LC with Electrochemical Detection. Recent Application to Pharmaceuticals and Biological Fluids

Many pharmaceutical compounds have electroactive groups and are readily measurable and detectable by liquid chromatography with electrochemical detection (LC–EC). LC–EC techniques have many advantages as measurement systems and new materials have been developed for working electrodes. Use of modern electroanalytical techniques for detection in LC of pharmaceutical compounds is discussed in this review. EC detection in LC often results in improved selectivity and detection limits for electroactive pharmaceutical compounds. Selected literature on the determination of pharmaceutical compounds in their dosage forms and in biological samples are reported.     

Electrochemical Detection of Retinoids Using Normal Phase HPLC

Abstract

Non-aqueous electrochemical (EC) detection of 13-cis-retinoic acid, all-trans retinoic, acitretin and vitamin A palmitate in non-aqueous solvents are reported.

Non-aqueous (EC) detection allows for normal-phase chromato-graphy of these compounds prior to detection. The normal phase system used a mobile phase of HEX/THF/AcOH for the separation of all four compounds. The stationary phase was either silica or PVA-sil. The lipophilic salts, t-butylammoniumtetrafluoroborate or t-butyl-ammoniumhexafluorophosphate necessary for EC detection were added post-column.

The limit of detection (LOD) for EC detection of these compounds is approximately 1 ng on column compared with an LOD by UV absorption of 2 ng on column.

The linear detection for these compounds with the EC detector was about two orders of magnitude.     

Determination of Trialkyl and Triaryl Phosphates by Narrow-bore Liquid Chromatography with On-line Thermionic Detection

Abstract

The potential of a previously reported on-line reversed-phase liquid chromatography-thermionic detector (LC–TID) system has been further evaluated. Several trialkyl (trimethyl to tri-octyl) and triaryl (triphenyl and tri-o-cresyl) phosphates were chosen as model compounds. LC was done on an alkyl-bonded silica column with methanol-water (80:20) at a flow-rate of 35—70μ/min as eluent. The band broadening in the interface-TID unit increased with decreasing volatility of the compounds and for the relatively non-volatile compounds, the band broadening decreased with increasing eluent flow-rate.

For most of the test compounds, detection limits of 40–100pg were obtained. As an illustration of the high selectivity of the LC[sbnd]TID system, the trace-level determination of two aryl phosphates in sediment samples is reported.     

Determination of phenolic compounds in dietary supplements and tea blends containing Echinacea by liquid chromatography with coulometric electrochemical detection

Analytical methodologies with ultrasonic extraction and liquid chromatography (LC) were developed for the determination of phenolic compounds in dietary supplements containing Echinacea. The phenolic compounds determined by these methods included caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid. Samples from tablets, capsules, and bags of tea blends were extracted by sonication for < or = 30 min with methanol-water (60 + 40). The extracts were centrifuged and filtered, and the filtrates were diluted and analyzed by LC using a reversed-phase column and coulometric electrochemical (EC) detection. The mobile phase was acetonitrile-ammonium formate buffer, pH 3.5 (15.3 + 84.7) containing tetrabutyl ammonium hydrogen sulfate as an ion-pairing reagent. Extraction conditions (e.g., composition of the extraction solvent and sonication time) were optimized for different types of samples. Intra- and interday analytical variations were determined, and intraday analyses were performed by 2 independent analysts using 2 different LC systems. Results were generally comparable. The LC method with EC detection showed better sensitivity and selectivity when compared with LC with ultraviolet detection, although results were similar for the 2 methods for major compounds, i.e., caftaric acid, echinacoside, and cichoric acid. The identities of these major compounds found in samples were confirmed by LC/electrospray ionization mass spectrometry.     

Determination of Sulfophenyl Carboxylic Acids in Agricultural Groundwater Samples by Liquid Chromatography with Fluorescence Detection

Abstract

We have developed an analytical method to determine six sulfophenyl carboxylic acids (SPC) in agricultural groundwater samples. It involves a solid-phase extraction (SPE) procedure and subsequent separation and determination using liquid chromatography with fluorescence detection (LC–FD).The quantification limits ranged between 0.7 and 1.2 ng ml^?1. The proposed method was proved satisfactorily for the detection and determination of these compounds in groundwater samples during a study into the biodegradation of linear alkylbenzene sulfonates (LAS) in an agricultural soil sampled from the irrigated plain to the west of Granada (Spain).     

Ochratoxin A in Wine: Its Determination and Photostability

Abstract

Due to the growing public concern regarding food safety, reliable, nondemanding and robust analytical methods are needed for quantitative determination of toxic compounds in complex matrices. Sample preparation is frequently a crucial step in determination of ochratoxin A (OTA) in wine, and a simplified and automated procedure is described, using solid‐phase extraction coupled on‐line to high pressure liquid chromatography (HPLC) with fluorimetric detection (λ_ex=333 nm, λ_em=460 nm). While the limit of quantitation is frequently better compared to off‐line procedures (30 ng/L), the decisive advantages of the new procedure are the absence of all sample manipulation during preconcentration and subsequent analysis, and consequentially no risk of analyte loss or sample contamination. Furthermore, using the standard addition method, matrix interferences can be avoided and the determination of extraction efficiency is unnecessary. These improvements have important consequences for the overall uncertainty of the analytical procedure. The developed method was applied for determination of OTA in 12 selected Slovenian wines. The typical relative standard deviation (RSD) was 10%. In none of the samples, did the OTA amount exceeded 2 µg/kg, the limit regulated by the EC.

The photo‐stability of the mycotoxin in solutions was examined. During irradiation of OTA solutions, its content was quickly reduced, while three fluorescent degradation products were detected. The degradation proceeds faster in water and 12% ethanolic solutions than in organic solvents or wine. Identification of the fluorescent degradation products was attempted using LC‐MS/MS with electrospray ionization.     

Statistical Model for Organic Chromatographic Trace Analysis of Complex Samples. A Case Study: Plant Extracts

Abstract

The use of pooled plant extracts is described in the estimation of matrix interference in HPLC (UV and EC) determinations of organic compounds in plant extracts. An extract from freeze dried leaves of 134 different plant species was used for this purpose. It was split in different subgroups with solid extraction clean-up procedures. UV, EC and chromatographic data of the subgroups were used in the calculation of minimum concentrations of organic compounds which are still accurately determinable in plant samples with HPLC methods. The UV and/or EC characteristics of the compound must be known. The contribution of the solid phase extraction procedures and of the analytical system to the selectivity of the method can be estimated. Information is also supplied which allows rapid comparison of the selectivity of the UV and EC (single, or dual parallel) detectors for the determination of a specified compound.     

Selective detection of unknown organic bromine compounds and quantification potentiality by negative-ion electrospray ionization mass spectrometry with induced in-source fragmentation

For the detection of unknown organic bromine compounds, a liquid chromatography–mass spectrometry (LC-MS) method with negative-ion electrospray ionization (NI-ESI) and induced in-source fragmentation (IISF) was established. After LC separation, the molecules are fragmentized in the source, and bromide is detected via m/z 79 and m/z 81 based on the isotopic occurrence of bromine. In this way, the retention times of the unknown organobromine compounds are determined, and this can be used to extract additional structural information (number of bound bromine atoms, molecular mass and fragmentation scheme) from measurements in the commonly used but less sensitive scan mode. The analysis of known organobromine compounds shows that LC/NI-ESI-IISF mass spectrometry with detection of m/z 79 and 81 is more sensitive than the detection of daughter ions (LC/ESI/MS-MS). Therefore, we present a method not only for the detection of unknown organic bromine compounds, but also for the selective and sensitive detection and quantification of known organobromine compounds.     

Simple Multiresidue Determination of Fluoroquinolones in Bovine Milk by Liquid Chromatography with Fluorescence Detection

Abstract

A simple and sensitive method for the simultaneous determination of five fluoroquinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, and sarafloxacin) in bovine milk was developed. Protein precipitation from milk samples was achieved by the addition of acetonitrile and o‐phosphoric acid. Acetonitrile was removed with dichloromethane, leaving the fluoroquinolones in the acid aqueous extract. The aqueous extract was analyzed by liquid chromatography with fluorescence detection (LC–FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile‐water (12∶88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found ranged from 1 to 6 ng · mL^?1 and were below the maximum residue limits (MRLs) established by the European Union. The proposed method was applied to the determination of these compounds in different bovine milk samples. Method validation was carried out by a recovery assay.     

A New Method for the Characterization of Organic Carbon in Aquatic Systems

Abstract

Some problems in organic carbon characterization are briefly discussed. A new chromatographic multi-method for the direct characterization of organic matter in original waters with the simultaneous determination of UV-Vis, fluorescence, DOC, TIC, and organically bound nitrogen (DON) is described. Some applications with UV-Vis and DOC (“CODOC”) detection are presented.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]⁺. ¹³C₂ acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

Determination of Coccidiostats in Milk Products by LC–MS and Its Application to a Fermentation Experiment

A liquid chromatographic tandem mass spectrometric (LC–MS–MS) method for the determination of five chemical coccidiostats (decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine) and five ionophore coccidiostats (maduramicin, monensin, narasin, salinomycin, and semduramicin) in yoghurt, kefir, and sour cream is presented. Lasalocid, the sixth ionophore listed in 124/2009/EC was not included because of its extremely dissimilar behavior during sample preparation. Main steps of the method include extraction with acetonitrile, centrifugation, clean-up on Oasis HLB solid phase extraction cartridge, evaporation under nitrogen stream, and LC–MS–MS determination. Selectivity, linearity, sensitivity, accuracy, repeatability, within-laboratory reproducibility, limit of determination, and limit of quantitation were determined during the validation procedure. The method proved to be applicable for both qualitative and quantitative determination of the ten above-mentioned target compounds. In our in-house fermentation experiments, milk fortified with coccidiostats was fermented to get yoghurt, kefir, and sour cream. Our results show that the coccidiostat content did not change significantly during fermentation for any of the target compounds.

     

Simultaneous Determination of Anthraquinones in Hedyotis diffusa by LC Coupled with UV Detection

A simple and reliable liquid chromatography method coupled with UV detection was developed and validated for the determination of the pharmacologically important anthraquinones in the total plants of Hedyotis diffusa. The separation of four anthraquinones was performed on a C₁₈ column using an isocratic mobile phase consisted of methanol-0.5% glacial acetic acid (58:42, v/v). The detection wavelength was set at 265 nm. The limits of detection obtained for the analytes were in the range of 0.004–0.012 μg mL^?1. The recovery of the developed method for the analysis of the four compounds was determined and ranged from 95.8 to 103.2%. According to the literatures, this is the first LC method reported for the simultaneous determination of the four anthraquinones and the method can be applied to determine the amounts of the bioactive compounds in Hedyotis diffusa.     

Spectrophotometric Determination of Cannabidiol

Abstract

Cannabidiol reacts with 2,4-dinitrophenol in alkaline medium to give a red colour having maximum absorbance at 500 nm. The reaction is specific for cannabidiol with 0.1 μg/10 ml as a minimum limit of detection. It also provides the basis for a new spectrophotometric determination of pure cannabidiol and in the presence of other organic compounds. The maximum tolerable limits of these are reported. The method is successfully applied to the determination of cannabidiol from leaves of Cannabis sativa growing wild and cultivated in Pakistan.     

New chiral liquid crystal cyclic monomers based on diosgenin: synthesis and mesomorphism

A series of new chiral liquid crystal (LC) intermediate compounds (LC-C₁~LC-C₄) containing diosgenyl groups and the corresponding cyclic LC aliphatic carbonate monomers (LC-M₁~LC-M₄) were synthesised to investigate the relationship between their structures and phase behaviour. The chemical structures of the compounds obtained in this study were characterised using FT-IR, ¹H NMR and ¹³C NMR measurement. The phase behaviour and mesophase structure were investigated with polarising optical microscopy, differential scanning calorimetry and X-ray diffraction measurement. As a result, LC-C₁ and LC-C₂ showed focal conic texture of the choleteric phase, while LC-C₃ and LC-C₄ with longer spacer length showed fan-shaped texture of a smectic A phase. The monomers (LC-M₁~LC-M₄) all exhibited focal conic texture of the choleteric phase on heating and cooling cycles. Furthermore, the effect of the spacer length on phase behaviour of the LC compounds was discussed.     

Evaluating polymer monolith in-tube solid-phase microextraction coupled to liquid chromatography/quadrupole time-of-flight mass spectrometry for reliable quantification and confirmation of quinolone antibacterials in edible animal food

A simple, rapid, and sensitive method is presented to determine seven trace quinolone antibacterials simultaneously in milk, egg, chicken and fish. This method is based on the combination of polymer monolith in-tube solid-phase microextraction with liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS). LC/ESI-QTOF-MS offers the capability of unequivocal identification of target compounds from complex matrices, as well as the possibility of quantitation at low-level concentrations in real samples. The extraction was performed with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic column. Under the optimized extraction conditions, good extraction efficiencies for the targets were obtained with no matrix interference in the subsequent LC/ESI-QTOF-MS. Good linearities were obtained for seven quinolones with the correlation coefficients (R) above 0.9951. The limits of detection (S/N = 3) for seven quinolones were found to be 0.3–1.2 ng/g in egg, 0.2–3.0 ng/mL in milk, 0.2–0.7 ng/g in chicken and 0.2–1.0 ng/g in fish. The recoveries of quinolones spiked in four different matrices ranged from 80.2 to 115.0%, with relative standard deviations less than 14.5%. The developed method was applied for the determination of quinolone residues in animal-producing food, and the positive samples were confirmed with high number of identification points (IPs) according to the IP system defined by the European Union (Commission Decision 2002/657/EC).     

Simultaneous Determination of CGS 10787B and Its Major Metabolite (CGS 12094) in Plasma by High Performance Liquid Chromatography

Abstract

A method for the simultaneous determination of CGS 10787B and its major, metabolite (CGS 12094) in plasma is described. The two compounds, and the internal standard (dichlorinated analog), are extracted from acidified plasma with ethyl acetate, taken to dryness, and reconstituted in chromatographic mobile phase. The analytes are determined automatically by high performance liquid chromatography in the reversed-phase mode as paired ions, using [N(Bu)₄]⁺ as the counterion. The separation of the compounds is achieved on a 3u C-8 column, with detection at 254 nm.

Recovery and reproducibility assessments indicate good accuracy and precision over the range of 1.0 to 250 ug/ml for CGS 10787B and 1.0 to 100 ug/ml for CGS 12094.

The method has a limit of detection of 0.2 ug/ml for both compounds, and has been shown to be adequate for studying the disposition kinetics of CGS 10787B.     

Determination of Formaldehyde in Waste Water by Lucigenin Chemilummescence

Abstract

A chemiluminescence analysis has been developed for the determination of formaldehyde based on its inhibition of the chemiluminescence reaction of lucigenin-C10^?-H₂o₂. The method is sensitive, convenient and selective with a detection limit of 0.05ng/ml. The linear dynamic range is 1.0ng/ml to 0.1 μg/ml. The variation coefficient of ten determinations for 2.Ong/ml formaldehyde is 1.2%. Applications to the trace determination of formaldehyde in industrial waste waters are discussed.     

The Application of Perdeuterated Polycyclic Aromatic Hydrocarbons (PAH) as Internal Standards for the Liquid Chromatographic Determination of PAH in a Petroleum Crude Oil and Other Complex Mixtures

Abstract

A sequential liquid chromatographic (LC) procedure for the determination of polycyclic aromatic hydrocarbons (PAH) in a petroleum crude oil and other complex mixtures is described. The procedure includes normal-phase LC on an aminosilane column to isolate fractions containing isomeric PAH and reversed-phase LC on a polymeric C₁₈ column to separate the individual PAH isomers. Appropriate perdeuterated PAH are added to the sample so that each isomeric fraction will contain one internal standard. The perdeuterated PAH are excellent internal standards for this sequential LC procedure. Perdeuterated PAH have normal-phase and reversed-phase LC retention characteristics similar to those of the parent PAH. In the normal-phase LC separation, the perdeuterated PAH elute in the same fraction as the parent PAH. In the reversed-phase LC separation, the perdeuterated PAH elute first and are generally resolved from the parent PAH. The optimized spectrofluorometric detection of each PAH analyte is accomplished by programming appropriate sets of excitation and emission wavelengths to correspond with the elation time of each analyte on the polymeric C₁₈ column. The analytical results obtained from this procedure for the analysis of a shale oil sample [Standard Reference Material (SRM) 1580] and a petroleum crude oil (SRM 1582) are compared to values obtained by gas chromatography - mass spectrometry.     

Quantitative Analysis of Persistent Organochlorines in Samples From Polar Regions a Challenge For The Analytical Chemist

Abstract

The study of the transport and fate of persistent polychlorinated compounds to remote areas such as polar regions requires analytical methods which are capable to detect fg to pg amounts in the main transport medium, the atmosphere, as well as their accumulation in the very short food chain. A list of requirements are given which have to be fulfilled for the detection of such compounds with sufficient reliability. Furthermore, problems observed during sample clean-up, separation and quantification of such low levels are discussed using practical examples, and proposals are made how to avoid them.