6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.[Pubmed:38297457]

Anal Chem. 2024 Feb 13;96(6):2666-2675.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (R(s) > 1.5 except for glutamic acid with R(s) = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section ((DT)CCS(N2)) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCS(N2) of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Quantitative determination of chondroitin sulfate with various molecular weights in raw materials by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.[Pubmed:38154285]

Food Chem. 2024 May 15;440:138273.

A simple and reliable HPLC method was developed for quantification of chondroitin sulfate (CS). The procedure is based on precolumn hydrolysis of CS to liberate galactosamine and subsequent derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate. Hydrolysis and derivatization conditions were optimized. A linear correlation coefficient of 0.9999 was calculated within the range of 10-1500 mug/mL from the standard curve. The method produces good precision and good accuracy (100.75 % recovery). An advantage over other common methods is its ability to quantify CS of all molecular weights and structures, as evidenced by the determination of CS fractions with narrow molecular weight distributions obtained through depolymerization by different methods, while enzymatic HPLC was proven to be infeasible. Extraction recoveries of CS from monosaccharide mixed samples were > 93 %. The reliability was also validated by a small difference (-1.95 % to 4.12 %) relative to enzymatic HPLC results in analysing representative CS samples of different animal origins and suppliers.

Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis.[Pubmed:37696129]

J Chromatogr A. 2023 Oct 11;1708:464349.

Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-(13)C(15)N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.

Comprehensive reversed-phasexchiral two-dimensional liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry with post-first dimension flow splitting for untargeted enantioselective amino acid analysis.[Pubmed:37464972]

J Sep Sci. 2023 Sep;46(18):e2300351.

This work describes a comprehensive achiral x chiral two-dimensional liquid chromatography separation for enantioselective amino acid analysis coupled to electrospray ionization-tandem mass spectrometry detection using data-independent acquisition. Flow splitting after the first and second dimension separation was utilized for volumetric flow reduction and for enabling a multi-detector approach (with ultraviolet, fluorescence, charged aerosol, and MS detection), respectively. Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate provided a chromophore, a fluorophore, and an efficient mass tag for efficient ionization in positive electrospray ionization-mass spectrometry. Chiral columns often have limitations in terms of their chemoselectivity, which may be a problem when complex sample mixtures with structurally related compounds need to be separated. It can be alleviated by a reversed-phasexchiral two-dimensional-liquid chromatography setup, in which the first dimension provides the chemoselectivity and a chiral tandem column constituted of quinine-carbamate derived weak anion-exchanger and zwitterionic ion-exchanger in the second dimension separation of D- and L-amino acid enantiomers. The method was used to control the stereointegrity of the therapeutic peptide octreotide. After hydrolysis, all amino acid constituents were detected with the correct configuration and composition. Some options for flow splitting and integration of destructive detectors in the first dimension separation are outlined.

Combination of SPE and fluorescent detection of AQC-derivatives for the determination at sub-mg/L levels of biogenic amines in dairy products.[Pubmed:36869470]

Food Res Int. 2023 Mar;165:112448.

Biogenic amines (BAs) are compounds generated by decarboxylation of their amino acid precursors. Their intake, even at low concentrations, can lead to several types of health problems in sensitive individuals. As they can be easily formed in fermented dairy products, their quantitative determination is very relevant. In the present paper, a method for the quantitative determination of four biogenic amines in different dairy products has been developed, validated and applied to 37 samples of milk, 23 of yogurt, and 14 of kefir. Amines were selectively extracted using solid phase extraction, subsequently derivatizatized with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and further determined by High Performance Liquid Chromatography with fluorescence detection. The method's sensitivity was highly satisfactory, with limits of detection lower than 0.2 mg/L. Optimal linearity and repeatability were also achieved. BAs were not detected in most of the milk samples, but they were found frequently at high levels in yogurt and kefir samples, reaching values of up to 79 mg/kg total BAs in kefir samples. Levels measured should not be a cause for concern for the population at large, but should be known by BAs-sensitive individuals.

A novel screening method for free non-standard amino acids in human plasma samples using AccQ.Tag reagents and LC-MS/MS.[Pubmed:36602091]

Anal Methods. 2023 Jan 26;15(4):445-454.

There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ.Tag derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.

Liquid Chromatographic Determination of o-Phosphoethanolamine in Human Plasma Using Fluorescent Derivatization.[Pubmed:36596506]

Chem Pharm Bull (Tokyo). 2023;71(1):10-14.

In this study, an HPLC analysis method using pre-column derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was developed for the determination of o-phosphoethanolamine (PEA), which is a potential biomarker for the diagnosis of major depressive disorder, in human plasma sample. After PEA was derivatized with AQC under mild conditions, the obtained derivative was subjected to purification with a titanium dioxide-modified monolithic silica spin column (MonoSpin((R)) TiO). The eluate from the MonoSpin((R)) TiO was directly injected into an amide-type hydrophilic interaction liquid chromatography (HILIC) column-equipped HPLC system, and the resulting derivative could be separated on the column under alkaline mobile phase conditions and subsequently detected fluorometrically at excitation and emission wavelengths of 250 and 395 nm, respectively. The limit of detection and limit of quantification for a 10 microL injection volume of PEA were 0.052 and 0.17 microM, respectively. The method was validated at 0.2, 1.0, and 5.0 nmol/mL levels in plasma sample, and the precision values were 2.0-6.6% as relative standard deviation and the correlation coefficient (r) of the calibration curve was 0.9995. Furthermore, applicability of this method was demonstrated by analyzing PEA levels in plasma samples from mental illness patients.

Determination of 23 Amino Acids and Amines in Plasma and Cortex of C57BL/6 Mice with HPLC-FLD.[Pubmed:36572394]

J Chromatogr Sci. 2023 Dec 30;62(1):44-57.

Using 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate as a pre-column derivatization reagent, optimized derivatization and chromatography parameters, a simple high-performance liquid chromatography fluorescence detector (HPLC-FLD) method was developed and validated to determine 23 related amines in plasma and cortex of C57BL/6 mice with cerebral ischemia-reperfusion injury. The prepared samples were separated on a ZORBAX SB-C18 column (4.6 mm x 250 mm, 5 mum) with 60% acetonitrile (ACN) and 20 mM sodium acetate solution (pH adjusted to 5.0 by phosphoric acid). All analytes achieved good separation within 1.2 h at a flow rate of 1 mL/min. The limits of detection and limits of detection quantitation of the method were ranged from (0.1-9.2) to (0.3-30.6) ng/mL, respectively. The analytical method was apt for simultaneously determining 23 amino acids in plasma and cortex. Our results revealed that the relevant amino acids were significantly altered (P < 0.05) in C57BL/6 mice.

Comprehensive Online Reversed-Phase x Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis.[Pubmed:36442145]

Anal Chem. 2022 Dec 13;94(49):17063-17072.

This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral x chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC ((1)D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) ((2)D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS(2). The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC x LC separation occurs within a total run time of 60 min ((1)D), with the chiral (2)D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-(13)C(15)N]-AA standard. The quantification performance of this LC x LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.

A rapid UHPLC-QDa method for quantification of human salivary amino acid profiles.[Pubmed:36274351]

J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15;1211:123485.

Abnormal salivary amino acid (AA) levels may indicate dysfunction of the body. Being noninvasive, sampling easily and cost-effective of saliva, a rapid, precise and simple analysis method has become very important for quantitative salivary AA profiles. After one-step to precipitate protein, the resultant extraction was derived with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) within 10 min. Quantitation of AA profile was achieved within 6 min in a single run by ultra-high performance liquid chromatography coupled with a single quadrupole mass spectrometry (UHPLC-QDA detector). The method was validated with acceptable accuracy ranging from 80.33 % to 122.31 %, appropriate linearity with the coefficient (R(2)) more than 0.991, good intra- and inter-day precision, repeatability and stability (RSD < 15 %). The recoveries at three different spiked concentrations ranged over 79.18 %-125.36 % while the matrix effect was from -19.86 % to 11.95 %. This simple, rapid and robust method was successfully applied to quantify human salivary 30 amino acids, in which the levels of taurine, gamma-aminobutyric acid, methionine and tryptophan in healthy people were close to the LOQs. Besides, the levels of histidine and cystine were not able to be measured due to their relatively high LOQs, which were considered as the limitations of this developed method.

Coumarin-based derivatization reagent for LC-MS analysis of amino acids.[Pubmed:36030735]

Talanta. 2023 Jan 15;252:123730.

In this paper, we report about the application of a sensitive fluorescent derivatization reagent Coumarin151-N-Hydroxysuccinimidyl Carbamate (Cou151DSC) for amino compounds using high-performance liquid chromatography (HPLC) compatible with ultraviolet (UV), fluorescence detector (FLD) and electrospray ionization - tandem mass spectrometry (ESI-MS/MS)-positive mode. We optimized derivatization procedure and validated an analytical method to determine 24 amino acids in Kvass drink using Norvaline as an internal standard. Compared to 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC), the derivatization with Cou151 DSC is faster and milder, for 5 min at 40 degrees C instead of 15 min at 55 degrees C. The limit of quantitation (LOQ, pmol on column) for 21 amino acids in this work is lower 1.1-30.0 times than values obtained with 6-AQC. The derivatives have excitation wavelength at 355 nm and emission wavelength at 486 nm. Their MS/MS fragmentation behaviors were examined together with 23 other amino compounds. We found three possibilities to lose a neutral group which can be Coumarin 151 isocyanate Cou151NCO (255 Da), amine Coumarin 151 (229 Da) or urea Cou151CONH(2) (272 Da). The accuracy of the proposed method was within 83-107% with good relative standard deviations (RSDs) of equal or less than 6%. The recoveries were from 82 to 120% in four spiked concentrations, repeatability was between 0 and 14%. The intra- and inter-day precision are less than 13% and 18%, respectively.

Determination of amino acids in food and feed by microwave hydrolysis and UHPLC-MS/MS.[Pubmed:36027706]

J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Oct 15;1209:123429.

The determination of amino acids in food and feed by chromatography has a long history and is described in several official methods, including standards from ISO, AOAC, and the European Commission (EC) regulation 152/2009. The procedure usually consists of labor- and time-consuming preparation techniques and ion-exchange chromatography with challenging chromatographic conditions. Consequently, several approaches have been published to overcome these drawbacks but the knowledge about their suitability for complex matrices such as food and feed is limited. In this paper, we describe the development of two new methods to determine amino acids in food and feed. These methods involve microwave hydrolysis and reversed-phase UHPLC-MS/MS with pre-column derivatization using 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Both methods provide streamlined sample preparations and a dramatic reduction in analysis time while offering a high degree of specificity and selectivity. Selectivity also enabled the simultaneous determination of the more uncommon substances hydroxyproline, hydroxylysine, taurine, ornithine, and gamma-amino butyric acid (GABA) along with amino acids typically present in food and feed. The results were all satisfactory with regards to sensitivity, accuracy, precision, and comparability with laboratories that use other methods, for example from ISO, AOAC, or regulation (EC) 152/2009. We therefore concluded that both methods provide a reliable and modern approach to overcome many of the drawbacks that occur with the conventional standard methods.

Matrix Effect of Diverse Biological Samples Extracted with Different Extraction Ratios on the Detection of beta-N-Methylamino-L-Alanine by Two Common LC-MS/MS Analysis Methods.[Pubmed:35737048]

Toxins (Basel). 2022 Jun 2;14(6):387.

Neurotoxin beta-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.

Detection of Decarboxylated Amino Acids after in Vitro Protease Digestion of the Hydrophilic Fraction of Crude Drug Extracts.[Pubmed:35110503]

Biol Pharm Bull. 2022;45(2):169-177.

Many constituents of crude drugs in Japanese Kampo formulas are thought to function as pro-drugs, whose pharmacological activity is manifested after oral administration. Proteins and peptides in crude drugs may be digested and metabolized in the digestive tract and liver. However, few studies have reported the pharmacological activity of peptides in crude drugs. Here, we applied an analysis using LC-tandem mass spectrometry (LC-MS/MS) to identify the compounds derived from six crude drugs that are assumed to have anti-inflammatory effects. To simulate in vivo protease digestion, each water-soluble fraction of the crude drug extracts was treated with proteases, including endoproteinases and exopeptidases. Amines in the resultant digests were modified by 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and analyzed using LC-MS/MS, which demonstrated the presence of four decarboxylated amino acids (primary amines). In the digest of the hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder (Taiso), isobutylamine, isoamylamine, and 2-methylbutylamine were identified, which may be derived from valinyl, leucinyl, and isoleucinyl residues, respectively. Additionally, tyramine possibly derived from tyrosyl residues was identified in the digests of all the crude drugs. In primary cultured rat hepatocytes treated with interleukin-1beta, all these decarboxylated amino acids suppressed the production of nitric oxide, a proinflammatory mediator. Our approach, i.e., in vitro protease digestion and LC-MS/MS analysis, suggests that decarboxylated amino acids may be formed in vivo from peptides and may be responsible for the anti-inflammatory effect of crude drugs included in Kampo medicine.