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 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.

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.

Determination of Ochratoxin A (OTA), Ochratoxin B (OTB), T-2, and HT-2 Toxins in Wheat Grains, Wheat Flour, and Bread in Lebanon by LC-MS/MS.

Cereals are prone to fungal infection during growth, harvesting, transportation, and/or storage. As a result, cereals such as wheat grains and wheat-derived products may be contaminated with mycotoxins leading to acute and chronic health exposure. The current study investigated the presence of the mycotoxins: ochratoxin A (OTA), ochratoxin B (OTB), T-2, and HT-2 toxins in samples of wheat grains (n = 50), wheat flour (n = 50), and bread (n = 37) from the main mills in Lebanon using LC-MS/MS. Accuracy ranged from 98-100%, recoveries from 93-105%, and intraday and interday precision were 5-7% and 9-12%, respectively. The tested wheat grains, wheat flour, and bread samples did not contain detectable levels of T-2 and HT-2 toxins and OTB. Four wheat flour samples (8% of flour samples) showed positive OTA levels ranging from 0.6-3.4 µg·kg-1 with an arithmetic mean of 1.9 ± 0.2 µg·kg-1. Only one sample contained an OTA concentration greater than the limit set by the European Union (3 µg·kg-1) for wheat-derived products. This study suggests that mycotoxin contamination of wheat grains, wheat flour, and bread in Lebanon is currently not a serious public health concern. However, surveillance strategies and monitoring programs must be routinely implemented to ensure minimal mycotoxin contamination of wheat-based products.