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d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (H

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (K

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and 13C-isotope coded derivatization (ICD) reagents, i.e., iDMT-(S)-A and iDMT-(S)-PO. The advantage of the method is the correction of all detected derivatives by parallel derivatization of the isotope-coded and noncoded reagents. The automatic data analysis platform using an MSDIAL and ICD discrimination program, called DINA, was also developed and used for the data analysis process. As a result, a 0.5–2.0% (d-/l-isomer) variation of the isomers was correctly recognized in the automatic data analysis step. Both the semiquantitative comparison and identification efficiency were improved as a result of the high resolution/accuracy of the MS and MS/MS spectra derived from the DDA analysis. This method was used for biomarker discovery in the cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD). Twenty-four biomarker candidates wer...

The dried saliva spot (DSS) seems to be a convenient sampling technique for bioanalysis. In our previous research, the DSS sampling was used for the determination of dl -lactic acid (DL-LA) and the D/L ratio of diabetic, pre-diabetic and healthy persons. However, the method required an expensive tandem MS/MS apparatus to obtain a highly sensitive detection. To avoid the use of such instrument, this paper proposes cheaper determination systems by ultra-performance liquid chromatography (UPLC) with fluorescence (FLR) and/or single mass spectrometry (MS). The carboxylic acids including DL-LA in the DSS were labeled with a chiral fluorescence reagent, (S)(+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-(S)(+)-APy), and determined by UPLC-FLR-MS. The limits of detection of LA by FLR and MS were approximately 1.0 μM (0.1 pmol) and 0.20 μM (0.02 pmol), respectively, whereas the limits of quantification were 5.0 μM (0.5 pmol) by FLR and 1.0 μM (0.1 pmol) by MS. The proposed DSS method was used for the determination of the enantiomers of LA of diabetic, pre-diabetic and healthy persons. The D/L-LA ratios obtained from the FLR were correlated with those of MS, although the ratios of the FLR were slightly lower than those of MS. Furthermore, the D/L-LA ratios of the present FLR or single MS were well correlated with those of the tandem MS/MS obtained from our previous study. The results indicated that the UPLC-FLR-MS was alterable to the previous UPLC-MS/MS method. Consequently, the proposed UPLC-FLR and/or UPLC-MS seem to be applicable as a cheaper screening method for diabetes mellitus using the DSS sampling.

The limitation and control of genotoxic impurities (GTIs) has continued to receive attention from pharmaceutical companies and authorities for several decades. Because GTIs have the ability to damage deoxyribonucleic acid (DNA) and the potential to cause cancer, low-level quantitation is required to protect patients. A quick and easy method of determining the liquid chromatography-mass spectrometry (LC/MS) conditions for high-sensitivity analysis of GTIs may prospectively accelerate pharmaceutical development. In this study, a quantitative structure-property relationship (QSPR) model was developed for predicting the ionization efficiency of compounds using liquid-chromatography-mass spectrometry (LC/MS) parameters and molecular descriptors. Before implementing the QSPR prediction model, linear regression analysis was performed to model the relationship between the ionization efficiency and the LC/MS parameters for each compound. Comparison of the predicted peak areas with the experimentally observed peak areas showed good agreement based on the coefficient of determination (R2 > 0.96). The machine learning-based QSPR approach begins with computation of the molecular descriptors expressing the physicochemical properties of a compound, followed by a genetic algorithm-based feature selection. Linear and nonlinear regression were performed, and support vector machine (SVM) was selected as the best machine learning algorithm for the prediction. The SVM algorithm was developed and optimized using 1031 experimental data points for nine compounds, including well-known GTIs. Validation of the model by comparison of the predicted and observed relative ionization efficiencies (RIE) showed a high coefficient of determination (R2 = 0.96) and low root mean squared error value (RMSE = 0.118). Finally, this established prediction model was applied to hydrophilic interaction liquid chromatography coupled with MS for a new compound in new mobile phase compositions and new MS parameter settings. The RMSE of the predicted versus observed RIE was 0.203. This prediction accuracy was sufficient to determine the starting point of the LC/MS method development. The methodology demonstrated in this study can be used to determine the LC/MS conditions for high sensitivity analysis of GTIs.

A sensitive analytical method was developed for the simultaneous detection of 11 chiral pharmaceuticals and their hepatic metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ovomucoid chiral column. After optimization of the LC conditions, all pharmaceuticals examined were enantio-separated with Rs of >0.82 in LC-MS/MS analysis. The limit of detections of all pharmaceuticals by MS/MS detection ranged from 1.2 to 92.3 nM, which is approximately 1000 - 25000 times lower than those obtained by UV detection. From hepatic metabolite analyses in P450-expressing cells, metabolites of three pharmaceuticals were detected and enantio-separated. By using the proposed method, changes in the optical isomer ratio of the hepatic metabolites chlorpheniramine and verapamil caused by differential cytochrome P450 enzyme expression for each isomer, could be successfully traced.

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm × 2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r2 > 0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N = 3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.

A novel computer-assisted methodology for the simultaneous optimization of aqueous pH and binary organic eluent composition through a broad range of analytical conditions of reversed-phase ultra high-performance liquid chromatography is proposed. Two of nonlinear prediction models were employed to fit into the retention time (tR) on a linear gradient elution with a predefined slope. One model was derived from Bernoulli-type probability distribution to predict the value of tR against the pH value of the aqueous eluent. This sigmoid-shaped model was successfully fitted for tR value shift in the presence of three levels of organic eluent compositions (volumetric mixing of acetonitrile/methanol ratios 1:0, 1:1, and 0:1). The resultant pH versus tR value models were subsequently combined into grid form by quadratic multiple regression models based on the solubility parameter theory and their binary organic composition axes. The predicted tR values afforded from grid models were highly accurate for 13 different acidic non-steroidal anti-inflammatory drugs [root mean square error (RMSE) ≤0.030] and 16 basic histamine H1-receptor blockers (RMSE ≤0.067) in a pH ranging from 2.5 to 9.0 and an acetonitrile/methanol volumetric mixing ratio ranging from 1:0 to 0:1. Each compatibility score was defined as the indicator of the peak separation. Scores were calculated for all combinations of aqueous pH values and binary organic compositions via the predicted tR values. A colored map generated from the calculated scores was greatly effective in determining optimal combinations of both mobile phase conditions. By employing this predictive data, all analytes in both acidic and basic sample mixtures were finally separated at their respective optimized conditions.

BACKGROUND Dexmedetomidine is a highly selective central α2-agonist used as a sedative in pediatric intensive care unit (PICU). However, little is known about the relationship between dexmedetomidine dose and its plasma concentration during long-term infusion. We have previously demonstrated that the sedative plasma dexmedetomidine concentration is moderately correlated with the administered dose in adults (r = 0.653, P = 0.001). We hypothesized that there would be a similar relationship between the sedative dexmedetomidine concentration and administered dose in infants. METHODS All patients admitted to the PICU at Nagoya City University Hospital, Japan, between November 2012 and March 2013 were eligible for inclusion in the study. Plasma dexmedetomidine concentration was measured by ultra-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS We measured the plasma dexmedetomidine concentration in 203 samples from 45 patients. Of these, 96 samples collected from 27 patients < 2 years old were included in this study. All patients received dexmedetomidine at 0.12-1.40 µg/kg/h. The median administration duration was 87.6 hours (range: 6-540 hours). Plasma dexmedetomidine concentration ranged from 0.07 to 3.17 ng/ml. Plasma dexmedetomidine concentration was not correlated with the administered dose (r = 0.273, P = 0.007). The approximate linear equation was y = 0.690x + 0.423. CONCLUSIONS In infants, plasma dexmedetomidine concentration did not exhibit any correlation with administered dose, which is not a reliable means of obtaining optimal plasma concentration.

Liquid chromatography coupled with mass spectrometry (LC-MS) is one of the most prominent analytical techniques due to its inherent selectivity and sensitivity. LC-MS is currently the first choice for high-throughput bioanalysis due to the advancements in MS instruments and the analytical software. Based on this situation, we are developing various types of derivatization reagents, including chiral reagents for MS and/or MS/MS detection. These developed reagents are adopted for the detection of biomarker candidates related to diseases. The biomarker candidates include not only achiral molecules, but also chiral ones. Although determining the already-identified chiral molecules is relative easy, it is very difficult to identify and/or determine unknown enantiomer(s) in real samples. To solve this difficulty, we proposed a new strategy to identify unknown enantiomeric biomarkers related to diseases. This review paper deals with the development of derivatization reagents for amines and carboxylic acids in LC-MS analysis and their application to bioanalysis.

As the use and development of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) increases, the need for accurate and robust bioanalytical methods is also increasing. Up to about a decade ago, bioanalysis of therapeutic mAbs was performed only by ligand-binding assay (LBA), and this was the only available method for absolute quantitation. Recent technological advances in liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) and LC-high-resolution mass spectrometry can realize specific, accurate, and multiplex bioanalysis of these with a wide dynamic detection range. In this review, we introduce recent analytical techniques in bioanalytical methods for therapeutic mAbs and ADCs using LC-MS with three (bottom-up, top-down, and middle-down) proteomics strategies from the viewpoint of improving detection sensitivity, assay accuracy, and selectivity.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d

A sensitive analytical method was developed for individual analyses of D- and L-glyceric acids by chiral derivatization - liquid chromatography-tandem mass spectrometry. To elucidate rapid and efficient optimization for derivatization we newly introduced a concept of design of experiments (DOE). The optimization of major 5 factors in the derivatization could be predicted with only 28 measurements. By applying DOE to optimization, the yields of desired derivatives increased five-fold against before optimization.

The determination of enantiomers of biological molecules is an important issue because a significant difference in the activity of the enantiomers is generally observed in biological systems. Chiral separations can be carried out by direct resolution using a chiral stationary column or by indirect resolution based on the derivatization with a chiral reagent. Many chiral-labeling reagents for ultraviolet-visible and fluorescence detections have been developed for various functional groups, such as amine and carboxylic acid. However, there are hardly any labeling reagents for LC-MS-specific detection. Based on this observation, we have developed several chiral-labeling reagents for LC-MS/MS analysis.This chapter describes methodologies and applications for the indirect LC-MS/MS determination of biological chiral molecules using triazine-based chiral-labeling reagents, i.e., (S and R)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S and R)-Apy) for carboxylic acids and (S and R)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S and R)-Pro-OSu) for amines and amino acids. A reliable method for the non-targeted chiral metabolomics is also described in this chapter.

We developed a highly sensitive and accurate differential method for analyzing chiral and achiral carboxylic acids in Japanese commercial beers, using ultra high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS), based on chiral derivatization combining three isotopic chiral reagents: 12C-DMT-3(S)-Apy, 13C2-DMT-3(S)-Apy, and d6-DMT-3(S)-Apy. By combining these reaction solutions and analyzing the resulting mixture, simultaneous comparative analyses of the three samples could be achieved while offsetting the matrix effects on the samples. Using this approach, it was possible to differentiate the beers according to the type (draft, low-malt, and non-alcoholic), manufacturer, and storage conditions (temperature and storage period), based on the concentrations of carboxylic acids in the beers. Furthermore, we identified α-ketoglutaric acid as a new marker for determining the storage condition of Japanese beers. The proposed method would provide insights into the identification of markers in various fields, such as in biological samples, as well as food and beverages.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification&ndash, high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 &mu, g/mL and showed good correlation coefficients (r2 &gt, 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 &mu, g/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.

We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A-HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 μg/mL, and showed a good correlation coefficient ( r2 = 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7 μm) (Rs = 1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The ratios of D/L-alanine in different yogurt products were successfully determined by the proposed method. The D/L ratios were almost comparable to those obtained from only using light reagent (i.e., L-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of DL-amino acids, not only in food products but also in biological samples.

The determination of enantiomers of biological molecules is an important issue because a significant difference in the activity of the enantiomers is generally observed in biological systems. Chiral separations can be carried out by direct resolution using a chiral stationary column or by indirect resolution based on the derivatization with a chiral reagent. Many chiral-labeling reagents for ultraviolet-visible and fluorescence detections have been developed for various functional groups, such as amine and carboxylic acid. However, there are hardly any labeling reagents for LC-MS-specific detection. Based on this observation, we have developed several chiral-labeling reagents for LC-MS/MS analysis.This chapter describes methodologies and applications for the indirect LC-MS/MS determination of biological chiral molecules using triazine-based chiral-labeling reagents, i.e., (S and R)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S and R)-Apy) for carboxylic acids and (S and R)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S and R)-Pro-OSu) for amines and amino acids. A reliable method for the non-targeted chiral metabolomics is also described in this chapter.

A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp

We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI-MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative's peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.

This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL(-1) (bevacizumab) and 1-10 μg mL(-1) (infliximab), and they had strong correlation coefficients (r(2) > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL(-1), and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL(-1), respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.

The presence of elevated uric acid (UA) levels is a sign of gout, that is, hyperuricemia. In this study the monitoring of the UA levels in less-invasive biological samples, such as the human fingernail, is suggested for the diagnosis and therapy of gout. Twenty-six healthy volunteers (HV) and 22 gout patients (GP) were studied. The UA was extracted from human fingernail samples, then separated on an Inertsil ODS-3 column (250 × 4.6 mm i.d., 4.0 μm, GL Sciences) by isocratic elution using methanol-74 mm phosphate buffer (pH 2.2) 2:98 (v/v). A UV detector was used to monitor the samples at 284 nm. Using the developed method, different UA concentrations were found in the GP and HV. When comparing the concentrations from GP with those from HV, a statistically significant correlation was observed between the UA (p < 0.01). In this study, the UA was confirmed as a potential biomarker for the diagnosis and therapy of gout. We have developed a novel sensitive, and simple method for the determination of UA in the fingernails of GP and HV. The human fingernail may serve as a noninvasive biosample for the diagnosis of gout. Copyright © 2016 John Wiley & Sons, Ltd.

In this study, a highly sensitive analysis method for the rapid detection of histamine (HA), imidazole-4-acetic acid (IAA) and 1-methylhistamine (MHA) in pregnant women's fingernails was developed using the ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). HA and MHA were connected with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) as the derivation reagent for the first time. IAA was derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) successfully. The derivative mixtures were simultaneously separated within 8 min on an ACQUITY UPLCTM BEH C18 column (1.7 μm, 100 × 2.1 mm i.d.) by isocratic elution using a mixture of 20 mM HCOONH4 and CH3CN (82:18) as the mobile phase, and sensitively detected by selected reaction monitoring (SRM). The quantitative analysis of HA, IAA, and MHA are performed by SRM using the fragmentation transitions of m/z 337.2 → 292.1, 420.6 → 375.1 and 351.2 → 306.0 under the positive ESI mode. The calibration curves for HA, IAA and MHA are presented herein, and their correlation coefficient were found to be above 0.9998, the measured detection limit for derivatized histamine and metabolites ranged from 0.06 to 0.15 fmol, and the relative standard derivation of intra-day and inter-day assays was 6.3%. Furthermore, the mean recoveries (%) of the standards added to human fingernails were in the range of 90.2 - 100.5%. The validated method was successfully applied to analyze human fingernail samples from three pregnant women and three healthy non-pregnant women. To the best of our knowledge, this report about the detection of histamine and metabolites in the fingernails of pregnant women's fingernails is the first published.

The increase in the use of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) has made the detailed bioanalysis of these drugs essential not only for planning optimal therapeutic programs for clinical practice, but also for evaluating the biological equivalencies in the development of other biosimilars. The ligand binding assays that are widely in use now are being replaced rapidly by the highly accurate, sensitive, and selective analytical method using a mass spectrometer. This review will discuss the progress in and challenges observed during the development of a mass spectrometry-based bioanalytical method for therapeutic mAbs and ADCs.

A rapid and simple analytical method for the quantification of uric acid (UA) in human fingernails by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. UA was extracted from human fingernail samples at 90°C for 20min, then separated on an Inertsil ODS-2 column (250×4.6mm I.D., 5.0μm, GL Sciences) by isocratic elution using methanol: 74mM phosphate buffer (pH 2.2) 2:98 (v/v). An UV detector was used to monitor at 284nm. The results indicated that under optimized measurement conditions results were achieved within 8.0min, and a good linearity was achieved from the calibration curves (r(2)>0.9999) in the range of 1.0-10000ng; the limit of detection (S/N=3) was 2.0pg, the inter-day and intra-day assay precisions were all less than 0.46% and the mean recoveries (%) of the uric acid spiked in the human fingernail were 101.95%. The amounts of UA in the fingernails of healthy volunteers were determined.

In this study including the field of metabolomics approach for food, the evaluation of untargeted compounds using HILIC-ESI/TOF/MS and multivariate statistical analysis method is proposed for the assessment of classification, contamination and degradation of infant formula. HILIC mode is used to monitor more detected numbers in infant formulas in the ESI-positive scan mode than the reversed phase. The repeatability of the non-targeted contents from 4 kinds of infant formulas based on PCA was less than the relative standard deviation of 15% in all groups. The PCA pattern showed that significant differences in the classification of types and origins, the contamination of melamine and the degradations for one week were evaluated using HILIC-ESI/TOF/MS. In the S-plot from the degradation test, we could identify two markers by comparison to standards as nicotinic acid and nicotinamide. With this strategy, the differences from the untargeted compounds could be utilized for quality and safety assessment of infant formula.

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.999, range from 10 pmol/mL to 50 nmol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus. Copyright © 2015 John Wiley & Sons, Ltd.

Early and definitive diagnosis of Alzheimer's disease (AD) can lead to a better and more-targeted treatment and/or prevention for patients. In the diagnostic biomarkers of AD, the blood sample represents a more non-invasive, inexpensive and acceptable sources for repeated measurements than the cerebrospinal fluid. In this study, the fingerprinting metabolomics was proposed for the challenge of the blood-based diagnosis of defined AD by hydrophilic interaction liquid chromatography mass spectrometry (HILIC/MS). These plasma samples were selected from postmortem specimens based on these pathological examinations. Firstly, we compared these HILIC columns for the non-targeted metabolic assay using pooled plasma. The principal component analysis plot of these seven columns was performed using the repeatability of these chromatograms, and can be used to visualize trends in data sets by three-dimensional dispersion, contributory standard deviation and the number of detections. Based on these results, TSK-Amide 80 and TSKgel-NH2 columns are used as a reliable HILIC/MS assay of blood-based AD metabolomics that showed metabolic profiling of the AD pathology in MS chromatograms that ranged from 1182 to 2284 compounds. A total of 54 peaks were evaluated in order to identify useful ion signal candidates using an orthogonal partial least-squares-discriminant analysis. These peaks were then specifically analyzed using the HILIC–tandem MS assay by a receiver operating characteristic curve and linear discriminant analysis for the diagnosis of the defined AD. The fingerprinting metabolomics can overcome the limitations of previous challenging blood-based diagnosis of AD, and directly evaluates the specific comparative statistical values from the raw data.

A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail. N1, N8-diacetylspermidine (DiAct-Spd), N1, N12-diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) [the internal standard (IS)] were extracted from human fingernail samples by a MeOH:5 M HCl solution, followed by 4-( N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min). The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLC-ESI-MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z 455.20 → 100.07, 737.25 → 100.07 and 567.10 → 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve is between 0.04 ng mL−1 and 10 ng mL−1 for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5–10 pg mL−1. A good linearity was achieved from the calibration curves ( r2 > 0.9999), and the intra-day and inter-day assay precisions were less than 7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18–97.11. The present method proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed method was used to analyze human fingernail samples from 15 lung-cancer patients and 22 healthy volunteers. Diacetylpolyamines were detected from the fingernails of the lung-cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer patient group tended to be higher than those in the healthy volunteers.

Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water-soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high-purity crocetin from gardenia yellow using high-speed countercurrent chromatography is described. High-speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans-crocetin is easily converted to about 15% cis-crocetin (85% trans-crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high-speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.

During liver injury, hepatic stellate cells (HSCs) are activated by various cytokines and transdifferentiated into myofibroblast-like activated HSCs, which produce collagen, a major source of liver fibrosis. Therefore, the suppression of HSC activation is regarded as a therapeutic target for liver fibrosis. Several epidemiological reports have revealed that caffeine intake decreases the risk of liver disease. In this study, therefore, we investigated the effect of caffeine on the activation of primary HSCs isolated from mice. Caffeine suppressed the activation of HSC in a concentration-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator, had no effect on the caffeine-induced suppression of HSC activation. None of the isoform-selective inhibitors of phosphodiesterase1 to 5 affected changes in the morphology of HSC during activation, whereas CGS-15943, an adenosine receptor antagonist, inhibited them. Caffeine had no effect on intracellular cAMP level or on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. In contrast, caffeine significantly decreased the phosphorylation of Akt1. These results suggest that caffeine inhibits HSC activation by antagonizing adenosine receptors, leading to Akt1 signaling activation.

The formation mechanisms of singly and multiply charged organophosphate metabolites by electrospray ionization (ESI) and their gas phase stabilities were investigated. Metabolites containing multiple phosphate groups, such as adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), and D-myo-inositol-1,4,5-triphosphate (IP3) were observed as doubly deprotonated ions by negative-ion ESI mass spectrometry. Organophosphates with multiple negative charges were found to be unstable and often underwent loss of PO3 –, although singly deprotonated analytes were stable. The presence of fragments due to the loss of PO3 – in the negative-ion ESI mass spectra could result in the misinterpretation of analytical results. In contrast to ESI, matrix-assisted laser desorption ionization (MALDI) produced singly charged organophosphate metabolites with no associated fragmentation, since the singly charged anions are stable. The stability of an organophosphate metabolite in the gas phase strongly depends on its charge state. The fragmentations of multiply charged organophosphates were also investigated in detail through density functional theory calculations.

An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 μM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.

RATIONALE Mecasermin is used to treat elevated blood sugar as well as growth hormone-resistant Laron-type dwarfism. Mecasermin isolated from inclusion bodies in extracts of E.coli must be refolded to acquire sufficient activity. However, there is no rapid analytical method for monitoring refolding during the purification process. METHODS We prepared mecasermin drug product, in-process samples during the oxidation of mecasermin, forced-reduced mecasermin, and aerially oxidized mecasermin after forced reduction. Desalted mecasermin samples were analyzed using MALDI-ISD. The peak intensity ratio of product to precursor ion was determined. The charge state distribution (CSD) of mecasermin ions was evaluated using ESI-MS coupled with SEC-mode HPLC. The drift time and collision cross-sectional areas (CCS) of mecasermin ions were evaluated using ESI-IMS-MS coupled with SEC-mode HPLC. RESULTS MALDI-ISD data, CSD values determined using ESI-MS, and the CCS acquired using ESI-IMS-MS revealed the relationship between the folded and unfolded proteoforms of forced-reduced mecasermin and aerially oxidized mecasermin with the free-SH: protein ratio of mecasermin drug product. The collision cross-sectional area, which is determined using ESI-IMS-MS, provided proteoform information through rapid monitoring (

A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all

Dicyandiamide is a compound for reducing the negative effects of greenhouse gas emissions and nitrate leaching into waterways. In this study, the trace contamination of dicyandiamide in infant formula was analysed by stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC–MS/MS). Dicyandiamide and a stable isotope internal standard were monitored by multiple reaction-monitoring with mass transitions: m/z 85 → 68/43 and m/z 89 → 71/45 in the electrospray positive ion mode. For sample preparation of the infant formula, a diluted/filtered procedure was developed for this assay. The calculated LOD and LOQ values were 0.01 or 0.05 ng/mL for the standard solution, respectively. The averaged recovery and precision were 110.8% and 7.4%, respectively. This assay was applied to monitor 23 infant formulas, and the dicyandiamide contamination in one sample was detected and quantified at 79.1 ± 1.2 ng/g (ppb) powder. We suggest that it is necessary to cautiously monitor the DCD in common products from international countries.

We established a simple, sensitive, and reproducible method to analyze the histamine and tyramine levels in Japanese soy sauce and its mash (called moromi) using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Histamine and tyramine quantification was performed using their stable isotopes for electrospray ionization-tandem mass spectrometry in the selected reaction monitoring mode. The sample pretreatment process was a simple, one-step liquid-liquid extraction. HILIC separation was performed with a gradient elution of aqueous ammonium formate and acetonitrile. Because of validation tests, the linearity, the accuracies, and precisions were sufficient. The limit of detection and the limit of quantification were 0.09 and 0.29 ppm for histamine and 0.13 and 0.42 ppm for tyramine, respectively. We successfully applied this method to histamine and tyramine determination in four kinds of commercial Japanese soy sauces and also in moromi samples during soy sauce production.