Glycine additive enhances sensitivity for N- and O-glycan analysis with hydrophilic interaction chromatography-electrospray ionization-mass spectrometry.

Glycosylation is critical for many biological processes and biotherapeutic development. One of the most powerful approaches for analyzing released glycans is hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry (HILIC-ESI-MS). The high sensitivity of MS is crucial for detecting low-abundance glycans and elucidating their structures. In this study, we presented a simple solution to boost MS response of procainamide (ProcA) labeled glycans for 2- to over 60-fold by including 1 mM glycine in ammonium formate mobile phases for HILIC-ESI-MS. The glycine additive increased charge states, enhanced ion intensities and signal-to-noise ratios, and improved tandem MS spectral quality of various N- and O-glycans without affecting chromatographic performance. Furthermore, more homogeneous ionization among different ProcA labeled glycans was achieved by using the glycine additive, resulting in more comparable quantitative results relative to fluorescence-based quantification. We demonstrated that ammonium formate caused ion suppression to ProcA labeled glycans, which were likely mitigated by glycine with enhanced ESI ionization. Overall, simple addition of glycine to mobile phases during HILIC-ESI-MS analysis significantly improves MS detection sensitivity and will facilitate future profiling and quantitation of glycans released from N- and O-glycoproteins.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

5-Aminosalicylic Acid Azo-Linked to Procainamide Acts as an Anticolitic Mutual Prodrug via Additive Inhibition of Nuclear Factor kappaB.

To improve the anticolitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local anesthetic, via an azo bond to prepare 5-(4-{[2-(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-ASA-azo-PA). 5-ASA-azo-PA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine, and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anticolitic effects occurred at least partly through the cooperative inhibition of NFκB activity.

Assignment of Core versus Antenna Fucosylation Types in Protein N-Glycosylation via Procainamide Labeling and Tandem Mass Spectrometry.

Fucosylation is an important feature of protein N-glycosylation as it has been reported to influence the efficacy of therapeutic proteins and as a potential disease biomarker. A common approach for characterizing protein N-glycans is to analyze the native glycans via tandem mass spectrometry (MS). However, tandem MS analysis of native N-glycans typically results in proton migration, which in turn leads to fucose residue migration from the glycan core to the antenna and vice versa. This phenomenon ultimately leads to ambiguous assignment of N-glycan fucosylation. Although the use of specific fucosidases has been successfully employed for assigning fucosylation, such strategies are often too cumbersome, expensive, and time-consuming for routine N-glycan analysis. As an alternative, we explore the influence of labeling N-glycans with procainamide hydrochloride to inhibit fucose migration during tandem MS analysis. The labeled N-glycan pool was separated and analyzed using ultraperformance liquid chromatography and a hydrophobic interaction liquid chromatography column coupled to a quadrupole time-of-flight mass spectrometer (UPLC-HILIC-QTOF-MS). The observation of the m/z 587.3 core fucose diagnostic peak corresponding to [GlcNAc + Fucose + Procainamide + H](+) in the tandem MS data of fucosylated N-glycans rapidly verifies core fucosylation while its absence signifies antennae fucosylation. This unique approach is here validated with human IgG (for core fucosylation) and human alpha-1-acid-glycoprotein (for antenna fucosylation). We further present a useful application toward the rapid verification of fucosylation types in a therapeutic protein (Rituximab).

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization-mass spectrometry.

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography-(ultra-)high-performance liquid chromatography [HILIC-(U)HPLC]. Here, we compare the data obtained by (U)HPLC-fluorescence (FLR) coupled to electrospray ionization-mass spectrometry (ESI-MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.

Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Peptide-N-glycosidase F or A treatment and procainamide-labeling for identification and quantification of N-glycans in two types of mammalian glycoproteins using UPLC and LC-MS/MS.

BACKGROUND: N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods. OBJECTIVE: To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins. METHODS: N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)]. The structure and quantity of each N-glycan were determined using UPLC and LC-MS/MS. RESULTS: The 21 N-glycans in fetuin and another 21 N-glycans in IgG by either PF-ProA or PA-ProA were identified using LC-MS/MS. The N-glycans in fetuin (8-13 N-glycans were previously reported) and in IgG (19 N-glycans were previously reported), which could not be identified by using the widely used PF-AB, were all identified by using PF-ProA or PA-ProA. The quantities (%) of the N-glycans (>0.1 %) relative to the total amount of N-glycans (100 %) obtained by AB- and ProA-labeling using LC-MS/MS had a similar tendency. However, the absolute quantities (pmol) of the N-glycans estimated using UPLC and LC-MS/MS were more efficiently determined with ProA-labeling than with AB-labeling. Thus, PF-ProA or PA-ProA allows for more effective identification and quantification of N-glycans than PF-AB in glycoprotein, particularly bovine fetuin. This study is the first comparative analysis for the identification and relative and absolute quantification of N-glycans in glycoproteins with PF-ProA and PA-ProA using UPLC and LC-MS/MS.

Rapid synthesis of new DNMT inhibitors derivatives of procainamide.

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.

Use of procaine and procainamide as derivatizing co-matrices for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Analysis of oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry often yields only alkali metal cation adducts, which results in lower fragmentation yields and difficulty to retrieve sequence information. Derivatization by reductive amination may be used to promote Y-type glycosidic cleavages. However, this involves time-consuming preparations and purifications with sample loss. Here, procaine and procainamide were used directly as co-matrices with 2,5-dihydroxybenzoic acid (DHB). METHODS: Acidified 10 g/L procaine hydrochloride or procainamide hydrochloride solutions in water/acetonitrile were added to the oligosaccharide solution one minute before preparing our MALDI targets using DHB with the dried-droplet method. This simple protocol resulted in deposits of very fine homogeneous crystals. RESULTS: Positive ion mass spectra, easily acquired in an automated mode, presented a high percentage of oligosaccharides derivatized as Schiff base or glycosylamine notably detected as protonated molecules [M + H](+). The high abundance of procaine or procainamide on the target did not impede the ionization process, improved the signal-to-noise ratio and eliminated the need to search for 'sweet spots'. Fragmentation of the protonated precursor ions of the derivatives largely favored Y-type glycosidic cleavages. CONCLUSIONS: This easy and fast sample preparation, involving low toxicity and easily accessible chemicals, allowed the selection of protonated molecules as precursor ions for post-source decay analyses. This opened the possibility of simplifying sequence retrieval in routine oligosaccharide analyses.

Synthesis and evaluation of a novel 68Ga-labeled DOTA-benzamide derivative for malignant melanoma imaging.

Malignant melanoma displays a highly aggressive metastasis. Thus, early diagnosis of malignant melanoma is important for patient survival. We designed and synthesized a novel (68)Ga-labeled benzamide derivative that specifically binds to melanoma as demonstrated by its ability to bind to melanin. (68)Ga-SCN-DOTA-PCA was synthesized with a radiochemical yield of ~80% and a radiochemical purity of >97% by analytical HPLC. The in vitro binding of (68)Ga-SCN-DOTA-PCA to melanin and its cellular uptake demonstrated the selective uptake in melanin. In addition, the biodistribution and micro-PET imaging of (68)Ga-SCN-DOTA-PCA in B16F10 tumor models showed the specific accumulation in melanoma. These results suggest that (68)Ga-SCN-DOTA-PCA would be a promising agent for melanoma diagnosis.

Fundamentals of ionic conductivity relaxation gained from study of procaine hydrochloride and procainamide hydrochloride at ambient and elevated pressure.

The pharmaceuticals, procaine hydrochloride and procainamide hydrochloride, are glass-forming as well as ionically conducting materials. We have made dielectric measurements at ambient and elevated pressures to characterize the dynamics of the ion conductivity relaxation in these pharmaceuticals, and calorimetric measurements for the structural relaxation. Perhaps due to their special chemical and physical structures, novel features are found in the ionic conductivity relaxation of these pharmaceuticals. Data of conductivity relaxation in most ionic conductors when represented by the electric loss modulus usually show a single resolved peak in the electric modulus loss M(")(f) spectra. However, in procaine hydrochloride and procainamide hydrochloride we find in addition another resolved loss peak at higher frequencies over a temperature range spanning across T(g). The situation is analogous to many non-ionic glass-formers showing the presence of the structural α-relaxation together with the Johari-Goldstein (JG) ß-relaxation. Naturally the analogy leads us to name the slower and faster processes resolved in procaine hydrochloride and procainamide hydrochloride as the primary α-conductivity relaxation and the secondary ß-conductivity relaxation, respectively. The analogy of the ß-conductivity relaxation in procaine HCl and procainamide HCl with JG ß-relaxation in non-ionic glass-formers goes further by the finding that the ß-conductivity is strongly related to the α-conductivity relaxation at temperatures above and below T(g). At elevated pressure but compensated by raising temperature to maintain α-conductivity relaxation time constant, the data show invariance of the ratio between the ß- and the α-conductivity relaxation times to changes of thermodynamic condition. This property indicates that the ß-conductivity relaxation has fundamental importance and is indispensable as the precursor of the α-conductivity relaxation, analogous to the relation found between the Johari-Goldstein ß-relaxation and the structural α-relaxation in non-ionic glass-forming systems. The novel features of the ionic conductivity relaxation are brought out by presenting the measurements in terms of the electric modulus or permittivity. If presented in terms of conductivity, the novel features are lost. This warns against insisting that a log-log plot of conductivity vs. frequency is optimal to reveal and interpret the dynamics of ionic conductors.

Drug-induced protein free radical formation is attenuated by unsaturated fatty acids by scavenging drug-derived phenyl radical metabolites.

Aromatic amine drugs like aminoglutethimide (AG) and related congeners have been shown to produce phenyl radicals through metabolism by myeloperoxidase (MPO)/H(2)O(2), which has been proposed to play a role in drug-induced agranulocytosis. AG has also been shown to induce MPO protein radical formation, but the ultimate fate of these metabolically generated phenyl radicals is still unknown. We tested the reactivity of linoleic acid (LA) and GSH with aniline-based compounds in the presence of horseradish peroxidase (HRP)/H(2)O(2) by measuring oxygen consumption. We found a qualitative correlation between drugs or xenobiotics that formed phenyl radical metabolites with the cooxidation of LA. Most compounds that reacted with LA did not react with GSH. Furthermore, an AG-derived phenyl radical was detected by EPR spin-trapping with MNP (2-methyl-2-nitrosopropane), in a reaction containing AG and HRP/H(2)O(2); these spectra were attenuated in the presence of LA and docosahexaenoic acid (DHA) indicating that phenyl radical scavenging occurred. Since it has been proposed that the phenyl radical metabolite leads to protein radical formation on MPO, we investigated the effect of LA and DHA in immuno-spin trapping experiments with MPO-containing HL-60 cell lysate. Using anti-DMPO, a protein radical was detected on a putative MPO fragment from the reaction of DMPO, AG, and glucose/glucose oxidase. When LA or DHA was included in this reaction, protein radical formation was significantly inhibited. Our results show that certain polyunsaturated fatty acids (PUFAs) act as scavengers of aromatic amine drug-derived phenyl radicals which in turn prevent protein radical formation. However, the interaction of phenyl radical drug metabolites with PUFAs will be dictated by their relative concentrations compared to those of other targets. Most importantly, it is possible to differentiate peroxidase substrates that generate phenyl radical metabolites from N-centered radicals on the basis of their reactivity toward GSH vs PUFAs, and PUFAs are targets for metabolically generated phenyl radicals.

Use of Exoglycosidases for the Structural Characterization of Glycans.

The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly specific exoglycosidases cleave monosaccharides from the nonreducing end of an oligosaccharide and yield information about the linkage, stereochemistry and configuration of the anomeric carbon. Here we use combinations of exoglycosidases to precisely characterize glycans on the Fc domain of therapeutic antibodies and dimeric fusion proteins. The workflow described includes glycan release with Rapid™ PNGase F (NEB #P0710), direct labeling of released glycans with procainamide (PCA) or 2-aminobenzamide (2AB), cleanup of labeled glycans and a 3 h enzymatic digestion with exoglycosidases. This protocol is designed for completion within an 8 h time frame to allow for subsequent LC-FLD, LC-MS, or CE analysis overnight.

Procainamide-SAHA Fused Inhibitors of hHDAC6 Tackle Multidrug-Resistant Malaria Parasites.

Epigenetic post-translational modifications are essential for human malaria parasite survival and progression through its life cycle. Here, we present new functionalized suberoylanilide hydroxamic acid (SAHA) derivatives that chemically combine the pan-histone deacetylase inhibitor SAHA with the DNA methyltransferase inhibitor procainamide. A three- or four-step chemical synthesis was designed starting from cheap raw materials. Compared to the single drugs, the combined molecules showed a superior activity in Plasmodium and a potent inhibition against human HDAC6, exerting no cytotoxicity in human cell lines. These new compounds are fully active in multidrug-resistant Plasmodium falciparum Cambodian isolates. They target transmission of the parasite by inducing irreversible morphological changes in gametocytes and inhibiting exflagellation. The compounds are slow-acting and have an additive antimalarial effect in combination with fast-acting epidrugs and dihydroartemisinin. The lead compound decreases parasitemia in mice in a severe malaria model. Taken together, this novel fused molecule offers an affordable alternative to current failing antimalarial therapy.

Cation-triggered drug release from a porous zinc-adeninate metal-organic framework.

A porous anionic metal-organic framework, bio-MOF-1, constructed using adenine as a biomolecular building block is described. The porosity of this material is evaluated, its stability in biological buffers is studied, and its potential as a material for controlled drug release is investigated. Specifically, procainamide HCl is loaded into the pores of bio-MOF-1 using a simple cation exchange process. Exogenous cations from biological buffers are shown to affect the release of the adsorbed drug molecules.

Procainamide, but not N-acetylprocainamide, induces protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.

Procainamide (PA) is a drug that is used to treat tachycardia in postoperative patients or for long-term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here, we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with PA/H 2O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis.

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase.

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30 min to purify BChE from 1 ml plasma. The method uses a microfuge spin column to build a 0.2 ml procainamide affinity column. The eluted BChE contains 3-4 microg of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.

Variation of Human Salivary O-Glycome.

The study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularly abundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins.

Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS.

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.