Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates

Two methods for the on-line detection in HPLC eluates of analytes possessing radical scavenging activity were improved and compared. The instrumental set-up of the method that is based on on-line inhibition of luminol chemiluminescence (CL) by antioxidants was improved using better quality syringe pumps, employing a diode array detector, and introducing a mixing/neutralisation coil and a pulse damper. Sensitivity of the HPLC-CL detection increased by a factor of 4. Post-column neutralisation of eluates improved compatibility of this detection method with acidified HPLC eluents. The second method, which is based on the post-column quenching of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH*), was improved by readjusting composition and flow-rate of the reagent, mounting an additional pulse damper and detecting unreacted DPPH* with a detector equipped with a tungsten lamp. Purging of the DPPH* solution with He gas prior to analysis was introduced. This led to 30-fold better detection limits. The improved methods were compared with respect to limits of detection, the radical scavenging mechanism involved, compatibility with common HPLC solvents and pH range, and some technical aspects. The techniques described have high potential for the rapid identification of radical scavengers in complex samples like plant extracts.     

The in‐capillary DPPH‐capillary electrophoresis‐the diode array detector combined with reversed‐electrode polarity stacking mode for screening and quantifying major antioxidants in Cuscuta chinensis Lam

An in‐capillary 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH)‐CE‐the DAD (in‐capillary DPPH‐CE‐DAD) combined with reversed‐electrode polarity stacking mode has been developed to screen and quantify the active antioxidant components of Cuscuta chinensis Lam. The operation parameters were optimized with regard to the pH and concentration of buffer solution, SDS, β‐CDs, organic modifier, as well as separation voltage and temperature. Six antioxidants including chlorogenic acid, p‐coumaric acid, rutin, hyperin, isoquercitrin, and astragalin were screened and the total antioxidant activity of the complex matrix was successfully evaluated based on the decreased peak area of DPPH by the established DPPH‐CE‐DAD method. Sensitivity was enhanced under reversed‐electrode polarity stacking mode and 10‐ to 31‐fold of magnitude improvement in detection sensitivity for each analyte was attained. The results demonstrated that the newly established in‐capillary DPPH‐CE‐DAD method combined with reversed‐electrode polarity stacking mode could integrate sample concentration, the oxidizing reaction, separation, and detection into one capillary to fully automate the system. It was considered a suitable technique for the separation, screening, and determination of trace antioxidants in natural products.     

Use of DPPH⋅|DPPH Redox Couple for Biamperometric Determination of Antioxidant Activity

A novel electrochemical method for selective determination of antioxidant activity based on 2,2‐diphenyl‐1‐picrylhydrazyl | 2,2‐diphenyl‐1‐picrylhydrazin (DPPH?|DPPH) redox couple and biamperometric technique is proposed. Two identical glassy carbon disk electrodes were mounted in a classic electrochemical cell and the tested working potential difference was between 50 and 200 mV. The DPPH?|DPPH redox couple exhibited a high degree of reversibility. Selectivity of detector was tested by different antioxidant compounds in ethanol‐phosphate buffer solution, pH 7.40. The results of antioxidant activity of pure antioxidant compounds and of actual samples of beverages, expressed as Trolox equivalents and determined by proposed biamperometric and spectroscopic measurements, were in good correlation (R=0.9959). The detection limit for Trolox established by the applied biamperometry was 0.05 μM while the resulting current was linear up to 25 μM of Trolox.     

Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2'-diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.     

Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania

Since biological activity of medicinal plants is dependent on cultivation area, climatic conditions, developmental stage, genetic modifications and other factors, it is important to study flora present in different growing sites and geographical zones. This study was focused on screening of antioxidant activity of C. angustifolium harvested in six different locations in Lithuania. The total contents of phenolic compounds, flavonoids and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated by spectrophotometric methods. A correlation between radical scavenging activity and total phenolic compounds content was observed (correlation coefficient 0.98). HPLC with online post-column DPPH radical scavenging reaction detection was used for the separation of extracts. Oenothein B, rutin and one unidentified compound were predominant. Volatile compounds were analysed using solid-phase microextraction coupled with gas chromatography–mass spectrometry. Based on the analysis of volatiles, all samples were classified into two chemotypes: (I) with predominant α- and β-caryophyllenes and (II) with predominant anethole.     

Optimization of extraction process and the antioxidant activity spectrum–effect relationship of Angelica dahurica

Despite the large number of studies indicating that Angelica dahurica has strong antioxidant capacity, there are no clear details on the specific antioxidant components involved. In this study, the chromatograms and antioxidant activity of A. dahurica were obtained by gas chromatography–mass spectrometry (GC–MS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric ion reducing antioxidant power (FRAP) methods. The factors affecting free radical scavenging were investigated under different extraction conditions, on the basis of the single-factor experiment. The results showed that the optimum conditions for the DPPH method were ultrasonic extraction, using 80% methanol as extract the extraction solvent, a 20:1 (ml/g) ratio of liquid to material and an extraction time of 30 min. Furthermore, the spectrum–effect relationship between the GC–MS chromatograms and the antioxidant effect of A. dahurica was established to evaluate the antioxidant components of A. dahurica using multiple data analysis methods. Isoimperatorin and byakangelicol made the greatest contribution to scavenging DPPH free radicals and ferric-reducing antioxidant power. This result may provide the basis for developing new and effective products based on the antioxidant ingredients of A. dahurica.     

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 μM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.     

Antioxidant activity assays on-line with liquid chromatography

Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS(+) have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.     

Optimization of extraction and quantification technique for phenolics content of garlic (Allium sativum): An application for comparative phytochemical evaluation based on cultivar origin

A range of conventional, i.e. maceration, percolation, ultrasonic assisted, Soxhlet and Soxtec extraction (STE), to advanced extraction techniques of accelerated solvent extraction (ASE) was utilized for the first time in order to optimize the extract yield and recovery of phenolics—gallic acid (GA), rutin (RT) and quercetin (QT)—quantified via ultra-high performance liquid chromatography with diode array detector (UHPLC–DAD). The effect of solvents (n-hexane, dichloromethane and methanol) and temperature (60, 80 and 100°C) upon extraction yield, phenolic content and antioxidant activity (DPPH, ABTS and DPPH) was studied, and the method was validated in commercial food samples from Saudi Arabia, China and India. A high extract yield with percentage recovery was observed for STE (1221.10 mg/5 g; 24.42%) and ASE techniques (91.50 mg/1 g; 9.15%) in methanol at 100°C. UHPLC–DAD showed retention times (min) of 0.67, 1.93 and 1.90 for GA, RT and QT, respectively in the shortest runtime of 3 min. The yield for phenolics was higher for STE/ASE (ppm): 15.27/15.29 (GA), 85.24/37.56 (RT) and 52.20/33.40 (QT), respectively. In terms of antioxidant activities, low IC₅₀ values (μg/ml) of 1.09/1.18 (DPPH), 2.11/5.32 (ABTS) and 4.35/7.88 (phenazine methosulfate–nicotinamide adenine dinucleotide) were observed for STE and ASE, respectively. Multivariate analysis for STE showed a significant (P = 0.000) correlation for extraction type vs. extract yield and phenolics content; however, there was no significance for antioxidant activities vs. extraction type. ASE showed a positive correlation for solvent vs. extraction yield, phenolics and antioxidant activity; however, there was no correlation for extraction yield and DPPH activity. Principal component analysis for STE showed a major variability (52.02%) for extraction yield and phenolics in PC1 followed by PC2 (38.30%) for antioxidant activities. For ASE, PC1 (48.68%) showed a positive correlation for solvent vs. extraction yield and phenolics while PC2 (33.12%) showed a positive correlation for temperature and antioxidant activities. STE and ASE were the optimized extraction techniques for the garlic food sample while a significant effect of solvent and temperature was observed upon extraction yield, phenolics and antioxidant activity.     

Comparison of phenolic content and antioxidant activity of Actaea racemosa L. and Actaea cordifolia DC

Actaea racemosa L. is used as a component of drugs or dietary supplements to alleviate the menopause symptoms. Its biological activity is associated with the presence of phenolic compounds. In our work, the analysis of isoflavones and phenolic acids – caffeic acid (CA), ferulic acid and isoferulic acid (iFA) – both free and bonded in two species of Actaea, was conducted using HPLC-PAD technique. Moreover, the antioxidant effect of extracts from different parts of the investigated plants was determined on the basis of DPPH assay. Significant variation of CA and iFA content was observed. The highest content of CA was found in A. racemosa, while Actaea cordifolia contained the highest amount of iFA. Isoflavones were not found in the investigated plants. The antioxidant activity assay showed the high free radical-scavenging ability of the extracts obtained from different parts of the plant.     

An Improved On-Line HPLC-DPPH Method for the Screening of Free Radical Scavenging Compounds in Water Extracts of Lamiaceae Plants

An on-line HPLC-1,1-diphenyl-2-picrylhydrazyl (DPPH*) method has been improved for the detection of polar and nonpolar radical scavenging compounds from complex plant extracts. Eight water extracts were prepared from steam-distilled essential oil-extracted Lamiaceae plants (Origanum vulgare L., O. Onites L., O. Minutiflorum O. Schwartz et P. H. Davis, O. Syriacum L., Satureja cuneifolia Ten., Thymbra spicata L., Coridothymus capitatus (L.) Reichb. f., Majorana hortensis Moench). After the components within each extract had been separated by reverse phase chromatography using 10% to 100% methanol with 2% acetic acid as a mobile phase, analytes capable of scavenging a citric acid-sodium citrate buffered methanolic DPPH* solution were detected by post-column derivatization at 517 nm. The HPLC-DPPH* on-line method was applied to the qualitative and quantitative analysis of these Lamiaceae plant extracts. There was a strong correlation between the scavenging (negative) peak area and the concentration of the radical scavenging reference substances used. The radical scavenging compounds within the extracts were determined as benzoic acid and hydroxycinnamic acid derivatives, flavonoids and diterpenoids according to their retention time and UV spectral data. Rosmarinic acid and carnosic acid were identified as the dominant radical scavengers in these extracts by this method.     

Spectrum–effect relationship study between HPLC fingerprints and antioxidant of honeysuckle extract

Honeysuckle (Lonicera japonica flos) is a well‐known agent of edible and medicinal value in China and its antioxidative activity makes a major contribution to its dual use. However, the compounds responsible for its antioxidative activity are still unknown. In this study, 10 batches of honeysuckle were collected from different origins in China. The fingerprints were established by HPLC technique to investigate the compounds and a 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity assay was carried out to evaluate their antioxidant activity. partial least squares regression analysis was applied to set up the regression equation between DPPH radical scavenging activity and average peak area of common peaks of fingerprints. The results showed that peaks 10 (isochlorogenic acid B), 12 (isochlorogenic acid C), 11 (isochlorogenic acid A) and 9 (cynaroside) in the fingerprints were closely related to the antioxidant activity of 50% methanol extracts of honeysuckle. This study successfully established the spectrum–effect relationship between HPLC fingerprints and DPPH radical scavenging activity and provided a general model for exploring active components with a combination of chromatography and efficacy.     

Screening for antioxidants in complex matrices using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

The use of high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection to screen for antioxidants in complex plant-derived samples was evaluated in comparison with two conventional post-column radical scavenging assays (2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(+))). In this approach, acidic potassium permanganate can react with readily oxidisable compounds (potential antioxidants), post-column, to produce chemiluminescence. Using flow injection analysis, experimental parameters that afforded the most suitable permanganate chemiluminescence signal for a range of known antioxidants were studied in a univariate approach. Optimum conditions were found to be: 1×10(-3)M potassium permanganate solution containing 1% (w/v) sodium polyphosphates adjusted to pH 2 with sulphuric acid, delivered at a flow rate of 2.5 mL min(-1) per line. Further investigations showed some differences in detection selectivity between HPLC with the optimised post-column permanganate chemiluminescence detection and DPPH and ABTS(+) assays towards antioxidant standards. However, permanganate chemiluminescence detection was more sensitive. Moreover, screening for antioxidants in green tea, cranberry juice and thyme using potassium permanganate chemiluminescence offers several advantages over the traditional DPPH and ABTS(+) assays, such as faster reagent preparation and superior stability; simpler post-column reaction manifold; and greater compatibility with fast chromatographic separations using monolithic columns.     

Analysis of Cytotoxicity of Selected Asteraceae Plant Extracts in Real Time,Their Antioxidant Properties and Polyphenolic Profile

Plants from Asteraceae family are widely used for their therapeutic effects in the treatment of gastrointestinal diseases, but the consequences of excessive intake still need to be studied. The aims of this study were the evaluation of cytotoxicity, measurement of antioxidant properties and determination of polyphenolic profile of Tanacetum vulgare L. (tansy), Achillea millefolium L. (yarrow) and Solidago gigantea Ait. (goldenrod) ethanolic extracts. The cytotoxicity of extracts was monitored by xCELLigence system in real time by using porcine intestinal epithelial cell line (IPEC-1) and by measurement of changes in metabolic activity ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay). The antioxidant properties were measured by spectrophotometric methods and polyphenolic profiles were determined by HPLC-DAD for 50% ethanol extracts (10% w/v). Strong cytotoxic effect was recorded for tansy and yarrow extracts (125–1000 µg/mL) by xCELLigence system and MTS assay. Conversely, a supportive effect on cell proliferation was recorded for goldenrod extracts (125 µg/mL) by the same methods (p < 0.001). The antioxidant activity was in good correlation with total polyphenolic content, and the highest value was recorded for goldenrod leaves, followed by tansy leaves, goldenrod flowers and yarrow leaf extracts. The goldenrod extracts were abundant with flavonoids, whereas phenolic acid derivatives predominated in the polyphenolic profile of tansy and yarrow.     

New Antioxidant Phenolic Glucosides from Viburnum dilatatum

Chemical investigation of the fruits of Viburnum dilatatum Thunb. resulted in the isolation and characterization of four new phenolic glycosides, jiamiziosides A–D ( 1 – 4 ), together with five known compounds. Their structures were established by spectroscopic means and by comparison with the literature values. The antioxidant activities of the new isolates were determined against 2,2‐diphenyl‐1‐picrylhydrazyl (=2,2‐diphenyl‐1‐(2,4,6‐trinitrophenyl)hydrazinyl; DPPH) and superoxide radicals. Among the compounds tested, jiamizioside C ( 3 ) possesses the most potent inhibitory scavenging effect on DPPH and superoxide radicals with IC₅₀ values of 16.8 and 17.8 μM , respectively.     

Quantification and antioxidant and anti-HCV activities of the constituents from the inflorescences of Scabiosa comosa and S. tschilliensis

To investigate the bioactive constituents of the inflorescences of Scabiosa comosa and S. tschilliensis, which are used traditionally for liver diseases, we tested the antioxidant activity using 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), ferric reducing antioxidant potential (FRAP), and DPPH-ultra high performance liquid chromatography-mass spectrometer (UPLC-MS) assay. In addition, cell-based anti-HCV activity of the major compounds were evaluated. The plant extracts showed strong antioxidant activity. For the first time, 3,4-dicaffeoylquinic acid (DCQA), 3,5-DCQA and 4,5-DCQA were identified from genus Scabiosa. A UPLC-MS method in multiple reaction monitoring (MRM) mode was established to quantify 18 constituents in the inflorescences of Scabiosa. The 3,5-DCQA, chlorogenic acid and some glycosides of luteolin or apigenin were found to be the most abundant constituents. Chlorogenic acid and 3,5-DCQA showed excellent radical scavenging activity and demonstrated anti-HCV activity. These findings provided scientific evidences for the clinic use of this herbal medicine for liver diseases.     

Lagoecia cuminoides L., its antioxidant activity and polyphenolic constituents from Iran

Lagoecia cuminoides L. belongs to the family of Umbelliferae (Apiaceae), and known also as common wild cumin. The aerial parts of L. cuminoides were collected at the flowering stage and dried, then the methanolic extract was analyzed for polyphenol compounds identified by HPLC-DAD and antioxidant activity (DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay). It was found that the predominant phenolic constituents were chlorogenic acid, hesperidin, rosmarinic acid, hesperetin and vanillin. The antioxidant activity of methanolic extract from L. cuminoides was found 1597 μg/mL in DPPH scavenging assay. There is no strict positive relationship between the polyphenolic content and antioxidant activity of extracts.     

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods

Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R(2)=0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R(2)=0.893).     

Antioxidant and DNA damage protecting activities of newly synthesized thiol bridged bis-benzimidazole derivative and its dicationic analogue

Two new types of bis-benzimidazole derivatives containing thiol group have been prepared and characterized. The compounds contain sulfur with imidazole ring show promising biological activities such as antioxidant, anticancer, antimicrobial and etc. The aim of this study was synthesis of benzimidazole derivatives which not only show antioxidant activity but also protect DNA from oxidative damage. Antioxidant activities of the synthesized compounds were investigated with DPPH and hydrogen peroxide radical scavenging assays. DNA nicking assay was applied to establish activity of compounds to protect plasmid DNA from Fenton's reagent radicals. Both compounds had antioxidant activity, however, activity of dicationic analogue was greater than well-known antioxidant Vit C. IC₅₀ values calculated according to DPPH method were 14.5 μM for dicationic analogue ( 2 ) and 57.5 μM for 1,2-bis(1-methyl-1H-benzo[d]imidazol-2-ylthio)ethane ( 1 ). In hydrogen peroxide scavenge assay IC₅₀ values of compounds were 638.6 μg/mL for 1,2-bis(1-methyl-1H-benzo[d]imidazol-2-ylthio)ethane ( 1 ), 398.9 μg/mL for dicationic analogue ( 2 ). Furthermore, dicationic analogue promised an effective DNA protection due to its positive charge interacting with negatively charged DNA. Also the high solubility of the dicationic analogue in water due to its positive charge could provide a great advantage in biological applications.     

Synthesis and application of a novel solid-phase microextraction adsorbent: hollow fiber supported carbon nanotube reinforced sol-gel for determination of phenobarbital

The present study reports the development, validation and application of a new green liquid chromatographic method for the determination of glutathione (GSH) in vegetable samples. In this work we introduce—for the first time—ethyl propiolate (EP) as an advantageous post-column derivatization reagent for thiolic compounds. GSH (t_R = 6.60 min) and N-acetylcysteine (NAC, internal standard) (t_R = 11.80 min) were separated efficiently from matrix endogenous compounds by using a 100% aqueous mobile phase (0.1%, v/v CH₃COOH in 1 mmol L⁻¹ EDTA, Q_V = 0.5 mL min⁻¹) and a Prevail^® reversed phase column that offers the advantage of stable packing material in aqueous mobile phases. The parameters of the post-column reaction (pH, amount concentration of the reagent, flow rates, length of the reaction coil and temperature) were studied. The linear determination range for GSH was 1–200 μmol L⁻¹ and the LOD was 0.1 μmol L⁻¹ (S/N = 3). Total endogenous GSH was determined in broccoli, potato, asparagus and Brussels sprouts using the standards addition approach. The accuracy was evaluated by both recovery experiments (R = 91–110%) and comparison to an o-phthalaldehyde/glycine corroborative post-column derivatization fluorimetric method.