Characterization of low-molecular-weight metal species in plant extracts by using HPLC with pulsed amperometric detection and cyclic voltammetry

An analytical separation scheme is presented for the isolation of low-molecular-weight metal species (< 10 kDa) in plants. After ultrafiltration of the aqueous plant extracts isotachophoresis or gel chromatography is used for pre-separation and HPLC at a cyclodextrin-phase for further separation of metal containing fractions. Trace metals are detected off-line by using either adsorptive stripping voltammetry for platinum or AAS for zinc and magnesium. It is shown that platinum and zinc detection closely correlate with pulsed amperometric detection (PAD), while magnesium behaves differently. PAD is carried out after post-column addition of sodium hydroxide, thus enabling a sensitive and selective detection of carbohydrates. It is demonstrated that cyclic voltammetry is a useful tool for further characterization of the PAD-detectable metal species. By comparison of the cyclic voltammograms of carbohydrate standards, including carbohydrate-metal species, with the respective voltammograms obtained from HPLC fractions, carbohydrate species isolated from the samples can be considered as being either partly oxidized sugars (sugar alcohols or sugar acids) or glycosidically bound sugars. Received: 26 January 1998 / Revised: 19 June 1998 / Accepted: 24 June 1998     

Selective detection of metal species in HPLC and FIA by means of pulsed amperometric detection (PAD)

Triple-step pulsed amperometry is used to detect selectively metal complex species after HPLC separation in the presence of coeluting matrix compounds. Optimized pulse parameters enable accurate and selective detection of species even in the presence of electroactive compounds such as phenols or sulfurcontaining peptides. Complexes of tin(IV) with rutin and of platinum(II) with glutathione are presented as examples for these classes of compounds. It is demonstrated that by changing the pulse waveform direct and indirect detection modes can be realized. The method is applied to detect platinum species in a grass extract and iron(II)-lactate in fruit juice. Furthermore it is shown, that pulsed amperometry can be used as a detector in FIA for determining the fraction of metal complex formed after mixing of metal and excess ligand without separation.     

Analysis of tetracycline antibiotics using HPLC with pulsed amperometric detection.

The analysis of tetracycline, oxytetracyline, chlortetracycline and doxycycline by high-performance liquid chromatography with pulsed amperometric detection using an anodized boron-doped diamond thin film (BDD) electrode is originally reported. The analyses were carried out using the mobile phase, phosphate buffer (0.01 M, pH 2.5)-acetonitrile (80:20; v/v), on a C18 column (250 x 4.6 mm i.d., 5 microm) at a flow rate of 1.0 mL/min. The optimal PAD waveform parameters at the anodized BDD were 1.5 V (versus Ag/AgCl) detection potential (E(det)) for 290 ms (200 ms delay time and 90 ms integration time), 2.0 V (versus Ag/AgCl) oxidation potential (E(oxd)) for 200 ms oxidation time (t(oxd)) and 0.4 V (versus Ag/AgCI) reduction potential (E(red)) for 200 ms reduction time (t(red)). The proposed method showed the simultaneous determination of tetracycline, oxytetracyline, chlortetracycline and doxycycline with a linear range of 0.1 - 100 microg/mL, detection limits of 0.05 - 0.1 microg/mL and recoveries of 70.8 - 96.0%. The application of this method to real samples was demonstrated and validated using a shrimp sample.     

Analysis of saikosaponins in Bupleuri Radix and Caihu-shugan-san using reversed-phase HPLC with pulsed amperometric detection

A simple and sensitive reversed-phase (RP) HPLC coupled with pulsed amperometric detection (PAD) method was developed to determine the saikosaponin content in Bupleuri Radix or Caihu-shugan-san. Four saikosaponins in Bupleuri Radix and Caihu-shugan-san were extracted with a 6:4 solution of 10 mM sodium phosphate buffer (pH 8)/100% ethanol. Pulsed amperometric detection of carbohydrates in four major saikosaponins was highly sensitive when used with a water-acetonitrile gradient on an alkaline RP column with a post-column delivery system. The limits of detection (S/N=3) and of quantification (S/N=10) of saikosaponins were 0.01-0.02 and 0.03-0.05 μg/mL, respectively. The intra- and inter-day precision (RSDs) were each <9.7% and the average recoveries were 95.0-97.6% in Bupleuri Radix. This method can be used to analyze saikosaponins in Bupleuri Radix and Caihu-shugan-san.     

Speciation of iron coordinated by phytosiderophores by use of HPLC with pulsed amperometric detection and AAS

Phytosiderophores of the mugineic acid family, and the respective iron species, have been separated by anion-exchange chromatography with NaOH gradient elution. Two different detection methods were used in parallel, pulsed amperometry (PAD) for phytosiderophores and atomic absorption spectrometry (AAS) for iron. This combination enables identification of separated iron species. Up to five different iron species were separated and detected within 30 min - two different phytosiderophore species, two amino acid species, and one species which has not yet been identified but which is most probably a decomposition product of phytosiderophores. The detection limit was in the low micro mol L(-1) concentration range, which is sufficiently low for determination in real plant samples, even after dilution. The method has been applied to root washings of iron-deficient wheat and barley plants and to a xylem exudate of non-deficient maize.     

Platinum particles-modified electrode for HPLC with pulsed amperometric detection of thiols in rat striatum

A new chemically modified electrode based on the immobilization of Pt particles is fabricated and exhibits electrocatalytic oxidation for L-cysteine (L-Cys), glutathione (GSH) and penicillamine (PEN) with relatively high sensitivity. It is also adaptable to HPLC for pulsed amperometric detection (PAD) of these thiols. PAD largely improves the detection sensitivity because the alternated polarizations can effectively clean and reactivate the electrode surface. It is shown that the peak currents of L-Cys, GSH and PEN are linear to their concentrations, with the calculated detection limit of 1.1 x 10(-7), 1.8 x 10(-7) and 3.8 x 10(-7) mol L(-1), respectively (S/N = 3). The method has been successfully applied to assess the contents of L-Cys and GSH in rat striatal microdialysates. The average contents of the two analytes in rat striatum are 2.6 x 10(-6) and 2.8 x 10(-6) mol L(-1), respectively.     

Analysis of phenolic acids in plant materials using HPLC with amperometric detection at a platinum tubular electrode

The suitability of a simple amperometric platinum tubular detector for HPLC analysis of selected phenolic acids in yacon (Smallanthus sonchifolius, Asteraceae) is reported. The detector offers good overall limits of detection for the phenolic acids of interest. Three different types of extracts from yacon leaves were analyzed and compared with respect to their content of phenolic acids. Caffeic acid was found in all yacon extracts, whereas the content of chlorogenic acid depends more on the extraction procedure used. It has been demonstrated that no complicated and sophisticated equipment is needed and HPLC with amperometric detection seems to be a very useful technique for analysis of plant extracts containing phenolic acids.     

Determination of phytosiderophores by anion-exchange chromatography with pulsed amperometric detection

Phytosiderophores of the mugineic acid family are separated by anion-exchange HPLC using NaOH gradient elution. Separation of mugineic acid (MA), 2'-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA) and 3-epi-hydroxymugineic acid (epi-HMA) is obtained within 15 min. Detection of the underivatised phytosiderophores is performed using pulsed amperometric detection (PAD) at pH 13. The sensitivity of the detection increases in the order DMA < MA < HMA < epi-HMA and respective detection limits of 5 microM (DMA), 1 microM (MA) and < 0.5 microM (HMA, epi-HMA) are achieved. PAD is discussed in comparison with the well-established fluorimetric detection method after post-column derivatisation with ortho-phthaldialdehyde. The main advantage of PAD is the simplicity of the method (no derivatisation) and the high sensitivity for hydroxylated mugineic acids. The method is used for the determination of phytosiderophores in root washings of iron-deficient and non-deficient wheat and barley plants.     

Development of an analytical method for yam saponins using HPLC with pulsed amperometric detection at different column temperatures

Yam saponins (dioscin, gracillin, protodioscin, and protogracillin) were analyzed with three different C18 columns at incremental column temperatures from 15 to 45°C to investigate the effect of temperature on the retention and resolution of yam saponins. At low temperature, yam saponins showed decreased retention times and improved resolutions in the C18 columns. In the Kinetex C18 column at 15°C, the four saponins achieved baseline separation (Rs > 1.5) within 30 min. Pulsed amperometric detection was used to identify saponins with high sensitivity. The limits of detection and quantification of saponins were 0.11–0.31 and 0.33–0.95 ng, respectively. The correlation coefficients ranged 0.9986–1.0000. Intra‐ and inter‐day precisions were <4.2% of retention times and <9.5% of the calculated contents. Average recoveries ranged from 92.18 to 105.98%. Saponin contents in Dioscorea nipponica tubers and commercial yam foods were determined without sample purification or concentration. Among the ten commercial yam foods investigated, only three showed significant saponin contents.     

Determination of clotiazepam by HPLC with spectrophotometric and amperometric detection

Summary Spectrophotometric and amperometric detectors were compared for the determination of clotiazepam by HPLC. Methanol-water (6040) containing 0.01 mol/l ammonium acetate and methanol-water (7030) containing 0.01 mol/l acetate buffer pH 4 were used as mobile phase in the spectrophotometric and amperometric detection, respectively. The minimum detectability for the spectrophotometric detection was 20 ng injected and the method was applied for the determination of clotiazepam in serum, whereas for the amperometric detection a limit of detection of 2.5 g injected was obtained, using this technique for clotizepam determination in tablets.

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Bestimmung von Clotiazepam durch HPLC mit spektralphotometrischer und amperometrischer Detektion

     

Sub-millimolar determination of formalin by pulsed amperometric detection

Formalin, formaldehyde in the presence of methanol, was determined by pulsed amperometric detection (PAD). A triple waveform using E_det=−0.3 V (t_det=30 ms), E_oxd=+0.8 V (t_oxd=200 ms), and E_red=−0.8 V (t_red=350 ms) versus Ag/AgCl was applied at a Au electrode for detection in a flow injection (FI) system. The approach was rapid and yielded a sub-millimolar detection limit (0.0129 mM) with a dynamic range up to 100 mM. A precision of 8.8% R.S.D. at 1.0 mM for two hundred repetitive injections by the FI-PAD was obtained, whereas holding at a constant potential (−0.3 V versus Ag/AgCl) for anodic oxidation of formaldehyde caused the response to decrease dramatically after a few measurements. The method developed was used to analyze the formalin contents of water from rinsed samples of vegetables and fruit and ice-melt from seafood, and the method showed good agreement with the liquid chromatography (LC) method.     

Determination of zidovudine using anion exchange chromatography with integrated pulsed amperometric detection

A rapid and practical method for direct detection of zidovudine in high performance anion exchange chromatography(HPAEC) has been developed with integrated pulsed amperometric detection(IPAD).Dionex AS 18(250 mm×2 mm) and AG 18 (50 mm x 2 mm) columns and 11 mmol/L NaOH solution were used for separation.Multi-step potential waveform parameters were optimized to maximize the signal-to-noise ratio(S/N).Utilizing an optimized waveform,the repeatability(intra-day) precision and intermediate(inter-day) precision are obtained with relative standard deviation(RSD) of 1.88,2.27,respectively.The limit of quantification(LOQ) and limit of detection(LOD) were found to be 9.70,3.0 ng/mL,respectively,with correlation coefficients of 0.9992 over concentration range 0.01-10μg/mL.The present method was successfully applied to the determination of zidovudine in human plasma.The recoveries of plasma sample spiked by 0.7μg/mL,2.7μg/mL obtained were 95.3-101.5%, with relative standard deviation(RSD) of 2.54%,2.21%,respectively.     

Determination of lisinopril using anion exchange chromatography with integrated pulsed amperometric detection

A rapid and practical method for direct detection of lisinopril in anion exchange chromatography(AEC) has been developed with integrated pulsed amperometric detection(IPAD).Dionex AS 18(250 mm×2 mm) and AG 18(50 mm×2 mm) columns and 40 mmol/L NaOH solution were used for separation.Multi-step potential waveform parameters were optimized to maximize the signal-to-noise ratio(S/N).Utilizing the optimized waveform,the repeatability(intra-day) precision and intermediate(inter-day) precision were obtained with relative standard deviation(RSD) of 0.74,0.93,respectively.The limit of quantification(LOQ) and limit of detection(LOD) were found to be 0.37,0.12ng/mL,respectively,with the correlation coefficient of 0.9998 over concentration range 0.01-1μg/mL.The present method was successfully applied to the determination of lisinopril in human plasma.The recoveries of plasma sample spiked by 0.2μg/mL,0.8μg/mL lisinopril were 98.31-103.23%with RSD of 1.41%, 0.61%,respectively.     

Liquid chromatographic determination of ethylenethiourea using pulsed amperometric detection.

A liquid chromatographic method was developed using pulsed amperometric detection at a gold working electrode to measure residue levels of ethylenethiourea (ETU) in crops and groundwater. Use of the sequential pulsing program eliminates electrode fouling while preserving the sensitive and selective detection of ETU. Minimum detection limits in crops were 5-10 ppb (1.25-2.5 ng on-column) and 5 ppb (0.5 ng) in groundwater. The commercial availability of the pulsed electrochemical detector and its gold working electrode that remains functional with a minimum of conditioning is an improvement in method simplicity.     

Determination of aminosaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography (HPAC) was used for the determination of aminosaccharides in microbial polymers, chitin, animal waste, sewage sludge, plant residues and soil. The aminosaccharides, galactosamine, mannosamine and glucosamine were separated on a strong anion-exchange column with 1OmM sodium hydroxide as the eluent and determined by pulsed amperometric detection (PAD). The HPAC-PAD methodology was compared with high-performance liquid chromatography (HPLC) with refractive index detection (RI) in terms of selectivity and sensitivity for aminosaccharides. The results indicate that HPAC-PAD required less sample preparation, and was more precise and nearly two orders of magnitude more sensitive than HPLC-RI. HPAC-PAD was not subject to matrix interferences and was highly selective for aminosaccharides. More than 3% of the total nitrogen in alfalfa, and 20% of that in straw, was found to be present as aminosaccharides.     

Analysis of etimicin sulfate by liquid chromatography with pulsed amperometric detection

A new method for determination of etimicin's (ETM) purity and content is developed by liquid chromatography (LC) and pulsed amperometric detection (PAD). A reversed-phase ion-pair LC method with pulsed amperometric detection on a gold electrode after post-added NaOH is described. The mobile phase consisted of an aqueous solution containing 0.033 mol L(-1) oxalic acid, 0.012 mol L(-1) heptafluorobutyric acid, and 210 mL L(-1) acetonitrile with pH 3.40 adjusting by dilute NaOH solution. The total analysis time was not more than 30 min. The effects of the different chromatographic parameters on the separation were also investigated. A number of commercial samples of etimicin sulfate were analyzed using this method.     

Determination of aliphatic aldehydes by liquid chromatography with pulsed amperometric detection

An electrochemical detection method for short-chain saturated and unsaturated aliphatic aldehydes separated by liquid chromatography in moderately acidic medium is described. A triple-step waveform of the potentials applied to the polycrystalline platinum electrode, is proposed for sensitive detection of aliphatic aldehydes in flowing streams avoiding tedious pre- or post-column derivatization and/or cleanup procedures. The influences of the perchloric acid concentration and dissolved oxygen in the mobile phase, on the amperometric and chromatographic performance were evaluated and considered in terms of sensitivity and selectivity. Under the optimised experimental conditions (i.e., deoxygenated 50mM HClO4) the proposed analytical method allowed detection limits between 0.2 microM for acrolein and 2.5 microM for valeraldehyde. Regression analysis of calibration data indicates that responses for all investigated compounds are linear over about 2 orders of magnitude above the LOD, with correlation coefficients >0.990. The method was successfully applied to the determination of formaldehyde, acetaldehyde, propionaldehyde and acrolein in real matrices such as spiked water and red wines with good mean recoveries (81-97%).     

Enhanced determination of glucose by microchip electrophoresis with pulsed amperometric detection

The present report describes a new analysis strategy for microchip capillary electrophoresis with pulsed amperometric detection and its application to the determination of glucose. The addition of sodium dodecyl sulfate (SDS) to the mobile phase and detection reservoir stabilized flow rates and enhanced the detection signal for glucose. A higher pH (compared to the running buffer) was used at the waste reservoir in order to improve the detection performance while maintaining good separations. To our knowledge, this is the first report describing the use of post-column pH modification using microchip electrophoresis. Under optimum conditions, a linear relationship between the peak current and the concentration of glucose was found between 10⁻²-10⁻⁵ M, with a limit-of-detection of 1.2 μM. In addition, the separation of glucosamine and glucose was performed at pH 7.1 while the detection was performed at pH 11 to demonstrate the ability to use post-column pH modification.