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.

Analysis of long-lived sulfur mustard-human hemoglobin adducts in blood samples by red blood cells lysis and on-line coupling of digestion on an immobilized-trypsin reactor with liquid chromatography-tandem mass spectrometry.

As a highly alkylating chemical warfare agent, sulfur mustard reacts with blood proteins such as hemoglobin to form long-lived hydroxyethylthioethyl adducts that can be used as biomarkers of exposure. An optimized method was developed for the extraction of hemoglobin from blood samples. This procedure, involving the hemolysis of the red blood cells by freezing at -80 °C in two cycles of 1 h, followed by the purification of the lysate by ultrafiltration on 100 and 50 kDa cutoff centrifugal devices, was then applied to the extraction of hemoglobin from blood samples spiked with sulfur mustard at different concentrations (ranging from 0.014 to 28 µg mL-1). More than 75% of the protein was extracted from the blood samples and the method demonstrated a satisfying repeatability, with a RSD of 12.6%. The extracted hemoglobin was then digested on-line on a laboratory-made trypsin IMER coupled with the analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) of the resulting alkylated peptides. A linear response was observed for the 13 alkylated peptides targeted for the sulfur mustard concentration range studied, with RSD down to 0.1% for the digestion repeatabilty. The limit of quantification of the method was estimated to be 0.4 ng mL-1 as concentration of exposure to sulfur mustard in whole blood. Finally, a variation of the alkylation rates of hemoglobin was observed between the biological matrix and pure sample, since the preferential adduction sites in blood were the residues ß-His97 and ß-Val98, both located on the alkylated peptide ß-T11, while for purified hemoglobin in water, the residue ß-His77 was the main adduction site. Thus, even though blood samples require an additional sample treatment step compared to pure standards, carrying out the study with whole blood allowed to collect information that are more representative of the phenomena occurring in the organism upon exposure to sulfur mustard.

Development of an immobilized-trypsin reactor coupled to liquid chromatography and tandem mass spectrometry for the analysis of human hemoglobin adducts with sulfur mustard.

Sulfur mustard reacts with blood proteins, such as hemoglobin, to form stable adducts that can be used as long-lived biomarkers of exposure. These adducts can be analyzed by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) after an enzymatic digestion step. The objective of this study was to develop trypsin-based immobilized enzyme reactors (IMERs) in order to obtain a faster digestion of hemoglobin than the conventional in-solution digestion. Trypsin IMERs were synthetized by grafting the enzyme on a CNBr-Sepharose gel and the influence of several parameters on the digestion yields, such as the transfer volume between the injection loop and the IMER, the temperature and the digestion time was studied. The repeatability of the digestion on three laboratory-made IMERs was demonstrated for pure hemoglobin and hemoglobin previously exposed to different concentrations of sulfur mustard (RSD inferior to 13% and 21% respectively) and was better than that obtained for in-solution digestions (RSD inferior to 28% and up to 53% respectively). A preferential adduction of sulfur mustard on the histidine residues of hemoglobin was confirmed, for both in-solution and IMER digestion results. On a quantitative point of view, the performances of in-solution and IMER digestions were similar, with the theoretical possibility to detect peptides resulting from the in vitro incubation of hemoglobin in pure water with sulfur mustard at 7.5 ng⋅mL-1. However, digestion on IMER proved to be more repeatable and 32 times faster than in-solution digestion, and a given IMER could be reused at least 60 times.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard.

Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL-1) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL-1). Five new alkylation sites were accurately identified on the protein (α-His72, α-His87, α-His89, ß-His2 and ß-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide α-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 µg.mL-1. The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 µg.mL-1). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin ß. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL-1. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

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.

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography-tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma.

Human butyrylcholinesterase (HuBuChE) has been widely used as a biomarker of exposure to organophosphorus (OPs) warfare agents. Indeed, intoxication by OPs can be proven by LC-MS/MS analysis of a specific HuBuChE nonapeptide on which OPs covalently bind. Therefore, we developed a fast, selective and sensitive on-line set-up for the analysis of HuBuChE from plasma that combines immunoextraction by anti-HuBuChE antibodies, pepsin digestion on Immobilized Enzyme Reactors (IMER) and microLC-MS/MS analysis of the target nonapeptide, FGESAGAAS. Two pepsin-based IMERs were prepared and characterized in terms of grafting and digestion yields and were coupled on-line to microLC-MS/MS analysis. In addition, immunosorbents were prepared by covalent grafting of three anti-HuBuChE antibodies on CNBr-sepharose and epoxy-polymethacrylate supports and packed in precolumns. The best antibody grafting yields were obtained with sepharose-based supports, with grafting yields up to 98%. B2 18-5 monoclonal antibody grafted on sepharose led to the best immunosorbent, with HuBuChE recovery close to 100%. The immunosorbent was introduced upstream of the on-line digestion set-up and immunoextraction of HuBuChE was achieved in 14min while digestion was performed in 20min, allowing detection of the target nonapeptide in less than 1h. The global recovery of the nonapeptide was higher than 42% using the best immunosorbent with a RSD value lower than 7% (n=3). Finally, the limit of quantification evaluated in plasma sample was 2fmol of nonapeptide. This value, corresponding to 0.5fmol of HuBuChE tetramer, is well below the average amount of HuBuChE tetramer in 50µL of plasma (590fmol).

Development of immobilized-pepsin microreactors coupled to nano liquid chromatography and tandem mass spectrometry for the quantitative analysis of human butyrylcholinesterase.

Human butyrylcholinesterase is a serine hydrolase that reacts with organophosphorus compounds (OP) to form stable adducts. These adducts are valuable biomarkers for OP exposure and can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after a preliminary digestion step in solution. However, this digestion step is time-consuming and cannot be directly coupled with LC-MS set ups. Therefore, the aim of this work was to develop pepsin-based immobilized enzyme microreactors (IMERs) for the rapid digestion of human butyrylcholinesterase (HuBuChE). Various IMERs were synthesized by grafting different amounts of pepsin on a CNBr-sepharose gel and the grafting yield was measured by a bicinchoninic acid assay (BCA). A sensitive nanoLC-MS/MS method was developed to evaluate the digestion yields of HuBuChE on IMERs which was made possible by a synthetic peptide which was used as a calibrant. The digestion was optimized by studying the impact of different parameters such as the digestion time, the temperature and the amount of pepsin grafted on IMER. This optimization allowed HuBuChE to be digested with-in 20min without pretreatment and with digestion yields up to 20%. The repeatability of the IMER synthesis and HuBuChE digestion was highlighted with the characterization of 3 similar IMERs. Finally, the digestion yields of HuBuChE were higher with IMERs when compared to a typical in solution digestion.

Three-phase hollow fiber liquid-phase microextraction of organophosphorous nerve agent degradation products from complex samples.

Degradation products of chemical warfare agents are considered as important environmental and biological markers of chemical attacks. Alkyl methylphosphonic acids (AMPAs), resulting from the fast hydrolysis of nerve agents, such as sarin and soman, and the methylphosphonic acid (MPA), final degradation product of AMPAs, were determined from complex matrices by using an emergent and miniaturized extraction technique, the hollow fiber liquid-phase microextraction (HF-LPME), before their analysis by liquid chromatography coupled to mass spectrometry (LC-MS). After studying different conditions of separation in the reversed phase LC-MS analysis, the sample treatment method was set up. The three-phase HF-LPME was carried out by using a porous polypropylene (PP) hollow fiber impregnated with 1-octanol that separates the donor and acceptor aqueous media. Various extraction parameters were evaluated such as the volume of the sample, the effect of the pH and the salt addition to the sample, the pH of the acceptor phase, the extraction temperature, the stirring speed of the sample, the immersion time in the organic solvent and the time of extraction. The optimum conditions were applied to the determination of MPA and five AMPAs in real samples, such as surface waters and urine. Compounds were extracted from a 3 mL acidified sample into only 6 µL of alkaline water without any other pretreatment of the complex matrices. Enrichment factors (EFs) higher than 170 were obtained for three less polar AMPAs. Limits of quantification (LOQs) in the 0.013-5.3 ng mL(-1) range were obtained after microextraction of AMPAs from river water and in the range of 0.056-4.8 ng mL(-1) from urine samples with RSD values between 1 and 9%.