Use of the dummy approach for the synthesis of ion imprinted polymers with Ni(II) or Zn(II) as template ion for the solid-phase extraction of Cu(II).

There is a strong interest in monitoring copper in environmental waters, but its direct analysis suffers from strong matrix interferences. This is why, a sample pretreatment based on solid-phase extraction (SPE) is often used but conventional sorbents usually lack specificity. It is overcome with ion-imprinted polymers (IIPs). This work evaluates for the first time the use of the dummy approach for the synthesis of Cu(II)-targeting IIPs. Two analog ions Ni(II) and Zn(II) were tested as templates and the resulting IIPs were packed in SPE cartridges. The SPE procedure was designed by optimizing a washing step following the sample percolation, to eliminate the interfering ions retained on the IIP by non-specific interactions. To optimize the washing step, solutions at different pH or containing tris(hydroxymethyl)aminomethane as a complexing agent at different concentrations were tested and combined. Zn-IIP appeared more promising than Ni-IIP, showing excellent specificity and a high selectivity. Its retention capacity was determined to be 100 µg/g, and different isotherm models were evaluated to fit with the adsorption data. Finally, applications to mineral and sea waters were successfully completed and led to high and repeatable extraction recoveries for Cu(II) (88 ± 1% and 83 ± 3%, respectively).

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 setups for real-time analysis of the effluent of a microreactor by mass spectrometry.

Monitoring a synthesis reaction in real time could allow not only the detection of the intermediates involved in the synthesis, to better understand its mechanisms, but also the impurities. Spectroscopic methods could be performed but are not so performant when analyzing complex mixtures and could require specific properties for the detection of the molecules of interest, the presence of a chromophore moiety for example. Mass spectrometry (MS) may overcome these limitations and is able to reach the accuracy and sensitivity required to efficiently detect, quantify, identify, and characterize the reagents and species produced during the synthesis. This is why the hyphenation of a microreactor with MS has already allowed synthesis processes to be monitored, but most of the time it targets a specific reaction or compounds and involves solvents compatible with MS. In this study, a universal setup for the hyphenation of a microreactor with MS and based on two valves has been developed. This two-valve setup has proven itself for the analysis of molecules of different nature and hydrophilicity, soluble in a large number of solvents even in non-MS-compatible ones. The developed setup evidenced a good repeatability and a linear response for the detection of the studied compounds. In addition, the dilution step included in the two-valve setup allows the MS monitoring of compounds initially synthesized at different concentrations. Finally, it was successfully used to study an amination reaction allowing the detection of the reaction products in 4 min with good repeatability as RSD values of MS signals were lower than 17%.

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.

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.

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.

Untargeted Metabolomic Approach to Study the Impact of Aging on Salivary Metabolome in Women.

Despite the growing interest in salivary metabolomics, few studies have investigated the impact of aging on the salivary metabolome. The alterations in metabolic pathways that occur with aging are likely to be observed in pathologies affecting older people and may interfere with the search for salivary biomarkers. It is therefore important to investigate the age-related changes occurring in the salivary metabolome. Using reversed phase liquid chromatography and hydrophilic interaction chromatography coupled to mass spectrometry used in positive and negative ionization modes, the salivary metabolic profiles of young (22 to 45 years old) and older people (55 to 92 years old) were obtained. Those profiles were compared with the use of XCMS online to highlight the under or overexpression of some metabolites with aging. A total of 60 metabolites showed differential expression with age. The identification of 26 of them was proposed by the METLIN database and, among them, 17 were validated by standard injections. Aging seemed to affect most of the main metabolic pathways (amino acid metabolism, Krebs cycle, fatty acid synthesis, and nucleic acid synthesis). Moreover, most of the metabolites that were over- or under-expressed with age in this study have already been identified as being potential biomarkers of diseases affecting older people, such as in Alzheimer's disease. Special attention should be paid in the search for biomarkers of pathologies affecting the elderly to differentiate age-related changes from disease-related changes.

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.

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.

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.

226Ra and 137Cs determination by inductively coupled plasma mass spectrometry: state of the art and perspectives including sample pretreatment and separation steps.

Achieving precise and accurate quantification of radium (226Ra) and cesium (137Cs) by inductively coupled plasma mass spectrometry (ICP-MS) is of particular interest in the field of radiological monitoring and more widely in environmental and biological sciences. However, the accuracy and sensitivity of the quantification depend on the analytical strategy implemented. Eliminating interferences during the sample handling step and/or during the analysis step is critical since presence of matrix elements can lead to spectral and non-spectral interferences in ICP-MS. Consequently, before the ICP-MS analysis, multiple sample preparation approaches have been applied to purify and/or pre-concentrate environmental and biological samples containing radium and cesium through years, such as (co)-precipitation, solid phase extraction (SPE) or dispersive SPE (dSPE). Separation steps using liquid chromatography and capillary electrophoresis can also be useful in complement with the abovementioned sample preparation techniques. The most attractive sample handling technique remains SPE but efficiency of the extraction procedures is currently limited by sorbent specificity. Indeed, with the recent advances in ICP-MS instrumentation, it becomes indispensable to eliminate residual interferences and improve sensitivity. It is in this direction that it will be possible to meet analytical challenges, e.g. analyzing radium and cesium at concentrations below the pg L-1 range in complex matrices of small volumes, as they are found for instance in pore waters or in biological samples. Development of new innovative sorbents based for example on hybrid and nanostructured materials has been reported with the aim of enhancing sorbent specificity and/or capacity. In the present review, the performances of the different analytical approaches are discussed, followed by an overview of applications.

Parallel artificial liquid membrane extraction of organophosphorus nerve agent degradation products from environmental samples.

An emerging miniaturized high-throughput microextraction technique named Parallel artificial liquid membrane extraction (PALME) was, for the first time, investigated for the extraction of polar alkyl methylphosphonic acids (AMPAs) that are the degradation products of organophosphorus nerve agents. The effect of the key-parameters of the extraction method (nature of the membrane, of the extraction solvent, of the pH values of both donor and acceptor phases, agitation speed, extraction time, temperature and ionic strength) on the extraction recoveries was studied in spiked pure water samples. This led to extraction recoveries in the range of 25-102% for the 5 targeted analytes from water with enrichment factors in the range of 4.50-42.75. The developed PALME-LC-MS/MS method was first evaluated with spiked pure water. LOQs (S/N ≥ 10) were in the range of 0.009-1.141 ng mL-1, linearity above 0.9973 for all the AMPAs and with RSD values below 11%. This method was then applied on simulated waste water, river water and aqueous soil extracts. The achieved LOQs were in the range of 0.011-1.210, 0.013-1.196 and 0.016-6.810 ng mL-1, respectively. A detailed comparison of the performances of this PALME method with those of a previously developed hollow fiber liquid-phase microextraction methods already applied to AMPAs was done thus allowing to demonstrate the easy transfer of methods from HF-LPME to PALME. Moreover, the high-throughput potential of PALME was revealed since 192 samples were processed in parallel during 120 min (37.5 s/sample).

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.

Recent Advances in Lectin-Based Affinity Sorbents for Protein Glycosylation Studies.

Glycosylation is one of the most significant post-translational modifications occurring to proteins, since it affects some of their basic properties, such as their half-life or biological activity. The developments in analytical methodologies has greatly contributed to a more comprehensive understanding of the quantitative and qualitative characteristics of the glycosylation state of proteins. Despite those advances, the difficulty of a full characterization of glycosylation still remains, mainly due to the complexity of the glycoprotein and/or glycopeptide mixture especially when they are present in complex biological samples. For this reason, various techniques that allow a prior selective enrichment of exclusively glycosylated proteins or glycopeptides have been developed in the past and are coupled either on- or off- line with separation and detection methods. One of the most commonly implemented enrichment methods includes the use of lectin proteins immobilized on various solid supports. Lectins are a group of different, naturally occurring proteins that share a common characteristic, which concerns their affinity for specific sugar moieties of glycoproteins. This review presents the different formats and conditions for the use of lectins in affinity chromatography and in solid phase extraction, including their use in dispersive mode, along with the recent progress made on either commercial or home-made lectin-based affinity sorbents, which can lead to a fast and automated glycosylation analysis.

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.

Simplified miniaturized analytical set-up based on molecularly imprinted polymer directly coupled to UV detection for the determination of benzoylecgonine in urine.

A simple, selective, and sensitive method involving a miniaturized solid phase extraction step based on a monolithic molecularly imprinted polymer (MIP) directly coupled on-line to UV detection was developed for the determination of benzoylecgonine (BZE) in complex biological samples. Monolithic MIPs were prepared into 100 µm internal diameter fused-silica capillaries either by thermal or photopolymerization. While leading to similar selectivities with respect to BZE, photopolymerization has made it possible to produce monoliths of different lengths that can be adapted to the targeted miniaturized application. The homogeneous morphology of these monolithic MIPs was evaluated by scanning electron microscopy prior to measuring their permeability. Their selectivity was evaluated leading to imprinting factors of 2.7 ± 0.1 for BZE and 4.0 ± 0.6 for cocaine (selected as template for the MIP synthesis) with polymers resulting from three independent syntheses, showing both the high selectivity of the MIPs and the reproducibility of their synthesis. After selecting the appropriate capillary length and the set-up configuration and optimizing the extraction protocol to promote selectivity, the extraction of BZE present in human urine samples spiked at 150, 250, and 500 ng mL-1 was successfully carried out on the monolithic MIP and coupled directly on-line with UV detection. The very clean-baseline of the resulting chromatograms revealing only the peak of interest for BZE illustrated the high selectivity brought by the monolithic MIP. Limits of detection and quantification of 56.4 ng mL-1 and 188.0 ng mL-1 were achieved in urine samples, respectively. It is therefore possible to achieve analytical threshold in accordance with the legislation on BZE detection in urine without the need for an additional chromatographic separation.

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.

Molecularly imprinted polymers in miniaturized extraction and separation devices.

Molecularly imprinted polymers are highly selective and cost-effective materials, which have attracted significant interest in various areas such as sample pretreatment and chromatographic and electrophoretic separations. This review aims to present the state of the art concerning the miniaturization of these materials in order to meet the societal demand for reliable, fast, cheap, and solvent/sample saving analyses. The polymerization route specificities for the production of miniaturized molecularly imprinted polymers in capillaries or chip channels, such as open tubular, packed particles, magnetic nanoparticles, and in situ imprinted monoliths, are investigated. Their performances as selective supports in solid phase extraction and as stationary phases in electrochromatography and liquid chromatography, as well as their possible perspectives are discussed.