Post-column Derivatization Method Research Articles

Dopamine-supported HPLC post-column derivatization to fluorescence: Simultaneous and sensitive detection of eight tea polyphenols

The effective differentiation and detection of multiple tea polyphenols are often challenging due to their subtle structural similarities. Although post-column derivatization HPLC strategies are commonly employed to distinguish multiple targets, the short physical distance between chromatographic column and detector limits reaction time, thereby reducing the derivatization efficiency. Dopamine (DA) reacts rapidly with resorcinol to form fluorescent azamonardine products, making fast fluorometric derivatization of tea polyphenols containing resorcinol motifs possible. In this study, a DA-driven rapid and post-column fluorescence derivatization method has been applied to sensitively detect eight tea polyphenols. This method is based on fluorescence derivatization and possesses low background interference, high sensitivity, and excellent reproducibility. Moreover, the practical application of this proposed fluorometric derivatization platform was further validated by simultaneous identification of multiple tea polyphenols in different tea samples. This work has great potential to become an alternative to the National Standard method for tea polyphenols determination.

Whole blood and plasma taurine reference intervals in adult Cavalier King Charles Spaniels and correlations between taurine concentration, diet and mitral valve disease.

To determine breed-specific reference intervals for whole blood (WB) and plasma taurine concentrations in adult, overtly healthy Cavalier King Charles Spaniels (CKCSs) and determine whether taurine concentrations differ across preclinical myxomatous mitral valve disease (MMVD) stages or between CKCSs eating diets that meet World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. 200 privately owned CKCSs. Clinically healthy adult CKCSs were recruited prospectively. Diet and supplement history was collected. Dogs were staged by echocardiography using MMVD consensus guidelines. Taurine concentrations were measured in deproteinized lithium heparin WB and plasma samples with the postcolumn ninhydrin derivatization method on a dedicated amino acid analyzer. There were 12 stage A (6%), 150 stage B1 (75%), and 38 stage B2 (19%) CKCSs. Seventy-eight dogs (39%) were reported by their owners to be eating diets meeting WSAVA nutritional guidelines; 116 (58%) were not. Taurine concentrations in plasma (P = .444) and WB (P = .073) were not significantly different across MMVD stages or between CKCSs eating diets meeting WSAVA nutritional guidelines versus other diets (P = .345 and P = .527, respectively). Reference intervals for WB taurine (152 to 373 µM) and plasma taurine (51 to 217 µM) concentrations in CKCSs were generated. In CKCSs, taurine concentrations do not differ significantly based on preclinical MMVD stage, nor do they differ significantly based on consumption of a diet that does or does not meet WSAVA nutritional guidelines.

Analysis of impurity profiling of arbekacin sulfate by ion-pair liquid chromatography coupled with pulsed electrochemical detection and online ion suppressor-ion trap-time off light mass spectrometry

Ion-pair liquid chromatography with pulsed electrochemical detection (LC-PED) was established for the analysis of impurities in arbekacin (ABK) sulfate. APursuit pentafluorophenylpropyl (PFP) column was used as stationary phase. This novel method showed greater separation and sensitivity ability. In a representative ABK sample, 24 impurity peaks were detected in LC-PED, where of only 9 were monitored by a post-column derivatization method prescribed by the Japanese Pharmacopoeia (JP). For identification of the chemical structures of the impurities detected by LC-PED, LC-Mass Spectrometry (MS) was used. Two challenges had to be overcome in this work. The first was the transfer of the MS incompatible mobile phase to an MS compatibleone while maintaining the elution order of the peaks in the chromatograms. Previously reported approaches such as two-dimensional (2D)LC were hardly applicable in this case due to the lack of ultraviolet (UV) absorbing chromophores in ABK and its impurities. The sodium hydroxide solution was replaced by aqueous ammonia to adjust the pH of the mobile phase used in LC-PED. The other challenge encountered was the ion suppression effect caused by trifluoroacetic acid (TFA) and pentafluoroproponic acid (PFPA) in the mobile phase. Some strategies such as “TFA-fixed” and its modifications were tried, but they were inconvenient and severe contamination of the MS was observed. A cationself-regenerating suppressor (CSRS), which was originally designed for increasing analyte conductivityof ammonia and amines analysis in ion chromatography (IC), was coupled between the LC and Ion Trap-Time of Flight (IT-TOF)-MS and almost all TFA and PFPA in the mobile phase were removed. The limit of detection (LOD) of ABK in this integrated system improved significantly to 20 ng/mL. The chemical structures of the 28 impurities were elucidated and 15 impurities were reported for the first time.

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core–shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box–Behnken experimental design, and the method was validated using the total error concept. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L−1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of −2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.

Photochemical Formation of Methylhydroperoxide in Dissolved Organic Matter Solutions.

Although it is known that the solar irradiation of chromophoric dissolved organic matter (CDOM) solutions generates H2O2, whether or not organic hydroperoxides (ROOHs) are photochemically formed remains unclear. This study employs high-performance liquid chromatography with the postcolumn enzymatic derivatization method to examine whether ROOHs can be formed in CDOM solutions under simulated solar irradiation. Methylhydroperoxide (MHP) is the only identified ROOH under our experimental conditions, and the quantum yields of MHP (ΦMHP) vary from (1.09 ± 0.09) × 10-6 to (4.95 ± 0.11) × 10-6 in the tested CDOM solutions, including four reference natural organic matters and two effluent organic matters. The quantum yields of H2O2 (ΦH2O2) are simultaneously measured, and the ratios of ΦH2O2 to ΦMHP range from 147 to 676. The formation of MHP is highly related to the presence of superoxide radical ions (O2•-) and methyl radicals (CH3•); therefore, a photoformation mechanism of MHP has been proposed. The photochemically generated CH3• reacts with O2 to yield CH3OO•. Subsequently, CH3OO• is reduced to MHP by O2•-. Our results also suggest that the yield of CH3• to MHP under air-saturated conditions is 52% and increases to 98% under oxygen-saturated conditions. The decays of MHP and H2O2 are very similar in terms of photodegradation, hydrolysis, Fenton, and photo-Fenton reactions. This study can be useful to understand the photochemical formation of organic peroxides in surface waters.

Liver and kidney serum profile abnormalities in workers exposed to aflatoxin B1 in urban solid waste management centers.

Many workers are exposed to health problems arising from molds, fungi, and their toxins during waste processing. Aflatoxin B1 (AFB1) level in airborne and settled dust, aflatoxin B1-albumin (AFB1-Alb) adduct in serum, liver and kidney biochemical tests, and body redox change of workers in municipal dry waste-processing sites were investigated. The surface, personal, and area air dust and the blood of workers' samples were collected from the plastic and bread waste-sorting sections in three recycling municipal dry waste sites. Digestion (only for serum samples), passed through SPE cartridge, elution, and collection with methanol, immune-affinity column clean-up, and HPLC system equipped with post-column derivatization method and fluorescence detection were performed for determination of AFB1 and AFB1-Alb levels in the samples. The mean level of dust and AFB1 in the personal and area air, and in the settled dust and the AFB1-Alb in the serum of workers in the bread waste sorting, was higher than plastic waste-sorting samples, in all of the sites. The differences in the biochemical profiles of subjects exposed to aflatoxin B1 as compared to the control group especially in liver and kidney function parameters as well as antioxidant factors of the serum were significant. The workers in handling of municipal waste may be exposed to potentially hazardous levels of aflatoxin B1. The adverse effects of AFB1 on the kidney and liver may be caused by changes in the redox system.

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma

Here we describe a new HPLC–MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds.Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

HPLC-CUPRAC post-column derivatization method for the determination of antioxidants: a performance comparison between porous silica and core-shell column packing

BackgroundAn HPLC method employing a post-column derivatization strategy using the cupric reducing antioxidant capacity reagent (CUPRAC reagent) for the determining antioxidants in plant-based materials leverages the separation capability of regular HPLC approaches while allowing for detection specificity for antioxidants.MethodsThree different column types, namely core-shell and porous silica including two chemically different core-shell materials (namely phenyl-hexyl and C18), were evaluated to assess potential improvements that could be attained by changing from a porous silica matrix to a core-shell matrix. Tea extracts were used as sample matrices for the evaluation specifically looking at catechin and epigallocatechin gallate (EGCG).ResultsBoth the C18 and phenyl-hexyl core-shell columns showed better performance compared to the C18 porous silica one in terms of separation, peak shape, and retention time. Among the two core-shell materials, the phenyl-hexyl column showed better resolving power compared to the C18 column.ConclusionsThe CUPRAC post-column derivatization method can be improved using core-shell columns and suitable for quantifying antioxidants, exemplified by catechin and EGCG, in tea samples.

Determination of hexavalent chromium concentration in industrial waste incinerator stack gas by using a modified ion chromatography with post-column derivatization method

An ion chromatography with post-column derivatization with 1,5-diphenylcarbazide (IC-DPC) analytical method was modified to enable measurement of trace-level hexavalent chromium (Cr(VI)) in air. One of the difficulties in determining trace levels of Cr(VI) in air with conventional IC-DPC methods is co-elution of the solvent and ion peaks due to high concentrations of ionic compounds in the extract. However, by using gradient elution rather than isocratic elution we were able to fully resolve the Cr(VI) ion peak from the solvent peak without the need for diluting the extract, which would have reduced the minimum quantifiable level of the method. With this method, we were able to detect Cr(VI) in air at concentrations of 5.3ng/m3 (assuming a sampling volume of 1m3 and a final solution volume of 10mL). Recovery tests at three different concentrations of Cr(VI) (50, 250, 1000ng) were performed with or without fly ash; recovery rates at all the concentrations of Cr(VI), with or without fly ash, ranged from 68% to 110% (mean±relative standard deviation, 96%±11%), and there were no differences in recovery rates with respect to the presence or absence of fly ash. Finally, we used the developed method to determine the concentration of Cr(VI) in stack gases collected from eight industrial waste incinerators located in Japan. The concentration of Cr(VI) in the stack gases ranged from below the method quantification limit to 3100ng/m3. The highest concentrations of Cr(VI) detected in the stack gases were two to three orders of magnitude higher than that in ambient air in Japan.

Postcolumn derivatization of amino acids using reaction flow chromatography columns with fluorescence detection: A fast new approach to selective derivatization techniques

ABSTRACTReaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yielded R2 ≥ 0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously.

A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC–MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide.Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5μm) and isocratic elution using the mobile phase acetonitrile – 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3μm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v).Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1–2000μg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12–2000μg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

Vitamin K2-4 and K2-7 Estimation in Nutraceutical Solid Dosage Forms by Post Column Derivatization with Fluorescence Detection

A post column derivatization method for the estimation of fat soluble vitamins K2-4 and K2-7 was developed by reverse phase HPLC and validated as per ICH guidelines. The compounds were extracted by solvent extraction with acetone followed by evaporation in a rotary evaporator. The residue was dissolved in ethanol and injected in to a chromatograph consists of C18 column (Waters symmetry, 150×4.6, 3.5 μ) and fluorescence detector. The derivatization reagent was prepared by dissolving 136 mg of zinc chloride, 40 mg of sodium acetate and 0.1 ml of glacial acetic acid in methanol. The mobile phase comprises of methanol: isopropyl alcohol: acetonitrile: zinc chloride buffer solution 850:90:50:10 with a flow rate of 1 ml/min. The overall percentage recovery of five different levels were found to be 99.85 and 100.5%, respectively. The linearity of the analytical method was determined from 10% to 120% level and the linear regression coefficients were 0.9991 and 0.9995 which is well within the acceptance criteria of 0.999. The limits of detection and limits of quantification was determined based on signal to noise ratio.The established values were 0.050, 0.50 μg/ml and 0.005, 0.047 μg/ml which is much lesser than available literatue limits. The developed method can be more suitable for the estimation of K2-4 and K2-7 present in drug substances as well as drug product formulations.

Occurrence of Aflatoxins in Imported Rice at Supermarkets of Ahvaz City, Khuzestan Province, Iran

Background: Rice is one of the major cereals that are primarily consumed by humans. It may become contaminated with aflatoxins (AFs). Aflatoxin B 1 (AFB 1 ) is known as one of the most potent environmental mutagens and carcinogens. Objectives: This study was carried out to determine the concentration of AFs in the rice currently sold at the supermarkets in the city of Ahvaz, Khuzestan Province, Iran. Materials and Methods: The levels of AFs in 90 collected imported rice samples after clean-up by AflaTest columns were measured by using a High-Performance Liquid Chromatographer equipped with a C18 column, fluorescence detector (excitation 360 nm and emission 440 nm) and post-column bromide derivatization method, mobile phase of water-acetonitrile-methanol (600:200:200 v/v) +119 mg potassium bromide +100 µL con. HNO 3 at a flow rate of 1 mL/min. Results: The results showed that the highest concentration of AFB 1 and total AFs in the rice samples were 2.3500 and 2.7040 ng/g, respectively. The different mean concentration of AFB 1 and total AFs in three brands of the rice samples was significantly lower than the maximum tolerable level (MTL) of AFB 1 (5 ng/g) and total AFs (30 ng/g) set by the Institute of Standards and Industrial Research of Iran. Conclusions: In all of the investigated imported rice samples, the level of AFB 1 and total AFS were found to be lower than the Iranian MTL, and no health risk for consumers were detected at these levels of contamination.

Determination of fumonisins B<SUB>1</SUB> and B<SUB>2</SUB> in corn by high performance liquid chromatography with post-column derivatization method

A high performance liquid chromatography-fluorescence detection with post-column derivatization method was developed to detect fumonisin B1 (FB1) and fumonisin B2 (FB2) in corn. Several factors, such as the pH of derivatization buffer, concentration and flow rate of derivatization reagents, excitation wavelength, emission wavelength, which affected the detection of fumonisins were optimized. The separation was performed on a ZORBAX SB C18 column operated at 40 degrees C with the gradient elution by two mobile phases of 0.1 mol/L sodium dihydrogen phosphate solution (pH 3.3) and methanol at a flow rate of 0.8 mL/min. The derivatization was performed at ambient temperature. The o-phthalaldehyde (OPA) flow rate was 0.4 mL/min. The results showed that the optimum conditions were pH 10.5 of the derivatization reagent, OPA concentration at 2 g/L, and excitation wavelength of 335 nm, emission wavelength of 440 nm. The linear plots of FB1 and FB2 were obtained between 0.2 to 20 mg/L, with the correlation coefficients above 0.999 for both FB1 and FB2. The limits of detection of fumonisins B1 and B2 were 0.02 mg/kg. The mean recoveries at the three spiked levels of 0.1 - 4.0 mg/kg were 82.5% - 89.8%. This method is accurate, simple, rapid and suitable for the determination of fumonisins B1 and B2 in corn.

Determination of fumonisins B<SUB>1</SUB> and B<SUB>2</SUB> in corn by high performance liquid chromatography with post-column derivatization method

Determination of fumonisins B<SUB>1</SUB> and B<SUB>2</SUB> in corn by high performance liquid chromatography with post-column derivatization method

Post-column labeling techniques in amino acid analysis by liquid chromatography

Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV-Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis.

Amino acid analysis: current topics and trends

The history of amino acids begins with the discovery of aspartic acid in asparagus sprouts in 1806. Thereafter, various naturally occurring amino acids of more than 360 species were detected from living organisms. Amino acid analysis was first performed by a postcolumn derivatization method using ninhydrin, which was originally developed by Moore and Stein [1]. They analyzed 124 amino acids and the ribonuclease structure using an automatic and systematic analyzer, and were awarded the Nobel Prize in Chemistry in 1972. Amino acid analysis is an integral part of analytical biochemistry. Amino acid analysis generally includes derivatization, i.e., precolumn, on-column, and postcolumn, coupled with chromatographic separation. A wide variety of separation techniques, including ion-exchange chromatography, reversed-phase chromatography, hydrophilic interaction chromatography, gas chromatography, capillary electrophoresis, and microchip electrophoresis, have been used. Various detection techniques, such as ultraviolet–visible, fluorescence, chemiluminescence, and electrochemical detection, have also been developed for amino acid analysis. On the basis of recent advances in the software and hardware used in mass spectrometry (MS), the variety of amino acid analysis methods has dramatically evolved and use has shifted to various types of MS detection techniques. In the meantime, a number of relatively old techniques, based on derivatization and conventional separation, have acquired a new look. On the basis of these observations, The Japan Society of Amino Acid Analysis (JSA3) was established in the autumn of 2011 to promote and follow the bioanalytical and/or technological advances relating to analysis of amino acids and related molecules. Various labeling techniques using ultraviolet–visible and fluorescent reagents are still used for the trace analysis of amino acids by the combination of chromatographic separation and MS detection. Of course, electrospray ionization MS allows the analysis of underivatized amino acids. Another new aspect is high-throughput analysis, such as the use of a high-pressure liquid-chromatographic system such as ultraperformance liquid chromatography using a sub-2-μm particle column, and miniaturization analysis such as the use of a microfluidic system. However, the most important issue in amino acid analysis today seems to be in the context of metabolomics studies as part of systems biology. Besides, naturally occurring D-amino acids, such as D-serine and D-aspartic acid, were found in awide variety of living organisms, both in their free form and as isomeric residues in many peptides. Their detection is due to the tremendous advances in the analytical techniques in recent years. Stereoselective metabolism of amino acids has also been gradually unveiled, and D-amino acids are now considered as novel physiologically active substances and biomarkers even in mammals. In view of the growing importance of the enantioselective analysis of amino acids, articles related to D-amino acid analysis are provided in this issue. Several articles regarding the enantioseparation of DL-amino acids by direct methods using a chiral stationary phase column are also provided. This topical collection covers a broad range of amino acid analysis, with contributions from the most prominent researchers in the field. The review articles are focused on the analytical methods applied to amino acids and related molecules using liquid chromatography, capillary electrophoresis, and microchip electrophoresis. The topics of the original research articles are expanded to the research field of peptides and proteins. Clarification of both the quantity and the distribution of amino acids in biological samples, including physiological fluids and foods, is also presented. Several research articles describe new methods of amino acid analysis, such as the use of a pillar column and a novel immunometric assay. This topical collection is focused on analysis of amino acids, including peptides, and provides a glimpse of the current research activity in the field. As a guest editor, it is my pleasure to present this collection of research articles and critical reviews which address the many challenging areas of amino acid analysis, including chiral recognition. Thanks are due to all of the authors for their excellent and timely contributions as well as the editorial staff of Published in the topical collection Amino Acid Analysis with guest editor Toshimasa Toyo’oka.

Optimization of an Online Post-Column Derivatization System for Ultra High-Performance Liquid Chromatography (UHPLC) and Its Applications to Analysis of Biogenic Amines

We investigated the efficacy and utility of a post-column derivatization method for the detection of amines with o-phthalaldehyde (OPA) in ultra high-performance liquid chromatography (UHPLC). Since it was anticipated that a derivatization reaction system placed downstream of the column would give considerable band broadening to narrow peaks generated by a UHPLC column, we examined the contributions of the dimensions of the reaction tube, the fluorescence (FL) detector flow cell, and the column to the extra-column peak broadening, and optimized the post-column derivatization system in UHPLC. We successfully separated amines within 6 min with gradient elution by using the post-column derivatization system connected to a UHPLC system. The analysis time was reduced by a factor of 7 compared with that by conventional HPLC. The detection limits were 18.0 - 105.7 fmol, depending on the compounds; the reproducibilities were 0.11 - 0.82% RSD for the retention times and 0.78 - 1.66% RSD for the peak areas (n = 10), respectively. The linear dynamic ranges were up to 12, 20, and 40 pmol/μL, depending on the compounds, respectively.