In ultralow-temperature HPLC, analyte retention is often enhanced, inhibiting elution. To solve this problem, we have investigated the use of low-molecular-weight hydrocarbons, methane and ethane, as the mobile phase in a monolithic ODS column. Analyte retention was successfully reduced by the use of these mobile phases, and elution of mono- and di-chloromethane and n-octane, which were not eluted in our previous work using a liquid nitrogen based mobile phase, was achieved. The analysis of octane structural isomers revealed that, in cryogenic HPLC, the retention of branched octanes was significantly reduced compared to the retention of n-octane, i.e., the retention factor of iso-octane (2,2,4-trimethylpentane) was almost negligible. The retention factors of branched octanes were distributed between those of n-pentane and n-heptane in HPLC at -176°C, whereas, in gas chromatography at 50°C, these values were between those of n-heptane and n-octane.
This report presents two fluorescence labeling methods for therapeutic monoclonal antibody, bevacizumab, to increase its detection sensitivity for fluorescence detection. One method is high-temperature reversed-phase LC (HT-RPLC) following post-column fluorogenic derivatization using o-phthalaldehyde with thiol. Another method is pre-column derivatization using Zenon Alexa Fluor 488 protein-tag following size-exclusion chromatography (SEC). The calibration curves of bevacizumab were 1–50 μg/mL (post-column method) and 0.1–10 μg/mL (pre-column method). Both methods showed good correlation coefficients (r2 > 0.991). The LOD and the LOQ of bevacizumab were, respectively, 0.13 and 0.43 μg/mL (post-column method) and 0.03 and 0.1 μg/mL (pre-column method). The sensitivities were about 2 and 10 times higher than that of native fluorescence detection. The proposed methods were applied to bevacizumab spiked human plasma samples. The bevacizumab in plasma samples was purified selectively with immunoaffinity beads and detected as a single peak using HT-RPLC or SEC with fluorescence detection.
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.
Poly(butylene terephthalate)-coated silica (PBT) have been introduced as a stationary phase in liquid chromatography (LC) and the retention behavior of polycyclic aromatic compounds (PACs) was evaluated in reversed-phase LC. The trend for the retention was compared with that obtained on two types of commercially-available octadecylsilica (ODS) phases and phenylbutylsilica (PBS) phase. A good liner relationship between molecular size of planar PACs and the corresponding logarithmic retention factor was confirmed on the PBT stationary phase, and the trend is quite similar to that obtained on a conventional polymeric ODS stationary phase. In addition, a good molecular shape recognition capability of the PBT stationary phase was confirmed for several solute pairs consisted of planar and non-planar PACs with a similar two-dimensional molecular size. The selectivities to some planar/non-planar solute pairs on the PBT stationary phase were significantly better than conventional ODS phases, even when compared with that of typical polymeric ODS stationary phases operated in a similar experimental condition. In the case of structural isomers of dichlorobenzene and dibromobenzene, the elution order on the PBT stationary phase was o-, m- and p-, however the corresponding elution order in typical ODS phases, and PBS phase was different, o-, p- and m-. The results can be explained on the basis of the molecular-molecular interaction between the stationary phase ligand and the analyte molecule, because the PBT stationary phase has a similar partial chemical structure to these p-isomers on the silica support.
We have previously reported analytical methods for the quantification of catecholamines (norepinephrine, epinephrine, and dopamine) via simple pretreatment using a monolithic silica disk-packed spin column with an attached phenylboronate moiety. However, under certain conditions, splitting in the dopamine peak was observed. In this study, we investigated the reason for this peak splitting and found that anions in the basic buffer solution used in the extraction influenced the peak shape. The extraction could be improved via additionally washing the column with a low-concentration buffer. The extraction recoveries of the catecholamines via the improved method were in the range of 95.9–100.8%. Thus, the improved method is expected to be more reliable for the quantification of catecholamines in biological samples.
A photodiode array detector (PDA) is frequently utilized as a detector in high-performance liquid chromatography (HPLC) system for a detection of various compounds. A PDA emits a light of a wide range wavelength including ultraviolet light (UV) which has a possibility to induce photodegradation of analyzed compounds. If so, target compounds might be degraded when analyzed in this HPLC system. Therefore, photoprotection during HPLC analysis is required for the accurate analysis. In this study, the protective effect of a UV cut-off filter, which can cut off UV especially at shorter wavelength (< 240 nm), on the quantitativity of a photodegradable compound L-ascorbic acid (AA) was examined. A UV cut-off filter prevents AA from photodegradation, followed by the improvement of several parameters of a calibration curve. Furthermore, this protective potency was significant in the case that photodegradability of AA was enhanced with the conditions such as a small injection volume and a low flow rate. This study strongly suggests that the UV cut-off filter is a useful equipment when analyzing photodegradable compounds.
A reversed-phase high-performance liquid chromatographic (HPLC) method using pre-column derivatization with o-phthalaldehyde (OPA) plus N-tert-butyloxycarbonyl-D-cysteine (Boc-D-Cys) has been developed for the determination of aspartic acid (Asp), serine (Ser) and alanine (Ala) enantiomers. D-Amino acids in the real world samples are trace in most cases, and their small peaks should be eluted faster than the huge peaks of the L-forms in order to avoid overlapping. Amino acids were rapidly derivatized at room temperature with OPA plus Boc-D-Cys under simple conditions and were detected by their fluorescence. The target amino acid enantiomers were separated within 60 min on a reversed-phase column, CAPCELL PAK C18 MG II (4.6 x 200 mm), and their resolution values were higher than 2.14. The developed system was successfully validated using standard amino acids, and sufficient calibration lines (r2 > 0.9983) and precision (RSD < 5.29%) results were obtained. In a Japanese traditionally fermented amber rice vinegar, all of the target D-amino acids were observed, and their %D values were 21.5 for Asp, 6.8 for Ser and 22.9 for Ala.