High-performance anion-exchange chromatography of carbohydrates using a new resin and pulsed amperometric detection

Summary This paper reports the results of a study on the use of a new polymer-based, strong anion-exchange, stationary phase for rapid and selective separation of carbohydrates and related compounds by high-pH, anion-exchange chromatography with pulsed amperometric detection. The new adsorbent has been obtained by direct nitration of 2.8 μm, spherical non-porous highly cross-linked, styrene-divinylbenzene copolymer beads, followed by reduction of superficially introduced nitro groups with nascent hydrogen and quaternization of the resultant amino groups with iodomethane. It is reported that by optimizing the ionic strength of the mobile phase, columns packed with the new anion-exchanger can be successfully employed to separate, either in isocratic or gradient elution mode, oligosaccharides, positional isomers of gluco-disaccharides, as well as uronic acids and sugar monophosphates.     

Determination of glycuronic acids by high-performance anion chromatography with pulsed amperometric detection

Summary Acid hydrolysis (0.25M H₂SO₄) coupled with enzyme catalysis (pectolyase and β-D-glucuronidase) were employed to extract galacturonic and glucuronic acids from microbial polysaccharides, plant residues, animal wastes, sewage sludge and soil. The glycuronic acids were separated by high-performance anion chromatography (HPAC) on a strong anion-exchange column using 0.1M sodium hydroxide with 0.25M sodium acetate as the mobile phase and determined by pulsed amperometric detection (PAD). HPAC-PAD was found to be superior to high-performance liquid chromatography with ultra-violet (UV) detection in terms of resolution and sensitivity of glycuronic acids. HPAC-PAD was not subject to interferences present with low UV detection (210 nm) and was highly selective for glycuronic acids. Enzymatic hydrolysis after treatment with mild acid (0.25M H₂SO₄) released galacturonic acids from orange peel and pectin, while glucuronic acid was released from Acacia powder. Large amounts of glycuronic acids were also extracted from plant materials. Low levels of uronic acids were detected in poultry manure, sewage sludge and organic-amended soils.     

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

Summary High performance anion-exchange chromatography (HPAC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 13) separates neutral saccharides based upon their molecular size, saccharide composition, and glycosidic linkages. Carbohydrates were detected by oxidation with a gold-working electrode. HPAC-PAD was compared to high performance liquid chromatography (HPLC) with refractive index (RI) detection in terms of selectivity and sensitivity of saccharides. The results indicate that HPAC-PAD was more precise, two orders of magnitude more sensitive (pmol range) and gives better resolution of saccharides than HPLC-RI. HPAC-PAD required less sample preparation and was not subjected to matrix interferences. The use of HPAC-PAD was applied to the analysis of organic materials (plant residues, animal wastes and sewage sludge) and soil.     

Preparation of amino-zirconia bonded phases and their evaluation in hydrophilic interaction chromatography of carbohydrates with pulsed amperometric detection

A series of non-porous, microspherical zirconia-based stationary phases with surface bound amine functionality have been introduced and evaluated in hydrophilic interaction chromatography (HILIC) of underivatized, neutral carbohydrates and anion exchange chromatography of nucleotides using pulsed amperometric detection and ultraviolet detection, respectively. Three aminopropyl alkoxysilane compounds were used in the surface modification of the non-porous zirconia support, namely 3-aminopropyltrimethoxysilane (monoamine), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (diamine), and trimethoxysilylpropyldiethylenetriamine (triamine). Due to the relatively low specific surface area of the non-porous zirconia support used in this study (ca. 7.3 m²/g), zirconia with surface coating of the triamine type yielded the best results as far as the separations of chitooligosaccharides and maltooligosaccharides are concerned. Since a non-porous zirconia could be readily modified with amine functionality via Zr? O? Si bonds, it is expected that all the three aminopropyl alkoxysilane compounds would yield satisfactory results with porous zirconia microparticles because of their much higher specific surface areas. Although the non-porous zirconia exhibited some limitations, the present study has demonstrated that microspherical zirconia particles are suitable supports for the production of polar sorbents for HILIC of carbohydrates. Another surface modification, which involved the activation of the zirconia surface with aldehyde groups followed by reductive amination with tetraethylenepentamine, was also evaluated. Although this chemistry would in principle yield sorbents with higher concentration in amine groups, the conversion of the majority of the primary amine groups of the tetraethyle-nepetamine molecules to secondary amine functions in the course of the reductive amination reaction have provided a stationary phase that did not afford satisfactory resolution for carbohydrates. However, this same stationary phase behaved as a weak anion exchanger and allowed the high resolution separation of nucleoside-5′-mono-, -di-, and triphosphates. Overall, the results obtained with zirconia-based hydrophilic sorbents paralleled those obtained on amino-silica bonded phases.     

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.     

Detection of albiflorin and paeoniflorin in Paeoniae Radix by reversed-phase high-performance liquid chromatography with pulsed amperometric detection

We have developed a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method for the detection of albiflorin and paeoniflorin in Paeoniae Radix and Wu-ji-san. Albiflorin and paeoniflorin were completely separated using 10% acetonitrile in 5 mM sodium phosphate buffer (pH 3.0) as an eluent and detected by PAD under alkaline conditions after using a post-column delivery system. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) were 0.10 and 0.35 ng for albiflorin, and 0.20 and 0.50 ng for paeoniflorin, respectively. The coefficients of linear regression were 0.9995 and 0.9999 for concentrations between 0.035 and 100 μg/mL. The intra- and inter-day precision (RSDs) was less than 3.56% in Paeoniae Radix and Wu-ji-san. The average recoveries from Paeoniae Radix and Wu-ji-san were 99.01–100.94% and 99.46–100.64%. This method shows higher selectivity than HPLC–UV method for analyzing albiflorin and paeoniflorin in Chinese medicinal preparation.     

Determination of neomycin in water samples by high performance anion chromatography with pulsed amperometric detection

A simple, fast and reliable method, using high performance anion chromatography with pulsed amperometric detection, had been developed for the analysis of neomycin in water samples. The elution and separation were carried out with an isocratic mobile phase, containing 10 mmol/L NaOH. The influence of the concentration and pH of the mobile phase on the separation and detection was investigated. A quadruple-potential waveform used for the detection was optimized. The detection limit of neomycin was down to 0.027μg/mL. The linearity of neomycin calibration curve ranged from 0.050 to 0.505μg/mL with correlation coefficient of 0.9997. R.S.D. (n = 11) was 4.0%.     

Determination of several sugars in serum by high-performance anion-exchange chromatography with pulsed amperometric detection

In this paper, a sensitive, simple and direct method for simultaneous determination of glucose, ribose, isomaltose and maltose in serum sample by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection was developed. The four target analytes were easily and completely separated on an anion-exchange column at a flow-rate of 0.25 mL/min by binary step gradient elution in about 16 min and the two eluents were deionized water and 500 mM sodium hydroxide, respectively. The separated four analytes were detected directly by using a gold electrode and quadruple-potential waveform integrated pulsed amperometry without derivatization. Under the optimized conditions, when the injection volume was 25 microL, the detection limits (signal-to-noise ratio equal to 3) for glucose, ribose, isomaltose and maltose were 0.92, 7.50, 12.9 and 10.3 ng/mL, respectively. The calibration graphs of peak area for the four analytes were linear over two to three orders of magnitude with correlation coefficients greater than 0.998. R.S.D. of peak areas of the four analytes for five determinations were no more than 5.6%. The analytical method had been applied to the determination of glucose, ribose, isomaltose and maltose in real serum samples and good results with low relative standard deviation not more than 5.3% were obtained. The accuracy of the proposed method was tested by recovery measurements on spiked samples and good recovery results (98.1-107.9%) were obtained.     

HPLC determination of morphine with amperometric detection at low potentials under basic pH conditions

Summary A simple, sensitive, accurate and reliable method for morphine determination in biological samples has been developed. It uses reverse-phase HPLC on a polymeric column with an eluent (0.05 mol/L dibasic sodium phosphate: acetonitrile, 8515) at pH 9.5, allowing both the suppression of ionization of morphine amine and the promotion of oxidation of the phenolic group. Amperometric detection at mild oxidizing potential (350 mV) proves very selective, and, therefore, only a simple and rapid one-step liquid-liquid sample preparation is required. Under these conditions clean chromatograms are obtained even with complex biological matrices such as cadaveric blood, urine and hair.Minimum detectable amount of morphine is about 200 pg injected. A calibration line with a correlation coefficient of 0.99996 has been produced over the range 7.5–250 ng/mL. Precision results comparable to other HPLC methods.A preliminary report was presented at the 25th T.I.A.F.T. Meeting 1988, Groningen (NL), June 27–30 1988.     

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.     

Analysis of alkyl polyglucosides in industrial products by capillary electrophoresis with pulsed amperometric detection

Pulsed amperometric detection following micellar electrokinetic chromatography has been applied successfully to the direct detection of alkyl polyglucosides (APGs) in shampoos and other industrial products without prior conversion to highly absorbing or fluorescing derivatives. For electrochemical detection, it is necessary to dissociate the hydroxyl groups of the APGs. Thus, we used 0.1 M NaOH in the outlet vial to dissociate the APGs. The main problems associated with the combination of electrochemical detection and capillary electrophoresis are the need to isolate the detector from the electric field used in the capillary electrophoresis separation and the difficulty of aligning the working electrode with the end of the capillary. To overcome these problems, a simple capillary-electrode holder was constructed. This holder automatically aligns the capillary and the electrode in a wall-jet configuration without the aid of micropositioners and facilitates the replacement of electrodes and capillaries without reconstruction of the entire capillary/electrode setup. Special microcylindrical gold electrodes have been produced by sealing 300-μm-diameter gold wire into borosilicate-glass capillaries.     

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 carnosine in feed and meat by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Carnosine (beta-alanyl-L-histidine) is a dipeptide regarded as an important molecular marker of the presence of processed animal proteins including meat and bone meal in animal feed. For its identification and quantification a sensitive and selective method by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) was developed. The assay is based on isocratic elution with 100 mM NaOH as the mobile phase. Interferences of real matrices were efficiently removed; carnosine could be determined at the concentration ranges 0.1-100 microM with a rather low detection limit of 0.23 ng. Unlike feeds for dogs and cats, no carnosine peak was observed in all examined feeds for ruminants. The good analytical characteristics allowed camosine determination down to 5 microg/g of feed.     

Direct determination of hydrogen cyanide in cigarette mainstream smoke by ion chromatography with pulsed amperometric detection

The determination of hydrogen cyanide in cigarette mainstream smoke has been achieved by ion chromatography (IC) with pulsed amperometric detection (PAD). The proposed method of totally trapping whole cigarette mainstream smoke by Cambridge filters, which are treated with sodium hydroxide/ethanol solution, possesses the advantage of fast analysis time over the widespread used solution absorption method. The possible co-existing interferents are evaluated under the optimized detection conditions and excellent recoveries of cyanide are obtained. The cyanide content of absorption solution can be directly determined by the optimized IC-PAD method without any pretreatments. The linear range is 0.0147-2.45 μg/mL with R2 value of 0.9997. The limit of the detection is 3 μg/L for a 25 μL injection loop. The overall relative standard deviation of the method is less than 5.20% and the recovery range from 94.3% to 101.0%. The results obtained from the developed method are in good agreement with that of continuous flow analyzer (CFA) method.     

Simultaneous determination of 13 carbohydrates using high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry

A simple, accurate, and highly sensitive method was developed for the determination of 13 carbohydrates in polysaccharide of Spirulina platensis based on high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry. Samples were extracted with deionized water using ultrasonic‐assisted extraction, and the ultrasound‐assisted extraction conditions were optimized by Box–Behnken design. Then the extracted polysaccharide was hydrolyzed by adding 1 mol/L trifluoroacetic acid before determination by high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and confirmed by high‐performance anion‐exchange chromatography coupled with mass spectrometry. The high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection method was performed on a CarboPac PA20 column by gradient elution using deionized water, 0.1 mol/L sodium hydroxide solution, and 0.4 mol/L sodium acetate solution. Excellent linearity was observed in the range of 0.05–10 mg/L. The average recoveries ranged from 80.7 to 121.7%. The limits of detection and limits of quantification for 13 carbohydrates were 0.02–0.10 and 0.2–1.2  μg/kg, respectively. The developed method has been successfully applied to ambient samples, and the results indicated that high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry could provide a rapid and accurate method for the simultaneous determination of carbohydrates.     

Simplified current decoupler for microchip capillary electrophoresis with electrochemical and pulsed amperometric detection

There is a need to develop broadly applicable, highly sensitive detection methods for microchip CE that do not require analyte derivatization. LIF is highly sensitive but typically requires analyte derivatization. Electrochemistry provides an alternative method for direct analyte detection; however, in its most common form, direct current (DC) amperometry, it is limited to a small number of easily oxidizable or reducible analytes. Pulsed amperometric detection (PAD) is an alternative waveform that can increase the number of electrochemically detectable analytes. Increasing sensitivity for electrochemical detection (EC) and PAD requires the isolation of detection current (nA) from the separation current (muA) in a process generally referred to as current decoupling. Here, we present the development of a simple integrated decoupler to improve sensitivity and its coupling with PAD. A Pd microwire is used as the cathode for decoupling and a second Au or Pt wire is used as the working electrode for either EC or PAD. The electrode system is easy to make, requiring no clean-room facilities or specialized metallization systems. Sensitive detection of a wide range of analytes is shown to be possible using this system. Using this system we were able to achieve detection limits as low as 5 nM for dopamine, 74 nM for glutathione, and 100 nM for glucose.     

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.     

Highly sensitive determination of ectoine and other compatible solutes by anion-exchange chromatography and pulsed amperometric detection

In saline media prokaryotes compensate for the osmotic pressure of the surrounding medium by producing osmolytes. Although these osmolytes or osmoprotectors have quite diverse structures, most of them can be determined by anion-exchange chromatography combined with integrated pulsed amperometric detection. This technique offers the advantages of very high sensitivity and new opportunities to determine ectoine and 5-hydroxyectoine—two important osmolytes —after hydrolytic cleavage of the pyrimidine ring. It can even be used to screen bacterial colonies on agar for compatible solutes. Furthermore, it allows amino acids and osmolytes of this type to be determined without derivatization. To test the method we applied it to two halotolerant bacterial strains: Stenotrophomonas rhizophila DSM 14405^T and Halomonas elongata DSM 2581^T. The first strain produced trehalose and glucosylglycerol, and the second ectoine, as the main osmotic counterweight. The relationship between the content of these osmolytes in the bacterial biomass and the external salinity is described.     

Flow injection analysis of doxycycline or chlortetracycline in pharmaceutical formulations with pulsed amperometric detection

A flow injection with pulsed amperometric detection for determination of doxycycline or chlortetracycline in pharmaceutical formulations is described. Doxycycline or chlortetracycline were studied at a gold rotating disk electrode with cyclic voltammetry as a function of pH of supporting electrolyte solution. The optimized PAD waveform parameters were obtained with a flow injection system. The optimized pulsed conditions of doxycycline were 1150 mV (versus Ag/AgCl reference electrode) detection potential (E_det) for 220 ms (150 ms delay time and 70 ms integration time), 1500 mV (versus Ag/AgCl reference electrode) oxidation potential (E_oxd) for 70 ms oxidation time (t_oxd) and 250 mV (versus Ag/AgCl reference electrode) reduction potentail (E_red) for 400 ms reactivation time (t_red). The optimized pulsed conditions of chlortetracycline were 1050 mV (versus Ag/AgCl reference electrode) detection potential (E_det) for 300 ms (200 ms delay time and 100 ms integration time), 1300 mV (versus Ag/AgCl reference electrode) oxidation potential (E_oxd) for 70 ms oxidation time (t_oxd) and 250 mV (versus Ag/AgCl reference electrode) reduction potentail (E_red) for 400 ms reactivation time (t_red). The optimized PAD waveform was applied to the determination of doxycycline hydrochloride and chlortetracycline hydrochloride standard solution and in pharmaceutical formulations. The linear dynamic ranges of doxycycline hydrochloride and chlortetracycline hydrochloride were 1 μM–0.1 mM. The sensitivity of this method was found to be 23 μA/mM for doxycycline hydrochloride and 33.76 μA/mM for chlortetracycline hydrochloride. The detection limit for both compounds is 1 μM. The doxycycline hydrochloride and chlortetracycline hydrochloride content in commercially available tablet dosage forms by the proposed method was comparable to those specified by the manufacturer.     

Sensitive high-performance liquid chromatography method of non-polar ginsenosides by alkaline-enhanced pulsed amperometric detection

We determined the minute amount of non-polar ginsenosides in red ginseng with a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method. Non-polar ginsenosides efficiently extracted by ethyl acetate were well separated in 40 min using a water–acetonitrile gradient eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limits (S/N = 3) were 0.03–0.10 ng. The coefficients of linear regression were 0.9972–0.9990. Intra- and inter-day precision (RSDs) was less than 8.34% and average recovery was 98.06–102.73%. The total amount of non-polar ginsenosides in hairy root of red ginseng was slightly higher than in the main root.