An ion-exchange monomer including an anion exchange group and a cation exchange group, 2-methacryloyloxyethyl- phosphoryl choline (MPC) was introduced on the surface of uniformly sized, hydrophilic macro-porous polymer particle. We carefully studied several solvents utilized for the surface modification to elucidate solubility of the monomer as well as its polymer into each solvent. Based on the change in existing form of poly-MPC modified on the surface of stationary phases, ion-exchange properties of poly-MPC were controllable to result in interesting selectivity changes in HPLC.
An acid dissociation constant (pKa) of bupropion has been determined in an aqueous solution by capillary zone electrophoresis (CZE). Electrophoretic mobility of bupropion was measured by CZE over its acid-dissociating pH range, and a pKa value has been determined as 8.75±0.02 (mean ± standard error, ionic strength: 0.010 mol dm−3). Although bupropion degrades under heated and alkaline conditions and the degraded species from bupropion coexist in the aqueous solution, the pKa value of bupropion can also be determined by CZE with the residual bupropion without any interference from the degraded species. The pKa value determined after the heat-degradation was 8.74±0.02; the result agreed well with the one determined with the freshly prepared solution. Utilization of the separation characteristics of CZE has been demonstrated with bupropion for the analysis of the fast equilibrium of acid-base reaction under the coexistence of the degraded species.
Previously, we have developed a gradient elution system for pillar array columns, which achieved faster separation than isocratic elution. In this study, we validated gradient elution in microchip liquid chromatography (LC) and investigated the retention and bandwidth predictions of fluorescently labeled aliphatic amines in fast gradient elution chromatography using semi-empirical retention models. The retention times and peak widths under different gradient elution programs were predicted by three solvent strength models and compared with the experimental results. The relative errors of prediction for the retention times and peak widths were below 14 % and 12 %, respectively. The results showed that the solvent strength models could be utilized for predicting the retention times and peak widths under gradient elution in the microchip LC system and that the gradient elution program for pillar array columns worked efficiently. The prediction by the retention model promises to be a potential tool for essential compound identification in biological samples.
Based on capillary zone electrophoresis (CZE), we validated a method of confirming the identity and purity of the separated charge variants of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). The validation includes the evaluation of the specificity, linearity, quantitation limit, precision (repeatability and intermediate precision), accuracy, range, and robustness. The method is applicable to the majority of mAbs and ADCs (with pI from 7 to 9 and a drug to antibody ratio up to 8), requiring no modification to the method conditions. The proposed CZE method showed reproducible separation profiles, while cation exchange chromatography (CEX) showed low reproducibility and deficient separation profiles due to an undesirable interaction between the separation column and the low molecular weight drugs combined in the ADCs. Since CZE is able to minimize this undesirable interaction during the separation, it proved to be a useful separation methodology for evaluating charge variants of ADCs. The validation of CZE for assessing ADCs was successfully demonstrated for the first time, and show that CZE is suitable for the separation method for detecting the charge heterogeneity of ADCs.
A new analytical method was developed for the determination of 35 different amino acids in human pancreas tissue sections embedded in O.C.T. by liquid chromatography tandem mass spectrometry with precolumn derivatization. An AccQ-Tag was selected as a derivatization reagent because it could avoid the ion suppression caused by matrix compounds in the pancreas tissue. This new method uses two different internal standards during the pretreatment processes to achieve good accuracy and high sensitivity, as well as a multiple reaction monitoring time segment program during the acquisition process. This method was fully validated for 35 different amino acids and showed a good linearity of calibration curve (r2>0.993), with a lower limit of quantification in the range of 0.01–0.75 µM. Furthermore, this method was applied to the analysis of 35 different amino acids in human pancreas tissue sections, and successfully allowed for the determination of 27 of these compounds. The accuracy of our newly developed method was estimated to be in the range of 87%–115% based on the results of a recovery test. This method therefore represents a powerful analytical platform for the analysis of human tissue sections and could be used to investigate the mechanism of pancreatic disease.
This report describes the relation between peak capacity and mass spectrometric detectivity on peptides analysis with low-density octadecylsilylated monolithic silica capillary columns for several acidic additives in mobile phase using a nano-flow LC/MS. As the acidic additives, trifluoroacetic acid (TFA), 3,3,3-trifluoropropionic acid (TriFPA), formic acid (FA), acetic acid (AA), and cyanoacetic acid (CAA) were evaluated. Peak capacity and mass spectrometric detectivity were evaluated by using the peptides mixture in the gradient elution of water/acetonitrile with 0.1% (v/v) additives at 1-50% acetonitrile composition for 35 minutes on low-density octadecylsilylated monolithic silica capillary columns. Peak capacity is usually estimated by using the ratio of gradient time to average peak width of peptides. However, we estimated the peak capacity by using the ratio of t0 to the time of final peak to average peak width of peptides because the retention time of peptides was varied by difference of additives. As a result, peak capacity was increased by utilizing higher acidity additives, while the mass spectrometric detectivity was decreased. This result suggested that both high peak capacity and high detectivity for peptides analysis is irreconcilable. To overcome this relation, the impact of CAA was investigated because of its thermolysis behavior. In the condition of mobile phase with CAA at high temperature condition of mass spectrometry capillary inlet, higher peak capacity than those with TriFPA and even detectivity for those with FA were observed.
Determination of asperulosidic acid (AA) and deacetylasperulosidic acid (DAA) in rat plasma after administration of Morinda citrifolia juice by HPLC-UV detection was described. Deproteinized rat plasma (200 L) with MeOH for the HPLC analysis was injected. AA and DAA was separated on ZIC®-HILIC (250×4.6 mm, i.d., 5 μm) with a mixture of CH3CN/MeOH/0.1% formic acid aqueous solution (=70:25:5, v/v/v) at a flow rate of 0.75 mL/min. The absorbance of eluate was monitored at 235 nm. Under this condition, the separation of AA and DAA was achieved within 25 min. The calibration curves using plasma spiked with standards indicated good linearity (r≥0.996) in the range of 0.8-20 g/mL for AA and 4-100 μg/mL for DAA, respectively. The limits of quantitation for AA and DAA at a signal-to-noise ratio of 10 were 0.3 and 1.1 μg/mL, respectively. The validation parameters of the method such as recovery (94.9-101.7 %), precisions (less than 7.1 % for intra-day and less than 9.8 % for inter-day) and accuracy (95.0-101.7 %) were acceptable. The analytes in rat plasma were stable after three freeze-thaw cycles or storage at room temperature for up to 6 h. Furthermore, monitoring of AA and DAA after administration of the Morinda citrifolia juice in which AA and DAA were determined, was successfully demonstrated. This is the first report to determine AA and DAA in rat plasma after administration of Morinda citrifolia juice.
Biological thiols – homocysteine (Hcy), cysteine (Cys), γ-glutamylcysteine (γGluCys), glutathione (GSH), and cysteinylglycine (CysGly) – in plasma samples of cystathionine β-synthase (CBS, EC 22.214.171.124)-deficient mice were quantified using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection. Mice deficient in CBS provide a model mouse of homocystinuria, an inherited metabolic disease characterized by the abnormal accumulation of Hcy in urine and blood. For quantification, the thiols were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by separation with an amide column containing a mobile phase of acetonitrile–40 mM ammonium formate buffer (pH 3.0) (75/25, v/v). The total concentrations of Hcy in the plasma samples of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were 24.7, 29.3, and 237.7 μmol/L, respectively; 236.4, 178.7, and 78.5 μmol/L for Cys; 5.80, 4.87, and 0.75 μmol/L for γGluCys; 64.6, 74.0, and 51.2 μmol/L for GSH; and 2.48, 3.98, and 0.90 μmol/L for CysGly. The concentration of Hcy was significantly increased, while Cys, γGluCys, and CysGly concentrations were significantly decreased in CBS-KO mice compared to those in CBS-WT mice. The results indicated that biological thiols other than Hcy could also be utilized for the evaluation and investigation of homocystinuria pathologies.
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