Post-column Fluorescence Derivatization Research Articles

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.

Some Insights about 1,6-Diphenyl-1,3,5-hexatriene—Lipid Supramolecular Assemblies by Steady-State Fluorescence Measurements

1,6-Diphenyl-1,3,5-hexatriene (DPH) is the most widely proposed molecular probe for the post-column fluorescence derivatization of lipids after liquid chromatography separation. This kind of detection consists of a supramolecular combination of DPH and eluted lipids. The detection is optimally performed in a mainly aqueous environment (over 80% v/v) because the weak fluorescence of DPH in water is drastically enhanced upon formation of supramolecular assemblies with lipids. In the present study, and in order to obtain better spectroscopic insights into the nature of these supramolecular assemblies, two different lipids were tested, 1,2,3-tridodecanoylglycerol (LLL) as a model triglyceride (nonpolar lipid) and dimyristoylphosphatidylcholine (DMPC) as a model phosphatidylcholine (charged amphiphilic lipid). Stoichiometry and association constants were determined on the basis of the variation of fluorescence intensity in the presence of various concentrations of lipids. LLL(60)-DPH(2) and DMPC(200)-DPH(2) complexes were identified with association constants as high as K(2) = (5.8 +/- 0.5) x 10(13) M(-2) and (17.3 +/- 2.0) x 10(13) M(-2) for LLL and DMPC, respectively. The fluorescence intensity of DPH in the presence of LLL is greater than in the presence of DMPC. An attempt to characterize the insertion mode of DPH in the lipidic supramolecular assemblies is also made.

Accumulation and Elimination Profiles of Paralytic Shellfish Poison in the Short-necked Clam Tapes japonica Fed with the Toxic Dinoflagellate Gymnodinium catenatum

The paralytic shellfish poison (PSP)-producing dinoflagellate Gymnodinium catenatum (Gc) was fed to the short-necked clam Tapes japonica, and the accumulation, transformation and elimination profiles of PSP were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested most of the Gc cells (4 x 10(6) cells) supplied as a bolus at the beginning of the experiment, and accumulated a maximal amount of toxin (181 nmol/10 clams) after 12 hr. The rate of toxin accumulation at that time was 16%, which rapidly decreased thereafter. During the rearing period, a variation in toxin composition, derived presumably from the transformation of toxin analogues in the clams, was observed, including a reversal of the ratio of C2 to C1, and the appearance of carbamate (gonyautoxin (GTX) 2, 3) and decarbamoyl (dc) derivatives (decarbamoylsaxitoxin (dcSTX) and dcGTX2, 3), which were undetectable in Gc cells. The total amount of toxin contained in clams and residue (remaining Gc cells and/or excrement in the rearing tank) gradually declined, and only about 1% of the supplied toxin was detected at the end of the experiment.

Rapid Determination of Vitellogenin in Fish Plasma by Anion Exchange High Performance Liquid Chromatography Using Postcolumn Fluorescence Derivatization with o-Phthalaldehyde

An analytical method involving anion exchange high performance liquid chromatographic determination of vitellogenin (Vtg) in fish plasma after postcolumn fluorescence derivatization with o-phthalaldehyde (OPA) was developed. The retention time of Vtg was about 11 min. The reagent variables for derivatization were optimized. The fluorophore was excited at 335 nm and detected at 435 nm. A calibration curve was established ranging from 0.13 to 11.28 microg. The determination limit of Vtg was found to be as low as 0.13 microg. The spiked recovery was 93.6% and interassay variability was less than 4%. The method developed was used to determine Vtg in fish plasma obtained from red sea bream (Pagrosomus major), black porgy (Sparus macrocephalus) and skew band grunt (Hapalogenys nitens), without complicated sample pretreatment. The results confirmed that the method showed advantages of being simple, rapid, reproducible and sensitive.

Uronic acid determination by high performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric high performance liquid chromatography (HPLC) technique for uronic acid microanalysis, a saline mobile phase and the postcolumn fluorimetric determination were combined. The detection limits of d-glucuronic, d-galacturonic and d-mannuronic acids were 7.19, 23.88 and 7.08 pmol, respectively. The proposed method was successfully applied to uronic acid microanalysis in a polysaccharide hydrolysate and a drink.

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol–water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 °C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5–100 μg/ml. The lower limit of detection (LLOD) was found to be 1.0 μg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.

The uptake, distribution and elimination of paralytic shellfish toxins in mussels and fish exposed to toxic dinoflagellates

We exposed green-lipped mussels Perna viridis and black sea breams Acanthopagrus schlegeli to toxic dinoflagellates Alexandrium fundyense to evaluate the accumulation, distribution, transformation, and elimination of paralytic shellfish toxins (PSTs) in a controlled environmental condition. The mussels were fed A. fundyense for 7 days followed by 3 weeks of depuration, and the fish were fed toxic clams (pre-exposed to the dinoflagellates) for 5 days followed by 2 weeks of depuration. The toxin content and the compartmental distribution of PSTs were monitored throughout the experiments by high-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD). This is the first report to assess the biokinetics of PSTs in marine fish under dietary exposure. The hepatopancreas in the mussels and the viscera in the fish accumulated most of the PSTs. Differential elimination of each toxin was observed in the mussels. The C2 toxins were eliminated rapidly in all organs; except in hepatopancreas, the more potent toxins such as GTX4, were eliminated slower during the depuration period. The relative proportions of various PSTs in the mussels changed over time, suggesting toxin-specific uptake and elimination rates, or biotransformation preferences between toxins. In the fish, the ratio of C1/C2 was 3.0 times ( p < 0.01) higher when compared to the clam tissues, indicating that conversion from C2 to C1 might have occurred when the toxin was transferred from the clams to the fish. In summary, species differences in uptake, distribution and elimination of PSTs were observed between mussels and fish, and this may influence trophic transfer of algal toxins in marine organisms.

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella ( Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4×10 7 cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12 h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5 h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5 h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.

Determination of Ophiopogon japonicus polysaccharide in plasma by HPLC with modified postcolumn fluorescence derivatization

Interest in antimyocardial ischemic activity of a graminan-type fructosan with an average molecular weight of 5000 Da from Ophiopogon japonicus (FOJ-5) has necessitated the development of a sensitive and specific method to study its pharmacokinetics. An HPLC method with modified postcolumn fluorescence derivatization to determine FOJ-5 in plasma was developed in this study. The Shodex Sugar KS-802 high-performance gel column was chosen for separating FOJ-5 from its degradation products and endogenous carbohydrates. The postcolumn procedure involved acid hydrolysis of the column eluate at 150 °C, which decreased the detection limit for FOJ-5 from 1 μg to 25 ng, followed by fluorometric reaction with guanidine in an alkaline medium at 90 °C. The clearance of FOJ-5 from the bodies of rats following intravenous injection displayed a complex type of kinetics involving at least two compartments, and the half-life of the elimination of FOJ-5 from plasma administered at 15 mg/kg (18.1 min) was quicker than that administered at 50 mg/kg (28.9 min). This modified approach can also be used for microanalysis of both nonreducing oligosaccharides and other neutral polysaccharides.

Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

We report the use of a sheath flow reactor for post-column fluorescence derivatization of proteins. The derivatization reaction employed naphthalene-2,3-dicarboxaldehyde (NDA) and β-mercaptoethanol, which were added in the sheath buffer. The labeled proteins were detected by laser-induced fluorescence with an argon-ion laser beam at 488 nm. The performance of this detection scheme was evaluated by separation of some protein standards. A column efficiency of 450,000 plates/m was obtained without stacking. The limits of detection for those standard proteins were determined to be from 8 to 32 nM. Excellent linear relationship was obtained with correlation coefficient of 0.9998 for α-lactalbumin concentration ranging from 3.91×10 −7 to 1.25×10 −5 M. Separation of protein standards at low pH was also demonstrated by reversing the electroosmotic flow (EOF) with addition of cetyltrimethylammonium bromide (CTAB) to the running buffer. Different separation selectivity was achieved, but the sensitivity is poorer than that at high pH. This post-column derivatization detection system was applied successfully to analyze the protein extract from HT29 human colon cancer cells as well as tryptic peptides.

Determination of bovine blood oleandrin by high-performance liquid chromatography and postcolumn derivatization.

A high-performance liquid chromatographic procedure with a postcolumn fluorescence derivatization is developed for the analysis of oleandrin in bovine blood. Oleandrin is separated by an octadecylsilane-bonded column with a mobile phase containing dehydroascorbic acid. The effluent of the column is mixed with concentrated hydrochloric acid and passed through poly(tetrafluoroethylene) tubing maintained at 70 degrees C. The resultant fluorophores are detected at 465 nm with excitation at 348 nm. Simple solid-phase extraction using Sep-Pak tC2 is effective for sample purification. We found the minimal detectable quantity of oleandrin in plasma to be 1.5 ng/mL at a signal-to-noise ratio of 3:1.

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric determination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of d-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.

Study of the feasibility of focused microwave-assisted Soxhlet extraction of N-methylcarbamates from soil

A study of the feasibility of a focused microwave-assisted Soxhlet device for the extraction of N-methylcarbamates from soil has been performed. The main factors contributing to the extraction efficiency—namely microwave power, irradiation time and number of cycles—were optimized by means of a two-level full factorial design. The extracts were analysed by HPLC–post-column fluorescence derivatization–detection at excitation wavelength 340 nm and emission wavelength 445 nm. The method has allowed the extraction of carbamate pesticides from contaminated soil with quantitative recoveries, similar to those provided by the US Environmental Protection Agency method 8318, without degradation of the target compounds during the extraction and using less organic solvent, as 75–80% of the extractant was recycled.

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction.

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.

A Modified Method for Measuring N.TAU.-Methylhistidine in Chiken Feed and Excreta.

Analysis of Nτ-methylhistidine is intricate and time consuming. In the present study, a modified technique using high-performance liquid chromatography, which utilizes a reversed-phase separation with ion pairing and post column fluorescence derivatization for the analysis of Nτ-methylhistidine in chicken feed and excreta is described. Two ml of the hydrolyzed sample was applied directly into the ion-exchange resin column without evaporation of HCl to roughly separate Nτ-methylhistidine from acidic and neutral amino acids. The Nτ-methylhistidine fraction was eluted with 1M pyridine and then evaporated, and the residue was dissolved in the mobile phase and subjected to high-performance liquid chromatography. The present method shortened the time needed to complete the assay by about 30min per sample and improved the separation of Nτ-methylhistidine.

High-Performance Liquid Chromatography Determination of N- and O-Demethylase Activities of Chemicals in Human Liver Microsomes: Application of Postcolumn Fluorescence Derivatization Using Nash Reagent

Formaldehyde is liberated in the process of cytochrome P450 (CYP) mediated demethylation of a wide variety of compounds containing the CH3N or CH3O functionality. A highly sensitive method using a high-performance liquid chromatography (HPLC) system with postcolumn derivatization was developed to measure the liberated formaldehyde as N- and O-demethylase activity of drugs in human liver microsomes. Following the chromatographic separation of formaldehyde on a C18 column, the formaldehyde was reacted with the Nash reagent in the postcolumn reactor at 100°C and detected by the fluorescence method. The results showed that the present method has excellent precision and accuracy. The intra- and interassay variances of this method were less than 10%. The newly developed HPLC method was found to be about 80-fold more sensitive than the colorimetric method in detection of formaldehyde. The N-demethylase activity of sertraline in rat liver microsomes determined by the present method did not differ from those detected by previous methods quantifying produced desmethyl metabolite. The present method has been successfully applied to determine the N-demethylase activities of several drugs, including aminopyrine, erythromycin, fluoxetine, S-mephenytoin, and sertraline, in human liver microsomes. This assay should be useful for generic analysis of N- and O-demethylase activities of xenobiotic and endobiotic chemicals by CYP enzymes.

Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal

Montargil reservoir, located in a dry flat area in the centre of Portugal, was filled in 1958 to fulfil agricultural, electric and industrial requirements. In May 1996, an intensive bloom of phytoplankton was detected. The algal community was strongly dominated by cyanobacteria with predominance of Aphanizomenon flos-aquae from May to June and Microcystis aeruginosa from July to August. Extracts of samples collected during the bloom period showed high toxicity by mouse bioassay. During the M. aeruginosa predominance period, the toxicity was ascribed to the presence of hepatotoxins, but clear symptoms of paralytic shellfish poison were observed when A. flos-aquae was the dominant species. In order to confirm the production of neurotoxins a strain of A. flos-aquae was isolated and established in culture. In this manuscript, we show the morphological characteristics and confirm paralytic shellfish toxins production by the strain isolated and maintained in culture. Identification of the saxitoxin analogs was achieved using high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) and liquid chromatographic mass spectrometry technique (LC-MS). The toxins found in the culture extract were GTX5 (64.5 mol%), neoSTX (23.0 mol%), dcSTX (6.1 mol%), STX (5.4 mol%) and GTX6 (1.1 mol%). This is, to our knowledge, the first report of unambiguous evidence of paralytic shellfish toxins produced by freshwater cyanobacteria in Portugal. The toxin profile is rather different from the previously reported PSP producing A. flos-aquae and demonstrates its diversity in terms of toxin production.

Analysis of O-phosphoamino acids in biological samples by gas chromatography with flame photometric detection.

The separation and determination of O-phosphoamino acids has been carried out by thin-layer chromatography (, , , , , , , , thin-layer electrophoresis (,,,, , , , , ), gel electrophoresis (), amino acid analyzer (, , ), high-performance liquid chromatography (HPLC) (, , , , , , , , , , , , , , ), capillary zone electrophoresis (CZE)(,) and immunoassay (, , ). However, most of these methods require 32P-labeling for detecting and quantifying O-phosphoamino acids, and therefore cannot be used for analysing nonradiolabeled O-phosphoamino acids in biological samples. Furthermore, some of them show poor resolution or low sensitivity. HPLC methods based on the precolumn ultraviolet (UV) derivatization with phenyl isothiocyanate (, , ), dabsyl chloride (), and N-α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (), the postcolumn fluorescence derivatization with o-phthalaldehyde (, , , , ), the precolumn fluores cence derivatization with 9-fluorenylmethyl chloroformate (,), and CZE methods based on the UV derivatization with phenyl isothiocyanate or dabsyl chloride (,) were highly sensitive, but some of these methods lack specificity. Furthermore, many of these methods require lengthy separation time, and clean-up of the sample to remove the excess reagent and coexisting substances. On the other hand, the immunoassays by antibodies raised against each O-phosphoamino acid have been employed for measuring endogeneously phos-phorylated amino acids, but these methods involve some problems in the specificity and crossreactivity of the antibodies. Current methods for the determination of O-phosphoamino acids have also been described in detail in ref. , and the review in ref. .

RP–HPLC tryptic mapping of IgG1 proteins with post-column fluorescence derivatization

Peptide mapping is an important analytical technique widely used to study the primary structure of proteins. In quality control settings, it is employed as an identity test to probe for small changes in protein primary structure. A great challenge in peptide mapping is to minimize the detection limit for peptides due to the low detectability of smaller peptides based on their ultraviolet absorbance. The detection of peptide fragments can be enhanced by pre- or post-column derivatization with fluorescent tags. The use of post-column o-pthalaldehyde (OPA) and fluorescamine chemistries for on-line derivatization of peptide fragments from the RP–HPLC tryptic maps of several IgG1 monoclonal antibodies was explored. This paper describes the simple and sensitive peptide mapping technique for structural confirmation of proteins using picomoles of samples by post-column fluorescence derivatization. A comparison of UV and fluorescence detection of a peptide map is also presented. The method includes post column OPA derivatization of tryptic peptides from RP–HPLC tryptic maps with fluorescence detection. The conclusion reached that fluorescence detection gave relative detectability for tryptic peptides that range from 10- to 100-fold better than those observed with UV detection. The sensitivity of the peptide map increased by about 200–500 fold, i.e. peptide maps could be obtained using 2–5 pmol of digest instead of 1 nmol of digest. A roughly equal fluorescence response for all peptides (equal peak areas) was generally observed.

Simultaneous determination of 7-methylguanine and O6-methylguanine in DNA by liquid chromatography with postcolumn fluorescence derivatization using 3,4-dimethoxyphenylglyoxal

3,4-Dimethoxyphenylglyoxal was found to react sensitively and selectively with guanine-containing compounds to yield their fluorescent derivatives. This reaction and postcolumn fluorescence derivatization liquid chromatography were used to determine the production of 7-methylguanine and O 6-methylguanine in DNA after alkylation with N-methyl- N-nitrosourea. In this method, nucleic acid bases produced by acid hydrolysis were separated on a reversed phase column (TSK-gel ODS-120T) and subjected to postcolumn derivatization involving demethylation followed by fluorescence reaction with 3,4-dimethoxyphenylglyoxal. The detection limits (S/N =3) for 7-methylguanine and O 6-methylguanine were 6.7 and 0.7 pmol respectively in a 20 μl injection volume.