Liquid chromatography/mass spectrometry (LC/MS) method is becoming an important approach for therapeutic antibody assays as an alternative to the ligand-binding assay (LBA) method. The LC/MS method has some advantages over the LBA method, such as a wider dynamic range and short developing time. However, the development of the LC/MS method is often challenging because of complicated sample preparation processes involving affinity purification, denaturation, reduction and methylation, enzymatic digestion, and peptide purification. In addition, it is difficult to select a sensitive and specific surrogate peptide that allows the determination of the lower limit of quantitation of the analytical target. Another issue remains in the bioanalytical method validation (BMV) of the LC/MS method for large molecules. The BMV guideline on the LC/MS method for small molecules and that on the LBA method are helpful while developing a bioanalytical method for large molecules using LC/MS; however, these guidelines lack inherent characteristics related to bioanalysis of large molecules by the LC/MS method. In this review, we describe points to be considered regarding selection of surrogate peptides and optimization of the sample preparation processes in the LC/MS method for therapeutic antibody assays. Furthermore, we propose criteria for BMV of the LC/MS method. We expect that this review will aid in the development of sensitive, specific, and robust bioanalytical LC/MS methods for therapeutic antibodies.

We optimized several analytical conditions for more sensitive and precise HT-RPLC analysis of the therapeutic monoclonal antibody (mAb), bevacizumab. Specifically, we (1) optimized the sample preparation process to reduce adsorption and aggregation of bevacizumab, (2) introduced a sample concentration process using a centrifugal ultrafiltration unit to increase detection sensitivity, and (3) used another therapeutic mAb as an internal standard to improve analytical precision. The optimized method for bevacizumab analysis was shown to have low detection and quantification limits of 0.010 and 0.032 µg/mL, respectively, good correlation coefficients (r2 > 0.9997), and good intra- and inter-day precisions within < 12.0 %. This study provides an important methodology for the intact bioanalysis of therapeutic mAbs, not merely their LC measurement.

This paper reviews recent 5-years application researches using online sample preconcentration (OSP) methods in capillary electrophoresis (CE) coupled with mass spectrometry (MS). CE-MS has excellent analytical features such as high-resolution separation, rapid analysis time, low sample consumption, and small consumption of organic solvents. Recently, low flow-rate interface for electrospray ionization has been developed and zmol-level sensitivity has been achieved. However, application of CE-MS has still been prevented due to small capacity of sample injection volume. OSP methods have been developed to address this issue and up to 5,000-fold sensitivity improvement was obtained so far. Various application has been carried out such as proteomics, metabolomics, and glycomics, and CE-MS attracts much attention as the highest-performance analytical tool for small-volume bioanalysis. Taking account of further progress in CE-MS, smart and practical application of OSP methods will be more important in various analytical fields.

Sodium dehydroacetate (SDA) was extracted from cosmetic powders using a Na2CO3-methanol-H2O system. After the extract was neutralized, the resulting dehydroacetic acid (DAA) reacted with 4-nitrophenylhydrazine∙HCl (4-NPH∙HCl) to give the corresponding hydrazone, which was determined by an HPLC-VIS (400 nm) system. Using 4-NPH∙HCl in great excess was suitable for detecting the presence of DAA, which almost reacted with the hydrazine to yield the corresponding hydrazone. As a result, it was determined that contaminants in the cosmetic powder did not interfere with analysis of the hydrazone. Importantly, the molar absorptivity of the hydrazone (1.29 × 104 at 400 nm) is five times that of DAA (2.57 × 103 at 230 nm). Accordingly, detection of the hydrazone at 400 nm in a new analytical method might have fivefold higher sensitivity compared with that of DAA at 230 nm in the standard method.

The circadian profiles of D-alanine (Ala) amounts in human serum and urine have been clarified using a two-dimensional HPLC system combining a microbore reversed-phase C18 column and a Pirkle-type enantioselective column. The two-dimensional HPLC system enables the determination of trace amounts of D-Ala in the clinical samples without severe interference from intrinsic substances in the human physiological fluids. In the volunteers having normal sleep-awake patterns, the amounts of D-Ala in the serum were high in the late evening and low in the morning. On the other hand, in the volunteers having the reversed sleep-awake patterns, the amounts of D-Ala in the serum were high in the morning and low in the nighttime. The similar circadian changes of D-Ala amounts were observed in the urine. These 24-h profiles of D-Ala are almost the same as those observed in rats and mice, suggesting that D-Ala has fundamental physiological functions related to rest-active conditions in mammals, and could also be useful as the biomarker for sleep-awake profiles.

A novel method for the determination of semi-volatile polycyclic aromatic hydrocarbons (PAHs) in gaseous samples was developed using a partition-based extraction device. The target compounds were collected and concentrated on the basis of partitioning between air and octadecyl (C18) chains bound to macroporous silica particles. The extracted PAHs were rapidly desorbed by just passing 10 mL of acetone. The desorption solvent was then subjected to gas chromatography-mass spectrometry (GC-MS) to determine PAHs. On the basis of the quantitative evaluation of the extraction and desorption conditions for 15 PAHs, the benefits of the partition-based extraction for typical airborne PAHs were clearly indicated. Finally, the developed method was successfully applied for the determination of PAHs in tobacco side-stream smoke.

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.

Perfluoroalkyl-containing compounds show a unique “fluorous” property, which is related to the specific affinity, i.e. fluorophilicity, between perfluoroalkyl groups. Therefore, fluorous compounds can be easily and selectively isolated from non-fluorous compounds utilizing liquid‒liquid extraction, solid phase extraction, and liquid chromatographic techniques via their fluorophilicity. We have also developed novel methods for the selective analysis of biogenic-related compounds using fluorous separation techniques. These methods involve the attachment of perfluoroalkyl groups to target analytes, followed by their selective separation from endogenous biological matrices with appropriate fluorous separation techniques. Because the perfluoroalkylated compounds are generally not included in biological matrices, highly selective analysis of perfluoroalkyl-attached target analytes is possible. This review summarizes our recent works concerning biogenic-related compound analysis methods using fluorous separation techniques.

Biological thiols – homocysteine (Hcy), cysteine (Cys), γ-glutamylcysteine (γGluCys), glutathione (GSH), and cysteinylglycine (CysGly) – in plasma samples of cystathionine β-synthase (CBS, EC 4.2.1.22)-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.

Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the peak widths (or half-widths) on the flow rate is reported. A new approach to quantify the deterioration of column performance for slow and fast flow rates and to characterize chromatographic columns is proposed. The approach makes use of the width (or half-widths) for a set of compounds with similar interaction kinetics and does not require knowledge of the extra-column contributions to the total variance. The chromatographic data of two sets of compounds of different natures (sulfonamides and β-blockers), eluted from Spherisorb and Chromolith columns with acetonitrile-water mixtures, are used to illustrate the approach.