Mass spectrometry-based proteomics platforms have been widely used as ‘proteome sequencers’ to characterize the proteomes of a wide range of organisms. The work-flow generally involves multiple steps of sample preparation, peptide purification/concentration/pre-fractionation, and nanoLC/MS/MS measurement. This review focuses on our contributions to the technical development of current proteomics platforms, and includes a consideration of the limitations of these systems, together with the prospects for developing superior new-generation proteome sequencers.
To construct liquid chromatography (LC)-based bioanalytical method for therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), twelve commercially available therapeutic mAbs and one ADC were chemically reduced, and the generated fragments were analyzed by high-temperature reversed-phase LC. For most therapeutic mAbs, single peaks of light and heavy chains were detected, indicating a possibility of homogeneous LC analysis using light chains. However, characteristic fragmentations were observed in infliximab, pembrolizumab, ramucirumab, and trastuzumab emtansine. We also performed a simple validation using the fragmented light chains for the bioanalysis of bevacizumab. The limit of detection (LOD) and limit of quantification (LOQ) of bevacizumab were 0.63 and 2.10 µg/mL, respectively, with dithiothreitol reduction, and 0.74 and 2.48 µg/mL, respectively, with tris (2-carboxyethyl) phosphine reduction. These results indicate that both the reductants confer sufficient linearity, LOQ, and LOD for the light chain analysis of bevacizumab. Thus, this method, combined with affinity purification, can be used for the bioanalysis of bevacizumab.
A quality assessment method for eight commercially available optically active flavor compounds: menthol, menthyl acetate, perillaldehyde, 1,8-cineole, α-pinene, limonene, neomenthol, and neomenthyl acetate, was developed for commercial foods. The simultaneous determination of the eight flavor compounds and the optical purity test for the complete enantiomeric separation of each flavor compound were achieved by gas chromatography-mass spectrometry (GC/MS) equipped with a tandem capillary column consisting of a fused silica column (DB-17MS) coupled with a cyclodextrin chiral column (β-DEX). The extraction of the flavor compounds from the food samples was carried out by a multiple headspace (MHS) solid-phase microextraction (SPME) technique in order to improve the quantitativeness of SPME. Optimization of MHS-SPME using a mathematical technique with repeated extraction yielded the total peak area of each flavor compound and excellent recoveries from the food samples in three repeated measurements. Of the twelve commercial samples subjected to the optical purity test, some were found to have undesirable enantiomers that are not designated as flavor compounds by the Food Hygiene Law of Japan. The results show the efficiency of the developed method, and suggest a need to conduct further optical purity tests for the quality assessment of flavor compounds in commercial foods.
Curcumin has been shown to be pharmacologically active in the prevention and treatment of various human diseases. In this study, we developed a fluorous derivatization method for selective analysis of curcumin with liquid chromatography (LC)tandem mass spectrometry (MS/MS). Curcumin was derivatized with the thiol-containing fluorous reagent (1H,1H,2H,2Hperfluoro-1-decanethiol) under mild conditions via Michael addition reaction, and the obtained derivative was introduced to a fluorous LC column (Fluofix-II 120E, 150 × 2.0 mm i.d., 5 μm, Wako). The selectively retained fluorous-derivatized curcumin on the column was also enabled highly sensitive detection with negative electrospray ionization MS/MS. Pretreatment of human serum sample was used protein precipitation with CH3CN. The calibration curve obtained by the present method showed good linearity (r2 = 0.9998) in the range of 7.4-442 ng/mL serum, and the limit of detection (S/N = 3) and limit of quantification (S/N = 10) were 1.8 ng/mL serum and 6.1 ng/mL serum, respectively. The present method was successfully applied to the analysis of curcumin in human serum sample.
Graphene was dispersed in an aqueous solution with poly(sodium 4-styrenesulfonate) as a dispersant. The charge of the graphene came to be apparently negative by the adsorption of poly(4-styrenesulfonate) ion (PSS). Two kinds of PSS were examined: the average molecular masses of 70,000 and 1,000,000 (PSS 70,000 and PSS 1,000,000, respectively). Capillary electrophoresis was used to evaluate the dispersion of the apparently anionic graphene in an aqueous solution. A broad signal corresponding to the dispersed graphene was detected in the electropherograms. The effective electrophoretic mobility of the dispersed graphene was somewhat larger at higher concentrations of PSS 70,000, suggesting that the adsorbed amount of PSS 70,000 increased. Even when the separation buffer did not contain PSS, the broad signal of the anionic graphene was still detected. The peak height and/or the peak area, as well as the effective electrophoretic mobility of the graphene decreased little at the reduced applied voltages, i.e., at longer separation/detection time. Therefore, the adsorption of PSS is irreversible or the desorption of PSS from the graphene surface is very slow. Accordingly, the dispersed graphene with PSS would be separated from the matrix PSS by the electrophoretic separation.
A simplified method for analyzing tryptophan (Trp) and its metabolites in human plasma was developed using liquid chromatography–electrospray ionization tandem mass spectrometry. Trp and its metabolites have various chemical properties but have no common functional group for derivatization. Using a reversed-phase pentafluorophenyl (PFP) column for liquid chromatography separation, Trp and its 15 metabolites (3-hydroxyanthranilic acid, 3-hydroxy-kynurenine, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 5-hydroxy-L-tryptophane, anthranilic acid, indole-3-lactic acid, kynurenine, kynurenic acid, melatonin, nicotinic acid, picolinic acid, quinolinic acid, serotonin, and xanthurenic acid were successfully separated within 15 min without derivatization. Neopterin which is known as a biomarker for inflammation and is often evaluated with Trp metabolites in several reports could also be simultaneously analyzed. With this method, Trp and its metabolites were detected with good sensitivity and selectivity without derivatization and solid-phase extraction. The method was validated in this study, showing that the relative standard deviation of 14 Trp metabolites was <15%, and accuracy was within 100% ± 20%, with the exception of nicotinic acid. The quantification range was optimized to 0.0150–100 µM covering the concentration of Trp metabolites in human plasma. Overall, 12 Trp metabolites in healthy human plasma were quantified with good precision and accuracy.
Determination of coumarin in kerosene was studied. Kerosene is discriminated from taxed fuel oil by the addition of coumarin in Japan. A fluorescence detection method has been widely used to check the legal distribution of kerosene, however, the method is inevitably resulting waste fluid consisted both of the aqueous alkaline solution and the organic solvent. By introducing a heart-cutting analysis with a set of on-line coupled packed-capillary and open-tubular columns, coumarin in kerosene was successfully determined in gas chromatography. The developed technique showed several advantageous features such as a significantly reduced waste, quick and easy separation/determination, along with a reduced sample volume required in the determination process.
Peculiar ion-pairing elution behavior of analyte anions with counter cations in electrostatic ion chromatography (EIC) using water as a mobile phase was theoretically elucidated based on the Donnan membrane equilibrium principle and charge balance condition. New parameters, fanion and fcation, containing a fluctuating coefficient, x, which corresponded to capacity factors, were derived from the Donnan equation. The fanion and fcation of the constituent anion and cation in ion-pairing elution were connected with each other by the x, and the capacity factor, kion pair (of co-eluting anion and cation) could be predicted by solving the relation of fanion = fcation for x. The elution volumes for various ion pairs thus semiempirically calculated were in good agreement with those experimentally obtained. In addition, the selective ion-pairing formation in EIC could reasonably be explained by applying the parameters to a modified classical plate theory.
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.
Change of the dosage form of the medicine is a useful method for the improvement of the medicine-taking compliance. However, the photostability of the medicine may be decreased on account of the change of the dosage form, followed by the change of the quality of it. On the other hand, there is few information focused on the photostability of the dosage-changed medicine. In this study, the effects of the change of the dosage form on the photostability of some non-steroidal anti-inflammatory drugs (NSAIDs) are investigated. Photo-exposure by means of the black light, containing ultraviolet (UV) at mainly 365 nm, induced the change of the color of naproxen tablets although the content of the active compound monitored by the high-performance liquid chromatography (HPLC) was not changed. However, the change of the dosage form of naproxen tablets to the powder and the suspension induced the change of the photostability followed by the decrease of the content of the active compound and the generation of two photoproducts. By means of the nuclear magnetic resonance (NMR) analysis, structures of two naproxen photoproducts were determined as 2-acetyl-6-methoxy-naphthalene and 1-(6-methoxy-2-naphthyl) ethanol. This is the first report evaluated the generation of naproxen photoproducts induced by the long-wavelength UV irradiation in the formulation.
A method of tube radial distribution chromatography (TRDC) based on an annual flow with inner and outer phases created through phase transformation in an open-tubular capillary was developed. The outer phase works as a pseudo-stationary phase under laminar flow conditions in TRDC. Two model fluorescent analytes, hydrophobic perylene and hydrophilic Eosin Y, were separated by TRDC using a water/acetonitrile/ethyl acetate mixed solution (3:8:4 volume ratio, organic solvent-rich or 4:3:1 volume ratio, water-rich) as an eluent solution and a fused-silica capillary tube (75 μm inner diameter and 120 cm length/100 cm effective length) as a separation column. We observed for the first time microfluidic behavior of fluorescent analytes that distributed and separated in the capillary tube visually by fluorescence microscope-charge coupled device camera. The separation mechanism in the TRDC was confirmed with visual fluorescence data and the obtained chromatographic data with UV detection. Furthermore, the elution times of perylene and Eosin Y were calculated based on their retention factors for the two phases and laminar flow conditions. The data and results obtained support the proposed separation mechanism in TRDC.
Enantiomeric separation ability on high-performance liquid chromatography (HPLC) of novel chiral stationary phase (CSP) was evaluated. A chiral selector of the CSP investigated in this study (SUMICHIRALTM OA-SHELL P1) is one of helical poly(diphenylacetylene) derivatives which is coated on core-shell silica support. OA-SHELL P1 showed excellent chiral separation ability for a wide range of aromatic chiral alcohols and it was also effective for separation of some chiral carboxylic acids, ketones and lactones. It is considered to be a new option for enantiomeric separation of these chiral compounds. OA-SHELL P1 is used in normal phase mode with a mobile phase of simple composition and one of its features is easy method development. The analysis time is relatively short even with conventional HPLC system since a core-shell support with particle size of 2.6 μm is used.