O-phthalaldehyde Method Research Articles

Impact of homolactic fermentation using Lactobacillus acidophilus on plant-based protein hydrolysis in quinoa and chickpea flour blended beverages

In this study, three beverages formulated with quinoa and chickpea flour blends were fermented using Lactobacillus acidophilus LA-5 to assess the effect of lactic acid fermentation on the degree of hydrolysis of plant-based proteins. Additionally, the impact of quinoa and chickpea blends on the protein content and protein solubility in the beverages was evaluated. Fermentation was completed within 10 h, resulting in a decrease in the pH (<4.3) and an increase in titratable acidity and lactic acid (>0.37 % and > 1.7 g/L), respectively. SDS-PAGE and the O-phthalaldehyde method revealed hydrolysis of quinoa and chickpea proteins. A quinoa-to-chickpea ratio of 50 % exhibited the highest protein content (>2 %), solubility (43.6 %), and hydrolysis (35.9 %) after fermentation, indicating that an increase in chickpea improved these parameters in the prepared PBBs. Overall, fermentation using Lactobacillus acidophilus increased plant protein hydrolysis, and legume addition improved the protein content and the nutritional value of plant-based beverages.

The Expression Levels of Genes Responsible for the Enzymatic Activity of Bile Salt Hydrolase (BSH) and the Relationship of Cholesterol Assimilation in L. plantarum and L. paracasei.

The bile salt hydrolase (BSH) activity is responsible for the cholesterol-lowering effect of the probiotic strains. The present study aimed to investigate the relationship between bsh gene-expression (GE) levels responsible for the BSH activity and the parameters of bile salt resistance of different Lactobacillaceae species. Accordingly, 11 Lactobacillaceae family strains with high cholesterol assimilation ratio (49.21-68.22%) determined by the o-phthalaldehyde method selected from 46 Lactobacillaceaespecies was evaluated for their features including acid tolerance, bile tolerance, and BSH activity. All tested strains survived at pH 2 medium and 0.3% (w/v) bile salt and showed positive BSH activity for glycocholic acid (GCA) and taurocholic acid (TCA). BSH gene expression was performed to provide clear information and to identify the key genes responsible for BSH activity. bsh3 genes were found highest GE level (P < 0.05) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains. The results showed that high cholesterol assimilation ratio were closely correlated with BSH activity and the parameters of bile salt resistance. The results of this study will support the development of a new approach based on phenotypic and genetic analysis to determine the bile salt parameters. The study will be useful for the selection of Lactobacillus strains with high bile salt resistance.

Comparison of in vitro digestibility and DIAAR between vegan and meat burgers before and after grilling

Plant-based meat alternatives of high quality and digestibility could be a way to reduce meat consumption and, consequently, the environmental impact. However, little is known about their nutritional characteristics and digestion behaviour. Therefore, in the present study, the protein quality of beef burgers, known as excellent source of protein, was compared with the protein quality of two highly transformed veggie burgers, based on soy or pea-faba proteins, respectively. The different burgers were digested according to the INFOGEST in vitro digestion protocol. After digestion, total protein digestibility was determined, either based on total nitrogen (Kjeldahl) analysis, or after acid hydrolysis based on total amino groups (o-phthalaldehyde method) or total amino acids (TAA; by HPLC). The digestibility of individual amino acids was also determined, and the digestible indispensable amino acid score (DIAAS) was calculated based on in vitro digestibility. The impact of texturising and grilling on in vitro protein digestibility and the digestible indispensable amino acid ratio (DIAAR) was evaluated at the level of the ingredients and the finished products. As expected, the grilled beef burger had the highest in vitro DIAAS values (Leu 124 %), and grilled soy protein-based burger reached in vitro DIAAS values that could be rated as good (soy burger, SAA 94 %) protein source, according to the Food and Agriculture Organization. The texturing process did not significantly affect the total protein digestibility of the ingredients. However, grilling led to a decrease in digestibility and DIAAR of the pea-faba burger (P < 0.05), which was not observed in the soy burger, but led to an increase in DIAAR in the beef burger (P < 0.005).

Characterization of Short-chain fatty acid-producing and cholesterol assimilation potential probiotic Lactic acid bacteria from Chinese fermented rice

Probiotic strains are phrased as beneficial for life because this bacterial association has substantial effects on human and animal health when administrated orally, among which bio-active metabolite short-chain fatty acids production and cholesterol assimilation are most significant. All the short-chain fatty acids, mainly acetate, propionate, and butyrate reveal valuable effects on human health. In this research, the ability of 9 the potential lactic acid bacteria food-isolated probiotics was investigated to produce bio-active metabolite short-chain fatty acids directly or indirectly, and cholesterol-lowering ability through various mechanisms. Their 16S rRNA gene sequences were analyzed. short-chain fatty acids were quantified by gas chromatography, while cholesterol assimilation was measured using the o-phthalaldehyde method with slight modifications. All isolated strains through this study have the ability to optimize cholesterol content in the culture broth, short-chain fatty acid production, and survival in extreme alkaline and acidic conditions. The highest resistance to bile salt concentrations, γ-hemolysis, and strong antibacterial activity against pathogenic indicators, especially Escherichia coliATCC25922. The highest Lipid accumulation inhibitory effect was noticed in Lentilactobacillus hilgardii LMG 07934 as a potential probiotic isolate on the basis of cholesterol assimilation capacity (i.e., 91.4%), along with the ability to produce abundant i.e., butyric acid, propionic acid, and acetic acid, followed by Lacticaseibacillus paracasei ATCC 334 which has 85.3% cholesterol assimilation ability. These findings demonstrated that these isolated strains from Chinese fermented rice can be used as a potential probiotic and alternate therapeutic supplement because of their ability to produce short-chain fatty acids and their cholesterol-lowering ability.

The production of collagen type I hydrolyzate derived from tilapia (Oreochromis sp.) skin using thermoase PC10F and its in silico analysis

ABSTRACT The main focus of this study was to produce the angiotensin-I converting enzyme (ACE) inhibitory peptides from collagen type I using new combination of Thermoase PC10F and tilapia skin as a protein collagen source. In silico evaluation of amino acids profile of collagen type I, ACE-inhibitory peptides and sensory profile of the hydrolyzate were performed using Protparam and BIOPEP, respectively, to initially predict the potential peptide produced. Hydrolysis conditions were studied based on the best preliminary design of degree hydrolysis (DH) using o-phthalaldehyde (OPA) method. The temperature of 60°C, pH of 5 and an enzyme to substrate ratio of 1:100 were observed as the best conditions to obtain the highest DH using Thermoase PC10F. Peptide chain length (PCL) showed a proportional decrease as DH increased. In silico study of alpha 1 and alpha 2 collagen type I indicated a significant amount of glycine and proline, which are known to stabilize the collagen structure. The profile of potential biological activity suggested that 247 ACE-inhibitory peptides could be obtained from the parent protein sequence of collagen type I alpha 1 and alpha 2. In silico hydrolysis by thermolysin is believed to release potent theoretical peptides with IC50 less than 5 μM for both collagen type I subunits with a high number of occurrences. Further analysis on its potential sensory peptides had shown that both alpha 1 and alpha 2 contained mainly sweet amino acid, bitter peptides, and bitter amino acids.

In vitro assessment of pediococci- and lactobacilli-induced cholesterol-lowering effect using digitally enhanced high-performance thin-layer chromatography and confocal microscopy.

The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19-59% in the absence and 14-69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study. Graphical abstract (A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB.

Glucose Glycation of α-Lactalbumin and β-Lactoglobulin in Glycerol Solutions.

The glucose glycation of α-lactalbumin and β-lactoglobulin at 50 °C in a glycerol-based liquid system was investigated to evaluate the effect of water activity on glycation and site-specificity in a glycerol matrix. Glycation extent during the reaction was determined using the o-phthalaldehyde (OPA) method as well as ultraperformance liquid chromatography combined with electro-spray ionization mass spectrum (UPLC-ESI-MS) analysis. Glycation sites were identified by data-independent acquisition LC-MS (LC-MSE). The surface potential achieved by PyMOL and the tertiary structure determined by circular dichroism (CD) were used to assist the analysis of the glycation site-specificity in the glycerol matrix. The water activity of glycerol solutions was negatively correlated to the glycerol concentration. Results showed that the initial glycation rate in glycerol matrix was fitted to a linear equation in the first 48 h. Glycation accelerated with the increase of glycerol concentration, namely, the decrease of water activity, regardless of the native structure of the protein. The glycation sites were identical at a similar DSP although achieved at different water activities, with 4 and 7 sites detected in α-lactalbumin and β-lactoglobulin, respectively. However, compared with the glycation sites in a water-based matrix, the site-specificity of glycation was affected by the glycerol matrix, depending on the native structure of the proteins. Glycation was prone to occur at the reactive sites distributed on the surface of the proteins, particularly in the region with positive potential.

&lt;i&gt;In vitro&lt;/i&gt; antioxidative activity of moss extract, and effect of moss on serum lipid level of mice fed with high-fat diet

Purpose : To evaluate the potential of active compounds derived from moss in the prevention and treatment of various diseases. Methods : Three species of moss were extracted with deionized water at 95 °C, and with 70.5 % ethanol at 85 °C. Analysis of total phenolic contents (TPC) of the extracts were performed by Folin- Ciocalteu (FC) method. The antioxidant activity of the extracts were determined using three methods, namely, by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). In vivo effects were evaluated in mice fed high fat diet (HFD) supplemented with 20 % ground moss. Cholesterol levels in HFD were evaluated by ophthalaldehyde method. Serum triglyceride levels were measured using triglyceride (TG) kit, while blood insulin level and leptin concentration were measured by enzyme-linked immunosorbent assay (ELISA) kit. Results : The moss extracts exhibited antioxidative effects, as evidenced of . TPC of 47.20 ± 11.20 to 119.87 ± 11.51 mg GAE/mg, respectively. ABTS scavenging activity was 1078.11 ± 18.95 to 2587.33 ± 46.19 μmol Trolox/mg, DPPH scavenging activity of were 42.11 ± 8.22 to 298.78 ± 20.02 μmol Trolox/mg, and FRAP value of 393.19 ± 24.64 to 1070.14 ± 17.92 μmol Trolox/mg, respectively. Mice fed with 20 % ground moss did not show any significant effect (p < 0.05) on visceral weight and blood lipid levels of HFD, while leptin concentrations reduced significantly to 4.74 ± 0.00 and 0.20 ± 0.00 ng/dL) relative to HFD alone (26.72 ± 6.53 ng/dL). Conclusion : Moss can potentially be used as an antioxidative ingredient, for the improvement of overall human health, suggesting that important medical benefits associated with moss consumption. However, further investigations are required to ascertain this. Keywords : Moss, total phenolic content, antioxidant activity, insulin, leptin

Studies on the Resistance of Cholesterol-Lowering of Lactic Acid Bacteria to Simulated Gastrointestinal Circumstance &lt;i&gt;In Vitro&lt;/i&gt;

Acid and bile are primary natural barriers in the gastrointestinal tract, and tolerances of the probiotics help to survive and exert their beneficial effects. Eight lactic acid bacteria strains were selected based on cholesterol-lowering ability from Chinese traditional kefir grain, namedlactococcus lactis subsp lactisKS4, J4, P1 and P5,lactobacillus caseiKTx, KL1 and J1 andstreptococcus thermophilusTx. The strains were cultured in MRS medium (pH 3.0 and 0.3% bile salt). Acid and bile salt tolerances were evaluated by measuring and comparing optical density values at 600nm (OD600). The o-phthalaldehyde method (OPA) was used. The cholesterol-lowering rate of strains KL1 and J1 are 60.1% and 55.3% to simulated gastrointestinal adverse circumstance in vitro. The results indicate thatlactobacillus caseiKL1 and J1 are effective as probiotics with cholesterol-lowering activities.

The effect of NaCl substitution with KCl on proteinase activities of cell-free extract and cell-free supernatant at different pH levels and salt concentrations: Lactobacillus acidophilus and Lactobacillus casei

The aim of this study was to investigate the effect of substitution of NaCl with KCl at different pH levels and salt concentrations on proteinase activity of cell-free extract and cell-free supernatant of the probiotics Lactobacillus acidophilus and Lactobacillus casei. de Man, Rogosa, and Sharpe broth aliquots were mixed with 2 pure salts (NaCl and KCl) and 2 salt concentrations at 2 concentration levels (5 and 10%), inoculated with Lactobacillus acidophilus or Lactobacillus casei, and incubated aerobically at 37°C for 22h. The cultures were then centrifuged at 4,000×g for 30min, and the collected cell pellets were used to prepare cell-wall proteinases and the supernatants used as a source of supernatant (extracellular) proteinases. The proteolytic activity and protein content of both portions were determined. After incubation of both portions with 3 milk caseins (α-, β-, κ-casein), the supernatants were individually subjected to analysis of angiotensin-converting enzyme (ACE)-inhibitory activity and proteolytic activity using the o-phthalaldehyde method. Significant differences were observed in ACE-inhibitory and proteolytic activities between salt substitution treatments of cell-free extract and cell-free supernatant from both probiotic strains at the same salt concentration and pH level.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Assessment of Different Fluorimetric Reactions for Cyanide Determination in Flow Systems

Several fluorimetric reactions for the determination of cyanide by means of flow injection systems were tested. The first reaction is based on the displacement of 8-hydroxyquinoline-5-sulfonic acid (HQS) from the Pd(HQS) 2 complex by cyanide ion and the subsequent reaction of free HQS with Mg II to form a fluorescent species. Secondly, the inhibitory effect of cyanide on the reaction between iodine and fluorescein was also evaluated as a method to quantify cyanide. The third method is based on the detection of an isoindole derivative formed by the reaction of o-phthalaldehyde (OPA) or 2,3-naphthalenedialdehyde (NDA) with glycine in the presence of cyanide. Chemical and hydrodynamic parameters were optimized for each system and the analytical performance of the methods was established. Detection limits of 5 µg l - 1 (fluorescein–iodine method), 0.40 µg l - 1 (Pd–HQS–Mg method), 0.25 µg l - 1 (OPA method) and 0.03 µg l - 1 (NDA method) were obtained.

Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of γ-glutamylcysteine and glutathione: application to assay of γ-glutamylcysteinyl synthetase activity and glutathione concentration in liver

The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic γ-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivitization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and γ-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at −20°C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic γ-glutamylcysteine synthetase in the same liver preparation was found to be 4.85±0.47 nmol min −1 mg −1 protein by the OPA method and 4.42±0.52 nmol min −1 mg −1 protein by the MB method. GSH concentrations were found to be 90.4±6.5 nmol/mg protein for the OPA method and 92.5±3.4 for the MB method.

A comparative study of the O‐phthalaldehyde method for the neurotoxin 3‐N‐oxalyl‐l‐2,3‐diaminopropanoic acid as modified by various laboratories

AbstractThe optimization of the quantitative photometric estimation of the neurotoxin 3‐N‐oxalyl‐L‐2,3‐diaminopropanoic acid (ODAP) after hydrolysis and reaction with o‐phthalaldehyde (OPT) reagent is described.Six modifications of the OPT procedure (as practiced in different laboratories) and the original Rao method were compared in estimating the ODAP content of two cultivars of Lathyrus sativus. Considerable variation in the ODAP content of the same sample was observed even when determined by the same procedure in some cases. The desired reproducibility was observed with the procedure of the Agriculture Canada Research Station, Mordsen, Manitoba, Canada (Campbell method). The use of 0.5 M tetraborate as buffer further significantly improves the reproducibility. The Campbell modification includes a number of controls which reduce variation. The present study recommends the Campbell modification of the OPT method but with tetraborate buffer as standard procedure for the screening of the toxicity of L. sativus cultivars.

Fluorescent Activity Assay of the Enzymes Hydrolyzing Acidic Polysaccharides

Based on the amide linkage formation catalyzed by water-soluble carbodiimide, EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), various acidic polysaccharides could be labeled by the fluorescent probe of EDAN (N-1-ethylenediaminonaphthalene). The pectinase reaction was evaluated by the EDC-OPA (o-phthalaldehyde) method [Anal. Biochem., 189, 122–125 (1990)]. Moreover, paper chromatographic purification of the glucuronic acid-EDAN adduct allowed a 57-fold increase in the sensitivity of detection limit, i.e., about 0.19 nmol of glucuronate could be measured. Pectin–EDAN adduct worked as the substrate for pectinase and changes in the molecular weight distribution during the course of pectinase digestion were monitored by gel filtration with a Sepharose 6B column. In the case of hyaluronic acid–EDAN adduct, the fluorescent substrate was unfavorable to the hyaluronidase action and 2 to 5-fold higher Kms were obtained with the hyaluronic acids of lower and higher contents of EDAN, respectively.

Determination of Methionine and Its Oxides in Gelatin

Methionine, methionine sulphoxide and methionine sulphone in gelatin were determined simultaneously by hydrolysis with p-toluenesulph- onic acid and then HPLC using the o-phthalaldehyde method. The IAG gelatins, oxidized gelatins, heat- coagulable substances (HC) and sol fractions (SF) of some gelatins were analyzed. Almost all the gelatins examined were found to have been oxidized. The oxidation ratios of the HC and SF were higher than the gelatins from which they were derived.

Automated Quantitation of Polyamines by Improved Cation-Exchange High-Performance Liquid Chromatography Using a Pump Equipped With a Plunger Washing System

Abstract An improved method for simple, precise, and sensitive quantitation of polyamines[putrescine, spermidine, spermine, cadaver-ine(in part) and 1, 6-diaminohexane] by cation-exchange high performance liquid chromatography is described. Postcolumn deriv-atization using the o-phthalaldehyde method was employed to detect them. Analysis took 45 minutes and the minimum detectable amount of each polyamine was 10 – 20 pmol/μ1. An isocratic pump equipped with a plunger washing system and a mobile phase of pH 5.3 are the advantages of this improved method. Consequently, an operator can carry out routine and automated polyamine analysis without any hesitation regarding maintenance of the pump or column damage due to crystal precipitation from the mobile phase on or in the pump, which has been a problem previously encountered with cation-exchange resin. Reproducibility of the day to day precision and duplicate determination, and simultaneous reproducibility of the standard mixture were calculated (CV<2.2%).

Magnesium and sulfur in mast cell and basophil granules of lower vertebrates in relation with histamine

Mast cell and basophil granules of the vertebrate contain heparin or related sulfated proteoglycans. Histamine is also present in mammalian mast cells and basophils. However, no histamine is detected in mast cell granules of the amphibian or fish, while it is shown in those of reptiles and birds A quantitative x-ray microanalysis of mast cell granules of fresh frozen dried ultrathin sections of the tongue of Wistar rats and tree frogs disclosed high concentrations of sulfur in rat mast cell granules and those of sulfur and magnesium in the tree frog granules. Their concentrations in tree frog mast cell granules were closely correlated (r=0.94).Fresh frozen dried ultrathin sections and fresh air-dried prints of the tree frog tongue and spleen and young red-eared turtle (ca. 6 g) spleen and heart blood were examined by a quantitative energy-dispersive x-ray microanalysis (X-650, Kevex-7000) for the element constituents of the granules of mast cells and basophils. The specimens were observed by transmission electron microscopy (TEM) (80-200 kV) and followed by scanning transmission electron microscopy (STEM) under an analytical electron microscope (X-650) at an acceleration voltage of 40 kV and a specimen current of 0.2 nA. A spot analysis was performed in a STEM mode for 100 s at a specimen current of 2 nA on the mast cell and basophil granules and other areas of the cells. Histamine was examined by the o-phthalaldehyde method.

Localization and quantification of histamine in injured skin as parameters for the timing of wounds

Localization and estimation of the histamine (HA) content in skin wound edges in 86 Sprague-Dawley rats and three cases of human injuries were carried out by a microfluorimetric method specific for this amine which forms a complex with o-phthalaldehyde (OPT). Distribution and density of the mast cells in the same areas were observed at the same time by toluidine blue stain. In all skin specimens with antemortem wounds, both the epidermis and upper dermis exhibit extracellular yellowish fluorescence of the HA-OPT complex. The fluorescent zone spreads in the wound edges with the lapse of time in vital injuries. The HA content increases gradually up to 30 min and then the yellow histamine fluorescence in areas 0–200 μm from the wound edge decreases. None of these features can be observed in normal skin and postmortem-injured skin. Mast cell degranulation can be demonstrated in all antemortem-injured skin. No statistical relationship exists between the number of mast cells and the HA-OPT fluorescence in either ante- or postmortem-injured groups. This study indicates that skin HA microfluorimetry by the OPT method is of practical value for distinguishing ante- from the postmortem wounds and for timing antemortem wounds.

Field method for determination of traces of thiols in natural waters

Derivatization with 2,2′-dithiobis(5-nitropyridine) (DTNP) was used to stabilize thiols in field samples and to determine these compounds by liquid chromatography (LC) with ultraviolet detection. Alternatively, the thiols can be regenerated from DTNP derivatives using tributylphosphine (TBP) and derivatized with o-phthalaldehyde (OPA). The DTNP derivatives were stable at pH 5 and 6 for more than 2 weeks in the reaction mixture, but the stability was considerably lower at pH values > 8. Derivatization at pH 6 was routinely done and the reaction was complete within 5 min. The derivatives can be extracted quantitatively on commercial C 18 cartridges, which allows several-fold sample enrichment. The cartridge-adsorbed derivatives are stable at 0–5 °C for long periods and, therefore, can be stored for LC separation at a later date. DTNP derivatives of ten low-molecular-weight thiols were separated on a C 18 microbore column using gradient elution and a flow-rate of 200 μl min −1. The relative standard deviation based on repeated analyses of standards is about 5% in the range 0.5–2 nM. Without C 18 cartridge enrichment, the detection limits of the studied thiols are in the range 50–100 nM. These limits are further reduced by about 100-fold by using the OPA method after regeneration of thiols from the DTNP derivatives using TBP. Applications of the method to coastal sediment pore water and anoxic Black Sea water are illustrated.