Analytical methods based on liquid chromatography for the analysis of albumin adducts involved in retrospective biomonitoring of exposure to mustard agents.

The objective of the present review is to list, describe, compare, and critically analyze the main procedures developed in the last 20 years for the analysis of digested alkylated peptides, resulting from the adduction of albumin by different mustard agents, and that can be used as biomarkers of exposure to these chemical agents. While many biomarkers of sulfur mustard, its analogues, and nitrogen mustards can easily be collected in urine such as their hydrolysis products, albumin adducts require blood or plasma collection to be analyzed. Nonetheless, albumin adducts offer a wider period of detectability in human exposed patients than urine found biomarkers with detection up to 25 days after exposure to the chemical agent. The detection of these digested alkylated peptides of adducted albumin constitutes unambiguous proof of exposure. However, their determination, especially when they are present at very low concentration levels, can be very difficult due to the complexity of the biological matrices. Therefore, numerous sample preparation procedures to extract albumin and to recover alkylated peptides after a digestion step using enzymes have been proposed prior to the analysis of the targeted peptides by liquid chromatography coupled to mass spectrometry method with or without derivatization step. This review describes and compares the numerous procedures including a number of different steps for the extraction and purification of adducted albumin and its digested peptides described in the literature to achieve detection limits for biological samples exposed to sulfur mustard, its analogues, and nitrogen mustards in the ng/mL range.

Identification of potential salivary biomarkers for Sjögren's syndrome with an untargeted metabolomic approach.

INTRODUCTION: Despite the rise of metabolomics over the past years, and particularly salivary metabolomics, little research on Sjögren's syndrome (SS) biomarkers has focused on the salivary metabolome. OBJECTIVES: This study aims to identify metabolites that could be used as biomarkers for SS. METHODS: Using the software called XCMS online, the salivary metabolic profiles obtained with liquid chromatography coupled to high-resolution mass spectrometry for 18 female SS patients were compared to those obtained for 22 age-matched female healthy controls. RESULTS AND CONCLUSION: A total of 91 metabolites showed differential expression in SS patients. A putative identification was proposed with the use of a database for 37 of these metabolites and, of these, 16 identifications were confirmed. Given the identified metabolites, some important metabolic pathways, such as amino acid metabolism, purine metabolism, or even the citric acid cycle seem to be affected. Through the analyses of the ROC (receiver operating characteristic) curves, three metabolites, namely alanine, isovaleric acid, and succinic acid, showed both good sensitivity (respectively 1.000, 1.000, and 0.750) and specificity (respectively 0.692, 0.615, and 0.692) for identifying SS and could then be interesting biomarkers for a potential salivary diagnosis test.

Synthesis and characterization of molecularly imprinted polymers for the selective extraction of oxazepam from complex environmental and biological samples.

Oxazepam, one of the most frequently prescribed anxiolytic drugs, is not completely removed from wastewater with conventional treatment processes. It can thus be found at trace levels in environmental water, with human urine constituting the major source of contamination. This study focused on the development and characterization of molecularly imprinted polymers (MIPs) for the selective solid-phase extraction of oxazepam at trace levels from environmental water and human urine samples. Two MIPs were synthesized, and their selectivity in pure organic and aqueous media were assayed. After optimizing the extraction procedure adapted to a large sample volume to reach a high enrichment factor, the most promising MIP was applied to the selective extraction of oxazepam from environmental water. Extraction recoveries of 83 ± 12, 92 ± 4 and 89 ± 10% were obtained using the MIP for tap, mineral and river water, respectively, while a recovery close to 40% was obtained on the corresponding non-imprinted polymer (NIP). Thanks to the high enrichment factors, a limit of quantification (LOQ) of 4.5 ng L-1 was obtained for river water. A selective extraction procedure was also developed for urine samples and gave rise to extraction recoveries close to 95% for the MIP and only 23% for the NIP. Using the MIP, a LOQ of 357 ng L-1 was obtained for oxazepam in urine. The use of the MIP also helped to limit the matrix effects encountered for the quantification of oxazepam in environmental samples and in human urine samples after extraction on an Oasis HLB sorbent.

Development of analytical methods to study the salivary metabolome: impact of the sampling.

Advances in metabolomics have allowed the identification and characterization of saliva metabolites that can be used as biomarkers. However, discrepancies can be noted with the content of the same biomarker being increased or decreased for a given disease. Differences in the way saliva is collected, stored, and/or treated could cause these discrepancies. Indeed, there is no standardized method for saliva sampling and analysis. In this work, two chromatographic modes were used, i.e., RP-LC and HILIC both coupled to MS used in positive and negative ionization modes. The analytical conditions were optimized with a mixture of 90 compounds naturally present in saliva, representative of the wide range of molecular mass and polarity of salivary metabolites and being described as having a differential expression in various pathologies. These four methods were applied to the analysis of saliva samples collected by spitting, aspiration, or Salivette® with or without prior rinsing of the mouth. Rinsing had an effect on some metabolite concentrations. As it can induce an additional parameter of variability to the sampling, it seems therefore preferable to use methods without rinsing while effects of these parameters on the metabolites are investigated. Saliva obtained by spitting and aspiration gave statistically equivalent results for 84% of the metabolites studied. Conversely, Salivette® gave different results since the majority of the metabolites chosen for the study were not quantified in the samples. The Salivette® does not seem therefore to be a suitable sampling method for an untargeted analysis of the salivary metabolome, unlike aspiration and spitting.

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

Identification and semi-relative quantification of intact glycoforms by nano-LC-(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone.

Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations. Graphical abstract.

Salivary metabolites to detect patients with cancer: a systematic review.

Novel adjunctive screening aids are needed to reduce the morbidity and mortality related to cancer, and every effort should be made for early diagnosis. This systematic review aimed to evaluate salivary metabolites and their diagnostic value in patients with cancer.The systematic review was performed in two phases and included studies that focused on the diagnostic value of salivary metabolites in humans with solid malignant neoplasms. Five electronic databases were searched, and the risk of bias in individual studies was evaluated using the revised Quality Assessment of Diagnostic Accuracy Studies criteria (QUADAS-2). All procedures were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Of the 1151 studies retrieved, 25 were included; 13 studies used targeted and 12 untargeted metabolomics approaches. Most studies included patients with breast and oral cancer. Except for one, all studies had case-control designs, and none fulfilled all quality assessments. Overall, 140 salivary metabolites were described. The most frequently reported metabolites were alanine, valine, and leucine. Among the 11 studies that reported diagnostic test accuracy (DTA) values, proline, threonine, and histidine in combination and monoacylglycerol alone demonstrated the highest DTA for breast cancer. Combined choline, betaine, pipecolinic acid, and L-carnitine showed better discriminatory performance for early oral cancer.This systematic review highlights the current evidence on salivary metabolites that may be used as a future strategy to diagnose cancer. Further studies including larger sample sizes with confirmation of the results by untargeted analysis are warranted.

Development and application of water-compatible molecularly imprinted polymers for the selective extraction of carbamazepine from environmental waters.

A molecularly imprinted polymer (MIP) was designed in order to allow the selective solid-phase extraction of carbamazepine (CBZ), an anticonvulsant and mood-stabilizing drug, at ultra-trace level from aqueous environmental samples. A structural analog of CBZ was selected as a dummy template and different synthesis conditions were screened. The selectivity of the resulting imprinted polymers was evaluated by studying the retention of CBZ in a solvent similar to the one used for the synthesis. The presence of imprinted cavities in the polymers was then demonstrated by comparing the elution profiles (obtained by using MIP and a non-imprinted polymer, NIP, as a control) of the template, of CBZ, and of a structural analog of CBZ. Then, the extraction procedure was further optimized for the treatment of aqueous samples on the two most promising MIPs, with special attention being paid to the volume and composition of the percolation and washing solutions. The best MIP provided a highly selective retention in tap water with 81% extraction recovery for CBZ in the elution fraction of the MIP and only 14% for NIP. The repeatability of the extraction procedure was demonstrated for both tap and river waters (RSD below 4% in river water) for the drugs CBZ, oxcarbamazepine, and one metabolite (carbamazepine 10,11-epoxide). A MIP capacity of 1.15 µmol g-1 was determined. Finally, an analytical procedure involving the MIP was developed allowing the detection of CBZ at a concentration level of only a few nanograms per liter in river water. The selectivity provided by the MIP resulted in a 3000-fold increase of the signal-to-noise ratio in LC/MS analysis as compared to the use of conventional sorbent. Graphical abstract.

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma.

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL-1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

Telomere length, ATM mutation status and cancer risk in Ataxia-Telangiectasia families.

Recent studies have linked constitutive telomere length (TL) to aging-related diseases including cancer at different sites. ATM participates in the signaling of telomere erosion, and inherited mutations in ATM have been associated with increased risk of cancer, particularly breast cancer. The goal of this study was to investigate whether carriage of an ATM mutation and TL interplay to modify cancer risk in ataxia-telangiectasia (A-T) families.The study population consisted of 284 heterozygous ATM mutation carriers (HetAT) and 174 non-carriers (non-HetAT) from 103 A-T families. Forty-eight HetAT and 14 non-HetAT individuals had cancer, among them 25 HetAT and 6 non-HetAT were diagnosed after blood sample collection. We measured mean TL using a quantitative PCR assay and genotyped seven single-nucleotide polymorphisms (SNPs) recurrently associated with TL in large population-based studies.HetAT individuals were at increased risk of cancer (OR = 2.3, 95%CI = 1.2-4.4, P = 0.01), and particularly of breast cancer for women (OR = 2.9, 95%CI = 1.2-7.1, P = 0.02), in comparison to their non-HetAT relatives. HetAT individuals had longer telomeres than non-HetAT individuals (P = 0.0008) but TL was not associated with cancer risk, and no significant interaction was observed between ATM mutation status and TL. Furthermore, rs9257445 (ZNF311) was associated with TL in HetAT subjects and rs6060627 (BCL2L1) modified cancer risk in HetAT and non-HetAT women.Our findings suggest that carriage of an ATM mutation impacts on the age-related TL shortening and that TL per se is not related to cancer risk in ATM carriers. TL measurement alone is not a good marker for predicting cancer risk in A-T families.

Selective tools for the solid-phase extraction of Ochratoxin A from various complex samples: immunosorbents, oligosorbents, and molecularly imprinted polymers.

The evolution of instrumentation in terms of separation and detection has allowed a real improvement of the sensitivity and the analysis time. However, the analysis of ultra-traces of toxins such as ochratoxin A (OTA) from complex samples (foodstuffs, biological fluids…) still requires a step of purification and of preconcentration before chromatographic determination. In this context, extraction sorbents leading to a molecular recognition mechanism appear as powerful tools for the selective extraction of OTA and of its structural analogs in order to obtain more reliable and sensitive quantitative analyses of these compounds in complex media. Indeed, immunosorbents and oligosorbents that are based on the use of immobilized antibodies and of aptamers, respectively, and that are specific to OTA allow its selective clean-up from complex samples with high enrichment factors. Similar molecular recognition mechanisms can also be obtained by developing molecularly imprinted polymers, the synthesis of which leads to the formation of cavities that are specific to OTA, thus mimicking the recognition site of the biomolecules. Therefore, the principle, the advantages, the limits of these different types of extraction tools, and their complementary behaviors will be presented. The introduction of these selective tools in miniaturized devices will also be discussed.

Reproductive Decision-Making in MMR Mutation Carriers After Results Disclosure: Impact of Psychological Status in Childbearing Options.

Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003-2010, n = 100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years [22-59]). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

Exposure to ß-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Aptamer-based-sorbents for sample treatment--a review.

To improve selectivity during sample pretreatment, various selective tools inducing a molecular recognition mechanism during the extraction procedure have been developed, such as sorbents constituted of immobilized antibodies, i.e., immunosorbents, or molecularly imprinted polymers. More recently, as an alternative to both previous approaches, aptamers immobilized onto a solid support, i.e., oligosorbents, were proposed. Thanks to the high affinity and high selectivity of the interaction that some aptamers offer toward some target analytes, they also provide powerful techniques that make selective extraction and the concentration of a target analyte from liquid matrices in one step or sample purification of extracts from solid matrices possible. This review describes the development and the properties of these oligosorbents developed for different types of targets-pharmaceuticals, mycotoxins, proteins, cells, etc. After describing the immobilization procedures, we discuss different parameters characterizing the potential of aptamer-based supports as extraction sorbents. Close relations exist between extraction recoveries and the affinity and amounts of aptamers immobilized on the extraction device. In addition, analyte-aptamer interactions may be affected by matrix components and by additives in the samples. This may also lower extraction recoveries and affect the stability and the possible reusability of the aptamer-based sorbent. All these points are discussed and illustrated. Numerous examples of applications of these sorbents to the treatment of complex samples such as food samples, environmental samples, and biological fluids are also reported. Their association with analytical devices, from conventional to miniaturized analytical systems, is also discussed.

Development of an analytical procedure for quantifying the underivatized neurotoxin ß-N-methylamino-L-alanine in brain tissues.

The cyanotoxin ß-methylamino-L-alanine (BMAA) has received renewed attention as an environmental risk factor for sporadic cases of amyotrophic lateral sclerosis (ALS) (Nunn et al., Brain Res 410:375-379, 1987). The aim of the present study was to develop and to validate an analytical procedure that allows the quantification of native BMAA and of its natural isomer, 2,4 diaminobutyric acid (DAB), in brain tissues. An analytical procedure was previously reported by our group for the determination of underivatized BMAA in environmental samples. It included a step of sample clean-up by solid phase extraction (SPE) with a mixed-mode sorbent and the analyses were performed by LC/MS-MS using hydrophilic interaction chromatography and multiple reactions monitoring scan mode. As brain tissues have a higher lipid content, the crucial step of sample clean-up had been optimized by evaluating the efficiency of the addition of a liquid/liquid extraction step prior to the SPE procedure or alternatively, of washing steps to the SPE extraction procedure. The efficiency was checked by visualizing the complexity of the resulting chromatograms in LC/MS and their performance by using spiked brain samples. The optimized analytical procedure, including a washing step with cyclohexane to the SPE with a recovery yield close to 100%, was validated using the total error approach and allowed the quantification of BMAA in a concentration level ranging from 20 to 1,500 ng/g in brain samples. Finally, the feasibility of implementation of this procedure was verified in human brain samples from two patients who died of ALS.

Characterization of Concanavalin A-based lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry.

Human chorionic gonadotropin (hCG) is constituted of the hCGα and hCGß subunits and is a highly glycosylated protein. Affinity supports based on immobilized Concanavalin A (Con A) lectin were used in solid phase extraction (SPE) to fractionate the hCG glycoforms according to their glycosylation state. For the first time, the lectin SPE fractions were off-line analysed by a nano liquid chromatography - high-resolution mass spectrometry (nanoLC-HRMS) method keeping the glycoforms intact. For this, home-made Con A sorbents were prepared by immobilizing lectin on Sepharose with a mean grafting yield of 98.2% (relative standard deviation (RSD) of 3.5%, n = 15). A capacity of about 100 µg of purified urinary hCG (uhCG) per ml of sorbent, grafted with a density of 10 mg of Con A per ml, was estimated. Average extraction yields of around 60% for both hCGα and hCGß glycoforms were obtained after optimization of the extraction protocol. Intra- and inter-assay evaluation led to average RSD values of around 10%, indicating a repeatable extraction procedure. Similar results were obtained with commercial Con A-based sorbents but only after their 3rd use or after an extensive pre-conditioning step. Finally, the Con A SPE led to the fractionation of some glycoforms of uhCG, allowing the detection of an hCGα glycoform with two tetra-antennary N-glycans that couldn't be detected by direct analysis in nanoLC-HRMS without Con A SPE. Regarding a recombinant hCG, a fractionation was also observed leading to the detection of unretained hCGα glycoforms with tri-antennary N-glycans. Therefore, the combination of lectin SPE with intact protein analysis by nanoLC-HRMS can contribute to a more detailed glycosylation characterization of the hCG protein.

Solid-phase immunoextraction followed by liquid chromatography-tandem mass spectrometry for the selective determination of thyroxine in human serum.

In this work, an analytical method based on solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) has been developed for the selective determination of thyroxine (T4) in human serum. For this purpose, two immunosorbents (ISs) specific to T4 were synthesized by grafting two different T4-specific monoclonal antibodies on a cyanogen bromide (CNBr)-activated-Sepharose® 4B solid support. The grafting yields obtained from the immobilization of each antibody on the CNBr-activated-Sepharose® 4B were over 90%, demonstrating that most of the antibodies were covalently bound to the solid support. The SPE procedure was optimized by studying the retention capability and selectivity of the two ISs in pure media fortified with T4. Under the optimized conditions, high elution efficiencies were achieved in the elution fraction for both specific ISs (i.e., 85%), whereas low ones were obtained in the control ISs (ca. 2%), showing the selectivity of the specific ISs. The ISs were also characterized by studying extraction and synthesis repeatability (RSD <8%), and capacity (104 ng of T4 per 35 mg of ISs, i.e., 3 µg g-1). Finally, the methodology was applied to a pooled human serum sample in order to study its analytical utility and accuracy. Relative recovery (RR) values between 81 and 107% were obtained, showing no matrix effects during the global methodology. Furthermore, the need to perform the immunoextraction was evidenced by comparing the LC-MS scan chromatograms and RR values with and without applying the immunoextraction procedure on a serum sample submitted to protein precipitation. This works exploits, for the first time, the use of an IS on the selective determination of T4 in human serum samples.

Determination of nerve agent biomarkers in human urine by a natural hydrophobic deep eutectic solvent-parallel artificial liquid membrane extraction technique.

Alkyl methyl phosphonic acids (AMPAs) are the major metabolites of organophosphorus nerve agents. A method based on the use of natural hydrophobic deep eutectic solvents as supported liquid membrane in parallel artificial liquid microextraction (PALME) combined with LC-MS/MS analysis was developed and applied to their extraction from urine samples. PALME is a miniaturized liquid-phase extraction method performed in a multiwell plate format where the aqueous sample and the aqueous acceptor phase are separated by a flat membrane impregnated with an organic solvent. In this study, we investigated the possibility of replacing the harmful conventional organic solvent by an emerging green solvent, a coumarin/thymol-based deep eutectic solvent, in ordered to raise the greenness of the sample preparation method. Linear response was obtained in an interval of 0.5, 5 or 10-100 ng/ml depending on the AMPAs with a determination coefficients (R2s) ranging from 0.9751 to 0.9989 for their determination in not treated urine samples. Enrichment factors (EFs) up to 12.65 were obtained, and repeatability was within 8.90-16.28% RSD (n = 12). The limit of quantifications (LOQs: S/N ≥ 10) of the whole analytical procedure were in the range from 0.04 to 5.35 ng/ml. In addition to its good sensitivity, the presented method permitted the treatment of 192 samples in 120 min (equivalent to 37.5 s/sample), which places it as one of the most powerful preparation technique for biomonitoring of civilian or military people exposed to nerve agents in case of public health emergency. Indeed, the developed procedure combined sensitivity, high-throughput, greenness, simplicity and practicality for the determination of five acidic polar AMPAs in urine samples.