Improved high-performance liquid chromatographic determination of pilocarpine and its degradation products in ophthalmic solutions importance of octadecylsilane column choice

An improved high-performance liquid chromatographic (HPLC) method for the determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in ophthalamic solutions was developed. The new method was adapted from the USP HPLC method for pilocarpine base and has been shown to give superior resolution of pilocarpine related substances compared to any previously reported HPLC technique. However, evaluation of several brands of C₁₈ (octadecylsilane, ODS) columns revealed significant column-to-column variability: only to (YMC Pack ODS-AM and Supelco LC-18-DB) out of eight columns tested were capable of baseline resolution of these analytes. Additionally, optimization of the diluent was needed to prevent any significant interconversion of the carpic acids to their respective carpines. Analyses for pilocarpine and its degradation products in several commercial ophthalmic formulations were performed to demonstrate the precision, accuracy and general applicability of the method. The preparation of a stable resolution test solution is also described.     

HPLC/UV-spectrometric and electrochemical detection of lignin-decomposition products in alcoholic beverages

Summary The determination of compounds responsible for the flavour of alcoholic beverages, which were separated by HPLC-RP-chromatography, was performed with a combination of UV-diode-array and electrochemical detection. The recording and comparing of diode-array spectra on the rising and descending flanks of each peak was very suitable for identifying peak impurities. The selectivity of the detection depends strongly on the detection wavelength, detection potential and electrode material. For higher sensitivity the electrochemical detection is favoured. The lowest detection limit is 30 pg (absolute) for syringic acid.     

Chromatography of bis-(3-sulfopropyl) disulfide and its breakdown products by HPLC coupled with electrochemical detection

A chromatographic method for the detection of bis-(3-sulfopropyl) disulfide (SPS), a common additive in acidic copper plating baths, and its breakdown products is demonstrated. The detection scheme involves a combination of solid-phase extraction for sample pre-treatment, C(18) reversed-phase high-performance liquid chromatography column for separation, and electrochemical sensor for detection of all non-fully oxidized sulfur-containing compounds. We were able to achieve an effective separation and accurately assign chromatographic peaks to all detectable species. Owing to a high sensitivity of the utilized electrochemical detector, detection in low parts per billion range was possible. This can prove crucial for plating bath control, since minute amounts of certain by-products significantly affect the bath performance.     

HPLC of porphyrinogens with electrochemical detection

Summary A reversed-phase system is described for the separation of coproporphyrinogen I, II, III, IV isomers, deethylisocoproporphyrinogen and isocoproporphyrinogen. The porphyrinogens are detected electrochemically with high sensitivity. The relative retention of the prophyrinogens is mainly governed by the arrangement of the ethyl and methyl substituents around the macrocycle, although steric effect may also be an important parameter.     

Trace analysis of ioxynil and bromoxynil using HPLC with electrochemical and UV detection

Summary A method for the determination of residues of bromoxynil and ioxynil in barley matrices is described. Samples are extracted with acetone; partition between water and chloroform/dichloromethane, gel permeation chromatography on a polystyrene-divinylbenzene gel and liquid chromatography on nitrile-modified silica are used for clean up of the sample extracts.The compounds are determined by reversed phase HPLC with electrochemical and UV-detection.Determination limits are in the range of 50 ppb.

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Spurenanalyse von Ioxynil and Bromoxynil unter Verwendung von HPLC mit elektrochemischer und UV-Detektion

Zusammenfassung Eine Methode für die Bestimmung von Rückständen von Bromoxynil und Ioxynil in Gerstenmatrices wird beschrieben. Die Proben werden mit Aceton extrahiert; Verteilung zwischen Wasser und Chloroform/Dichlormethan, Gelchromatographie an Polystyrol-Divinylbenzol-Gel und Flüssigkeitschromatographie an nitrilmodifiziertem Kieselgel werden zur Reinigung der Extrakte verwendet. Die Verbindungen werden mittels Umkehrphasen — HPLC mit elektrochemischer und UV-Detektion bestimmt. Die Bestimmungsgrenzen liegen bei 50 ppb.

     

Determination of sugars, sugar degradation products and alcohols by high-performance liquid chromatography with electrochemical detection

Summary The use of an electrochemical, amperometric detector with nickel as a working electrode for the determination of carbohydrates and alcohols after separation by high-performance liquid chromatography (HPLC) is discussed. In order to get sensitive and stable signals pretreatment experiments were carried out and the combination of this detector with polymer-based cation-exchange columns as well as reversed phase columns is described.     

Separation of sugars by HPLC with electrochemical detection

Summary In order to develop new sensitive detectors for the determination of sugars after HPLC-separation, the voltammetric behaviour of several mono- and disaccharides has been investigated. Nickel electrodes turned out to be well suited for this problem. The properties of the electrodes were studied in a stationary system, in a flow-injection system and in combination with high-performance liquid chromatography. Parameters for optimisation are discussed and the application to the detection of several sugars is shown.

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Trennung von Zuckern durch HPLC mit elektrochemischer Detektion

Zusammenfassung Da für die Bestimmung von Zuckern nach HPLC-Trennung ein Bedarf an neuen empfindlichen Detektoren besteht, wurde das voltammetrische Verhalten einiger Mono- und Disaccharide untersucht. Nickel-Elektroden erwiesen sich als gut geeignet für dieses Problem. Die Eigenschaften der Elektrode wurden in einem stationÄren System, im flie\enden System und in Kopplung mit der Hochdruckflüssigkeitschromatographie untersucht. Parameter für die Optimierung werden diskutiert und die Anwendungen auf Detektion verschiedener Zucker gezeigt.

     

Monitoring degradation processes of explosives by HPLC analysis with UV- and amperometric detection

 The impact of spilled explosives, their by-products and degradation products on human beings and the environment has been recognised as a serious problem at areas of existing and former ammunition plants. In nature, aerobic and anaerobic degradation processes of explosives and their accompanying compounds yield polar contaminants with relatively high water solubilities. Most are potentially carcinogenic and mutagenic. An HPLC method applying UV-detection for nitroaromatic compounds and amperometric detection for aminoaromatic and phenolic compounds was used for monitoring the degradation of explosives in a polluted groundwater sample under natural conditions. Analysis was performed by direct injection of aliquots of the sample after exposition to daylight for different periods of time. Received: 6 January 1996/Revised: 7 March 1996/Accepted: 13 March 1996     

Monitoring degradation processes of explosives by HPLC analysis with UV- and amperometric detection

The impact of spilled explosives, their by-products and degradation products on human beings and the environment has been recognised as a serious problem at areas of existing and former ammunition plants. In nature, aerobic and anaerobic degradation processes of explosives and their accompanying compounds yield polar contaminants with relatively high water solubilities. Most are potentially carcinogenic and mutagenic. An HPLC method applying UV-detection for nitroaromatic compounds and amperometric detection for aminoaromatic and phenolic compounds was used for monitoring the degradation of explosives in a polluted groundwater sample under natural conditions. Analysis was performed by direct injection of aliquots of the sample after exposition to daylight for different periods of time. Presented as a poster at the Anakon ’95 Conference in Schliersee, April 24–26, 1995     

Speciation of iron using HPLC with electrochemical and flame-AAS detection

Summary A combined detection system for HPLC is presented, which consists of an electrochemical detector for iron(II) detection and an on-line flame-AAS detector for total iron. Detection limits are 5 ng iron for AAS and 1 ng iron(II) for electrochemical detection. Quantitative analysis of separated iron species is possible, even if iron(II) and iron(III) coelute. The proposed system is used for the investigation of coupled complexation and redox equilibria (e.g. Fe + oxalate). The application to fruit juice and wine is also presented. The predominant species found in apple juice are iron(II)-malate and iron(III)-citrate and in white wine iron(II)- and iron (III)-tartrates.     

Determination of sulphonamides in milk by HPLC with electrochemical detection

Summary A simple quantitative method for the analysis of the residues of sulphadiazine and sulphadimidine in milk is described. The method is based on a simple extraction step and high-performance liquid chromatography (HPLC) with electrochemical detection. The Chromatographic seperation is performed on a reversed phase column (RP-C2) and an aqueous eluent. With this analytical system 10 ng/ml can be detected. Recoveries of sulphonamides from milk are between 94.4% and 110% in the concentration range of 0.01–1.8 g/ml sample.

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Bestimmung von Sulfonamidrückständen in Milch mit Hilfe der HPLC und dem elektrochemischen Detektor

Zusammenfassung Es wird eine einfache Methode zum Nachweis von Sulfonamidrückständen in Milch am Beispiel von Sulfadiazin und Sulfadimidin beschrieben, mit der noch Rückstandsmengen von 10 ng/ml erfaßbar sind. Das Verfahren besteht aus einem einfachen Aufreinigungsschritt, der Trennung der Rückstände auf einer Umkehrphase (RP-C2) und der Detektion mit einem elektrochemischen Detektor. Die Wiederfindungsraten liegen zwischen 94,4% und 110% für einen Konzentrationsbereich von 0,01–1,8 g/ml Untersuchungsmaterial.

     

Quantitative reversed phase HPLC analysis of L-ascorbic acid (Vitamin C) and identification of its degradation products

Summary An isocratic ion suppression reversed phase method is utilized for the qualitative and quantitative determination of L-ascorbic acid in fresh fruit juices — complex sample matrices. The selectivity of the method, when a macroreticular poly(styrene-divinylbenzene) reversed phase adsorbent is used, is sufficient to resolve the isomers L-ascorbic acid and D-erythorbic acid (isoascorbic acid) to baseline in under 8 minutes. L-Ascorbic acid solution stability is monitored using the same analytical conditions with the degradation products sufficiently well resolved not to interfere in the quantification of L-ascorbic acid. By the use of commercial materials and the preparation of the proposed products of the oxidative degradation of L-ascorbic acid the degradation sequence and identification of the products in an aged L-ascorbic acid solution is accomplished.     

HPLC with electrochemical detection of metal-flavonoid-complexes isolated from food

Summary The electrochemical and chromatographic behaviour of flavonoid standards and of flavonoids extracted from food (green tea, black tea and onions) is investigated with respect to metalbinding properties. It is shown that metals such as iron, copper or aluminium are complexed by flavonoids, preferrably by those having an aromatic o-dihydroxy structure. This is confirmed by cyclic voltammetry on HPLC fractions (stopped flow) and by AAS measurement of metals. As the complexing sites of flavonoids are closely related to electrochemical properties, this is used for an indirect detection of metal species at low oxidation potentials. For iron species in particular a sensitive and selective detection is possible. For copper reductive detection can also be used.     

Development and optimization of a method for the analysis of Brazilein by HPLC with electrochemical detection

The aim of this study was to establish an easy and accurate method for the determination of Brazilein in plant samples due to its potential pharmacological activities. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used for the assay of Brazilein in this study for the first time. Crucial influence parameters including concentration of dodecane-1-sulfonic acid sodium salt (DSASS), inorganic modifier, tetrabutyl-ammonium hydroxide solution (TBAOH), and applied potential of proposed method were investigated. The proposed method is simple, rapid (analysis time: approximately 10 min), sensitive [(detection limit: 0.6 ng per injection (20 microl) at a signal-noise ratio 3:1)], highly selective and precise (intra- and inter-day precisions were within 5%, n = 7). The calibration graph of Brazilein was linear in the range 0.6-150 ng per injection 20 microl. Recovery of Brazilein was over 92% by standard addition method.     

Determination of fat-soluble vitamins in yogurt by HPLC with electrochemical detection

A method employing HPLC with electrochemical detection for the rapid and simultaneous determination of vitamins A, D(3) and E is described. The method uses a C-18 reverse phase column and 2.5 mM HAcO-NaAcO in methanol-water (99:1, v/v) solution as the mobile phase. The compounds are quantified using amperometric detection with a glassy carbon electrode at a potential of + 1300 mV (vs. Ag/AgCl) and the results are compared with those obtained using UV detection at a wavelength of 280 nm. The method was successfully applied to the analysis of vitamins A, D(3) and E in yogurt samples. After saponification, fat-soluble vitamins were extracted and the methanolic solution of the extracts was injected directly into the chromatographic system, avoiding the clean-up step which is necessary when no electrochemical detection is used. Good recovery percentages were obtained.     

Determination of galactose in human plasma by HPLC with electrochemical detection

Galactose in plasma from patients with hepatic diseases who had undergone low level galactose infusion was determined by using HPLC with electrochemical detection (LCEC). Agreement between galactose concentration determined by the LCEC and a fluorometric method was remarkably good at moderate levels of galactose in plasma. However, the fluorometric method is not suitable for samples containing very small amounts of galactose (blood from hepatic veins) and even for a few samples at moderate galactose content (blood from peripheral veins), suggesting the presence of an endogenous interference. There was no interference for the quantitation of galactose by the LCEC method, by virtue both of the specificity involved in the electrochemical detection and the separation by liquid chromatography. The detection limit of the LCEC method was 0.4 mg galactose/L blood.     

Kinetic study of the degradation of forskolin in aqueous systems by stability-indicating HPLC method and identification of its degradation products

The degradation kinetics of forskolin in aqueous solution was investigated qualitatively and quantitatively. Two degradation products were isolated and identified as isoforskolin and forskolin D by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of forskolin and its degradation products. Chromatographic separation was performed on a Luna C₁₈ column with acetonitrile–water (65:35, v/v) as the mobile phase. The flow rate was kept at 1 mL/min, and the detection wavelength was 210 nm. The kinetic study of forskolin was carried out in aqueous solutions of pH 1.5–8.5 at 37, 50, 65, and 80°C. The degradation rate of forskolin increases with increasing temperature. Forskolin is relatively stable in the pH range 3.5–6.5, but its stability decreases when the pH is outside this range. In the pH range 6.5–8.5, the forskolin degradation follows pseudo-first-order kinetics. Based on the structural identification and quantitative analysis of the degradation products, a possible pathway for forskolin degradation is proposed. Forskolin can be converted to isoforskolin rapidly, and both forskolin and isoforskolin can further decompose to forskolin D.     

Artificial neural networks in analysis of indinavir and its degradation products retention

Artificial neural networks (ANN) are biologically inspired computer programs designed to simulate the way in which the human brain processes the information. In the past few years, coupling of experimental design (ED) and ANN became useful tool in the method optimization. This paper presents the application of ED-ANN in analysis of chromatographic behavior of indinavir and its degradation products. According to preliminary study, full factorial design 2⁴ was chosen to set input variables for network training. Experimental data (inputs) and results for retention factors from experiments (outputs) were used to train the ANN with aim to define correlation among variables. For networks training multi-layer perceptron (MLP) with back propagation (BP) algorithm was used. Network with the lowest root mean square (RMS) had 4-8-3 topology. Predicted data were in good agreement with experimental data (correlation was higher than 0.9713 for training set). Regression statistics confirmed good ability of trained network to predict compounds retention.