Detailed profiling of polysorbate 80 oxidative degradation products and hydrolysates using liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant that is commonly used to stabilize proteins in biopharmaceutical formulations. PS80 undergoes oxidative and/or enzymatic degradation. However, because PS80 is a complex mixture consisting of many constituents, comprehensive evaluations of its oxidative degradation products are difficult and insufficient. METHODS: Our previously reported comprehensive liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based method for PS80 effectively provides an overall profile of PS80 components under simple LC conditions. In this study, we attempted to shorten the analysis time. Furthermore, PS80 was oxidatively degraded in a solution containing histidine and iron, and the oxidative degradation products were evaluated using a modified LC/MS/MS method. In addition, enzymatically hydrolyzed PS80 samples were analyzed. RESULTS: We succeeded in shortening the analysis time from 70 to 20 min while maintaining the resolution of the PS80 components of the same selected reaction monitoring transition. Both the previously reported oxidative degradation products and the newly discovered products were successfully detected, and their composition ratios and changes over time were observed. Changes in the hydrolysates over time are shown in the analysis of the hydrolyzed PS80 samples. CONCLUSIONS: This study clearly showed the presence of changes in PS80 oxidative and/or enzymatic degradation products, including those previously unreported. These results demonstrate that a detailed profiling of PS80 degradation products can be performed using LC/MS/MS, which is less expensive and more generally adopted than high-resolution MS.

Profiling polysorbate 80 components using comprehensive liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant commonly used in pharmaceutical protein formulations and is composed of fatty acid (FA) esters of polyethoxylated sorbitan. However, commercial PS80 products contain substantial amounts of by-products. The development of simple and reliable methods for PS80 component analysis is challenging given the inherent heterogeneity. METHOD: We developed a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to profile the components of PS80. Semi-comprehensive LC-MS/MS analyses of 11 subspecies in three commercial PS80 products were performed to estimate the average degree of polymerization of the ethylene oxide units (Avg-n) in the molecules. Furthermore, three subspecies (polyoxyethylene sorbitan monoester, polyoxyethylene isosorbide monoester, and polyoxyethylene monoester) were analyzed to estimate the composition ratios of the seven ester-bonded FAs present in PS80. RESULTS: The Avg-n values of five polyoxyethylene sorbitan esters (none, mono, di, tri, and tetra), three polyoxyethylene isosorbide esters (none, mono, and di), and three polyoxyethylene esters (none, mono, and di) were 26.5-30.6, 12.1-14.6, and 11.4-15.8, respectively. These values were comparable regardless of the number of ester-bonded FAs. Each product had a similar FA composition ratio regardless of the differences in the subspecies. However, the obtained C18:2 values were higher than those reported in the product certificates. CONCLUSION: The proposed LC-MS/MS method evaluated the overall PS80 components, revealing the possibility of underestimation of ester-bonded linoleic acid using the conventional gas chromatography-mass spectrometry method. The similarity of Avg-n values and FA compositions among subspecies suggested the high reliability of these results, indicating that the presented approach may help in the quality control of PS80 formulations.

Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype.

Change in fatty acid composition of plasma triglyceride caused by a 2 week comprehensive risk management for diabetes: A prospective observational study of type 2 diabetes patients with supercritical fluid chromatography/mass spectrometry-based semi-target lipidomic analysis.

AIMS/INTRODUCTION: Hypertriglyceridemia is common in patients with diabetes. Although the fatty acid (FA) composition of triglycerides (TGs) is suggested to be related to the pathology of diabetes and its complications, changes in the fatty acid composition caused by diabetes treatment remain unclear. This study aimed to identify short-term changes in the fatty acid composition of plasma triglycerides after diabetes treatment. MATERIALS AND METHODS: This study was a sub-analysis of a prospective observational study of patients with type 2 diabetes aged between 20 and 75 years who were hospitalized to improve glycemic control (n = 31). A lipidomic analysis of plasma samples on the 2nd and 16th hospital days was conducted by supercritical fluid chromatography coupled with mass spectrometry. RESULTS: In total, 104 types of triglycerides with different compositions were identified. Most of them tended to decrease after treatment. In particular, triglycerides with a lower carbon number and fewer double bonds showed a relatively larger reduction. The inclusion of FA 14:0 (myristic acid), as a constituent of triglyceride, was significantly associated with a more than 50%, and statistically significant, reduction (odds ratio 39.0; P < 0.001). The total amount of FA 14:0 as a constituent of triglycerides also decreased significantly, and its rate of decrease was the greatest of all the fatty acid constituents. CONCLUSIONS: A 2 week comprehensive risk management for diabetes resulted in decreased levels of plasma triglycerides and a change in the fatty acid composition of triglycerides, characterized by a relatively large reduction in FA 14:0 as a constituent of triglycerides.

Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry.

In the chiral separation of amino acids, liquid chromatography has been mainly used because of the physicochemical properties of the analytes. To date, only few reports of the use of supercritical fluid chromatography (SFC) for the analysis of chiral amino acids exist, and there is much room for improvement in terms of the number of measurable amino acids, peak shape, and analysis time. In this study, we developed a novel method for the chiral analysis of native amino acids using a system combining SFC and tandem mass spectrometry. Specifically, the separation of amino acid enantiomers was investigated using a CROWNPAK CR-I(+) column with a chiral stationary phase of optically active crown ether. Methanol/water mobile phase with trifluoroacetic acid as a modifier based on supercritical carbon dioxide (CO2) was used. At a low modifier concentration of 30% for the separation of hydrophilic compounds, 18 proteinogenic amino acid enantiomers except glycine and proline were successfully separated with resolution (Rs) = 1.96-33.62 within 6.5 min. In attempt to shorten the analysis time, the flow rate was increased; using a CO2/modifier ratio of 60/40 at a flow rate of 3 mL/min, ultrafast chromatography of 17 amino acid enantiomers, except histidine, was achieved with retention time ≤ 1 min and resolution ≥ 1.5. The developed ultrafast chiral separation method was verified by analyzing a commercially available black vinegar, which detected eight kinds of d-amino acids. The present method has thus confirmed to be successful and practical in terms of both analyte coverage and throughput.

Metabolomic investigation of cholestasis in a rat model using ultra-performance liquid chromatography/tandem mass spectrometry.

Metabolomics follows the changes in concentrations of endogenous metabolites, which may reflect various disease states as well as systemic responses to environmental, therapeutic, or genetic interventions. In this study, we applied metabolomic approaches to monitor dynamic changes in plasma and urine metabolites, and compared these metabolite profiles in Eisai hyperbilirubinemic rats (EHBR, an animal model of cholestasis) with those in the parent strain of EHBR - Sprague-Dawley (SD) rats - in order to characterize cholestasis pathophysiologically. Ultra-performance liquid chromatography/tandem mass spectrometry-based analytical methods were used to assay metabolite levels. More than 250 metabolites were detected in both plasma and urine, and metabolite profiles of EHBR differed from those of SD rats. The levels of antioxidative and cytoprotective metabolites, taurine and hypotaurine, were markedly increased in urine of EHBR. The levels of many bile acids were also elevated in plasma and urine of EHBR, but the extent of elevation depended on the particular bile acid. The levels of cytoprotective ursodeoxycholic acid and its conjugates were markedly elevated, while that of cytotoxic chenodeoxycholic acid remained unchanged, suggesting the balance of bile acids had shifted resulting in decreased toxicity. In EHBR, reduced biliary excretion leads to increased systemic exposure to harmful compounds including some endogenous metabolites. Our metabolomic data suggest that mechanisms exist in EHBR that compensate for cholestasis-related damage.

Development of a novel method for polar metabolite profiling by supercritical fluid chromatography/tandem mass spectrometry.

Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from -5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples.

Mechanistic study on the high-selectivity enantioseparation of amino acids using a chiral crown ether-bonded stationary phase and acidic, highly organic mobile phase by liquid chromatography/time-of-flight mass spectrometry.

The separation mechanism of amino acid enantiomers using a chiral crown ether-bonded stationary phase, CROWNPAK CR-I(+), and acetonitrile (ACN)-rich mobile phases (MPs) was studied. The retention factors of proteinogenic l-amino acids (except proline) formed U-shaped plots against the ACN content in the MP with a sharp increase at a high ACN content, while d-amino acids showed much smaller increases or monotonous decreases in retention within the same range. The use of an acidic, highly organic MP with trifluoroacetic acid (TFA) provided a high enantioselectivity with a short separation time from the contribution of the increased binding of the ammonium group of the analytes to the crown ether functionality of the stationary phase and electrostatic repulsion counteracting the hydrophilic partition mechanism. Optimizing the sample diluent and MP alleviated the peak distortion caused by a moving water band that accompanied the hydrophilic interaction liquid chromatography-like elution conditions. The liquid chromatography/time-of-flight mass spectrometry method with the optimized MP - ACN/ethanol/water/TFA = 80/15/5/0.5 (v/v/v/v) - enabled the determination of eighteen pairs of proteinogenic amino acid enantiomers within 10 min. The conditions also provided the following advantages: (i) fast and highly reproducible separations under isocratic conditions, (ii) high sensitivity and low backpressure using the MP with a high organic content, and (iii) highly reliable peak identification by combining two columns (CR-I(+) and CR-I(-)), reversing the elution orders of the enantiomers.

Novel high-throughput and widely-targeted liquid chromatography-time of flight mass spectrometry method for d-amino acids in foods.

Recently, the demand for d-amino acid profiling has been drastically increasing because the significance of d-amino acid in various biological events is suggested. However, the present methodologies for d-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for d-amino acid profiling must be developed. In this paper, we developed a novel method for d-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for d-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the d-amino acid research in the future.

Investigation of storage time-dependent alterations of enantioselective amino acid profiles in kimchi using liquid chromatography-time of flight mass spectrometry.

Although naturally abundant amino acids are represented mainly by l-enantiomers, fermented foods are known to contain various d-amino acids. Enantiospecific profiles of food products can vary due to fermentation by bacteria, and such alterations may contribute to changes in food properties that would not be dependent exclusively on l-amino acids. Therefore, more attention should be paid to the study of temporal alterations of d-amino acid profiles during fermentation process. However, there have been very few studies reporting time-dependent profiling of d-amino acids because enantioseparation of widely targeted d-amino acids is technically difficult. This study aimed to achieve high throughput profiling of amino acids enantiomers. Enantioselective profiling of amino acids using CROWNPAK CR-I(+) column, liquid chromatography, time of flight mass spectrometry, and principle component analysis was performed to investigate time-dependent alterations in concentrations of free d- and l-amino acids in kimchi stored at 4°C or 25°C. We demonstrated significant changes in d- and l-amino acid profiles in kimchi stored at 25°C. In particular, concentrations of the amino acids d-Ala, d-Ser, d-allo-Ile, d-Leu, d-Asp, d-Glu, and d-Met became higher in kimchi with storage time. This is the first report of time-dependent alterations of d- and l-amino acid contents in kimchi. This study showed that our analytical method of enantioselective detection of amino acids using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) with CROWNPAK CR-I(+) enables high throughput food screening and can be recommended for advanced studies of the relationship between d-amino acid content and food properties.

Development of a liquid chromatography-tandem mass spectrometry method for quantitative analysis of trace d-amino acids.

d-Amino acids have recently attracted much attention in various research fields including medical, clinical and food industry due to their important biological functions that differ from l-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. Thus, the aim of this research is to develop a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By optimizing MS/MS parameters, we established a quantification method that allowed the simultaneous analysis of 18 d-amino acids with high sensitivity and reproducibility. Additionally, we applied the method to food sample (vinegar) for the validation, and successfully quantified trace levels of d-amino acids in samples. These results demonstrated the applicability and feasibility of the LC-MS/MS method as a novel, effective tool for d-amino acid measurement in various biological samples.

Extra-facile chiral separation of amino acid enantiomers by LC-TOFMS analysis.

An extra-facile chiral liquid chromatography-time of flight mass spectrometry (LC-TOFMS) analytical method of amino acid enantiomers has been developed without a derivatization process. The enantioseparation of eighteen proteinogenic amino acids (except for proline) was simultaneously performed using a combination of a chiral column (CROWNPAK CR-I(+)) and a TOFMS within 15 min. An isocratic condition of a simple mobile phase comprising acetonitrile/water/trifluoroacetic acid (96/4/0.5) gave baseline separation of all underivatized amino acid enantiomers on the chiral column.