Electrochemical detection in high-performance liquid chromatography of organic compounds

The selectivity and sensitivity of analytical response in electrochemical (amperometric and conductometric) detection methods in the high-performance liquid chromatography of organic compounds is discussed in comparison with other detection techniques. The importance of electrode materials of different nature for extending the range of analytes is noted. Presented at the V All-Russian Conference with the Participation of CIS Countries on Electrochemical Methods of Analysis (EMA-99), Moscow, December 6–8, 1999.     

Porphyrin analysis by reversed-phase high-performance liquid chromatography: biomedical applications

Reversed-phase high-performance liquid chromatography has considerable advantages in porphyrin analysis since many of the extraction and esterification steps required for normal-phase high-performance liquid chromatography can be eliminated. This technique is of increasing importance for the diagnosis of porphyrias and in other clinical and experimental studies of porphyrins.     

Comprehensive multi-dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review

'Multi-dimensional' liquid separations have a history almost as long as chromatography. In multi-dimensional chromatography the sample is subjected to more than one separation mechanism; each mechanism is considered an independent separation dimension. The separations can be carried out either offline via fraction collection, or directly coupled online. Early multi-dimensional separations using combinations of paper chromatography, electrophoresis and gels, in both planar and columnar modes are reviewed. Developments in HPLC have increased the number of measurable analytes in ever more complex matrices, and this has led to the concept of 'global metabolite profiling'. This review focuses on the theory and practice of modern 'comprehensive' multi-dimensional liquid chromatography when applied to biomedical and pharmaceutical analysis.     

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection

Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm×4.6 mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min⁻¹. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.     

Electrochemical treatment of textile dyes and their analysis by high-performance liquid chromatography with diode array detection

Several textile dyes were individually exposed to electrochemical treatment. Chromaticity variation and the formation of degradation products were followed using a UV spectrophotometer and HPLC with diode array detection. Dyes studied belong to the azo (color index, C.I. 15,510), methine (C.I. 48,013), indigo (C.I. 73,040), natural (C.I. 75,760) and arylmethane (C.I. 42,000) classes. Aliquots of the solutions treated at constant potential were analyzed and compared with control dye solutions. The final electrolysis solutions obtained by using different electrode materials: Pt, Ti and diamond presented different chromatograms. It was found that the novel (in this application) diamond electrode is efficient in studying the degradation of various dyes. Possible fragmentation and molecule moiety rearrangement are proposed as a result of the electrochemical treatment.     

Electrochemical detection of tryptophan metabolites following high-performance liquid chromatography.

Electrochemical detection of tryptophan metabolites following separation on a reversed-phase high-performance liquid chromatography column was compared with other means of detection. Of 29 compounds studied, 26 could be detected at a sensitivity comparable to that of fluorescence derivatisation procedures. Response was linear over a wide range of concentrations and selectivity was shown to be superior to ultraviolet detection when analysing urine. Additionally, it was possible to control selectivity so that only those tryptophan metabolites from the tryptophan hydroxylase pathway were detected. This is of particular value in the study of distrubances of serotonin metabolism and is unique to this type of detector.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

Electrochemical detection of indole alkaloids of Catharanthus roseus in high-performance liquid chromatography

Hydrodynamic voltammograms for indole and five indole alkaloids with different amino functions were obtained in order to evaluate the applicability of high-performance liquid chromatography (HPLC) with coulometric detection to these compounds. With the exception of serpentine, which has a quaternary nitrogen in its structure, all the compounds were oxidised and gave net signals of greater than 25 nA pmol(-1) at potentials of between +0.2 and +0.85 V versus a solid Pd electrode in an acetate-buffered (pH 6.5) water-methanol-acetonitrile system. An HPLC assay for quantifying picomole amounts of catharanthine and ajmalicine in Catharanthus roseus cell culture samples is described.     

Characterisation of iota-carrageenans oligosaccharides with high-performance liquid chromatography coupled with evaporative light scattering detection

Enzymatically digested oligo-iota-carrageenans were separated with liquid chromatography, coupled to evaporative light scattering detection. As expected, compared to oligo-kappa-carrageenans, the additional sulphate group in the neocarrabiose unit of iota-carrageenans significantly modified the separation mechanisms on ion-exchange chromatography, porous graphitic carbon and ion-pair chromatography. The oligomers were then isolated and characterised off-line with electrospray ionisation mass spectrometry in positive-ion mode. The tetrasaccharide, hexasaccharide and octasaccharide that were identified were associated with protonated heptylamine molecules whose number depended on the number of sulphate groups.     

Light-scattering detection with a fluorimetric detector in high-performance liquid chromatography

Non-fluorescence compounds were detected by a fluorescence detector based on scattering light. The fluorescence detector was used without any modification, and the scattering light was observed at the wavelength twice as long as the excitation wavelength. Actually the wavelength of the observed scattering light was the same as that of the excitation light. The maximum signal was achieved at around 280 nm. The signal was increased with increasing molecular weight or size of analytes. Colloidal silica with nanometer sizes, ethylene glycol oligomers, saccharides and cyclodextrins could be visualized by the present detection method. The detection limit at S/N=3 for colloidal silica with 78 nm was 39 pg for 20-microL injection.     

Indirect detection in high-performance liquid chromatography

Indirect detection is a technically simple method to follow and quantify compounds without inherent detector response in high-performance liquid chromatography. The underlying principle can be expressed in simple equations. These equations indicate clearly how the detection sensitivity can be optimized and how disturbance effects can be avoided when the mobile phase gives detector response.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Determination of nalbuphine using high-performance liquid chromatography coupled to photodiode-array detection and gas chromatography coupled to mass spectrometry.

A procedure involving capillary column gas chromatography coupled to mass spectrometry and a method involving liquid chromatography coupled to a diode-array detector have been developed for the analysis of nalbuphine. The extraction step is the same for both techniques and involves extraction under alkaline conditions in chloroform-2-propanol-n-heptane (50:17:33, v/v/v) with levallorphan as the internal standard. After purification by acidic extraction and back alkaline extraction, drugs are derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane for gas chromatography-mass spectrometry and directly injected for high-performance liquid chromatography-diode-array detection. The limits of detection are 2.0 and 25.0 ng/mg, respectively.     

The potential of inductively coupled plasma mass spectrometry detection for high-performance liquid chromatography combined with accurate mass measurement of organic pharmaceutical compounds

Quantification of unknown components in pharmaceutical, metabolic and environmental samples is an important but difficult task. Most commonly used detectors (like UV, RI or MS) require standards of each analyte for accurate quantification. Even if the chemical structure or elemental composition is known, the response from these detectors is difficult to predict with any accuracy. In inductively coupled plasma mass spectrometry (ICP-MS) compounds are atomised and ionised irrespective of the chemical structure(s) incorporating the element of interest. Liquid chromatography coupled with inductively coupled plasma mass spectrometry (LC/ICP-MS) has been shown to provide a generic detection for structurally non-correlated compounds with common elements like phosphorus and iodine. Detection of selected elements gives a better quantification of tested 'unknowns' than UV and organic mass spectrometric detection. It was shown that the ultrasonic nebuliser did not introduce any measurable dead volume and preserves the separation efficiency of the system. ICP-MS can be used in combination with many different mobile phases ranging from 0-100% organic modifier. The dynamic range was found to exceed 2.5 orders of magnitude. The application of LC/ICP-MS to pharmaceutical drugs and formulations has shown that impurities can be quantified below the 0.1 mol-% level.     

Carbohydrate and alditol analysis by high-performance anion-exchange chromatography coupled with electrochemical detection at a cobalt-modified electrode

A cobalt oxyhydroxide film dispersed on a carbon electrode surface was characterized and proposed as an amperometric sensor for determination of alditols and carbohydrates in flowing streams. Complex mixtures of carbohydrates were separated by anion-exchange chromatography using a moderately alkaline solution as mobile phase. The cobalt modified electrode (GC-Co) was employed under a constant applied potential of 0.5 V (vs Ag/AgCl). Under these experimental conditions the detection limits (S/N=3) for all analyzed electroactive molecules ranged between 0.3 micromol L(-1) and 1.5 micromol L(-1) and the dynamic linear ranges spanned generally three orders of magnitude above the relevant detection limits. Analytical determinations of carbohydrates and alditols in red and white wines, are reported.     

Electrochemical detection of microcystins, cyanobacterial peptide hepatotoxins, following high-performance liquid chromatography

A novel amperometric HPLC detection method for the cyanobacterial (blue–green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were shown to originate in arginine and tyrosine residues of microcystins.