Gestational glyphosate exposure and early childhood neurodevelopment in a Puerto Rico birth cohort.

INTRODUCTION: N-(phosphonomethyl)glycine, or glyphosate, is a non-selective systemic herbicide widely used in agricultural, industrial, and residential settings since 1974. Glyphosate exposure has been inconsistently linked to neurotoxicity in animals, and studies of effects of gestational exposure among humans are scarce. In this study we investigated relationships between prenatal urinary glyphosate analytes and early childhood neurodevelopment. METHODS: Mother-child pairs from the PROTECT-CRECE birth cohort in Puerto Rico with measures for both maternal urinary glyphosate analytes and child neurodevelopment were included for analysis (n = 143). Spot urine samples were collected 1-3 times throughout pregnancy and analyzed for glyphosate and aminomethylphosphonic acid (AMPA), an environmental degradant of glyphosate. Child neurodevelopment was assessed at 6, 12, and 24 months using the Battelle Developmental Inventory, 2nd edition Spanish (BDI-2), which provides scores for adaptive, personal-social, communication, motor, and cognitive domains. We used multivariable linear regression to examine associations between the geometric mean of maternal urinary glyphosate analytes across pregnancy and BDI-2 scores at each follow-up. Results were expressed as percent change in BDI-2 score per interquartile range increase in exposure. RESULTS: Prenatal AMPA concentrations were negatively associated with communication domain at 12 months (%change = -5.32; 95%CI: 9.04, -1.61; p = 0.007), and communication subdomain scores at 12 and 24 months. At 24 months, four BDI-2 domains were associated with AMPA: adaptive (%change = -3.15; 95%CI: 6.05, -0.25; p = 0.038), personal-social (%change = -4.37; 95%CI: 7.48, -1.26; p = 0.008), communication (%change = -7.00; 95%CI: 11.75, -2.26; p = 0.005), and cognitive (%change = -4.02; 95%CI: 6.72, -1.32; p = 0.005). Similar trends were observed with GLY concentrations, but most confidence intervals include zero. We found no significant associations at 6 months. CONCLUSIONS: Our results suggest that gestational exposure to glyphosate is associated with adverse early neurodevelopment, with more pronounced delays at 24 months. Given glyphosate's wide usage, further investigation into the impact of gestational glyphosate exposure on neurodevelopment is warranted.

Study on the preparation of molecular imprinted polymer for analysis of N-phenylglycine in human urine.

N-phenylglycine (NPG) in human urine could be an important biomarker for predicting cancers, but its detection has difficulty due to its low abundance in urine. Herein, we report a molecular imprinted polymer (MIP) method to efficiently recognize NPG in urine. The MIP was prepared by precipitation polymerization, adopting NPG as the template, acrylamide (AM) as functional monomer, trimethylpropane triacrylate (TRIM) as crosslinking agent, and acetonitrile as porogen. The specificity and selectivity of MIP towards NPG in human urine were determined by comparing MIP's adsorption to the NPG and N-crotonylglycine (NTG) under the same conditions. The result ß = QMIP-NPG/QMIP-NTG = 4.7 indicated the satisfactory specificity and selectivity. Parameters affecting the extraction efficiency were further optimized. Under the optimum conditions, the linear range, limit of detection, and limit of quantification of NPG were 0.5-100 mg∙L-1, 1.6 × 10-2 mg∙L-1, and 5.5 × 10-2 mg∙L-1, respectively. Recoveries of NPG in human urine were in the range of 84.7-100.0% with RSDS of 3.8-10.8%. The developed method demonstrated superior selectivity to the target analyte, which can be applied to separate and enrich the NPG from urine samples.

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon.

BACKGROUND: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,ß-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. METHODS: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. RESULTS: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. CONCLUSIONS: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.

Comprehensive metabolomic profiling in early IgA nephropathy patients reveals urine glycine as a prognostic biomarker.

Identification of a urinary metabolite biomarker with diagnostic or prognostic significance for early immunoglobulin A nephropathy (IgAN) is needed. We performed nuclear magnetic resonance-based metabolomic profiling and identified 26 metabolites in urine samples. We collected urine samples from 201, 77, 47, 36 and 136 patients with IgAN, patients with membranous nephropathy, patients with minimal change disease, patients with lupus nephritis and healthy controls, respectively. We determined whether a metabolite level is associated with the prognosis of IgAN through Cox regression and continuous net reclassification improvement (cNRI). Finally, in vitro experiments with human kidney tubular epithelial cells (hTECs) were performed for experimental validation. As the results, the urinary glycine level was higher in the IgAN group than the control groups. A higher urinary glycine level was associated with lower risk of eGFR 30% decline in IgAN patients. The addition of glycine to a predictive model including clinicopathologic information significantly improved the predictive power for the prognosis of IgAN [cNRI 0.72 (0.28-0.82)]. In hTECs, the addition of glycine ameliorated inflammatory signals induced by tumour necrosis factor-α. Our study demonstrates that urinary glycine may have diagnostic and prognostic value for IgAN and indicates that urinary glycine is a protective biomarker for IgAN.

Glycine suppresses kidney calcium oxalate crystal depositions via regulating urinary excretions of oxalate and citrate.

An abnormal urine composition is a key reason for kidney stone formation, but little is known about the roles of small metabolites in the urine during kidney stone formation. Here, we found urine glycine in patients with kidney calcium oxalate (CaOx) stone was significantly lower than that in healthy people via 1 H NMR spectra detection, and investigated the role and underlying mechanism of glycine in the regulation of CaOx stone formation. Our results showed that glycine could significantly attenuate ethylene glycol-induced CaOx crystal depositions in rat kidney via decreasing urine oxalate and increasing urine citrate. Mechanism studies revealed that glycine could decrease urine oxalate through downregulating Slc26a6 expression, whereas increase urine citrate via inhibiting Nadc1 expression. Moreover, glycine decreased the protein expression of both Slc26a6 and Nadc1 via increasing the expression of miRNA-411-3p, which directly bound to the 3'-untranslated regions of Slc26a6 and Nadc1 messenger RNAs, in vitro and in vivo. Together, our results revealed a novel role of glycine in the regulation of kidney CaOx crystal formation and provided a potential target for the treatment of kidney CaOx stone.

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the determination of glyphosate in human urine after pre-column derivatization with 9-fluorenylmethoxycarbonyl chloride.

In 2015, glyphosate was classified as "Group 2A - probably carcinogenic to humans" by the International Agency for Research on Cancer (IARC). Therefore, public concerns about the environmental and health risks of this substance have rapidly increased. Considering its toxicokinetic characteristics, urinary levels of glyphosate could be a powerful tool for human biomonitoring. Nevertheless, the physicochemical properties of this molecule and the complexity of the matrix make this purpose particularly challenging. In order to solve this problem, the presented study describes a simple LC-MS/MS method for the quantification of glyphosate in human urine after pre-column derivatization with FMOC-Cl. Method development was focused on the optimization of the derivatization reaction in human urine, adjusting critical variables such as pH of borate buffer, FMOC-Cl concentration and derivatization time. Besides, chromatographic separation and spectrometric parameters were also established. The analytical method was fully validated according international guidelines for selectivity, carry over, linearity, accuracy, precision, lower limit of quantitation, matrix effect and stability under different conditions. All performance parameters were within the acceptance criteria. In addition, the method was successfully applied to 52 urine samples obtained from exposed subjects from northern Argentina, laying the foundation for future epidemiological studies.

Identification of Hypoxia-inducible factor (HIF) stabilizer roxadustat and its possible metabolites in thoroughbred horses for doping control.

Hypoxia-inducible factor (HIF) stabilizer belongs to a novel class of pharmacologically active substances, which are capable of inducing the endogenous erythropoietic system. The transcriptional activator HIF has been shown to significantly increase blood hemoglobin and is well set for the treatment of anemia resulting from chronic kidney disease. This research work reports a comprehensive study of the most popular HIF stabilizer roxadustat and its metabolites in thoroughbred horse urine after oral administration. The plausible structures of the detected metabolites were postulated using liquid chromatography-high-resolution mass spectrometry. Under the experimental condition 13 metabolites (7 phase I, 1 phase II, and 5 conjugates of phase I metabolism) were positively detected (M1-M13). The major phase I metabolites identified were formed by hydroxylation. Dealkylated and hydrolyzed phase I metabolites were also observed in this study. In phase II, a glucuronic acid conjugate of roxadustat was detected as the major metabolite. The sulfonic acid conjugates were observed to be formed from phase I metabolites. The characterized in vivo metabolites can potentially serve as target analytes for doping control analysis; hence, the result is an important tool for assessing its use and abuse in competitive sport.

Determination of glyphosate, glufosinate and their major metabolites in urine by the UPLC-MS/MS method applicable to biomonitoring and epidemiological studies.

The preoccupation concerning glyphosate (GLYP) has rapidly grown over recent years, and the availability of genetically modified crops that are resistant to GLYP or glufosinate (GLUF) has increased the use of these herbicides. The debate surrounding the carcinogenicity of GLYP has raised interest and the desire to gain information on the level of exposure of the population. GLYP and aminomethylphosphonic acid (AMPA) are commonly simultaneously analysed. GLUF is sometimes also monitored, but its major metabolite, 3-[hydroxy(methyl)phosphinoyl]propionic acid (3MPPA), is rarely present in the method. Using a pentafluorobenzyl derivative to extract the analytes from human urine, we present a method that contains four important analytes to monitor human exposure to GLYP and GLUF. The use of the flash freeze technique speeds up the extraction process and requires less organic solvent than conventional liquid-liquid extraction. The limits of detection in the low µg/L range enable the use of this method for epidemiological studies. The results obtained for 35 volunteers from the Quebec City area are presented with the results from multiple interlaboratory comparisons (G-EQUAS, HBM4EU and OSEQAS). This methodology is currently being used in the Maternal-Infant Research on Environmental Chemicals (MIREC-ENDO) study and in the Canadian Health Measures Survey (CHMS).

Solute carrier SLC16A12 is critical for creatine and guanidinoacetate handling in the kidney.

A heterozygous mutation (c.643C.A; p.Q215X) in the creatine transporter SLC16A12 has been proposed to cause a syndrome with juvenile cataracts, microcornea, and glucosuria in humans. To further explore the role of SLC16A12 in renal physiology and decipher the mechanism underlying the phenotype of humans with the SLC16A12 mutation, we studied Slc16a12 knockout (KO) rats. Slc16a12 KO rats had lower plasma levels and increased absolute and fractional urinary excretion of creatine and its precursor guanidinoacetate (GAA). Slc16a12 KO rats displayed lower plasma and urinary creatinine levels, but the glomerular filtration rate was normal. The phenotype of heterozygous rats was indistinguishable from wild-type (WT) rats. Renal artery to vein (RAV) concentration differences in WT rats were negative for GAA and positive for creatinine. However, RAV differences for GAA were similar in Slc16a12 KO rats, indicating incomplete compensation of urinary GAA losses by renal GAA synthesis. Together, our results reveal that Slc16a12 in the basolateral membrane of the proximal tubule is critical for the reabsorption of creatine and GAA. Our data suggest a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation. Furthermore, in the absence of Slc16a12, urinary losses of GAA are not adequately compensated by increased tubular synthesis, likely caused by feedback inhibition of the rate-limiting enzyme l-arginine:glycine amidinotransferase by creatine in proximal tubular cells.NEW & NOTEWORTHY SLC16A12 is a recently identified creatine transporter of unknown physiological function. A heterozygous mutation in the human SLC16A12 gene causes juvenile cataracts and reduced plasma guanidinoacetate (GAA) levels with an increased fractional urinary excretion of GAA. Our study with transgenic SLC16A12-deficient rats reveals that SLC16A12 is critical for tubular reabsorption of creatine and GAA in the kidney. Our data furthermore indicate a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation.

The effect of circuit resistance training on plasma levels of amino acids, alpha-hydroxybutyrate, mannose, and urinary levels of glycine conjugated adducts in obese adolescent boys.

Few studies have examined the improving effects of exercise on the association between metabolites of impaired protein metabolism and insulin resistance in obese children. Therefore, this study aims to investigate the effect of circuit resistance training (CRT) on plasma levels of amino acids, alpha-hydroxybutyrate (α-HB), mannose, and urinary levels of glycine conjugated adducts in obese adolescent boys. Forty obese adolescent boys (body mass index above the 95th percentile) with an age range of 14-17 years were randomly divided into the CRT group (n = 20) and control group (n = 20). The CRT program (3 times/week, 70%-80% of 1-repetition maximum) was performed for 8 weeks. The results indicated that the body composition and plasma levels of glucose, insulin resistance, valine, mannose, lysine, and the sum of branched-chain amino acids (BCAA) were decreased because of CRT. The plasma levels of asparagine, glycine, serine, and urinary levels of glycine conjugated adduct also increased in the CRT group. Although α-HB level decreased during CRT, it had no significant difference from that of the control group. It can be concluded that the improvement in obesity complications including insulin resistance in obese adolescent boys after CRT may be due to decrease in plasma levels of mannose and BCAA and increase urinary metabolites. Novelty: CRT improves glucose metabolism and insulin resistance in obese adolescent boys. CRT decreases plasma levels of mannose and BCAA and normalizes other amino acids. CRT increases urinary levels of glycine conjugated adducts.

Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

A recurring disease outbreak following litchi fruit consumption among children in Muzaffarpur, Bihar-A comprehensive investigation on factors of toxicity.

Litchi fruits are a nutritious and commercial crop in the Indian state of Bihar. Litchi fruit contains a toxin, methylene cyclopropyl-glycine (MCPG), which is known to be fatal by causing encephalitis-related deaths. This is especially harmful when consumed by malnourished children. The first case of litchi toxicity was reported in Bihar in 2011. A similar event was recorded in 2014 among children admitted to the Muzaffarpur government hospital, Bihar. Litchi samples sent to ICMR-NIN were analyzed and MCPG was found to be present in both the pulp and seed of the fruit. Diethyl phosphate (DEP) metabolites were found in the urine samples of children who had consumed litchi fruit from this area indicating exposure to pesticide. The presence of both MCPG in litchi and DEP metabolites in urine samples highlights the need to conduct a comprehensive investigation that examines all factors of toxicity.

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.

BACKGROUND: Glyphosate and its salt formulations are nonselective herbicides that have been extensively used worldwide, both for residential and agricultural purposes. The possible carcinogenicity and teratogenicity of glyphosate remain to be elucidated. We developed a sensitive and high-throughput analytical method for urinary glyphosate using liquid chromatography-tandem mass spectrometry with the aim of contributing to glyphosate exposure assessment in epidemiological studies. METHODS: After urine dilution (creatinine matching dilution to 0.05 g creatinine/L), glyphosate was extracted using two types of solid phase extraction columns (SCX and NH2) with automated sample preparation instruments. The eluate was dried and dissolved in the mobile phase, followed by liquid chromatography-tandem mass spectrometry analysis. The optimized method was applied to urine samples obtained from 54 Japanese adults and children. RESULTS: The results from the validation study demonstrated good recoveries (91.0-99.6%), within- and between-run precisions (< 15%), low detection limits (0.1 µg/L), and lower limit of quantification (0.3 µg/L). The detection frequency and median concentration of the urinary glyphosate in Japanese subjects were 59% and 0.25 µg/L (0.34 µg/g creatinine). CONCLUSIONS: Our reliable determination method was successful in measuring urinary glyphosate concentration. Moreover, this is the first biomonitoring report of urinary glyphosate levels in the Japanese general population.

Monitoring Methylmalonic Aciduria by NMR Urinomics.

The paper reports on monitoring methylmalonic aciduria (MMA)-specific and non-specific metabolites via NMR urinomics. Five patients have been monitored over periods of time; things involved were diet, medication and occasional episodes of failing to comply with prescribed diets. An extended dataset of targeted metabolites is presented, and correlations with the type of MMA are underlined. A survey of previous NMR studies on MMA is also presented.

Guanidinoacetic acid deficiency: a new entity in clinical medicine?

Guanidinoacetic acid (GAA, also known as glycocyamine or betacyamine) is a naturally-occurring derivative of glycine and a direct metabolic precursor of creatine, a key player in high-phosphate cellular bioenergetics. GAA is found in human serum and urine, with circulating GAA likely reflects an equilibrium between its endogenous production and utilization/excretion. GAA deficiency (as indicated by low serum GAA) has been reported in various conditions yet this intriguing clinical entity appears to be poorly characterized as yet, either as a primary deficit or a sequel of secondary disease. This minireview article summarizes the inherited and acquired disorders with apparent GAA deficiency and discusses a possible relevance of GAA shortfall in clinical medicine.

Analysis of glyphosate, aminomethylphosphonic acid, and glufosinate from human urine by HRAM LC-MS.

Aminomethylphosphonic acid (AMPA) is the main metabolite of glyphosate (GLYP) and phosphonic acids in detergents. GLYP is a synthetic herbicide frequently used worldwide alone or together with its analog glufosinate (GLUF). The general public can be exposed to these potentially harmful chemicals; thus, sensitive methods to monitor them in humans are urgently required to evaluate health risks. We attempted to simultaneously detect GLYP, AMPA, and GLUF in human urine by high-resolution accurate-mass liquid chromatography mass spectrometry (HRAM LC-MS) before and after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) or 1-methylimidazole-sulfonyl chloride (ImS-Cl) with several urine pre-treatment and solid phase extraction (SPE) steps. Fmoc-Cl derivatization achieved the best combination of method sensitivity (limit of detection; LOD) and accuracy for all compounds compared to underivatized urine or ImS-Cl-derivatized urine. Before derivatization, the best steps for GLYP involved 0.4 mM ethylenediaminetetraacetic acid (EDTA) pre-treatment followed by SPE pre-cleanup (LOD 37 pg/mL), for AMPA involved no EDTA pre-treatment and no SPE pre-cleanup (LOD 20 pg/mL) or 0.2-0.4 mM EDTA pre-treatment with no SPE pre-cleanup (LOD 19-21 pg/mL), and for GLUF involved 0.4 mM EDTA pre-treatment and no SPE pre-cleanup (LOD 7 pg/mL). However, for these methods, accuracy was sufficient only for AMPA (101-105%), while being modest for GLYP (61%) and GLUF (63%). Different EDTA and SPE treatments prior to Fmoc-Cl derivatization resulted in high sensitivity for all analytes but satisfactory accuracy only for AMPA. Thus, we conclude that our HRAM LC-MS method is suited for urinary AMPA analysis in cross-sectional studies.

Sensitive and selective quantification of glyphosate and aminomethylphosphonic acid (AMPA) in urine of the general population by gas chromatography-tandem mass spectrometry.

Glyphosate is the highest volume herbicide used worldwide, and its main biodegradation product is aminomethylphosphonic acid (AMPA), both are listed as priority substances in the Human Biomonitoring for Europe (HBM4EU) initiative which aims at improving policy by filling knowledge gaps by targeted research. The objective of the current study was to advance the sensitivity of an existing gas chromatography-tandem mass spectrometry analytical method to measure environmental population exposures. A 50% lower limit of quantification of 0.05 µg/L was achieved for both analytes by slight modifications in sample work-up, and use of another isotope labelled internal standard. In a pilot study, 41 urine samples from the general German population were analysed, of which glyphosate and AMPA could be quantified in 66% and 90% of the samples respectively, which is sufficient to reliably describe distributions of urinary concentrations in the non-occupationally exposed population.

Pilot study evaluating inhalation and dermal glyphosate exposure resulting from simulated heavy residential consumer application of Roundup®.

OBJECTIVES: The purpose of this study was to evaluate the individual contributions of inhalation and dermal exposures to urinary glyphosate levels following the heavy residential consumer application of a glyphosate-containing herbicide. METHODS: A pilot study was conducted in which each participant mixed and continuously spray-applied 16.3 gallons of a 0.96% glyphosate-containing solution for 100 min using a backpack sprayer. Twelve participants were divided evenly into two exposure groups, one equipped to assess dermal exposure and the other, inhalation exposure. Personal air samples (n = 12) and dermal patch samples (n = 24) were collected on the inhalation group participants and analyzed for glyphosate using HPLC-UV. Serial urine samples collected 30-min prior to application and 3-, 6-, 12-, 24-hr (inhalation and dermal groups) and 36-hr (dermal group only) post-application were analyzed for glyphosate and glyphosate's primary metabolite (AMPA) using HPLC-MS/MS. RESULTS: The mean airborne glyphosate concentration was 0.0047 mg/m3, and the mean concentrations of glyphosate for each applicator's four patch samples ranged from 0.04 µg/mm2 to 0.25 µg/mm2. In general, urinary glyphosate, AMPA, and total effective glyphosate levels were higher in the dermal exposure group than the inhalation exposure group, peaked within 6-hr following application, and were statistically indistinguishable from background at 24-hr post-application. CONCLUSIONS: This is the first study to characterize the absorption and biological fate of glyphosate in residential consumer applicators following heavy application. The results of this pilot study are consistent with previous studies that have shown that glyphosate is rapidly eliminated from the body, typically within 24 hr following application.

Glyphosate in Portuguese Adults - A Pilot Study.

BACKGROUND: Glyphosate is a broad-spectrum biocide and the active ingredient in the most widely used herbicides worldwide. Since 2015, when the International Agency for Research on Cancer classified it as a Class 2A carcinogen, global interest in this chemical spiked particularly as regards exposure of the general population. OBJECTIVE: An exploratory glyphosate exposure assessment was conducted among Portuguese adults. METHODS: Self-selected participants provided first morning urine which was tested for glyphosate and its metabolite aminomethylphosphonic acid (AMPA) at two distinct periods of time, by two different laboratories using gas chromatography with tandem mass spectrometry (GC-MS-MS) and high performance liquid chromatography linked to triple quadrupole mass spectrometry (HPLC-MS/MS), respectively. RESULTS: In the first round of testing 28% and 50% presented detectable levels of glyphosate and AMPA respectively, with median values of 0.25 and 0.16 µg/L. Systematically available internal dose values were 8.20E-06 mg/Kg (glyphosate) and 5.04-05 mg/Kg (AMPA). In the second round 73% and 97% presented detectable levels of glyphosate and AMPA respectively with median values of 0.13 and 0.10 µg/L. Systematically available internal dose values were 4.00E-06 mg/Kg (glyphosate) and 3.00E-06 mg/Kg (AMPA). CONCLUSIONS: Glyphosate exposure was detected among Portuguese adults, with percentages of glyphosate and AMPA contaminated urine in both rounds of testing and above values from previous studies in other European countries. Systematically available internal doses values were below EFSA's risk assessment values (ADI or AOEL), and as such, the concentration values measured in this study are not per se a human health problem. Even though there were study limitations, it is the first assessment in Portugal and contributes to the overall knowledge map of glyphosate exposure in Europe.

Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data.

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures.