A Method for the Analysis of Glyphosate, Aminomethylphosphonic Acid, and...

[Full title: A Method for the Analysis of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Human Urine Using Liquid Chromatography-Tandem Mass Spectrometry]

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Citation: Li, Z.-M.; Kannan, K. A Method for the Analysis of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Human Urine Using Liquid Chromatography-Tandem Mass Spectrometry Int. J. Environ. Res Public Health 2022 , 19 , 4966 https://doi.org/10.3390/ ijerph 19094966 Academic Editor: Paul B. Tchounwou Received: 24 March 2022 Accepted: 18 April 2022 Published: 19 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations Copyright: © 2022 by the authors Licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/) International Journal of Environmental Research and Public Health Article A Method for the Analysis of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Human Urine Using Liquid Chromatography-Tandem Mass Spectrometry Zhong-Min Li 1,2 and Kurunthachalam Kannan 1,2,3, * 1 Department of Pediatrics, New York University Grossman School of Medicine, New York, NY 10016, USA; zhongmin.li@nyulangone.org 2 Department of Environmental Medicine, New York University Grossman School of Medicine, New York, NY 10016, USA 3 King Fahd Medical Research Center, Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Abdulaziz University, Jeddah 80200, Saudi Arabia * Correspondence: kurunthachalam.kannan@nyulangone.org; Tel.: +212-263-1546 Abstract: The extensive use of herbicides, such as glyphosate and glufosinate, in crop production during recent decades has raised concerns about human exposure. Nevertheless, analysis of trace levels of these herbicides in human biospecimens has been challenging. Here, we describe a method for the determination of urinary glyphosate, its degradation product aminomethylphosphonic acid (AMPA), and glufosinate using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The method was optimized using isotopically labelled internal standards ( 13 C 2 , 15 N-glyphosate, 13 C, 15 N, D 2 -AMPA, and D 3 -glufosinate) and solid-phase extraction (SPE) with cation-exchange and anion-exchange cartridges. The method provides excellent chromatographic retention, resolution and peak shape of target analytes without the need for strong acidic mobile phases and derivatization steps. The instrument linearity was in the range of 0.1–100 ng/mL, with R > 0.99 in the matrix for all analytes. The method detection limits (MDLs) and the method quantification limits (MQLs) were in the ranges of 0.12 (AMPA and glufosinate)–0.14 (glyphosate) ng/mL and 0.40 (AMPA)–0.48 (glyphosate) ng/mL, respectively. The recoveries of analytes spiked into urine matrix ranged from 79.1% to 119%, with coefficients of variation (CVs) of 4–10%. Repeated analysis of samples for over 2 weeks showed intra-day and inter-day analytical variations of 3.13–10.8% and 5.93–12.9%, respectively. The matrix effects for glyphosate, AMPA, and glufosinate spiked into urine matrix averaged − 14.4%, 13.2%, and 22.2%, respectively. The method was further validated through the analysis of external quality assurance proficiency test (PT) urine samples. The method offers optimal sensitivity, accuracy, and precision for the urine-based assessment of human exposure to glyphosate, AMPA, and glufosinate Keywords: glyphosate; aminomethylphosphonic acid; glufosinate; urine; LC–MS/MS 1. Introduction Glyphosate ( N -(phosphonomethyl)glycine) and glufosinate (2-amino-4-(hydroxy(methyl) phosphoryl)butanoic acid) are non-selective, broad-spectrum herbicides used in both agricultural and non-agricultural sectors. Their use in agriculture has greatly increased since the development of crop strains genetically modified to tolerate them. The current annual use of glyphosate, the most widely used herbicide, is estimated at 600,000–750,000 tons of active ingredients and is expected to increase to 740,000–920,000 tons by 2025 [ 1 ]. The United States accounts for 19% of the global glyphosate usage and >100,000 tons of glyphosate have been applied annually in agriculture since 2010 [ 2 ]. Glufosinate is mainly used to Int. J. Environ. Res. Public Health 2022 , 19 , 4966. https://doi.org/10.3390/ijerph 19094966 https://www.mdpi.com/journal/ijerph

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 2 of 14 control glyphosate-resistant weeds. The United States is the world’s largest market for glufosinate with >4000 tons of active ingredients used annually since 2016 [ 3 ]. The large-scale use of glyphosate and glufosinate has resulted in their ubiquitous presence in food and environmental matrices [ 4 – 6 ]. Concerns over human exposure to glyphosate and its analogues are mounting due to potential health risks [ 7 , 8 ]. Studies have reported human exposure to glyphosate, its degradation product aminomethylphosphonic acid (AMPA), and glufosinate through food and water [ 9 , 10 ]. After ingestion, these compounds are primarily excreted unchanged in feces and urine [ 11 ]. Human biomonitoring studies have reported the occurrence of glyphosate and AMPA in urine collected from different countries, with average glyphosate concentrations ranging between 0.26 and 73.5 ng/mL in occupationally exposed individuals and between 0.16 and 7.6 ng/mL in the general population [ 9 ]. The acute oral toxicity of glyphosate in rats was low, with LD 50 values greater than 5000 mg/kg-bw. The health risks from exposure to glyphosate include oxidative stress [ 12 ], anti-estrogenicity [ 13 ], anti-androgenicity [ 13 ], reproductive toxicity [ 14 ], and carcinogenicity [ 15 ]. For glufosinate, the acute oral and dermal toxicity was low, with LD 50 values greater than 1500 mg/kg-bw in rats and mice [ 16 ]. The European Food Safety Authority concluded that there was no evidence for genotoxicity, carcinogenicity, or neurotoxicity of glufosinate. However, a reversible reduction in glutamine concentration in mammalian tissues was observed following exposure to high levels of glufosinate [ 17 ]. Additionally, glufosinate was shown to induce preand post-implantation loss, vaginal bleeding, abortion and dead fetus in rats [ 18 ]. The International Agency for Research on Cancer (IARC) classified glyphosate as a probable human carcinogen (Group 2 A) [ 19 ]. Nevertheless, controversies surround its carcinogenic potential [ 10 , 20 , 21 ]. There exists a need for additional research regarding human exposure to glyphosate, AMPA, and glufosinate, especially in the general population Accurate determination of urinary glyphosate, AMPA, and glufosinate is hampered by these chemicals’ high polarity, hydrophilicity, and low molecular weights. The high polarity affects chromatographic separation, and the low molecular weight, in a range where interferences are frequent, affects test specificity (creating a high risk for false positives). Liquid chromatography (LC) coupled with UV detection (LC–UV) [ 22 ], LC with fluorescence detection [ 23 ], and gas chromatography coupled with mass spectrometry (GC–MS) [ 8 ] have been used to analyze glyphosate in environmental samples. However, these methods require derivatization of the analytes, a tedious and time-consuming step involving toxic reagents. More recently, with the advent of the more sensitive and rugged liquid chromatography-tandem mass spectrometry (LC–MS/MS) method, it has become possible to determine glyphosate without a derivatization step. Different strategies have been used to improve chromatographic retention and peak shape, including cation-exchange [ 24 , 25 ], anion-exchange [ 26 ], and ion-pairing reversed-phase chromatography [ 27 ]. However, poor peak shape, lack of adequate sensitivity, and the use of strongly acidic mobile phases to enhance ionization (e.g., ≥ 1% formic or acetic acids) hamper the analysis of trace levels of these chemicals in human specimens such as urine, especially for application in large-scale human biomonitoring studies [ 26 – 30 ]. Furthermore, despite its significance as a widely used herbicide, glufosinate has rarely been measured in human specimens [ 31 , 32 ]. Our aim was to develop a method for sensitive and selective determination of urinary glyphosate, AMPA, and glufosinate using isotope dilution LC–MS/MS suitable for application in large-scale human biomonitoring studies. We optimized the method to improve chromatographic retention and peak shape, eliminate matrix effects, and improve sensitivity while using milder mobile phases (less corrosive conditions) and avoiding derivatization. We then validated its sensitivity, accuracy, precision, and matrix effects by using fortified human urine samples and analyzing external quality assurance proficiency test (PT) urine samples.

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 3 of 14 2. Materials and Methods 2.1. Reagents and Materials The molecular structures of the target analytes are shown in Figure 1 . Glyphosate (10 µ g/mL in water), 13 C 2 , 15 N-glyphosate (100 µ g/mL in water), AMPA (100 µ g/mL in water), and 13 C, 15 N,D 2 -AMPA (100 µ g/mL in water) with purities of 95–98% were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Glufosinate and D 3 -glufosinate (purity ≥ 95%) were from Toronto Research Chemicals (Toronto, ON, Canada). Primary stock solutions of glufosinate and D 3 -glufosinate (1 mg/mL) were prepared in water. Working standard solutions were diluted from stock solutions using water:acetonitrile (ACN) (95:5, v / v ) containing 0.1% formic acid. Formic acid (88%) and ammonium hydroxide (NH 4 OH; 28–30%) of analytical grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). LC passivation solution containing 10 M medronic acid was from Restek Corp (Bellefonte, PA, USA). Water, methanol (MeOH), and ACN were purchased from Fisher Scientific (Waltham, MA, USA). Oasis ® MAX cartridges (60 mg/3 mL) and Oasis ® MCX cartridges (60 mg/3 mL) were obtained from Waters Corp. (Milford, MA, USA) Int. J. Environ. Res. Public Health 2022 , 19 , x FOR PEER REVIEW 3 of 15 fortified human urine samples and analyzing external quality assurance proficiency test (PT) urine samples. 2. Materials and Methods 2.1. Reagents and Materials The molecular structures of the target analytes are shown in Figure 1. Glyphosate (10 µ g/mL in water), 13 C 2 , 15 N-glyphosate (100 µ g/mL in water), AMPA (100 µ g/mL in water), and 13 C, 15 N,D 2 -AMPA (100 µ g/mL in water) with purities of 95 – 98% were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Glufosinate and D 3 -glufosinate (purity ≥ 95%) were from Toronto Research Chemicals (Toronto, ON, Canada). Primary stock solutions of glufosinate and D 3 -glufosinate (1 mg/mL) were prepared in water. Working standard solutions were diluted from stock solutions using water:acetonitrile (ACN) (95:5, v/v ) containing 0.1% formic acid. Formic acid (88%) and ammonium hydroxide (NH 4 OH; 28 – 30%) of analytical grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). LC passivation solution containing 10 M medronic acid was from Restek Corp (Bellefonte, PA, USA). Water, methanol (MeOH), and ACN were purchased from Fisher Scientific (Waltham, MA, USA). Oasis ® MAX cartridges (60 mg/3 mL) and Oasis ® MCX cartridges (60 mg/3 mL) were obtained from Waters Corp. (Milford, MA, USA). A small number of archived human urine samples previously collected for other studies were analyzed [33]. Institutional Review Board approvals were obtained from New York State Department of Health for the analysis of de-identified urine samples (under exempt category) to demonstrate application of the method developed in this study. Figure 1. Molecular structures of the target analytes determined in this study. AMPA, aminomethylphosphonic acid. 2.2. Sample Preparation A 250 µ L aliquot of each urine sample was transferred into a 15 mL polypropylene (PP) tube. Urine samples were fortified with the target compounds and internal standards at 0.5, 1, and 5 ng/mL concentrations (in water: ACN [95:5 v/v ] containing 0.1% formic acid) for method optimization and validation. The sample was vortexed vigorously and kept at room temperature for 30 min. The mixture was loaded onto an Oasis MCX cartridge that had been preconditioned with 2 mL MeOH and 2 mL water. The eluate was collected immediately, as the target analytes were not absorbed by the cation-exchange cartridges (this step was for purification and removal of cationic interferences). The cartridge was then washed with 2 mL water, and the eluate was collected and combined. Thereafter, 2.5 mL of 3% NH 4 OH ( v/v ) aqueous solution was added and vortexed vigorously. The mixture (~5 mL in total) was then loaded onto an Oasis MAX cartridge preconditioned with 2 mL MeOH, 2 mL water, and 1 mL of 3% NH 4 OH. The cartridge was washed with 2 mL of 3% NH 4 OH and 2 mL MeOH, and moisture was removed using a vacuum pump for 3 min. The analytes were then eluted into a 15 mL PP tube with 3 mL of 3% formic acid in MeOH ( v/v ), and the eluate was evaporated to dryness under N 2 at 40 °C. The residue was reconstituted in 250 µ L of water: ACN (95:5, v/v ) containing 0.1% Figure 1. Molecular structures of the target analytes determined in this study. AMPA, aminomethylphosphonic acid A small number of archived human urine samples previously collected for other studies were analyzed [ 33 ]. Institutional Review Board approvals were obtained from New York State Department of Health for the analysis of de-identified urine samples (under exempt category) to demonstrate application of the method developed in this study 2.2. Sample Preparation A 250 µ L aliquot of each urine sample was transferred into a 15 mL polypropylene (PP) tube. Urine samples were fortified with the target compounds and internal standards at 0.5, 1, and 5 ng/mL concentrations (in water: ACN [95:5 v / v ] containing 0.1% formic acid) for method optimization and validation. The sample was vortexed vigorously and kept at room temperature for 30 min. The mixture was loaded onto an Oasis MCX cartridge that had been preconditioned with 2 mL MeOH and 2 mL water. The eluate was collected immediately, as the target analytes were not absorbed by the cation-exchange cartridges (this step was for purification and removal of cationic interferences). The cartridge was then washed with 2 mL water, and the eluate was collected and combined. Thereafter, 2.5 mL of 3% NH 4 OH ( v / v ) aqueous solution was added and vortexed vigorously. The mixture (~5 mL in total) was then loaded onto an Oasis MAX cartridge preconditioned with 2 mL MeOH, 2 mL water, and 1 mL of 3% NH 4 OH. The cartridge was washed with 2 mL of 3% NH 4 OH and 2 mL MeOH, and moisture was removed using a vacuum pump for 3 min. The analytes were then eluted into a 15 mL PP tube with 3 mL of 3% formic acid in MeOH ( v / v ), and the eluate was evaporated to dryness under N 2 at 40 ◦ C. The residue was reconstituted in 250 µ L of water: ACN (95:5, v / v ) containing 0.1% formic acid, vortexed vigorously, and transferred into a glass vial. Finally, 20 µ L of the sample was injected into the LC–MS/MS instrument.

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 4 of 14 2.3. LC–MS/MS Identification and detection of the target analytes were performed using an AB Sciex 5500 Q-trap mass spectrometer (Framingham, MA, USA) coupled with a Shimadzu LC-30 AD ultra-high-performance liquid chromatograph (Shimadzu Corp., Kyoto, Japan). Analytes were separated on a Gemini ® C 6-Phenyl column (150 × 4.6 mm, 5 µ m; Phenomenex, Torrance, CA, USA) connected to a Betasil C 18 guard column (20 × 2.1 mm, 5 µ m; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were water (A) and ACN (B) each containing 0.1% formic acid ( v / v ). The following mobile-phase gradient program was used: hold at 5% B for 2 min, linear ramp to 95% B over 8 min, hold at 95% B for 1 min, then return to initial conditions in over 1 min, and equilibrate at initial conditions for additional 2 min prior to the next injection. The column temperature was maintained at 40 ◦ C; the autosampler temperature was 15 ◦ C; and the mobile phase flow rate was 0.8 mL/min The target analytes were determined using negative-ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MRM parameters, including declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP), are shown in Table S 1. The IonSpray voltage was − 5.5 kV; the ionization source temperature was 500 ◦ C; and the curtain gas flow rate was 20 psi. Data were acquired and processed using the Analyst software, version 1.7.2 (AB Sciex, Framingham, MA, USA). Typical MS/MS chromatograms of the target compounds in standard solution are shown in Figure 2 . Int. J. Environ. Res. Public Health 2022 , 19 , x FOR PEER REVIEW 4 of 15 formic acid, vortexed vigorously, and transferred into a glass vial. Finally, 20 µ L of the sample was injected into the LC – MS/MS instrument. 2.3. LC – MS/MS Identification and detection of the target analytes were performed using an AB Sciex 5500 Q-trap mass spectrometer (Framingham, MA, USA) coupled with a Shimadzu LC- 30 AD ultra-high-performance liquid chromatograph (Shimadzu Corp., Kyoto, Japan). Analytes were separated on a Gemini ® C 6-Phenyl column (150 × 4.6 mm, 5 µ m; Phenomenex, Torrance, CA, USA) connected to a Betasil C 18 guard column (20 × 2.1 mm, 5 µ m; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were water (A) and ACN (B) each containing 0.1% formic acid ( v/v ). The following mobile-phase gradient program was used: hold at 5% B for 2 min, linear ramp to 95% B over 8 min, hold at 95% B for 1 min, then return to initial conditions in over 1 min, and equilibrate at initial conditions for additional 2 min prior to the next injection. The column temperature was maintained at 40 °C; the autosampler temperature was 15 °C; and the mobile phase flow rate was 0.8 mL/min. The target analytes were determined using negative-ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MRM parameters, including declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP), are shown in Table S 1. The IonSpray voltage was − 5.5 kV; the ionization source temperature was 500 °C; and the curtain gas flow rate was 20 psi. Data were acquired and processed using the Analyst software, version 1.7.2 (AB Sciex, Framingham, MA, USA). Typical MS/MS chromatograms of the target compounds in standard solution are shown in Figure 2. Figure 2. Representative MRM chromatograms of glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in neat standard solution (concentrations of the target analytes and internal standards were 10 ng/mL; injection volume was 20 µ L).

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 5 of 14 2.4. Method Validation The method was validated by following a protocol of the New York State Department of Health (Wadsworth Center, Laboratory of Organic Analytical Chemistry; available at: https://www.wadsworth.org/sites/default/files/WebDoc/NYS%20 DOH%20 MML- 301-06 SOP.pdf (accessed on 10 March 2022)). Calibration curves were constructed for standards prepared both in neat solution and in fortified urine matrix. Calibration standards ranged in concentrations from 0.05 to 100 ng/mL, with 10 ng/mL of labelled internal standards, diluted from stock solutions with HPLC-grade water: ACN (95:5, v / v ) containing 0.1% formic acid. Matrix-matched calibration curves were prepared by spiking various concentrations of the target analytes (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 ng/mL) into pooled urine Matrix effect was calculated as the percentage of signal enhancement or suppression, as shown in Equation (1): Matrix effect (%) = (A/B − 1) × 100 (1) where A and B are the slopes of analytes from the matrix-matched calibration curve and calibration curve prepared in neat solution, respectively The instrument detection limit (IDL) and instrument quantification limit (IQL) were defined as the concentrations of analytes in solvent that produced a peak with a signal-tonoise ratio ( S / N ) of 3 and 10, respectively. To estimate the method detection limit (MDL) and method quantification limit (MQL), six pooled urine samples were fortified with each target analyte individually at 0.5 ng/mL, a concentration that yielded peaks with S / N values of 11.3, 7.3, and 12.8 for glyphosate, AMPA, and glufosinate, respectively. MDL and MQL were calculated as 3 and 10 times the standard deviation (SD) measured in matrix, spiked at 0.5 ng/mL, respectively The accuracy of the method was determined as the recoveries of analytes spiked at three different concentrations (0.5, 1 and 5 ng/mL) in pooled urine. Procedural blank samples (water in place of urine) were included to monitor for background levels contamination. The precision of the method was assessed by intra-day and inter-day variations, which were calculated as the percentage of the coefficient of variation (%CV) of the measured concentrations in six pooled urine samples spiked at 0.5, 1, and 5 ng/mL, respectively. The inter-day CV was measured by repeated injection of fortified samples over a period of 2 weeks 3. Results and Discussion 3.1. Chromatography and Mass Spectrometry Reported LC–MS/MS methods for the determination of glyphosate and AMPA in urine are summarized in Table 1 . Due to the highly polar and hydrophilic nature of the target analytes, chromatographic retention and separation using conventional reversedphase columns (e.g., C 18 column) is arduous, resulting in their co-elution with other matrix components. Retention of such analytes can be improved by reversed-phase ion-pair chromatography [ 27 ], which is based on the addition of ion pair reagents in the mobile phase to promote the formation of ion pairs. The increase in the hydrophobic character of the electrically neutral ion pair results in a greater affinity for the reverse stationary phase. Because of their strong hydrophobic interactions, the ion pair reagents cannot be completely flushed out of the LC column even through extensive washing, and thus require the use of a dedicated column for a particular application. Hydrophilic interaction liquid chromatography (HILIC) columns enable the retention and separation of hydrophilic compounds, but they often lead to poor peak shape due to interactions with metals in the stationary phase or the chromatographic hardware [ 34 ]. Alternatively, considering the low pKa values of the analytes (0.8 for the first phosphonate of glyphosate, 0.9 for the first phosphonate of AMPA, and 0.8 for the phosphonate of glufosinate [ 26 , 30 ]), an anionexchange column was expected to offer efficient retention. However, a high concentration

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 6 of 14 of an acid (e.g., 1% formic acid) was needed in the mobile phases to maintain an optimal peak shape [ 26 ]. Other studies have employed a cationic ( − H + ) guard column. Although this resulted in separation, glyphosate was eluted within a short retention time, while the peak shape of AMPA was poor [ 35 , 36 ]. In this study, we compared the performance of different chromatographic columns, including reversed-phase (C 18-, C 8-, C 6-Phenyl), HILIC, and anion-exchange (polymer-based NH 2 , hydroxide-selective anion-exchange) columns (data not shown), and found that the C 6-Phenyl column exhibited the best chromatographic performance. All analytes were well separated, and the peak shape of AMPA and glufosinate was symmetrical (Figures 2 and 3 ). However, peak tailing was observed for glyphosate (Figure 3 ), probably due to the chelation of glyphosate with metal ions in the LC system [ 37 ]. Hsiao et al. recommended the addition of 5 µ m medronic acid in the mobile phase (passivation solution) to eliminate chelation by metal ions and improve peak shape for metal-sensitive compounds [ 37 ]. Nevertheless, we observed a reduced intensity (by ~2-fold) for all analytes when medronic acid was added in mobile phases, indicative of ionization suppression. As an alternative, we passivated the LC system by injecting 10 mM medronic acid before analyzing real samples, i.e., we injected 20 µ L of 10 mM medronic acid at the beginning of the analytical run (with the mobile phases directed to waste instead of the mass spectrometer). After this passivation, no ionization suppression was found and all analytes including glyphosate exhibited sharp and symmetrical peaks (Figure 3 ) for at least 300 subsequent injections Int. J. Environ. Res. Public Health 2022 , 19 , x FOR PEER REVIEW 6 of 15 low pKa values of the analytes (0.8 for the first phosphonate of glyphosate, 0.9 for the first phosphonate of AMPA, and 0.8 for the phosphonate of glufosinate [26,30]), an anion-exchange column was expected to offer efficient retention. However, a high concentration of an acid (e.g., 1% formic acid) was needed in the mobile phases to maintain an optimal peak shape [26]. Other studies have employed a cationic ( − H + ) guard column. Although this resulted in separation, glyphosate was eluted within a short retention time, while the peak shape of AMPA was poor [35,36]. In this study, we compared the performance of different chromatographic columns, including reversed-phase (C 18-, C 8-, C 6-Phenyl), HILIC, and anion-exchange (polymer-based NH 2 , hydroxide-selective anion-exchange) columns (data not shown), and found that the C 6-Phenyl column exhibited the best chromatographic performance. All analytes were well separated, and the peak shape of AMPA and glufosinate was symmetrical (Figures 2 and 3). However, peak tailing was observed for glyphosate (Figure 3), probably due to the chelation of glyphosate with metal ions in the LC system [37]. Hsiao et al. recommended the addition of 5 µ M medronic acid in the mobile phase (passivation solution) to eliminate chelation by metal ions and improve peak shape for metal-sensitive compounds [37]. Nevertheless, we observed a reduced intensity (by ~2-fold) for all analytes when medronic acid was added in mobile phases, indicative of ionization suppression. As an alternative, we passivated the LC system by injecting 10 mM medronic acid before analyzing real samples, i.e., we injected 20 µ L of 10 mM medronic acid at the beginning of the analytical run (with the mobile phases directed to waste instead of the mass spectrometer). After this passivation, no ionization suppression was found and all analytes including glyphosate exhibited sharp and symmetrical peaks (Figure 3) for at least 300 subsequent injections. Figure 3. HPLC – MS/MS chromatograms of glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in neat standard solution before ( A ) and after ( B ) passivation of the LC system with medronic acid (analyte concentrations were 100 ng/mL; injection volume was 20 µ L) 3.2. Optimization of Sample Cleanup Because of their low pKa values, we expected anion-exchange cartridges, which are positively charged and can bind negatively charged target analytes, to be effective for this application [26,38]. We first optimized a mixed-mode anion-exchange cartridge (Oasis ® MAX cartridge), which contains sorbents having both hydrophobic and anion-exchange functionalities. Indeed, MAX cartridges provided excellent recoveries for all target analytes after optimization of elution solvents. However, matrix components were not completely removed, as we observed strong ionization suppression of glyphosate and glufosinate, which resulted in poor sensitivity. For example, the S/N values of glyphosate Figure 3. HPLC–MS/MS chromatograms of glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in neat standard solution before ( A ) and after ( B ) passivation of the LC system with medronic acid (analyte concentrations were 100 ng/mL; injection volume was 20 µ L) 3.2. Optimization of Sample Cleanup Because of their low pKa values, we expected anion-exchange cartridges, which are positively charged and can bind negatively charged target analytes, to be effective for this application [ 26 , 38 ]. We first optimized a mixed-mode anion-exchange cartridge (Oasis ® MAX cartridge), which contains sorbents having both hydrophobic and anion-exchange functionalities. Indeed, MAX cartridges provided excellent recoveries for all target analytes after optimization of elution solvents. However, matrix components were not completely removed, as we observed strong ionization suppression of glyphosate and glufosinate, which resulted in poor sensitivity. For example, the S / N values of glyphosate and glufosinate in pooled urine spiked at 0.5 ng/mL were <3 and 3.8, respectively (Figure S 1) Therefore, we introduced an additional purification step to reduce matrix effects. We compared several cartridges for cleanup, including reversed-phase cartridges (hydrophilic lipophilic balanced (HLB) solid-phase extraction (SPE) cartridges, C 18, and graphitized

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 7 of 14 non-porous carbon) and mixed-mode strong cation-exchange cartridges (Oasis ® MCX) (data not shown). We found that a pre-cleanup step in which samples were passed through MCX cartridges (as described above) significantly reduced matrix effects, and thus increased the method sensitivity. The responses of all analytes, especially that of glyphosate, increased considerably after MCX pre-cleanup (Figure S 1). The peak area of glyphosate was >10-fold higher in urine sample passed through MCX and MAX cartridges than in those that passed only through MAX (Table S 2). These results highlighted the efficacy of MCX SPE as a pre-cleanup step for the improvement of method sensitivity. We believe that this is due to the efficient removal of cationic components from the matrix. An earlier study reported the successful use of MCX cartridges for pre-cleanup in the analysis of glyphosate and AMPA in foodstuffs [ 38 ]. LC–MS/MS chromatograms obtained following a combination of cation-exchange and anion-exchange SPE cartridges in the preparation of urine samples showed well-resolved peaks in samples fortified at 0.5 ng/mL (Figure 4 ). Int. J. Environ. Res. Public Health 2022 , 19 , x FOR PEER REVIEW 7 of 15 and glufosinate in pooled urine spiked at 0.5 ng/mL were <3 and 3.8, respectively (Figure S 1). Therefore, we introduced an additional purification step to reduce matrix effects. We compared several cartridges for cleanup, including reversed-phase cartridges (hydrophilic lipophilic balanced (HLB) solid-phase extraction (SPE) cartridges, C 18, and graphitized non-porous carbon) and mixed-mode strong cation-exchange cartridges (Oasis ® MCX) (data not shown). We found that a pre-cleanup step in which samples were passed through MCX cartridges (as described above) significantly reduced matrix effects, and thus increased the method sensitivity. The responses of all analytes, especially that of glyphosate, increased considerably after MCX pre-cleanup (Figure S 1). The peak area of glyphosate was >10-fold higher in urine sample passed through MCX and MAX cartridges than in those that passed only through MAX (Table S 2). These results highlighted the efficacy of MCX SPE as a pre-cleanup step for the improvement of method sensitivity. We believe that this is due to the efficient removal of cationic components from the matrix. An earlier study reported the successful use of MCX cartridges for pre-cleanup in the analysis of glyphosate and AMPA in foodstuffs [38]. LC – MS/MS chromatograms obtained following a combination of cation-exchange and anion-exchange SPE cartridges in the preparation of urine samples showed well-resolved peaks in samples fortified at 0.5 ng/mL (Figure 4). Figure 4. Representative MRM chromatograms of glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in pooled human urine spiked with 0.5 ng/mL native compounds and 10 ng/mL isotope-labelled internal standards (injection volume: 20 µ L) 3.3. Method Validation We assessed the linearity of the instrument by injecting analytical standards prepared both in solvent (0.05 – 100 ng/mL) and urine matrix (0.1 – 100 ng/mL). An excellent linearity was found for all analytes with R values >0.99 (Table 2). We assessed the accuracy Figure 4. Representative MRM chromatograms of glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in pooled human urine spiked with 0.5 ng/mL native compounds and 10 ng/mL isotope-labelled internal standards (injection volume: 20 µ L) 3.3. Method Validation We assessed the linearity of the instrument by injecting analytical standards prepared both in solvent (0.05–100 ng/mL) and urine matrix (0.1–100 ng/mL). An excellent linearity was found for all analytes with R values >0.99 (Table 2 ). We assessed the accuracy of the method using the recoveries of analytes fortified at three different concentrations (0.5, 1 and 5 ng/mL) in a pooled urine matrix and analyzed in six replications. The recoveries of glyphosate, AMPA, and glufosinate were 79.1–84.4% (mean: 81.6%), 100–109% (mean: 103%), and 106–119% (mean: 112%), respectively, with CV values of 8.4–9.6% (mean: 9.3%),

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 8 of 14 4–8% (mean: 6%), and 5–10% (mean: 8%), respectively (Table 2 ). We also assessed the intra-day and inter-day precision of the method by analyzing fortified samples (0.5, 1 and 5 ng/mL) repeatedly for six times over a period of two weeks. The intra-day CVs were 3.13–8.83% (mean: 6.38%), 3.19–10.8% (mean: 7.70%), and 3.46–10.1% (mean: 6.06%) for glyphosate, AMPA, and glufosinate, respectively, and the inter-day CVs were 7.22–9.09% (mean: 8.52%), 5.93–7.85% (mean: 7.10%), and 6.61–12.9% (mean: 10.0%), respectively (Table 2 ). We determined the sensitivity of the method as IDLs/IQLs as well as MDLs/MQLs through the injection of standards and fortified urine samples. The respective IDLs and IQLs were 0.01 and 0.05 ng/mL for all target analytes. The MDLs/MQLs were 0.14/0.48, 0.12/0.40, and 0.12/0.41 ng/mL for glyphosate, AMPA, and glufosinate, respectively The sensitivity of our method is comparable to those found in several previous studies [ 11 , 39 – 42 ], and slightly higher than those of others [ 27 , 43 ] (Table 1 ). We expect that further improvements in MDLs/MQLs could be accomplished through inclusion of additional sample volumes available for extraction Table 1. Reported analytical methods for the measurement of glyphosate, AMPA, and glufosinate in urine Sample Type Analytes Internal Standards Sample Cleanup LC Condition MS/MS LODs/LOQs (ng/mL) Ref(s). Cation-exchange column Human urine Glyphosate, AMPA D 2 , 13 C 3 - Glyphosate; 13 C, 15 N- AMPA SPE cleanup using Oasis HLB cartridges (3 cc, 60 mg) Bio-Rad Micro-Guard Cation-H + column (30 × 4.6 mm, 9 µ m); A: water B: 0.2% formic acid in ACN Glyphosate: 168 / 63 , 168/150; AMPA: 110/79, 110 / 63 ; IDL: 0.02–0.04 IQL: 0.05–0.1 a [ 25 ] Human urine Glyphosate, AMPA 13 C 3 , 15 N- Glyphosate; D 2 , 13 C 15 N- AMPA Diluted with 0.1% formic acid, shaken and centrifuged Bio-Rad Micro-guard Cation-H + column (30 × 4.6 mm, 9 µ m); A: 0.1% formic acid in water B: ACN Glyphosate: 168 / 63 , 168/79 AMPA: 110 / 63 , 110/79 MDL: 0.023–0.041 MQL: 0.1 [ 36 , 44 ] Human urine Glyphosate, AMPA 13 C 2 , 15 N- Glyphosate; D 2 , 13 C, 15 N- AMPA Refer to Jensen et al. [ 36 ] Glyphosate: 168 / 63 , 168/126 AMPA: 110 / 63 , 110/79 MDL: 0.05–0.09 MQL: 0.20 [ 45 ] Anion-exchange column Pet urine (dogs and cats) Glyphosate, AMPA 13 C 2 , 15 N- Glyphosate; D 2 , 13 C, 15 N- AMPA (1) Sample basified with 1% NH 4 OH; (2) Cleanup using Oasis MAX SPE cartridge (3 cc, 60 mg) Dionex IonPac AS 21 IC column (250 × 2.0 mm, 7 µ m); Isocratic elution: 1% formic acid in ACN/water (5/95) Glyphosate: 168 / 63 , 168/79; AMPA: 110 / 63 , 110/79; MDL: 0.15 a MQL: 0.5 [ 26 ] Human urine Glyphosate 13 C 2 , 15 N- Glyphosate Sample diluted with 1% formic acid, then filtered Dionex IonPac AS 21 (250 × 2.0 mm, 7 µ m); Isocratic elution: 1% formic acid in ACN/water (5:95) MDL: 0.1 a MQL: 0.33 [ 46 ] Hybrid-phase column Human urine Glyphosate 13 C 2 - N - Glyphosate − Obelisc-N mixed-mode column (100 × 2.1 mm, 5 µ m); Isocratic elution: 1% formic acid in water 168 / 63 , 168/81 MDL: 0.1 MQL: 0.5 [ 47 ]

[Find the meaning and references behind the names: Cont, Amac, Sim, Back, Plate, Moc, Wash, Lower, Sax, Positive, German, Common]

Int. J. Environ. Res. Public Health 2022 , 19 , 4966 9 of 14 Table 1. Cont Sample Type Analytes Internal Standards Sample Cleanup LC Condition MS/MS LODs/LOQs (ng/mL) Ref(s). Reversed-phase column Human urine Glyphosate 13 C 2 , 15 N- Glyphosate (1) Sample diluted with water; (2) SPE: Strata SAX (1 cc, 100 mg) Zorbax SB-C 3 column (150 × 4.6 mm, 5 µ m), or Zorbax XDB-C 8 column (150 × 4.6 mm, 5 µ m) A: 1% acetic acid in water B: ACN 168/63 MQL: 0.5 [ 11 , 40 – 42 ] Human urine Glyphosate 13 C 2 , 15 N- Glyphosate (1) Sample diluted with H 2 O; (2) SPE: ISOLUTE-96 SCX plate (25 mg), then ISOLUTE-96 NH 2 plate (100 mg) Scherzo SM-C 18 MF column (100 × 2 mm, 3 µ m) A: MeOH/water (5:95) containing 0.1% formic acid and 5 µ m medronic acid B: MeOH and 20 mM ammonium formate (20:80) with 5 µ m medronic acid 170 / 88 , 170/60, 170/42 b MDL: 0.1 MQL: 0.3 [ 39 ] Human urine Glyphosate − − SUPELCO Discovery C 18 column (50 × 2.1 mm, 5 µ m) − MDL: 1 MQL: 2 [ 43 ] Reversed-phase column (Ion-pairing chromatography) Human urine Glyphosate, Glufosinate 13 C 2 , 15 N- Glyphosate; D 3 -Glufosinate (1) Dilute with water; (2) Back wash with dichloromethane Agilent ZORBAX SB-Aq column (100 × 2.1 mm, 1.8 µ m) A: 15 mM HFBA; B: ACN Glyphosate: 170 / 88 , 170/60; Glufosinate: 182 / 136 , 182/119 MDL: 0.1 [ 31 ] Human urine Glyphosate, AMPA 13 C 3 , 15 N- Glyphosate; 13 C, 15 N- AMPA Sample diluted with HFBA Gemini C 6-Phenyl column (150 × 4.6 mm, 5 µ m) A: 15 mM HFBA in water B: ACN Glyphosate: 170 / 88 , 170/60; AMPA: 112/30 b MDL: 2.5 MQL: 5 [ 27 ] Reversed-phase column (derivatization) Human urine Glyphosate AMPA, Glufosinate 13 C 3 , 15 N- Glyphosate; D 2, 13 C, 15 N- AMPA; D 3 -Glufosinate (1) EDTA pre-treatment; (2) SPE: Strata-X; (3) Derivatization; (4) SPE: C 18 Kinetex C 18 column A: 5 mM AmAc (pH 9):MeOH:ACN (90:5:5) B: MeOH: ACN (50:50) ESI positive, SIM mode Glyphosate- Fmoc: 392.08937 AMPA-Fmoc: 334.083890 Glufosinate- Fmoc: 404.12575 MDL: 0.1–0.3 [ 32 ] Abbreviations: ACN, acetonitrile; AMPA, aminomethylphosphonic acid; HFBA, heptafluorobutyric acid; HLB, hydrophilic-lipophilic balanced; MDL, method detection limit; MQL, method quantification limit; SPE, solidphase extraction; SIM, selective ionization mode; F-moc: 9-fluorenylmethoxycarbonyl chloride a The authors did not specify whether the values are instrument detection limits/instrument quantification limit (IDL/IQLs) or MDL/MQLs b Analytes were measured under ESI positive-ionization mode. Italicized transitions indicate quantitative ions monitored The matrix effect is a common phenomenon in LC–MS analysis, especially in the ESI mode, that involves enhancement or suppression of analyte responses by matrix components [ 48 ]. We observed an ionization suppression for glyphosate (matrix effect: − 14.4%) and ionization enhancements for AMPA (13.2%) and glufosinate (22.2%) (Table 2 ). The ionization suppression may explain the lower recoveries of glyphosate in fortified samples, which were in the range of 79.1–84.4%. However, the addition of labelled internal standards for quantification enabled correction for matrix effects We also validated our method by analyzing external quality assurance proficiency test (PT) urine samples, offered by the German External Quality Assessment Scheme (G-EQUAS) and the Quebec External Quality Assessment Scheme for Organic Substances in Urine (OSEQAS). Our results were within the acceptable ranges of assigned values, indicating high accuracy of our method (Table 3 ).

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 10 of 14 Table 2. Optimized analytical parameters for the analysis of glyphosate, AMPA, and glufosinate in human urine. AMPA, aminomethylphosphonic acid; IDL, instrument detection limit; IQL, instrument quantification limit; MDL, method detection limit; MQL, method quantification limit Glyphosate AMPA Glufosinate R in solvent a 0.9995 0.9999 0.9999 R in matrix b 0.9982 0.9993 0.9998 IDL (ng/mL) 0.01 0.01 0.01 IQL (ng/mL) 0.05 0.05 0.05 MDL (ng/mL) 0.14 0.12 0.12 MQL (ng/mL) 0.48 0.40 0.41 Spike recovery (%), n = 6 0.5 (ng/mL) 84.4 ± 9.6 109 ± 8 110 ± 8 1 (ng/mL) 79.1 ± 9.8 100 ± 6 106 ± 10 5 (ng/mL) 81.2 ± 8.4 100 ± 4 119 ± 5 Matrix effect (%) –14.4 13.2 22.2 Intra-day variation (%), n = 6 0.5 (ng/mL) 8.83 10.8 10.1 1 (ng/mL) 3.13 3.19 3.46 5 (ng/mL) 7.18 9.10 4.61 Inter-day variation (%), n = 6 0.5 (ng/mL) 9.09 7.51 12.9 1 (ng/mL) 9.25 5.93 10.6 5 (ng/mL) 7.22 7.85 6.61 a The instrument linearity for all anlaytes in solvent (0.05–100 ng/mL) b The instrument linearity for all analytes in urine matrix (0.1–100 ng/mL) Table 3. Glyphosate and AMPA concentrations measured in external quality assurance proficiency test urine samples using the method developed in this study and compared with the assigned values. Glyphosate and AMPA were assigned in the OSEQAS Round 2021-01 PT samples, while the G- EQUAS PT samples only include glyphosate. OSEQAS, Quebec External Quality Assessment Scheme for Organic Substances in Urine; G-EQUAS, German External Quality Assessment Scheme OSEQAS Round 2021-01 ID Glyphosate (ng/mL) AMPA (ng/mL) Assigned value Acceptable range Our results Assigned value Acceptable range Our results OS-U- E 2101 1.24 0.713–1.77 1.55 1.65 0.954–2.35 1.65 OS-U- E 2102 1.67 0.949–2.39 2.18 6.62 3.72–9.52 6.94 OS-U- E 2103 2.23 1.24–3.22 2.80 2.2 1.31–3.09 2.15 G-EQUAS Round 66/2020 ID Glyphosate (ng/mL) Assigned value Acceptable range Our results 9 A 0.64 0.49–0.79 0.78 9 B 1.2 0.93–1.47 1.37 Abbreviations: AMPA, aminomethylphosphonic acid Next, we applied the validated method to the determination of concentrations in twenty human urine samples randomly collected from the populations of the US states of Iowa ( n = 10) and New York ( n = 10). In samples from Iowa, we detected glyphosate in six out of ten samples (mean: 1.18 ng/mL) and AMPA in five out of ten samples (mean: 0.88 ng/mL). In samples from New York, we found glyphosate in only one sample (0.53 ng/mL) and did not detect AMPA in any samples (Table 4 ). Samples from Iowa were

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 11 of 14 collected from adult males living in a rural farming region, whereas those from New York were from a population of office workers including adult males and females. Glufosinate was not found in any of the samples, probably due to its rapid metabolism, and low usage (~200-fold lower than glyphosate) [ 1 , 2 , 17 ]. However, with the exponential increase in glufosinate usage, its concentration in human urine may increase in the future. Overall, our results suggest the feasibility of measuring glyphosate, AMPA, and glufosinate in biomonitoring studies using the current method In comparison to previous studies (Table 1 ), our method has several advantages for application in human biomonitoring studies: (1) Excellent chromatographic retention, resolution, and peak shape, which were achieved through the use of less corrosive (i.e., less acidic) mobile phases. Previous studies used very high concentrations of acids in mobile phases (i.e., 1% formic acid or acetic acid) [ 26 , 40 ], used ion-pairing reagents (i.e., heptafluorobutyric acid) [ 27 , 31 ], or applied derivatization steps [ 32 ] to enhance sensitivity and selectivity. Such techniques are tedious, time-consuming, or corrosive; (2) Our method provides excellent sensitivity and uses a smaller sample volume (250 µ L) compared with other methods that used 0.5–2.5 mL urine [ 25 , 36 ]. Although one method reported relatively higher sensitivity [ 36 ], that method used a dilute-and-shoot method, which can affect selectivity and sensitivity due to matrix interferences. Furthermore, it was not clear if the reported detection limit for that method was that of the method or the IDL. (3) Our method has been validated through various QC parameters and successful participation in external assurance schemes while previously reported method did not report such external validation protocols (Tables 3 and 4 ). Table 4. Glyphosate, AMPA, and glufosinate concentrations measured in twenty human urine samples randomly collected from the general populations in Iowa ( n = 10) and New York ( n = 10), USA. Calculated concentrations are provided for those between MDL and MQL ID Location Glyphosate (ng/mL) AMPA (ng/mL) Glufosinate (ng/mL) 1 Iowa, USA 0.54 0.50 <MDL 2 Iowa, USA <MDL <MDL <MDL 3 Iowa, USA 0.91 0.39 (<MQL) <MDL 4 Iowa, USA 3.04 1.21 <MDL 5 Iowa, USA 0.36 (<MQL) 0.44 <MDL 6 Iowa, USA <MDL <MDL <MDL 7 Iowa, USA 0.70 0.85 <MDL 8 Iowa, USA 1.40 1.42 <MDL 9 Iowa, USA 0.49 0.19 (<MQL) <MDL 10 Iowa, USA 0.27 (<MQL) 0.20 (<MQL) <MDL 11 New York, USA <MDL <MDL <MDL 12 New York, USA <MDL <MDL <MDL 13 New York, USA <MDL <MDL <MDL 14 New York, USA <MDL <MDL <MDL 15 New York, USA <MDL <MDL <MDL 16 New York, USA <MDL <MDL <MDL 17 New York, USA <MDL <MDL <MDL 18 New York, USA <MDL <MDL <MDL 19 New York, USA <MDL <MDL <MDL 20 New York, USA 0.53 0.39 (<MQL) <MDL Abbreviations: MDL, method detection limit; MQL, method quantification limit; AMPA, aminomethylphosphonic acid 4. Conclusions We have developed and validated an LC–MS/MS method for the determination of glyphosate, AMPA, and glufosinate in human urine. Passage of samples in sequence through a combination of cationand anion-exchange solid-phase extraction cartridges for purification reduced matrix effects and increased sensitivity. Chromatographic and

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 12 of 14 MS/MS methods were optimized for the separation of analytes from interferences as well as to improve peak shape and sensitivity. The analytical parameters, including linearity, sensitivity, accuracy, and precision, were suitable for trace analysis of glyphosate, AMPA, and glufosinate in human urine and for use in large-scale biomonitoring studies of exposure in the general population Supplementary Materials: Supplementary material associated with this article can be found online at: https://www.mdpi.com/article/10.3390/ijerph 19094966/s 1 . Table S 1 . MRM parameters for the determination of glyphosate, AMPA, and glufosinate. For each compound SRM ion transitions are shown as m / z for the parent ion and a product ion. Optimized values include retention time (t R ), declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) Table S 2. Improvement in signals of glyphosate, AMPA, and glufosinate following pre-cleanup using Oasis MCX cartridge prior to Oasis MAX cartridge. Pooled urine samples were fortified with 0.5 ng/mL target analytes and 10 ng/mL internal standards Figure S 1. Overlaid chromatograms of fortified urine sample (0.5 ng/mL; internal standards: 10 ng/mL) following MAX cartridge cleanup only (red line) and the two-step procedure (MCX cartridge pre-cleanup and MAX cartridge post-cleanup) (black line). Respective signal-to-noise ratio (S/N) of the target analytes are given in the figure Author Contributions: Z.-M.L.: methodology, data curation, formal analysis, and writing—original draft. K.K.: conceptualization, funding acquisition, supervision, and writing—review and editing All authors have read and agreed to the published version of the manuscript Funding: This research was funded, in part, by the US National Institute of Environmental Health Sciences (NIEHS) under grant number U 2 CES 026542 (K.K.) Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the New York State Department of Health for the analysis of de-identified urine samples (under exempt category) to demonstrate application of the method developed in this study Informed Consent Statement: Informed consent was waived as the study was deemed exempt human study and only de-identified specimens were analyzed Acknowledgments: The research reported here was supported, in part, by the US National Institute of Environmental Health Sciences (NIEHS) under award number U 2 CES 026542 (K.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS Conflicts of Interest: The authors declare no conflict of interest References 1 Maggi, F.; la Cecilia, D.; Tang, F.H.M.; McBratney, A. The global environmental hazard of glyphosate use Sci. Total Environ 2020 , 717 , 137167. [ CrossRef ] [ PubMed ] 2 Benbrook, C.M. Trends in glyphosate herbicide use in the United States and globally Environ. Sci. Eur 2016 , 28 , 3. [ CrossRef ] [ PubMed ] 3 Takano, H.K.; Dayan, F.E. Glufosinate-ammonium: A review of the current state of knowledge Pest Manag. Sci 2020 , 76 , 3911–3925. [ CrossRef ] [ PubMed ] 4 Battaglin, W.A.; Meyer, M.T.; Kuivila, K.M.; Dietze, J.E. Glyphosate and its degradation product AMPA occur frequently and widely in U.S. soils, surface water, groundwater, and precipitation J. Am. Water Resour. Assoc 2014 , 50 , 275–290. [ CrossRef ] 5 Thompson, T.S.; van den Heever, J.P.; Limanowka, R.E. Determination of glyphosate, AMPA, and glufosinate in honey by online solid-phase extraction-liquid chromatography-tandem mass spectrometry Food Addit. Contam. Part A Chem. Anal. Control Expo Risk Assess 2019 , 36 , 434–446. [ CrossRef ] 6 Bai, S.H.; Ogbourne, S.M. Glyphosate: Environmental contamination, toxicity and potential risks to human health via food contamination Environ. Sci. Pollut. Res. Int 2016 , 23 , 18988–19001. [ CrossRef ] 7 Connolly, A.; Coggins, M.A.; Koch, H.M. Human biomonitoring of glyphosate exposures: State-of-the-art and future research challenges Toxics 2020 , 8 , 60. [ CrossRef ] 8 Conrad, A.; Schroter-Kermani, C.; Hoppe, H.W.; Ruther, M.; Pieper, S.; Kolossa-Gehring, M. Glyphosate in German adults—Time trend (2001 to 2015) of human exposure to a widely used herbicide Int. J. Hyg. Environ. Health 2017 , 220 , 8–16. [ CrossRef ]

[Find the meaning and references behind the names: Van Gerwen, Zhang, Liu, Foods, Lee, Rupp, Natural, Santos, Morra, Chagnon, June, Shimada, Guyton, Mcdade, January, Level, Rana, Europa, Fernandez, Holmes, Mcgowan, Silver, Life, Zhao, Sabino, Hermann, Hsu, Wang, Rosario, Vega, Kenny, Morse, Kroes, Uren, Honda, Clair, Hao, Gov, Webster, Berry, Ware, Nau, Guha, Cordero, Neubert, Meeker, Ananda, Brown, Echa, Hori, Tsao, Sheppard, Benbrahim, Gasnier, Franke, Lines, Epa, Blood, Dumont, Lai, Hirose, Sato, Grosse, Rico, Lyon, Mrls, France, Galea, Trout, Geiser, Kikuchi, Swiss, Chen, Velez, Zoller, Case, Loomis, Johannesen, Jones, Schulte, Shaffer, Benachour, Nunn, Puerto, Half, Washington, Lin, Yang, Cheng, Springer, Bmc]

Int. J. Environ. Res. Public Health 2022 , 19 , 4966 13 of 14 9 Gillezeau, C.; van Gerwen, M.; Shaffer, R.M.; Rana, I.; Zhang, L.; Sheppard, L.; Taioli, E. The evidence of human exposure to glyphosate: A review Environ. Health 2019 , 18 , 2. [ CrossRef ] 10 EFSA. Conclusion on the peer review of the pesticide risk assessment of the active substance glyphosate EFSA J 2015 , 13 , 4302 [ CrossRef ] 11 Connolly, A.; Jones, K.; Basinas, I.; Galea, K.S.; Kenny, L.; McGowan, P.; Coggins, M.A. Exploring the half-life of glyphosate in human urine samples Int. J. Hyg. Environ. Health 2019 , 222 , 205–210. [ CrossRef ] [ PubMed ] 12 Uren Webster, T.M.; Santos, E.M. Global transcriptomic profiling demonstrates induction of oxidative stress and of compensatory cellular stress responses in brown trout exposed to glyphosate and Roundup BMC Genom 2015 , 16 , 32. [ CrossRef ] [ PubMed ] 13 Gasnier, C.; Dumont, C.; Benachour, N.; Clair, E.; Chagnon, M.C.; Seralini, G.E. Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines Toxicology 2009 , 262 , 184–191. [ CrossRef ] 14 Silver, M.K.; Fernandez, J.; Tang, J.; McDade, A.; Sabino, J.; Rosario, Z.; Velez Vega, C.; Alshawabkeh, A.; Cordero, J.F.; Meeker, J.D. Prenatal exposure to glyphosate and its environmental degradate, aminomethylphosphonic acid (AMPA), and preterm birth: A nested case-control study in the PROTECT cohort (Puerto Rico) Environ. Health Perspect 2021 , 129 , 57011. [ CrossRef ] 15 Guyton, K.Z.; Loomis, D.; Grosse, Y.; El Ghissassi, F.; Benbrahim-Tallaa, L.; Guha, N.; Scoccianti, C.; Mattock, H.; Straif, K. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate Lancet Oncol 2015 , 16 , 490–491. [ CrossRef ] 16 Hoerlein, G. Glufosinate (Phosphinothricin), A Natural Amino Acid with Unexpected Herbicidal Properties. In Reviews of Environmental Contamination and Toxicology: Continuation of Residue Reviews ; Ware, G.W., Ed.; Springer: New York, NY, USA, 1994; pp. 73–145 17 Schulte-Hermann, R.; Wogan, G.N.; Berry, C.; Brown, N.A.; Czeizel, A.; Giavini, E.; Holmes, L.B.; Kroes, R.; Nau, H.; Neubert, D.; et al. Analysis of reproductive toxicity and classification of glufosinate-ammonium Regul. Toxicol. Pharmacol 2006 , 44 , S 1–S 76 [ CrossRef ] [ PubMed ] 18 EFSA. Reasoned opinion on the review of the existing maximum residue levels (MRLs) for for glufosinate according to Article 12 of Regulation (EC) No 396/2005 EFSA J 2015 , 13 , 3950–4030. [ CrossRef ] 19 IARC. Some Organophosphate Insecticides and Herbicides. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans ; IARC: Lyon, France, 2015; Volume 112, ISBN 978-92-832-0178-6. Available online: https://publications.iarc.fr/549 (accessed on 1 January 2022) 20 ECHA. Glyphosate Not Classified as a Carcinogen by ECHA. 2017. Available online: https://echa.europa.eu/-/glyphosate-notclassified-as-a-carcinogen-by-echa (accessed on 1 January 2022) 21 EPA Glyphosate Issue Paper: Evaluation of Carcinogenic Potential ; United States Environmental Protection Agency: Washington, DC, USA, 2016. Available online: https://www.epa.gov/sites/production/files/2016-09/documents/glyphosate_issue_paper_ evaluation_of_carcincogenic_potential.pdf (accessed on 25 June 2020) 22 Hori, Y.; Fujisawa, M.; Shimada, K.; Sato, M.; Kikuchi, M.; Honda, M.; Hirose, Y. Quantitative determination of glufosinate in biological samplesby liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization J. Chromatogr. B 2002 , 767 , 255–262. [ CrossRef ] 23 Hori, Y.; Fujisawa, M.; Shimada, K.; Sato, M.; Honda, M.; Hirose, Y. Enantioselective analysis of glufosinate using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography J. Chromatogr. B 2002 , 776 , 191–198. [ CrossRef ] 24 Zoller, O.; Rhyn, P.; Rupp, H.; Zarn, J.A.; Geiser, C. Glyphosate residues in Swiss market foods: Monitoring and risk evaluation Food Addit. Contam. B Surveill 2018 , 11 , 83–91. [ CrossRef ] 25 Zoller, O.; Rhyn, P.; Zarn, J.A.; Dudler, V. Urine glyphosate level as a quantitative biomarker of oral exposure Int. J. Hyg. Environ Health 2020 , 228 , 113526. [ CrossRef ] [ PubMed ] 26 Karthikraj, R.; Kannan, K. Widespread occurrence of glyphosate in urine from pet dogs and cats in New York State, USA Sci Total Environ 2019 , 659 , 790–795. [ CrossRef ] [ PubMed ] 27 Jaikwang, P.; Junkuy, A.; Sapbamrer, R.; Seesen, M.; Khacha-ananda, S.; Mueangkhiao, P.; Wunnapuk, K. A dilute-and-shoot LC–MS/MS method for urinary glyphosate and AMPA Chromatographia 2020 , 83 , 467–475. [ CrossRef ] 28 Hao, C.; Morse, D.; Morra, F.; Zhao, X.; Yang, P.; Nunn, B. Direct aqueous determination of glyphosate and related compounds by liquid chromatography/tandem mass spectrometry using reversed-phase and weak anion-exchange mixed-mode column J Chromatogr. A 2011 , 1218 , 5638–5643. [ CrossRef ] [ PubMed ] 29 Granby, K.; Johannesen, S.; Vahl, M. Analysis of glyphosate residues in cereals using liquid chromatography-mass spectrometry (LC-MS/MS) Food Addit. Contam 2003 , 20 , 692–698. [ CrossRef ] 30 Wang, K.C.; Chen, S.M.; Hsu, J.F.; Cheng, S.G.; Lee, C.K. Simultaneous detection and quantitation of highly water-soluble herbicides in serum using ion-pair liquid chromatography-tandem mass spectrometry J. Chromatogr. B 2008 , 876 , 211–218 [ CrossRef ] 31 Tsao, Y.C.; Lai, Y.C.; Liu, H.C.; Liu, R.H.; Lin, D.L. Simultaneous determination and quantitation of paraquat, diquat, glufosinate and glyphosate in postmortem blood and urine by LC-MS-MS J. Anal. Toxicol 2016 , 40 , 427–436. [ CrossRef ] 32 Franke, A.A.; Li, X.; Lai, J.F. Analysis of glyphosate, aminomethylphosphonic acid, and glufosinate from human urine by HRAM LC-MS Anal. Bioanal. Chem 2020 , 412 , 8313–8324. [ CrossRef ]

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Int. J. Environ. Res. Public Health 2022 , 19 , 4966 14 of 14 33 Martinez-Moral, M.P.; Kannan, K. How stable is oxidative stress level? An observational study of intraand inter-individual variability in urinary oxidative stress biomarkers of DNA, proteins, and lipids in healthy individuals Environ. Int 2019 , 123 , 382–389. [ CrossRef ] 34 Myint, K.T.; Uehara, T.; Aoshima, K.; Oda, Y. Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent Anal. Chem 2009 , 81 , 7766–7772. [ CrossRef ] 35 Guo, H.; Riter, L.S.; Wujcik, C.E.; Armstrong, D.W. Direct and sensitive determination of glyphosate and aminomethylphosphonic acid in environmental water samples by high performance liquid chromatography coupled to electrospray tandem mass spectrometry J. Chromatogr. A 2016 , 1443 , 93–100. [ CrossRef ] 36 Jensen, P.K.; Wujcik, C.E.; McGuire, M.K.; McGuire, M.A. Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS J. Environ. Sci. Health B 2016 , 51 , 254–259 [ CrossRef ] [ PubMed ] 37 Hsiao, J.J.; Potter, O.G.; Chu, T.W.; Yin, H. Improved LC/MS methods for the analysis of metal-sensitive analytes using medronic acid as a mobile phase additive Anal. Chem 2018 , 90 , 9457–9464. [ CrossRef ] [ PubMed ] 38 Nagatomi, Y.; Yoshioka, T.; Yanagisawa, M.; Uyama, A.; Mochizuki, N. Simultaneous LC-MS/MS analysis of glyphosate, glufosinate, and their metabolic products in beer, barley tea, and their ingredients Biosci. Biotechnol. Biochem 2013 , 77 , 2218–2221 [ CrossRef ] [ PubMed ] 39 Nomura, H.; Hamada, R.; Saito, I.; Nakane, K.; Sawa, R.; Ukai, M.; Shibata, E.; Sato, M.; Kamijima, M.; Ueyama, J. Optimization and validation of a highly sensitive method for determining glyphosate in human urine by solid-phase extraction and liquid chromatography with tandem mass spectrometry: A methodological study Environ. Health Prev. Med 2020 , 25 , 83. [ CrossRef ] 40 Connolly, A.; Jones, K.; Galea, K.S.; Basinas, I.; Kenny, L.; McGowan, P.; Coggins, M. Exposure assessment using human biomonitoring for glyphosate and fluroxypyr users in amenity horticulture Int. J. Hyg. Environ. Health 2017 , 220 , 1064–1073 [ CrossRef ] 41 Connolly, A.; Basinas, I.; Jones, K.; Galea, K.S.; Kenny, L.; McGowan, P.; Coggins, M.A. Characterising glyphosate exposures among amenity horticulturists using multiple spot urine samples Int. J. Hyg. Environ. Health 2018 , 221 , 1012–1022. [ CrossRef ] 42 Connolly, A.; Leahy, M.; Jones, K.; Kenny, L.; Coggins, M.A. Glyphosate in Irish adults—A pilot study in 2017 Environ. Res 2018 , 165 , 235–236. [ CrossRef ] 43 Mesnage, R.; Moesch, C.; Grand, R.L.G.; Lauthier, G.; Vend ô mois, J.S.d.; Gress, S.; S é ralini, G.-E. Glyphosate exposure in a farmer’s family J. Environ. Prot 2012 , 3 , 1001–1003. [ CrossRef ] 44 Fagan, J.; Bohlen, L.; Patton, S.; Klein, K. Organic diet intervention significantly reduces urinary glyphosate levels in U.S. children and adults Environ. Res 2020 , 189 , 109898. [ CrossRef ] 45 Soukup, S.T.; Merz, B.; Bub, A.; Hoffmann, I.; Watzl, B.; Steinberg, P.; Kulling, S.E. Glyphosate and AMPA levels in human urine samples and their correlation with food consumption: Results of the cross-sectional KarMeN study in Germany Arch. Toxicol 2020 , 94 , 1575–1584. [ CrossRef ] [ PubMed ] 46 Trasande, L.; Aldana, S.I.; Trachtman, H.; Kannan, K.; Morrison, D.; Christakis, D.A.; Whitlock, K.; Messito, M.J.; Gross, R.S.; Karthikraj, R.; et al. Glyphosate exposures and kidney injury biomarkers in infants and young children Environ. Pollut 2020 , 256 , 113334. [ CrossRef ] [ PubMed ] 47 Parvez, S.; Gerona, R.R.; Proctor, C.; Friesen, M.; Ashby, J.L.; Reiter, J.L.; Lui, Z.; Winchester, P.D. Glyphosate exposure in pregnancy and shortened gestational length: A prospective Indiana birth cohort study Environ. Health 2018 , 17 , 23. [ CrossRef ] [ PubMed ] 48 Van Eeckhaut, A.; Lanckmans, K.; Sarre, S.; Smolders, I.; Michotte, Y. Validation of bioanalytical LC-MS/MS assays: Evaluation of matrix effects J. Chromatogr. B 2009 , 877 , 2198–2207. [ CrossRef ]

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