Estimation of tetrahydrobiopterin and other pterins in cerebrospinal fluid using reversed-phase high-performance liquid chromatography with electrochemical and fluorescence detection

We describe an isocratic, reversed-phase high-performance liquid chromatographic method for the simultaneous measurement of fully oxidised, dihydro- and tetrahydropterins in cerebrospinal fluid. Tetrahydrobiopterin is detected electrochemically using an ESA Coulochem detector in the redox mode. Dihydropterins are detected by fluorescence following post-column electrochemical oxidation, and fully oxidised pterins by their natural fluorescence. Apart from addition of antioxidants, no sample preparation is required. Comparison is made with methods requiring chemical oxidation for detection of tetrahydrobiopterin. Some results on children with neurological disease are presented.     

Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography

An efficient LC method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins based on ion-exchange chromatographic separation of the toxins followed by electrochemical post-column oxidation and fluorescence detection as well as mass spectrometric (MS) detection. The method can be applied to the determination of PSP toxins in phytoplankton and to control seafood for PSP content.     

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection

Unmodified silica columns together with non-aqueous ionic eluents give stable yet flexible systems for the analysis of basic drugs by high-performance liquid chromatography. Low-wavelength UV and fluorescence detection may be used, and fluorescence may be optimised by, for example, post-column pH change or derivatisation of some primary aliphatic amines with o-phthaldialdehyde. A novel feature is that electrochemical oxidation can be used for the detection of most analytes and this detection mode is thus discussed in detail. Retention and relative response data (UV, 254 nm and electrochemical, +1.2 V) have been generated for 462 compounds using a 125-mm Spherisorb S5W silica column and methanolic ammonium perchlorate (10 mM, pH 6.7) as eluent. This system can be used isocratically in qualitative analyses and also for quantitative work, when either the wavelength or the applied potential can be adjusted to optimise the response.     

离子色谱-在线柱后电化学衍生-荧光法测定酪氨酸

采用自制电解池作为电化学衍生装置,建立了离子色谱一电化学衍生一荧光法测定饮料中的酪氨酸。在碱性淋洗液作用下,酪氨酸在阴离子交换柱上被分离,到达自制电解池的阳极室,在阳极上被氧化,氧化后的产物因具有较强的荧光而被荧光检测器检测。离子化试剂既可以做色谱分离所需的淋洗液,又可以作为电化学反应优良的支持电解液,因此,离子色谱和电化学衍生具有较好的兼容性。最佳的实验条件为：淋洗液NaOH（10mmol／L）＋乙腈（ACN,1＋9）,流动相流速1．0mL／min,电解池电压1．0V,激发／发射波长320／420NM。在优化的实验条件下,酪氨酸的线性范围为0．01～10mg／L,检出限为1．2μg／L（信噪比S／N=3）。50μg／L的酪氨酸标准溶液进样7次,得到的色谱峰面积相对标准偏差为2．5％。方法具有快速,灵敏和选择性好的特点,并成功用于饮料中酪氨酸的测定。     

Improvements to EPA method 531.1 for the analysis of carbamates that resulted in the development of U.S. EPA Method 531.2

This project is undertaken to fully optimize the U.S. Environmental Protection Agency Method 531.1 post-column chemistries and to incorporate recent advances in liquid chromatographic separation, post-column derivatization, and detection techniques. Sample preservation and storage stability studies establish citric acid as a suitable replacement for the caustic monochloroacetic acid in the current method and confirm its antimicrobial effectiveness. Performance of an alternate set of commercially available post-column reagents is also investigated. This research has resulted in the publication of Method 531.2, a high-performance liquid chromatographic direct injection method for the analysis of N-methylcarbamoyloximes and N-methylcarbamates using post-column derivatization and fluorescence detection.     

Singlet oxygen (O2(1Deltag)) quenching by dihydropterins

Pterins belong to a class of heterocyclic compounds present in a wide range of living systems. They participate in relevant biological functions and are involved in different photobiological processes. Dihydropterins are one of the biologically active forms of pterins. The photoinduced production and quenching of singlet oxygen (1O2) by a series of dihydropterins (7,8-dihydrobiopterin (DHBPT), 7,8-dihydroneopterin (DHNPT), 6-formyl-7,8-dihydropterin (FDHPT), sepiapterin (SPT), 7,8-dihydrofolic acid (DHFA), and 7,8-dihydroxanthopterin (DHXPT)) in aqueous solution at physiological pH ( approximately 7) were investigated, and the quantum yields of 1O2 production (PhiDelta) and rate constants of total quenching (kt) of 1O2 were determined. Studied compounds do not produce 1O2 under UV-A irradiation and are very efficient 1O2 quenchers. The chemical reactions between 1O2 and dihydropterin derivatives were investigated, and the corresponding rate constants (kr) were found to be particularly high. The oxidized pterin derivatives, biopterin (BPT), neopterin (NPT), 6-formylpterin (FPT), and folic acid (FA), were identified and quantified during the reaction of 1O2 with DHBPT, DHNPT, FDHPT, and DHFA, respectively. Besides the oxidation of the dihydropyrazine ring to yield the corresponding oxidized pterins, a second oxidation pathway, leading to fragmentation of the dihydropterin and formation of non-pterinic products, was identified. Mechanisms and biological implications are discussed.     

Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

Assay of 1-hydroxy-3-aminopropylidene-1,1-bisphosphonate and related bisphosphonates in human urine and plasma by high-performance ion chromatography

A method is described for the analysis of 1-hydroxy-3-aminopropylidene-1, 1-bisphosphonate and related bisphosphonates in human urine and plasma. Samples are spiked with 1-hydroxy-5-aminopentylidene-1,1-bisphosphonate as an internal standard and calcium chloride is added to precipitate the bisphosphonates. Following centrifugation the precipitate is redissolved in acetic acid, and the bisphosphonates are separated by high-performance ion chromatography on a Dionex AS7 column using nitric acid as mobile phase. The bisphosphonates are oxidised to orthophosphate using post-column addition of ammonium persulphate and this is followed by post-column reaction with molybdenum-ascorbate to yield the phosphomolybdate chromophore which is detected at 820 nm. A detection limit of 10 ng/ml is possible.     

Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination

A commercially available system is described for the fully automated clean-up and high-performance liquid chromatographic (HPLC) analysis of aflatoxins in foods and animal feeds. The system marketed primarily for handling solid-phase extraction columns has modified software to facilitate use with immunoaffinity columns. Sample extract clean-up followed by injection onto an HPLC column with post-column iodination and fluorescence detection is carried out completely unattended. A coefficient of variation of 5.1% for aflatoxin B1 analysis was obtained, and the accuracy of the system was demonstrated by the analysis of peanut butter certified reference material.     

Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection

A method for the simultaneous quantification of reduced and oxidized glutathione in human plasma employing a two-dimensional chromatographic system with parallel porous graphitized carbon (PGC) columns coupled with fluorescence (FLD) and coulometric electrochemical detection (ED) has been developed. Post-sampling oxidation of reduced glutathione (GSH) was prevented by derivatizing the -SH group with monobromobimane (MBB) and the glutathione-bimane adduct (GSMB) was detected by FLD. Oxidized glutathione (GSSG) was detected by ED optimized to give lowest possible limits of detection (LOD). The method is fully validated and is currently used for determination of GSH, GSSG and its redox potential in different clinical studies.     

Fluorescence probe assisted post-column detection for lipid analysis in microbore-LC

A general approach, still few exploited so far and never associated with microbore-LC, consisting of detection of various lipid classes (i.e. phospholipids, triglycerides, ceramides and glycosphingolipids) by non-covalent association with 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe is developed. This mode of detection was coupled with non-aqueous reversed-phase microbore-LC (C18) by using classical post-column fluorescence detection. The classical LC system was first adapted to microbore-chromatography (internal diameter 1 mm) without apparatus miniaturization of the solvent delivery system and the detection cell. For this purpose, the detection parameters (probe concentration, post-column flow rate, post-column reactor length and post-column system temperature) were optimized by a central composite design (CCD) using a mixture of phosphatidylcholine (PC) species as a lipid model and DPH (lambda(ex) = 350 nm, lambda(em) = 430 nm) as a fluorescence probe. The optimal conditions of detection for the various molecular species of PC were determined for a DPH concentration of 3.35 micromol/L, a post-column flow rate of 0.5 mL/min, a reactor length of 1.4 m and a temperature of 35 degrees C. The fluorescence response was linear over a wide range of PC species from 5 microg/mL to 100 microg/mL and the lower limit of detection (signal/noise = 3) was about 1 microg/mL, that is equivalent to evaporative light scattering detection (ELSD). Others molecular species of various classes of lipids, i.e. triglycerides, ceramides and glycosphingolipids were also easily detected. Thus, this study demonstrated the versatility of the proposed system of detection which was shown to be sensitive, easy to perform, non-destructive and allowed, in contrast to ELSD, for a linear response with various polarity lipid classes.     

HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization

A liquid chromatography method with indirect fluorescence detection has been developed for simultaneous detection of cysteine, cystine, homocysteine, homocystine, glutathione and glutathione disulfide. After separation in their native forms, a post-column solution of tris(2-carboxyethyl)phosphine (TCEP) at 32 mM, pH 8 is added to reduce the disulfides on-line to the corresponding thiol. The effluent is then merged with a second post-column solution of the highly fluorescent complex Cd(HQS)(2)(2-). The cadmium is complexed by the eluting thiols, effectively quenching the fluorescence. Optimization of the separation, the on-line reduction and the indirect fluorescence detection are discussed. Detection limits from 0.3-4.3 microM (0.04 to 2.6 ppm) are achieved for the six analytes in a 20 min separation.     

Study of the tautomerization of quinonoid dihydropterins by liquid chromatography/electrochemistry

An investigation of the tautomerization of quinoid dihydropterins by dual-electrode liquid chromatography/electrochemistry (l.c.e.c.) is presented. The effect of various side-chains in the 6-position of the pterin revealed that wide variations in the rate of tautomerization occur with different pterin species. The effects of two buffers (phosphate and acetate) with low pK_a values were also studied over the pH range 2–8. The rate of tautomerization was much faster for quinonoid dihydrobiopterin in the phosphate buffer than in the acetate buffer whereas the rate of rearrangement of the other quinonoid dihydropterins was little affected by the type of buffer. This report also illustrates the advantages of l.c.e.c. for this type of investigation. Variations in pH are readily studied without changes in experimental conditions. Species that cannot be resolved chromatographically are shown to be resolvable by the electrochemical detector. Stability to rearrangement can be compared to stability to oxidation. Finally, only very little sample is required.     

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.     

Ionspray mass spectrometry of marine toxins. III. Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry.

Ionspray mass spectrometry has been used to monitor the purification of saxitoxin, the parent compound in the family of toxins responsible for paralytic shellfish poisoning (PSP), from a strain of the dinoflagellate Alexandrium excavatum. Quantitative results obtained by flow-injection analysis are compared to those obtained by high-performance liquid chromatography with post-column oxidation and fluorescence detection. The coupling of liquid chromatography and capillary electrophoresis with ionspray mass spectrometry is described for the separation of mixtures of PSP toxins and the highly potent pufferfish toxin tetrodotoxin. Tandem mass spectrometry is used to provide the structural information, and the ability to distinguish isomeric PSP toxins both chromatographically and mass spectrometrically is demonstrated.     

Indirect detection of substituted phenols and cannabis based on the electrochemical adaptation of the Gibbs reaction

The voltammetric behaviour of 2,6-dichloro-p-aminophenol (PAP) in aqueous solution at an edge plane pyrolytic graphite electrode was explored and its sensitivity to additions of substituted phenols examined. Proof of concept is shown for the electrochemical adaptation of the Gibbs reaction, where reaction of the oxidised form of PAP with substituted phenols provides an indirect methodology for the analytical detection of these compounds. This indirect protocol provides an attractive alterative to the direct electrochemical oxidation of phenolic compounds, since the latter is plagued by electrode passivation, leading to low sensitivity. It is observed that phenol, 4-phenoxyphenol, methylphenol (para and meta), nitrophenol and most importantly, tetrahydrocannabinol, can be detected voltammetrically. Such a protocol is particularly attractive for roadside testing for cannabis in drug drivers.     

Analysis of Pterins in Urine by HILIC

Pterins are a class of compounds excreted in urine. Levels of the pterins are found to be significantly elevated in a variety of diseases. A new method involving hydrophilic interaction chromatography with fluorescence detection has been developed for analysis of neopterin, biopterin, and isoxanthopterin in urine samples. Separation of these pterins on an aminopropyl hydrophilic interaction column was achieved by isocratic elution. The effects of the organic modifier content, ionic strength, and pH of the mobile phase on the hydrophilic behavior of the pterins were studied and the mechanism of their separation was also investigated. Under the optimum chromatographic conditions the linearity (r ≥ 0.9995) and repeatability (relative standard deviation < 4.0%) of the method are good. Compared with reversed-phase high-performance liquid chromatography, the method is simple and convenient. The method was applied to the analysis of pterins in urine samples with satisfactory results.     

Redox Mechanisms of Nodularin and Chemically Degraded Nodularin

The electrochemical behaviour of Nodularin (NOD), a hepatotoxic cyclic pentapeptide, was studied at a glassy carbon electrode. NOD electrochemical oxidation is an irreversible, pH‐independent process, involving the transfer of one electron. Upon incubation in different pH electrolytes, chemical degradation of NOD was electrochemically detected by the appearance of a new oxidation peak. The chemically degraded NOD (cdNOD), undergoes an irreversible, pH‐dependent oxidation, and its redox products are reversibly oxidised. The charge transfer properties of cdNOD as well as of its redox metabolites were investigated. Mechanisms for NOD oxidation, NOD chemical degradation and oxidation of cdNOD and its metabolites were proposed.     

Simple method for the quantitative examination of extra column band broadening in microchromatographic systems

In recent years capillary chromatography has gained popularity for trace analyses. Most often UV or electrochemical detection is employed because the small peak volumes make post-column derivatization challenging. We have developed a simple method based on flow injection for determining contributions to peak broadening from post-column reactors. The only requirement for application of our methodology is that diffusion be in the Taylor regime so that radial concentration gradients are relaxed enabling mixing purely by diffusion.     

Determination of H2O2 and organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection

A method for the determination of hydrogen peroxide and several organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection is described. By means of post-column UV irradiation in the presence of water organic peroxides are converted into hydrogen peroxide and organic hydroperoxides, which react rapidly with the post-column derivatization agent p-hydroxyphenylacetic acid (PHPAA) under catalysis of horseradish peroxidase to yield the fluorescent PHPAA dimer that is detected at excitation and emission wavelengths of 285 and 400 nm, respectively. The detection limit for hydrogen peroxide is 14 ng/mL, for organic peroxides between 34 ng/mL and 5 μg/mL. No interference by other compounds was observed when their concentrations were below 10 mg/mL except ethers and phenols. Received: 6 August 1997 / Revised: 11 December 1997 / Accepted: 15 December 1997