Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, ß-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). ß-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

Separation and determination of alpha-synuclein monomeric and oligomeric species using two electrophoretic approaches.

Parkinson's disease (PD) is a frequent degenerative disorder that is diagnosed based on clinical symptoms. When the first symptoms appear, more than 70% of the dopaminergic cells are already lost. Therefore, it is of utmost importance to have reliable biomarkers to diagnose much earlier PD. In this context, alpha-synuclein (aSyn) is a protein of high interest because of its tendency to form oligomers and amyloid fibrils. The oligomeric forms seem to play a critical pathological role in PD. To date, most of studies aiming at detecting and quantifying aSyn oligomers were performed by immunoassays, mainly by ELISA using specific antibodies. In this study a capillary gel electrophoresis (CGE) coupled with fluorescence detection method was developed to detect and quantify the oligomeric forms of aSyn formed in vitro. All the results obtained were supported by SDS-PAGE analysis, a widely used and well-known technique but exhibiting a main drawback since it is not an automated technique. The repeatability and the intermediate precision of the method were evaluated, as well as the stability of the labeled and non-labeled aSyn samples. After careful screening and optimization of various labeling reagents, 4-fluoro-7-nitrobenzofurazan (NBD-F) was selected and used to establish a calibration curve with monomeric fluorescently-labeled aSyn. Finally, the method was used to study the effect of doxycycline on the oligomerization process. Altogether, our results show that CGE is a very promising automated technique to analyze aSyn monomers, as well as small oligomers.

Analysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis.

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non-aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit® gave the best results, providing the separation of the four major protamine peptides within 25 min.

Improvement of chemo- and stereoselectivity for phosphorothioate oligonucleotides in capillary electrophoresis by addition of cyclodextrins.

Phosphorothioate (PS) modification is one of the most widely used oligonucleotide chemical alterations in the oligonucleotide backbone. It has proven to be crucial in the field of therapeutic oligonucleotides regarding the optimization of their physicochemical and biological properties. In this study, a capillary electrophoresis (CE) method with an acidic background electrolyte (BGE) containing a combination of ß- and γ-cyclodextrins derivatives as chiral selectors is proposed for the diastereomeric separation of 5-mer oligonucleotides containing 0, 1, 2, or 3 phosphorothioate linkages (5´-TCGTG-3´). The effects of the BGE pH, organic modifier addition, and type of cyclodextrin (CD) on chemo- and stereoselectivity and resolution were studied. A mixture of 25 mM (2-hydroxy-3-N,N,N-trimethylamino)propyl-γ-CD and 10 mM carboxymethyl-ß-cyclodextrin in a pH 3 buffer was found to be the most appropriate system for the qualitative evaluation of the short oligonucleotides investigated. These phosphorothioate oligonucleotides were separated with high efficiency in less than 11 min with no capillary treatment. The suggested approach can be the basis for purity testing of this new generation of therapeutics.

Separation of phosphorothioated oligonucleotide diastereomers using multiplexed drift tube ion mobility mass spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power. The entrance voltage providing the best two-peak resolution was -1000V with 3-bit multiplexing. Under optimized conditions, the eight diastereomers of an oligonucleotide with three PS linkages (5'-TC∗G∗T∗G-3') could be separated unambiguously. Indeed, those diastereomers differed in their collision cross section (CCS) values. The minimal CCS values difference between two adjacent diastereomers was 0.9% with maximal RSD on CCS values of 0.3%. The use of multiplexed ion mobility and the novel high-resolution demultiplexing tool represents a real breakthrough for resolution enhancement of diastereomers in linear DTIMS.

Analytical techniques currently used in the pharmaceutical industry for the quality control of RNA-based therapeutics and ongoing developments.

The number of RNA-based therapeutics has significantly grown in number on the market over the last 20 years. This number is expected to further increase in the coming years as many RNA therapeutics are being tested in late clinical trials stages. The first part of this paper considers the mechanism of action, the synthesis and the potential impurities resulting from synthesis as well as the strategies used to increase RNA-based therapeutics efficacy. In the second part of this review, the tests that are usually performed in the pharmaceutical industry for the quality testing of antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) and messenger RNAs (mRNAs) will be described. In the last part, the remaining challenges and the ongoing developments to meet them are discussed.

Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein.

In this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals.

Hyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces.

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE-MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest.

Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides.

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks. In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency. In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides.

Dibenzoylthiamine Has Powerful Antioxidant and Anti-Inflammatory Properties in Cultured Cells and in Mouse Models of Stress and Neurodegeneration.

Thiamine precursors, the most studied being benfotiamine (BFT), have protective effects in mouse models of neurodegenerative diseases. BFT decreased oxidative stress and inflammation, two major characteristics of neurodegenerative diseases, in a neuroblastoma cell line (Neuro2a) and an immortalized brain microglial cell line (BV2). Here, we tested the potential antioxidant and anti-inflammatory effects of the hitherto unexplored derivative O,S-dibenzoylthiamine (DBT) in these two cell lines. We show that DBT protects Neuro2a cells against paraquat (PQ) toxicity by counteracting oxidative stress at low concentrations and increases the synthesis of reduced glutathione and NADPH in a Nrf2-independent manner. In BV2 cells activated by lipopolysaccharides (LPS), DBT significantly decreased inflammation by suppressing translocation of NF-κB to the nucleus. Our results also demonstrate the superiority of DBT over thiamine and other thiamine precursors, including BFT, in all of the in vitro models. Finally, we show that the chronic administration of DBT arrested motor dysfunction in FUS transgenic mice, a model of amyotrophic lateral sclerosis, and it reduced depressive-like behavior in a mouse model of ultrasound-induced stress in which it normalized oxidative stress marker levels in the brain. Together, our data suggest that DBT may have therapeutic potential for brain pathology associated with oxidative stress and inflammation by novel, coenzyme-independent mechanisms.

Thiamine and benfotiamine protect neuroblastoma cells against paraquat and ß-amyloid toxicity by a coenzyme-independent mechanism.

BACKGROUND: Benfotiamine (BFT) is a synthetic thiamine precursor with high bioavailability. It is efficient in treating complications of type 2 diabetes and has beneficial effects in mouse models of neurodegenerative diseases. The mechanism of action of BFT remains unknown, though it is sometimes suggested that it may be linked to increased thiamine diphosphate (ThDP) coenzyme function. METHODS: We used a mouse neuroblastoma cell line (Neuro2a) grown in thiamine-restricted medium. The cells were stressed by exposure to paraquat (PQ) or amyloid ß1-42 peptide in the presence or absence of BFT and the cell survival was measured using the MTT method. In each case, BFT was compared with sulbutiamine (SuBT), an unrelated thiamine precursor, and thiamine. Metabolites of BFT were determined by HPLC and mass spectrometry. RESULTS: At 50 µM, BFT protects the cells against PQ and amyloid ß1-42 peptide-induced toxicity with the same efficacy. Protective effects were also observed with SuBT and with higher concentrations of thiamine. The main metabolites of BFT were thiamine and S-benzoylthiamine (S-BT). Treatment with both precursors induces a strong increase in intracellular content of thiamine. Protective effects of BFT and SuBT are directly related to thiamine (but not ThDP) levels in Neuro2a cells. CONCLUSIONS: BFT, SuBT and thiamine all protect the cells against oxidative stress, suggesting an antioxidant effect of thiamine. Our results are not in favor of a direct ROS scavenging effect of thiamine but rather an indirect effect possibly mediated by some antioxidant signaling pathway. It is however not clear whether this effect is due to thiamine itself, its thiol form or an unknown metabolite. GENERAL SIGNIFICANCE: Our results suggest a role of thiamine in protection against oxidative stress, independent of the coenzyme function of thiamine diphosphate.