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.

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.

Development of ion-imprinted polymers for the selective extraction of Cu(II) ions in environmental waters.

Several ion-imprinted polymers (IIPs) were synthesized via bulk polymerization with Cu(II) as template ion, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent, and azobisisobutyronitrile as initiator in acetonitrile or methanol as porogen solvent. Non-imprinted polymers (NIPs) were similarly synthesized but without Cu(II). After grounding and sieving, the template ions were removed from IIPs particles through several cycles of elimination in 3 M HCl. All NIPs were equally subjected to this acid treatment with the exception of one NIP, called unwashed NIP. The resulting IIP/NIP particles were packed in solid phase extraction (SPE) cartridges for characterization. The SPE protocol was designed by optimizing a washing step following the sample percolation to eliminate potential interfering ions prior to the elution of Cu(II), all fractions analyzed by inductively coupled plasma mass spectrometry. The best IIP showed a high specificity (recovery of Cu(II) vs. interfering ions) and a good selectivity (retention on IIP vs. NIP). Its adsorption capacity was determined to be 63 µg g-1. Then, a volume of 50 mL was percolated with 30 mg of IIP, thus giving rise to an enrichment factor of 24. Finally, applications to real samples (mineral and sea waters) were successfully performed. In addition, Brunauer-Emmett-Teller analyses showed that the surface area of the washed NIP was almost double that of the unwashed one (140.70 vs. 74.49 m2 g-1), demonstrating for the first time that the post-treatment of a NIP after its synthesis may have a significant impact on its porous structure, and thus need to be more precisely detailed by authors in the future papers.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry.

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGß, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGß glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGß glycoforms, which allowed for the first time the detection of 33 hCGß glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Using an Untargeted Metabolomics Approach to Identify Salivary Metabolites in Women with Breast Cancer.

Metabolic alterations are a hallmark of the malignant transformation in cancer cells, which is characterized by multiple changes in metabolic pathways that are linked to macromolecule synthesis. This study aimed to explore whether salivary metabolites could help discriminate between breast cancer patients and healthy controls. Saliva samples from 23 breast cancer patients and 35 healthy controls were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry and a bioinformatics tool (XCMS Online), which revealed 534 compounds, characterized by their retention time in reverse-phase liquid chromatography and by the m/z ratio detected, that were shared by the two groups. Using the METLIN database, 31 compounds that were upregulated in the breast cancer group (p < 0.05) were identified, including seven oligopeptides and six glycerophospholipids (PG14:2, PA32:1, PS28:0, PS40:6, PI31:1, and PI38:7). In addition, pre-treatment and post-treatment saliva samples were analyzed for 10 patients who experienced at least a partial response to their treatment. In these patients, three peptides and PG14:2 were upregulated before but not after treatment. The area under the curve, sensitivity, and specificity for PG14:2 was 0.7329, 65.22%, and 77.14%, respectively. These results provide new information regarding the salivary metabolite profiles of breast cancer patients, which may be useful biomarkers.

Selective solid-phase extraction of organophosphorus pesticides and their oxon-derivatives from water samples using molecularly imprinted polymer followed by high-performance liquid chromatography with UV detection.

A molecularly imprinted polymer was synthesized and characterized to be used as solid-phase extraction sorbent for simultaneous chlorpyrifos and diazinon and their oxon derivatives. Several imprinted polymers were prepared and evaluated in a retention study of these analytes compared with a non-printed polymer. Several parameters affecting the extraction of imprinted polymer such as washing solvent, composition and volume of the eluting solvent and sample volume, were also investigated. Under the optimum conditions, the developed method provided satisfactory limits of detection ranging between 0.07 µg L-1 to 0.12 µg L-1 and the material showed an excellent reusability (> 50 reuses). The method was applied to the extraction and preconcentration of these analytes in water samples. The average recoveries ranged from 79 ± 6 to 104 ± 3 %.

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.

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.

Synthesis and application of molecularly imprinted silica for the selective extraction of some polar organophosphorus pesticides from almond oil.

The aim of this work was to prepare and evaluate molecularly imprinted polymers obtained by a sol-gel approach for the selective solid-phase extraction (SPE) of organophosphorus pesticides (OPs) from almond oil. The performances of molecularly imprinted silicas (MISs), prepared using different conditions of synthesis, were studied by applying different extraction procedures in order to determine the ability of the MISs to selectively extract ten target OPs. For this, the retention of OPs on MISs in pure media was compared with the retention on a non-imprinted silicas (NISs), used as control sorbent, to prove the presence of specific cavities. The most promising MIS allowed the selective extraction of the 3 most polar OPs among the 10 studied. The capacity was studied and the repeatability of the extraction recovery yield was demonstrated both in pure and real media. This MIS was able to selectively extract fenthion sulfoxide and dimethoate contained in almond oil extract after applying the optimized extraction procedure with recovery yields between 100 and 114%. The estimated limit of quantification (LOQ, S/N = 10), thanks to LC/MS analysis in MRM mode, between 1.2 and 4.6 µg/kg for those OPs in the almond fruits, was more than 10 times lower than the Maximum Residue Levels (MRLs) established by the European Commission. This MIS therefore shows a high potential for the analysis of those two polar OPs at trace levels from almond oils.

Synthesis and application of molecularly imprinted polymers for the selective extraction of organophosphorus pesticides from vegetable oils.

The increasing use of pesticides in agriculture causes environmental issues and possible serious health risks to humans and animals. Their determination at trace concentrations in vegetable oils constitutes a significant analytical challenge. Therefore, their analysis often requires both an extraction and a purification step prior to separation with liquid chromatography (LC) and mass spectrometry (MS) detection. This work aimed at developing sorbents that are able to selectively extract from vegetable oil samples several organophosphorus (OPs) pesticides presenting a wide range of physico-chemical properties. Therefore, different conditions were screened to prepare molecularly imprinted polymers (MIPs) by a non-covalent approach. The selectivity of the resulting polymers was evaluated by studying the OPs retention in pure media on both MIPs and non-imprinted polymers (NIP) used as control. The most promising MIP sorbent was obtained using monocrotophos (MCP) as the template, methacrylic acid (MAA) as the monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker with a molar ratio of 1/4/20 respectively. The repeatability of the extraction procedure and of the synthesis procedure was demonstrated in pure media. The capacity of this MIP was 1mg/g for malathion. This MIP was also able to selectively extract three OPs from almond oil by applying the optimized SPE procedure. Recoveries were between 73 and 99% with SD values between 4 and 6% in this oil sample. The calculated LOQs (between 0.3 and 2µg/kg) in almond seeds with a SD between 0.1 and 0.4µg/kg were lower than the Maximum Residue Levels (MRLs) established for the corresponding compounds in almond seed.

Selective solid phase extraction of lanthanides from tap and river waters with ion imprinted polymers.

For the first time, an ion imprinted polymer (IIP) able to selectively extract simultaneously all the lanthanide ions was successfully synthesized in acetonitrile using Nd3+ as a template ion, methacrylic acid as a complexing monomer, and ethylene glycol dimethacrylate as a cross-linker. A non-imprinted polymer (NIP) was synthesized under the same conditions as those of the IIP, but in the absence of the template ion. After the removal of the template ions, grounding and sieving, the IIP particles were packed in solid phase extraction (SPE) cartridges. The selectivity of the IIP was evaluated by comparing its behavior with the one of the NIP. Each SPE step (percolation, washing, and elution) was optimized in order to find the best compromise between the selectivity and the extraction recoveries. Using the optimized SPE conditions, the extraction recoveries of eight lanthanide ions representative of the lanthanide family were higher than 77% with an average value of 83% with the IIP, whereas, in the case of the NIP, they ranged between 14 and 36% and they were below 3% for the interfering ions from alkali, transition, and post-transition metal families with the IIP. A first evaluation of the reproducibility of the SPE profiles was carried out by performing statistical tests on the data obtained with several cartridges filled with particles obtained from two different IIP and NIP syntheses. Promising results were obtained. The specific capacity, i. e. the adsorption capacity of Nd3+ ions by the specific cavities of the imprinted polymer, was about 9 mg of Nd3+ per gram of IIP (60 µmol g-1), which is more than enough for the extraction of the lanthanide ions at trace levels. The breakthrough volume was about 1 mL per mg of IIP, leading to an enrichment factor of 15, which allows not only to selectively extract the lanthanides but also to concentrate them. Finally, the imprinted polymer was successfully used to selectively extract lanthanides from tap and river waters spiked at 1 µg L-1.

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.

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%.

Selective immunoclean-up followed by liquid chromatography for the monitoring of a biomarker of exposure to polycyclic aromatic hydrocarbons in urine at the ng l-1 level.

A selective clean-up procedure using an immunosorbent (IS) was developed for the trace-level determination, in water and urine samples, of 3-benzo(a)pyrene-glucuronide (3-BP-G), a biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). First, three sorbents used for the immobilization of antibodies were evaluated for their ability to limit the risk of non-specific interactions and to provide a high bonding density. The best sorbent, i.e. sepharose, was used for the immobilization of two different monoclonal antibodies. The most specific antibody for 3-BP-G was applied to the selective extraction from urine providing a clean extract, an easy and reliable quantification by comparison with a classical SPE process. The sensitivity of the fluorescence associated with the selectivity of the IS provides a limit of detection up to 1.2 ng l(-1) in urine for 3-BP-G.