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.

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.

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.

Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction.

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.

A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin-like growth factor-1 (mecasermin).

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 area (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 CCS area, which is determined using ESI-IMS-MS, provided proteoform information through rapid monitoring (<2 min) of in-process samples during the manufacture of mecasermin. CONCLUSIONS: ESI-IMS-MS coupled with SEC-mode HPLC is a rapid and robust method for analyzing the free-SH:protein ratio of mecasermin that allows proteoform changes to be evaluated and monitored during the oxidation of mecasermin. ESI-IMS-MS is applicable as a process analytical technology tool for identifying the "critical quality attributes" and implementing "quality by design" for manufacturing mecasermin.

Highly sensitive glycosylamine labelling of O-glycans using non-reductive ß-elimination.

When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.

Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors.

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.

Retention of glycopeptides analyzed using hydrophilic interaction chromatography is influenced by charge and carbon chain length of ion-pairing reagent for mobile phase.

Characterization of the glycans of glycoproteins is essential for the development and production of biologics. Numerous methods are available for analyzing the glycans of glycoproteins directly and labeled glycans. Nevertheless, glycopeptides are difficult to resolve because of their exceptional complexity and the microheterogeneity of glycans. These properties represent technical challenges to efforts to insure the accurate characterization of biopharmaceuticals to comply with regulatory requirements. Therefore, we investigated the retention behavior of peptides and glycopeptides in hydrophilic interaction chromatography-mode HPLC in the presence of ion-pairing reagents. Anionic ion-pairing reagents decreased the retention times of glycopeptides and improved resolution in the presence of higher concentrations or hydrophobicities of ion-pairing reagent. Anionic ion-pairing reagents increased retention times of larger glycans because of their increased hydrophilicity. In contrast, in the presence of cationic ion-pairing reagents, the retention times of glycopeptides with greater numbers of sialic acid residues decreased. It is appropriate to add an anionic ion-pairing reagent to the mobile phase for good separation of glycopeptides. The collision cross-sectional area values of glycopeptides determined using electrospray ionization-ion mobility spectrometry-mass spectrometry correlated with retention times. These findings support the implementation of hydrophilic interaction chromatography-mode HPLC to improve the characterization of glycosylated biopharmaceuticals.

The great importance of normalization of LC-MS data for highly-accurate non-targeted metabolomics.

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating co-eluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS.

Dried Saliva Spot (DSS) as a Convenient and Reliable Sampling for Bioanalysis: An Application for the Diagnosis of Diabetes Mellitus.

This paper proposes the dried saliva spot (DSS) as a convenient sampling technique for bioanalysis. The analytical method with the DSS was used for the determination of D,L-lactic acid (D,L-LA) and the D/L ratio of diabetic patients and prediabetic persons for the simple screening of the disease. The D,L-LA in the DSS was labeled with a chiral reagent (DMT-3(S)-Apy) for carboxylic acids and determined by UPLC-ESI-MS/MS. The limits of detection (signal-to-noise ratio (S/N) = 3) for the DSS analysis were on the amol level (∼30 amol). Because good stability, recovery, accuracy, and precision of the D,L-LA for the DSS method was also obtained from the proposed procedure, the DSS method was applied to the determination of the D- and L-isomers of LA of diabetic patients, and prediabetic and healthy persons. The D/L-LA ratio by the present DSS method and the HbA1c value in blood were well-correlated to the serious diabetic patients, whereas the relation in the prediabetic persons was not very good. The reason seems to be due to the rough saliva sampling, and not to the DSS method, because strict regulation was not requested for the prediabetic and healthy persons. In order to have a successful DSS analysis, the stability of the target molecule, the detection sensitivity to the target molecule, and the validated determination method are important.

Diagnostic Approach to Disease Using Non-invasive Samples Based on Derivatization and LC-ESI-MS/MS.

The determination of biologically-active molecules is very important in order to understand biological functions. A novel approach for the highly sensitive and specific determination seems to be essential for this purpose. Based on this consideration, we synthesized various types of fluorogenic and fluorescent reagents for the derivatization of chiral and achiral molecules. The fluorescence analysis is excellent for the analysis of target molecules and generally provides good expected results. However, the trace analysis of the bioactive molecules in complex matrices, such as plasma and urine, is not always satisfactory even using high-performance fluorometry. In such a situation, mass spectrometry (MS) is another technique for the selective and sensitive determination of biological components. Therefore, various derivatization reagents for MS/MS detection were developed and used for the determination of amines and carboxyls including chiral molecules. These newly developed reagents were also adopted for the biomarker detection related to diseases using non-invasive samples (i.e., saliva, nail, hair). Although the determination of the targeted chiral molecules is relatively easy, it is very difficult to identify and/or determine the enantiomeric biomarker in real samples. To solve this difficulty, we proposed the strategy called "chiral metabolomics," which means the total analysis of the enantiomers of various chiral metabolites in complex matrices. This review paper focused on the development of various new derivatization reagents for amines and carboxyls by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and the detection of the biomarker candidates related to several diseases in non-invasive samples (i.e., hair, nail, saliva) using these reagents.

Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

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 (r(2) > 0.999, range from 10 pmol/mL to 50 mol/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.

Uric acid quantification in fingernail of gout patients and healthy volunteers using HPLC-UV.

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.

Principal component analysis of molecularly based signals from infant formula contaminations using LC-MS and NMR in foodomics.

BACKGROUND: The challenge in developing analytical assessment of unexpected excess contaminations in infant formula has been the most significant project to address the widespread issue of food safety and security. Foodomics based on metabolomics techniques provides powerful tools for the detection of tampering cases with intentional contaminations. However, the safety and risk assessments of infant formula to reveal not only the targeted presence of toxic chemicals, but also molecular changes involving unexpected contaminations, have not been reported. In this study, a huge amount of raw molecularly based signals from infant formula was analysed using reversed phase and hydrophilic interaction chromatography with time-of-flight MS (LC-MS) and (1) H nuclear magnetic resonance (NMR) and then processed by a principal component analysis (PCA). RESULTS: PCA plots visualised signature trends in the complex signal-data batches from each excess contamination of detectable chemicals by LC-MS and NMR. These trends in the different batches from a portion of excess chemical contaminations such as pesticides, melamine and heavy metals and out-of-date products can be visualised from spectrally discriminated infant formula samples. CONCLUSION: PCA plots provide possible attempts to maximise the covariance between the stable lot-to-lot uniformity and excess exogenous contaminations and/or degradation to discriminate against the molecularly based signals from infant formulas. © 2015 Society of Chemical Industry.

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

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 <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells.

A Reliable Method for the Separation and Detection of Synthetic Cannabinoids by Supercritical Fluid Chromatography with Mass Spectrometry, and Its Application to Plant Products.

A reliable method using supercritical fluid chromatography with mass spectrometry (SFC-MS) was developed for cannabinoids using compressed carbon dioxide (CO2) and methanol as the mobile-phase. The cannabinoids, i.e., cannabicyclohexanol (CCH: cis-isomer), trans-CCH, 5-(1,1-dimethylheptyl)-2-[(1R,3S)-3-hydroxycyclohexyl]-phenol (CP-47497), 5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol (CP-55940), 3-(1,1'-dimethylheptyl)-6aR,7,10,10aR-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol (HU-210), 2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol (CBD), (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-018), (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073) and 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), were determined within 12 min using a conventional column (2-EP) for SFC. Furthermore, two optical isomers of CCH and trans-CCH were completely and rapidly separated by a chiral stationary phase column (AMY1). A highly sensitive detection (0.002-3.75 ppb) was also obtained by these methods using 2-EP and AMY1 columns. These methods were applied to the qualitative and quantitative determination of cannabinoids in dried plant products. Although the concentration and species were different in the products, JWH-018, JWH-073 and CCH, including the cis-isomer, trans-isomer and the optical isomers, were detected in the products. Therefore, the proposed SFC-MS method seems to be useful as an alternative method to GC-MS and LC-MS for illegal drugs, such as cannabinoids.

Simultaneous determination of post-translational racemization and isomerization of N-terminal amyloid-ß in Alzheimer's brain tissues by covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry.

Typical markers of protein aging are spontaneous post-translational modifications such as amino acid racemization (AAR) and amino acid isomerization (AAI) during the degradation of peptides. The post-translational AAR and AAI could significantly induce the density and localization of plaque deposition in brain tissues. Alzheimer's disease (AD) is reliably related to the formation and aggregation of amyloid-ß peptide (Aß) plaques in the human brain. No current analytical methods can simultaneously determine AAR and AAI during the degradation of Aß from AD patients. We now report a covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry (CCD-UPLC-MS/MS) method for the determination of post-translational AAR and AAI of N-terminal Aß (N-Aß1-5) in human brain tissues. When subjected to tryptic N-Aß1-5 from post-translationally modified natural Aß in focal brain tissues by the CCD procedure, it was monitored at m/z 989.6→637.0/678.9 during electrospray collision-induced dissociation. These N-Aß1-5 fragments with l-aspartic acid (l-Asp), d-Asp, l-isoAsp, and d-isoAsp could be separated using the UPLC system with a conventional reversed-phase column and mobile phase. The quantification of these peptides was determined using a stable isotope [(15)N]-labeled Aß1-40 internal standard. The CCD-UPLC-MS/MS assay of potential N-Aß1-5 allowed for the discovery of the present and ratio levels of these N-Aß1-5 sequences with l-Asp, d-Asp, l-isoAsp, and d-isoAsp.

Evaluation of a series of prolylamidepyridines as the chiral derivatization reagents for enantioseparation of carboxylic acids by LC-ESI-MS/MS and the application to human saliva.

Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the mass spectrometry (MS) detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-MS/MS analysis is very limited. According to these observations, we are currently in the process of developing novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the electrospray ionization-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring. Based upon these considerations, a series of prolylamidepyridines ((S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-3-yl)amide, and (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-4-yl)amide) was synthesized as ideal labeling reagents for the enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity by ultra-performance LC (UPLC)-MS/MS. Among the synthesized reagents, PCP2 was the most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs, which were selected as the representative carboxylic acids, were in the range of 2.52-6.07 and 49-260 amol, respectively. The sensitive detection of biological carboxylic acids (detection limits, 32-520 amol) was also carried out by the proposed method using PCP2 and UPLC-MS/MS. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), 3-hydroxybutylic acid, maric acid, succinic acid, α-ketoglutalic acid, and citric acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal subjects. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only of chiral, but also achiral, carboxylic acids in real samples.