A monolithic silica capillary column suitable for intact protein separation was developed for the high-resolution separation of proteins contained in biological samples by LC or LC/MS analysis. While investigating of the stationary phases, monolithic silica capillary columns modified with C4 or phenyl groups showed less carryover for high-molecular-weight monoclonal antibodies than C8 modification. Furthermore C4-modified monolithic silica capillary columns with lower adsorption are suitable for protein analysis containing unknown and low concentration proteins. In addition, we can evaluate the thermal degradation and denaturation of proteins using monoclonal antibodies, since a C4-modified monolithic silica capillary column can perform LC separation of proteins at relatively low temperature. As a result, it was suggested that it was important to keep the column temperature below 60 °C for the quantitative analysis of proteins. The C4-modified monolithic silica capillary column is useful for intact protein separation analysis, such as high-speed LC/MS analysis of monoclonal antibodies and high-resolution separation analysis of human cell-derived proteins.
In this study, the method for separetion and measurement using normal phase LC/MS/MS for levofloxacin and (R)-ofloxacin, which are enantiomers that are difficult to separete by reversed phase LC, was investigated. The mobile phase is t-butyl methyl ether/ethanol/acetic acid/ethylenediamine (30 : 70 : 0.2 : 0.2, v/v/v/v), and the column is phenomenex Lux i-Cellulose-5 (150 × 2.0 mm i.d., 3 μm). The two substances could be separeted under this condition. The calibration curves for both levofloxacin and (R)-ofloxacin showed good linearity in the range of 0.10 to 100 ng mL−1, and the lower limit of instrument detection was levofloxacin: 0.22 ng L−1, (R)-ofloxacin: 0.33 ng L−1. As a result of applying this method to the eluates extracted from a water sample with a polymer-based reversed-phase solid-phase column and ion-exchange reversed-phase mixed solid-phase column, levofloxacin 420 ng L−1 and (R)-ofloxacin 1.7 ng L−1 were detected from sewage treated water, sensitive enantio-separetion. For then, a determination that can be applied to environmental water sample measurements has been made possible.
Electrospray ionization (ESI), the most general ionization method in LC/MS, is a representative example of soft ionization, and usually does not generate fragment ions. However, in some compounds which have a hydroxyl group, [M+H]+, [M+Na]+, [M+NH4]+, etc. are not generated by positive ions, and dehydration from [M+H]+ and [M+H-H2O]+ are mainly observed. If such ions are observed in the measurement of an unknown sample, it will cause inconvenience when acquiring mass information of the analyte. In this study, in the ESI used for on-line LC/MS, the dehydration ions [M+H-H2O]+ from [M+H]+ are mainly observed at normal desolvation temperatures. This paper reports a method for obtaining mass information of an analyte correctly from changes in mass spectral patterns by lowering the desolvation temperature. The performance was demonstrated using cholesterol, eudesmol and hinesol.
In this study, a rapid target screening method for organic pollutants in water environments by liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed using a rapid pretreatment cartridge (the cartridge method). A salting-out pretreatment was carried out, with acetonitrile as the extraction solvent. The recovery tests of 71 (octanol-water partition coefficient (Log Pow): –0.46-8.20) out of 85 pesticides, using river and sea water, were at acceptable levels (recovery rate, 54-132 %) compared with the target recovery rates (50-150 %). There were 14 pesticides with lower recovery rates, which were polar chemicals (Log Pow: –0.91-3.18). We focused on a direct injection method that uses LC/MS/MS measurements, after filtering samples and adding internal standards to improve the low recovery rates of those 14 pesticides. The results of the direct method showed that the 14 pesticides were at acceptable levels (recovery rate, 73-114 %). Furthermore, in the direct injection method, the range of Log Pow was investigated by recovery tests of 85 pesticides. As a result, some hydrophobic chemicals (Log Pow, 2.58-8.20) adsorption to the filter was estimated. It was possible to measure chemicals with a Log Pow of –0.91 to 8.20 by using both the cartridge method and direct injection methods. The analysis time of the developed rapid screening method was shorter than that of the conventional solid-phase extraction method, and the total time required for our analytical procedure was approximately 10 min.
In two-dimensional LC (2D-LC), a combination of different separation modes is effective for increasing the peak capacity. However, for example, in the combination of reversed-phase chromatography (RPC) and hydrophilic interaction chromatography (HILIC), the elution strength of the organic solvent in the mobile phase is contradictory, so that the compatibility of the mobile phase, such as deterioration of the peak shape in the second dimension, is often a problem. On-line diluting of an eluate from the first dimension is a well-known procedure, but it has drawbacks, such as a complicated system. Recently, a new valve, which has the function of splitting the second-dimensional mobile phase was developed. By using this valve, we investigated the effect on improving the second-dimensional peak shape. As a result, it was confirmed that the flow split/on-line dilution method is effective for improving the peak shape of the second dimension in 2D-LC, such as RPC → HILIC and gel permeation chromatography (GPC) → gradient polymer elution chromatography (GPEC).
In this study, LC-MS/MS analysis was carried out using ammonia solution as an eluent for pesticide residues. The detection sensitivity was compared with an ammonium acetate solution by the notification test method using the S/N ratio as an index. As a result, the detection sensitivity of most pesticide residues was improved by 3.9 times on average when using an ammonia solution. We considered that the electrospray of an ammonia solution is easier than that of an ammonium acetate solution as a reason for the improved detection sensitivity. Moreover, the matrix effect was the same as that of the notification test method as a result of confirming about carrot. LC-MS/MS analysis using an ammonia solution can be expected as a simple analytical method that enables high-sensitivity detection without the need for concentration operation in the analysis of pesticide residues.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for structure determination. However, NMR spectra sometimes suffer from severe signal overlaps. To avoid such signal overlaps, LC-DAD-NMR systems have been used. These systems have enabled the separation of components on a column before NMR detection. However, DAD detection alone is insufficient when the sample does not contain chromophores. To increase the detection coverage, we used a corona charged aerosol detector (CAD) that responded to analytes independent of their physical/chemical characteristics. The established LC-DAD/CAD NMR system was evaluated. Its calculated detection and quantitation limits were 0.25 and 1.0 μg/peak, respectively. This system was used to analyze rubus extract. Along with glucose and fructose, a small amount of rubusoside was detected by corona CAD and identified by NMR without needing a chromophore.