticillioides or Fusarium proliferatum, which present in food and feed. It causes hazardous effects on human and animal health. A monoclonal antibody (mAb) against FB₁ was produced and a simple, reliable and sensitive, competitive, indirect enzyme-linked immunosorbent assay (ci-ELISA) for detection of FB₁ was developed and the experiment conditions were optimized. The coating concentration of FB₁-ovalbumin (FB₁-OVA) was 500 ng mL^?1, the action concentrations of anti-FB₁ mAb and goat anti-mouse IgG were 1.28 × 10⁴ and 1:5000, respectively. The 50% inhibitory concentration (IC₅₀) was 11 ng mL^?1, with a detectable range of 1.25–250 ng mL^?1, and a limit of determination (LOD) of 1.15 ng mL^?1. The cross-reactivity (CR) of the antibody against fumonisin B₂ (FB₂) was 60.4, and <1% against deoxynivalenol (DON), aflatoxin B₁ (AFB₁), ochratoxin A (OTA) or zearalenone (ZEN). In spiked samples (250 ng g^?1, 500 ng g^?1, 1000 ng g^?1), the mean recoveries ranged from 86.7 ± 5% to 102 ± 4%, and the coefficient of variation (CV) ranged from 3% to 10%. A survey of 96 corn samples from Bozhou, Fuyang, Bengbu, and Hefei, in Anhui province, China, was performed. Frequencies of FB₁ contamination were 83.3%, 95.8%, 20.8% and 91.7%, and the mean concentrations of positive samples were 0.702 μg kg^?1, 0.883 μg kg^?1, 0.074 μg kg^?1, and 0.276 μg kg^?1, respectively. The results of this study suggest that the ci-ELISA developed in this study can be used to identify FB₁ in corn, furthermore, further study is needed to investigate FB₁ contamination in food and feed to prevent its harmful health effects.     

Rapid and Sensitive LC/MS/MS Direct Injection Method for the Determination of Trace Level Corexit EC9500A Oil Dispersant in Seawater

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the determination of trace dioctyl sulfosuccinate (DOSS) concentrations in seawater samples has been established. The method is well suited to aquatic environment impact monitoring following application of the dispersant Corexit EC9500A. Linearity of the method was demonstrated down to 0.05 ng/mL^?1 (0.05 µgL^?1) DOSS in seawater, with a 2.4% relative standard deviation precision for preparation replicates. A US EPA method limit of detection of <0.02 ng/mL^?1 (<0.02 µgL^?1) was calculated and specificity was confirmed by monitoring of two qualifier ions at 291.1 m/z and 227.1 m/z. These transitions were confirmed by QToF analysis to be associated with the DOSS precursor ion at 421.2 m/z. For application to seawater samples and samples containing oil particulates, a practical and repeatable calibration range of 0.5 ng/mL^?1 (0.5 µgL^?1) to 25.0 ng/mL^?1 (25.0 µgL^?1) DOSS is reported. The method was shown to have excellent precision and accuracy, with a consistent ≤1.6% relative standard deviation for system suitability standards at 0.5 ng/mL^?1 (0.5 µgL^?1) and linear weighted (1/x) regression coefficients of determination ≥0.995. The surfactant nature of the analyte is discussed in relation to detection limit and loss of analyte. Speculation of a relationship between DOSS in association or aggregation with divalent cations, such as Ca²⁺ present in salt water and hard water, is suggested. The consequent effects on cell ionic balance and membrane function are discussed.     

Analysis of accumulation, extractability, and metabolization of five different phenylarsenic compounds in plants by ion chromatography with mass spectrometric detection and by atomic emission spectroscopy

Phenylated arsenic compounds occur as highly toxic contaminants in former military areas where they were formed as degradation products of chemical warfare agents. Some phenylarsenic compounds such as roxarsone and aminophenylarsonic acids were applied as food additive and veterinary drugs in stock-breeding and therefore pose an environmental risk in agricultural used sites. Very few data exist in the literature concerning uptake and effects of phenylarsenic compounds in plants growing on contaminated soils. In this study, the accumulation, extractability, and metabolization of five different phenylarsenic compounds, phenylarsonic acid, p- and o-aminophenylarsonic acid, phenylarsine oxide, and 3-nitro-4-hydroxyphenylarsonic acid called roxarsone, by the terrestrial plant Tropaeolum majus were investigated. Ion chromatography coupled to inductively coupled plasma mass spectrometry was used to differentiate these arsenic compounds, and inductively coupled plasma atomic emission spectroscopy was used for total arsenic quantification. All compounds considered were taken up by the roots and transferred to stalks, leaves, and flowers. The strongest accumulation was observed for unsubstituted phenylarsonic acid followed by its trivalent analogue phenylarsine oxide that was mostly oxidized in soil whereas the amino- or nitro- and hydroxy-substituted phenylarsonic acids were accumulated to a smaller degree.The highest extraction yield of 90% for ground leaf material was achieved by 0.1 M phosphate buffer, pH 7.7, in a two-step extraction with a total extraction time of 24 h. The extraction of higher amounts of arsenic (50–70% of total arsenic present in leaves depending on arsenic species application) from non-ground intact leaves with deionized water in comparison with the buffer (20–40% of total arsenic) is ascribed to osmotic effects. The arsenic species analysis revealed a cleavage of the amino groups from the phenyl ring for plants treated with aminophenylarsonic acids. A further important metabolic effect consisted in the production of inorganic arsenate and arsenite from the phenylated arsonic acid groups.     

Validation and global uncertainty of a gas chromatographic with mass spectrometry method for fenamidone analysis in grapes and wines

Fenamidone is an imidazolinone fungicide recently introduced in viticulture practices. This work reports the validation and assessment of global uncertainty of a gas chromatographic with mass spectrometry method to analyze fenamidone in grapes and wines. This method consists in a simple and fast liquid-liquid extraction step followed by chromatographic determination. Limits of detection for fenamidone in grapes and wines were, respectively, 0.05 mg/kg and 0.06 mg/L, precision was below 9.4% and average recovery was 89 ± 5%. In the concentration range from 0.05 to 1.00 mg/kg (or mg/L) of fenamidone, global uncertainty calculated following the EURACHEM/CITAC rules, and also by the Horwitz function, was below 25%. The EURACHEM/CITAC global uncertainty budget used gave lower estimates than those obtained from the Horwitz function.     

Suitability of a magnetic particle immunoassay for the analysis of PBDEs in Hawaiian euryhaline fish and crabs in comparison with gas chromatography/electron capture detection-ion trap mass spectrometry

A gas chromatograph/electron capture detector-ion trap mass spectrometer (GC/ECD-ITMS) was used for the determination of polybrominated diphenyl ethers (PBDEs) in euryhaline fish and crabs. GC/ECD-ITMS results showed that average recoveries from the spiked fish samples are in a range of 58-123% with relative standard deviations (RSDs) of 5-19%. PBDE concentrations obtained from GC/ECD-ITMS ranged from 28 ng/g to 1845 ng/g lipid weight (lw) in all aquatic species collected from Hawaiian brackish waters. The general BDE congener concentration profile observed in this study is BDE-47 > BDE-100 > BDE-154 > BDE-99 > BDE-153 > BDE-28 > BDE-183. The ELISA results expressed as BDE-47 equivalents correlated well with those of GC/ECD-ITMS, with a correlation coefficient (R² = 0.68) and regression coefficient (slope = 0.82). Comparison of ELISA with GC/ECD-ITMS results demonstrated that ELISA provides a timely and cost-effective method to screen PBDEs in fish and crab samples.     

Development of a fast instrumental method for the analysis of pharmaceuticals in environmental and wastewaters based on ultra high performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS)

This work describes the development, optimization and validation of an analytical method for the simultaneous detection and identification of 74 pharmaceutically active compounds (PhACs), from various therapeutic groups, in both environmental (ground and surface water) and wastewaters (WW). The method is based on the simultaneous extraction of all target compounds by solid phase extraction (SPE), using a hydrophilic-lipophilic balanced polymer followed by ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS). Two selected reaction monitoring (SRM) transitions have been monitored per compound in order to fulfil the EC guidelines, as well as to ensure an accurate identification of target compounds in the samples. Quantification is performed by internal standard approach, applying 24 specific isotopically labeled compounds. The main advantages of the developed method, besides the selectivity and reliability of the results, is its high throughput. All compounds are extracted in a single step and the instrumental analysis lasts 5 min (NI mode) + 8 min (PI mode), allowing fast throughput of samples. The limits of detection range from 0.01 to 50 ng L⁻¹, depending on the matrix, for most of the compounds. Finally, the method developed has been applied to the analysis of pharmaceuticals in the Ebro river basin (NE Spain).