Amlodipine is a dihydropyridine drug that is used to lower high blood pressure. In this study, the photostabilities of orally disintegrating (OD) amlodipine tablets, both original and generic versions, and their powdered and suspended forms were evaluated. The concentration of the active pharmaceutical ingredient (API) was monitored by high-performance liquid chromatography (HPLC), revealing that residual amounts of API in powders and suspensions significantly decreased after ultraviolet light (UV) irradiation for 24 hr. Amlodipine OD tablets and their powders were packaged and stored at a house and a pharmacy for up to 90 days for the evaluation of their photostabilities in clinical use, and that amounts of API in the powdered form significantly decreased when they were stored in the house. ESI-LC/MS/MS analysis was performed to determine the chemical structure of photoproducts generated by photo-irradiated amlodipine OD tablets. It revealed that mainly a dehydrogenated form of amlodipine was generated concomitant with the photodegradation by UV and sunlight irradiation. This is the first study that evaluated the photostabilities of original amlodipine OD medicines, their generic OD versions and their altered forms.

We elucidated factors for the chiral conversion of α-lipoic acid enantiomers in α-lipoic acid-containing dietary supplements. Samples were cleaned up by the solid-phase dispersive extraction method using Oasis MAX and MCX as the solid-phase gel. The α-lipoic acid enantiomers were reciprocally converted by heating at 180°C, and finally became a racemate. The chiral conversion rate changed depending on sample purity, particularly the presence or absence of coexisting components in the dietary supplements. As candidates for coexisting components, neutral nonionic and highly polar substances were suggested, such as sugars. We found that chiral conversion was promoted by heating in the presence of glucose. Oftentimes, a relatively large amount of S-(–)-α-lipoic acid is detected in dietary supplements claiming to contain R-(+)-α-lipoic acid on the bottle label. We speculate that the proportion of S-form may have been increased by coexisting components such as glucose when heat treatment is performed during the manufacturing process.

For the HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags to achieve quantitative separation. However, the influence of such fluorescent tags on the structure and retention of glycans is not completely understood. Here, to investigate the separation of labeled glycans, we prepared four types of derivatives (2-aminopyridine, 2-aminobenzoic acid, 2-aminobenzamide, and ethyl 4-aminobenzoate) of three model glycans (high-mannose oligosaccharides from ribonuclease B, complex oligosaccharides from bovine fetuin, and an isomaltooligosaccharide mixture). The glycan derivatives were separated on newly developed stationary phases, namely, an amino- and amide-bonded hydrophilic (AAH) phase and a capillary-packed monolithic ODS phase without end-capping treatment. The performance of these phases was compared with typical amide hydrophilic interaction chromatography (HILIC) and end-capped C18 packed columns. In the HILIC separation mode, the AAH column exhibited superior resolution for isomaltooligosaccharides, compared to those obtained on a conventional amide column. Nevertheless, the plate numbers were the same for glycoprotein glycans and the three linkage isomers of Man7GlcNAc2 could not be resolved, whereas they could be separated on the amide column. However, the cationic amino groups of the AAH column enhanced the resolution of sialylated complex glycans that could not be separated on the amide HILIC column. In the reversed-phase separation mode, the retention of glycan derivatives was mainly dependent on the hydrophobicity of the labeling groups. In contrast to the conventional packed ODS column, the monolithic capillary ODS column showed good resolution for neutral glycans.

Herein, we report an intact LC–native fluorescence analysis method for therapeutic monoclonal antibodies (mAbs) based on a centrifugal filtration device with adsorption suppression treatment. Coating the centrifugal filtration device with MPC monomer suppressed the non-specific adsorption of mAbs, especially in the low concentration range; trastuzumab could be quantitatively and sensitively analyzed in the 0.2–10 μg/mL range. In this analysis, the average concentration factor over the entire concentration range was approximately 25 times. The other mAbs (bevacizumab, rituximab, nivolumab) also showed good linearity with R2 ≥ 0.996, and the average concentration factors were similar to that obtained for trastuzumab. This method can potentially be used in combination with affinity purification for simple and sensitive bioanalysis.

A pillar array column with a perfectly ordered internal structure has a higher separation efficiency than a particle-packed column used in conventional high-performance liquid chromatography. However, applying the pillar array column technology to quantitative analysis is challenging as the injection volume of the sample varies for each injection. This occurs because the sample volumes are in the order of nanoliters and the sample is injected manually. In this study, an automated sample injection system was developed to solve this problem. The system was composed of two pumps (one for the sample and other for the mobile phase), a six-way valve that can be controlled by a PC, and an autosampler. The peak height deviation, which is more than 20% in the conventional manual injection system, was improved to 1.2% and 0.4% for the two coumarin dye samples. This result indicated that the automated sample injection method developed in this study could be applied to pillar array columns to allow for quantitative analysis.

Quantification of four extracellular matrices commonly found in foods with functional claims was conducted using capillary zone electrophoresis with direct ultraviolet (UV) absorbance detection at 200 nm. The four polymeric compounds, namely, proteoglycan, chondroitin sulfate, hyaluronic acid, and collagen, were separated with 100 mM borate buffer (pH 10.0) using capillaries coated with poly-N,N-dimethylacrylamide on the inner wall, which almost completely suppressed the electroosmotic flow to give reproducible migration times for the analytes. The addition of 5 mM sodium dodecyl sulfate to the running solutions improved the shapes of the peaks and the linearity of the calibration curves. Some commercial products, including the extracellular matrices under investigation, were analyzed with the method developed.

Parkinson’s disease (PD) is diagnosed by neurological examinations, as well as by scintigraphy for the dopamine transporter and metaiodobenzylguanidine (MIBG). We studied possible application of the skin gas in diagnosis of PD. We analyzed chemical substances emanated from the skin by gas chromatograph/mass spectrometer (GC/MS) after on-line-pre-concentrator. We analyzed the skin gas in 61 PD patients and 61 controls. The GC/MS chromatograms were sectionalized every 30 sec. The retention time drift was shifted every 5 sec, and a similarity coefficient (Z score) between a reference chromatogram and each shifted chromatogram was calculated. Chromatograms with high Z scores were excluded from our analysis. Models were made with partial least square (PLS), support vector machine (SVM), and support vector regression (SVR) analyses. PLS modeling to predict the Unified Parkinson’s Disease Rating Scale part 3 (UPDRS3), representing motor deficits in PD, with all the detected mass numbers yielded 50 mass numbers with high PLS coefficients. Among the 50 mass numbers, nine mass numbers (m/e 48, 63, 67, 70, 81, 93, 96, 104, and 105) had dependable signal-to-noise ratios. We then generated an SVM model to differentiate PD and controls. Our SVM model had a sensitivity of 90.2% and a specificity of 85.2% by leave-one-out cross-validation (LOOCV) analysis. We next generated an SVR model to predict UPDRS3 with the nine mass numbers, and obtained a Pearson’s correlation coefficient of 0.834. LOOCV analysis of our SVR model similarly gave rise to a correlation coefficient of 0.710. We propose that chemical substances in the skin gas potentially serve as biomarkers for PD.

Quantitative analysis of monosaccharides or glycoprotein glycans by high-performance liquid chromatography (HPLC) often involves labeling of the saccharide aldehyde groups with fluorescent tags to enhance sensitivity and selectivity. However, the methods required to remove the large excess of labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods often hinder the quantitative analysis of the labeled samples. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminobenzoic acid (2-AA)-labeled monosaccharides or oligosaccharides using a ten-port valve and mini columns. An online purification system using a combination of short HLB columns with the valve was proposed for the analysis of 2-AA-labeled monosaccharides utilizing reversed-phase modes. In the analysis of 2-AA-labeled glycans derived from glycoproteins, a short CN column with the valve was proposed utilizing hydrophilic interaction liquid chromatography (HILIC) modes. Optimized conditions enabled the direct injection of the diluted labeling reaction mixture into the chromatographic system without any prior removal of the excess labeling reagents. These methods were successfully applied to the analysis of various monosaccharides and N-linked glycans released from specific glycoproteins.

In this study, we reveal the suppression of non-specific hydrophobic interaction in poly(ethylene-co-glycidylmethacrylate) (PEGM) based spongy monolith (SPM), PEGM-SPM, which has recently be reported as a new platform of separation medium for affinity chromatography in our previous study, by an simple acidic treatment. Additionally, the immobilization procedures of protein-A toward the PEGM-SPM and the separation conditions for immunoglobulin G (IgG) were optimized for further effective affinity separations. As a result of treatment by a mixture of trifluoroacetic acid and acetonitrile, the hydrophobicity was dramatically suppressed in the PEGM-SPM. The optimizations for the density of PEGM in the PEGM-SPM, the protein A immobilization, and the binding/releasing conditions showed that variety of proteins and peptides were not retained on the protein A immobilized spongy column at all while IgG was absolutely separated by a simple stepwise pH gradient condition.

An on-line coupled comprehensive two-dimensional liquid chromatography (2D-LC) system was developed for the separation of triglycerides (TGs) in this work. Taking advantage of two-dimensional separation on the basis of two separation columns with different selectivities, where an silver-ion column and an octylsilica column were employed as the columns in the 1st and 2nd dimensions, respectively, all the TGs in perilla and tuna oils were well separated. Introducing the systematic qualitative analysis in gas chromatography (GC), it was possible to make the assignments for all the fatty acid compositions in the TGs that were fractionated after the comprehensive 2D-LC separations. In the subsequent GC analysis, 23 and 76 types of TGs were determined for perilla and tuna oils, respectively. The results clearly showed an excellent performance of the two-dimensional separation system for the separation of TGs in oils and fats, suggesting a future applications to the high performance separation of other types of fish oils with the on-line coupled comprehensive 2D-LC system developed in this work.