Development of an on-site therapeutic drug monitoring method using a portable spectrometer.

We report on the development of an on-site therapeutic drug monitoring (TDM) method for vancomycin (VCM) utilizing a portable spectrometer and commercially available immunoturbidimetric assay reagents designed for automated clinical chemistry analyzers. The method enables the quantification of VCM in plasma samples within 10 min, with a good correlation between the measured values and the theoretical values (r2 = 0.995). The intra and inter-day precisions were found to be below 12.5% and 17.7%, respectively. Moreover, we established a correlation between the quantitative values using this method and those measured through HPLC-UV and automated clinical chemistry analyzers, showing good reliability (R2 = 0.970 and 0.951, respectively). This method allows anyone to rapidly perform TDM at the bedside and is expected to be used to evaluate appropriate drug therapy.

Development of a novel method for analysing N-acetyl-DL-leucine enantiomers in human fingernail by UPLC-ESI-MS/MS and the evaluation in diabetes mellitus.

BACKGROUND: Recent research has been reported that N-acetyl-leucine content is significantly reduced in the saliva of diabetic patients, but no reports of detection in human nails have been found. This study aims to develop a novel method for the chiral separation of N-acetyl-DL-leucine (Ac-DL-Leu) in human fingernails to investigate the differences between healthy volunteers (HVs), prediabetes (PDs) and diabetic patients (DPs), and to verify its effectiveness in early warning of diabetes. METHOD: Chiral resolution was performed using DBD-Apy pre-column derivatization on a C18 column (2.1 × 150 mm, 1.9 µm) at 40 °C, and detected by UPLC-ESI-MS/MS. RESULTS: The resolution and the limit of detection (LOD) of Ac-DL-Leu were 1.75 and 1.50 fmol, respectively. The linear range of Ac-DL-Leu was 10-2000 fmol and the determination coefficient (R2) was above 0.9997. The recovery of Ac-DL-Leu in human nails was 96.92-105.69 %. The contents of Ac-D-Leu and Ac-L-Leu were analyzed in 18 HVs, 13 PDs and 16 DPs fingernails. The results showed that their contents were significantly lower in DPs than in PDs and HVs (p < 0.0001). CONCLUSIONS: A method for evaluating the effectiveness of Ac-DL-Leu enantiomers in human fingernails as a biomarker for diabetes was firstly developed.

Precolumn derivatization LC/MS method for observation of efficient hydrogen sulfide supply to the kidney via d-cysteine degradation pathway.

d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (H2S) by d-amino acid oxidase (DAO)/3-mercaptopyruvate sulfurtransferase pathway. The pathway is required for H2S supplementation that ameliorates acute kidney injury after the oral administration of d-Cys in mice. However, whether the rate-limiting activity of DAO regulates the tissue-selectivity or the extent of d-Cys degradation and H2S supplementation remains unclear. Here, to analyze the levels of d-Cys and H2S, we use two derivatization methods, a new method with no detectable isomerization of Cys and an established method for H2S. The derivatives were determined by LC/MS using a C18 column. With the methods, we show that inhibition of DAO significantly suppresses the H2S supplementation and d-Cys degradation in the mouse kidney. Additionally, we found that d-Cys is more efficiently metabolized into H2S than l-Cys in the kidney. Our results reveal the utility of the method and support the advantage of d-Cys administration in improving the supply of H2S to the kidneys.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

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 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

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 <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

Machine learning guided prediction of liquid chromatography-mass spectrometry ionization efficiency for genotoxic impurities in pharmaceutical products.

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.

Development of a selective and sensitive analytical method to detect isomerized aspartic acid residues in crystallin using a combination of derivatization and liquid chromatography mass spectrometry.

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-ß-Asp, d-α-Asp, and d-ß-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation. Of three candidate derivatization reagents tested for the separation of peptides containing isomerized aspartic acid, 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazine-2-yl) pyrrolidine-2-carboxylate (DMT-(R)-Pro-OSu) was the most suitable reagent for separating isomerized peptides and improved the sensitivity of mass spectrometry by 50-fold. This method was applied to analyze heat-denatured crystallin. Asp58 and Asp151 residues in αA-crystallin (AAC) exhibited the highest isomerization rate in heated crystallin. Furthermore, the analysis of α-crystallin extracted from bovine eye lens identified isomerized Asp residues (Asp24/35, Asp58, and Asp151 in AAC and Asp140 in αB-crystallin (ABC)). These results indicate that the newly developed method using chiral derivatization provides selective and sensitive analysis of isomerized Asp sites in α-crystallin protein. This novel method will allow for the identification and quantification of isomerized amino acids in crystallin proteins.

Bioanalytical methods for therapeutic monoclonal antibodies and antibody-drug conjugates: A review of recent advances and future perspectives.

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.

An accurate differential analysis of carboxylic acids in beer using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry based on chiral derivatization combining three isotopic reagents.

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.

DL-Amino Acid Analysis Based on Labeling with Light and Heavy Isotopic Reagents Followed by UPLC-ESI-MS/MS.

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.

Introducing an Experimental Design Approach for Efficient Optimization of Chiral Derivatization Conditions for D- and L-Glyceric Acids.

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.

Chiral Metabolomics Using Triazine-Based Chiral Labeling Reagents by UPLC-ESI-MS/MS.

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.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification⁻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 µg/mL and showed good correlation coefficients (r² > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 µg/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.

Isotope Corrected Chiral and Achiral Nontargeted Metabolomics: An Approach for High Accuracy and Precision Metabolomics Based on Derivatization and Its Application to Cerebrospinal Fluid of Patients with Alzheimer's Disease.

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 were successfully determined, including 8 chiral ones.

High-Throughput Bioanalysis of Bevacizumab in Human Plasma Based on Enzyme-Linked Aptamer Assay Using Anti-Idiotype DNA Aptamer.

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.

Development of Highly Sensitive Analysis Method for Histamine and Metabolites in Pregnant Women's Fingernail by UPLC-ESI-MS.

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.

Sensitive and Comprehensive LC-MS/MS Analyses of Chiral Pharmaceuticals and Their Hepatic Metabolites Using Ovomucoid Column.

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.

Current Mass Spectrometric Tools for the Bioanalyses of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugates.

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 novel, simplified strategy of relative quantification N-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q.

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2=0.9999; d8/d0, R2=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC-MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.

Gas-Phase Stability of Negatively Charged Organophosphate Metabolites Produced by Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization.

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. Graphical Abstract.