Liquid chromatographic determination of vanillin and related aromatic compounds

This paper describes a reversed-phase liquid chromatographic method for the determination of vanillin, associated natural aromatic compounds and/or synthetic precursors, ethyl vanillin, and coumarin, a commonly encountered adulterant in nonbeverage and beverage alcohol products using a ternary gradient mobile phase. The compounds were separated on a Nova-Pak C18 column by using water, methanol, and tetrahydrofuran as the mobile phase. Measurements were made by using a photodiode array detector at 275 nm. The choice of the mobile phase and the column provides baseline resolution of vanillin and the associated aromatic compounds commonly found in vanilla-flavoring material. Because this method provides low-level detection/quantitation, it is suitable for the characterization of vanilla flavoring materials that are currently added to vanilla flavored beverage alcohol products.     

Trace-level determination of aromatic sulfonates in water by on-line ion-pair extraction/ion-pair chromatography and their behavior in the aquatic environment

A new HPLC method for the fully automatic determination of aromatic sulfonates in aqueous samples is presented. The analytical procedure consists of an on-line combination of ion-pair extraction (IPE) and ion-pair chromatography (IPC), both using RP-C18 solid-phase material and a tetrabutylammonium salt as ion pairing reagent. Experimental details and performance data are given. This method is suited for the trace-level determination of a wide variety of benzene, naphthalene, anthraquinone and stilbene sulfonates. Detection limits for surface water using a diode-array detector are in the sub-ppb range. For naphthalene sulfonates a very good selectivity and minimal detectable limits of 0.02 μg/L or even lower can be achieved. So far, this method has been successfully applied to waste water, river water, bank filtrate, and water from different steps of drinking water production. The fate of several aromatic sulfonates has been studied beginning at the effluents of industrial waste water treatment plants and ending after activated carbon filtration in a water works. Napthalene-1,5-disulfonate (NDS, Armstrong acid) and cis-4,4′-dinitrostilbene-2,2′-disulfonate (DNS) appear in the raw water of the investigated water works and therefore have to be termed as relevant to water works. In contrast to other disulfonates NDS is extremely stable to biodegradation and ozonation and it is even desorbed from a highly loaded activated carbon filter.     

Improved detection limits for electrospray ionization on a magnetic sector mass spectrometer by using an array detector

Array detection was compared with point detection for solutions of hen egg-white lysozyme, equine myoglobin, and ubiquitin analyzed by electrospray ionization with a magnetic sector mass spectrometer. The detection limits for samples analyzed by using the array detector system were at least 10 times lower than could be achieved by using a point detector on the same mass spectrometer. The minimum detectable quantity of protein corresponded to a signal-to-background ratio of approximately 2∶1 for a 500 amol/μL solution of hen egg-white lysozyme. However, the ultimate practical sample concentrations appeared to be in the 10–100 fmol/μL range for the analysis of dilute solutions of relatively pure proteins or simple mixtures.     

Liquid chromatographic separation and UV determination of certain antihypertensive agents

Three antihypertensive agents were extracted and isolated from commercial formulations. These were purified and characterized by melting point, lambdamax and IR. The percentage recovery by extraction process was in the range 81-91%. Active ingredients from binary formulations were separated by RP-HPLC using methanol-water (50:50 v/v) and by TLC using CHCl3-CH3OH (6:1) as mobile phase. Detection was by UV at 210 nm in HPLC, and by iodine vapors in TLC. The solvent conditions from TLC were transferred to open column chromatographic separation. Quantitative determination was carried out using TLC and column chromatography supplemented with UV spectrophotometry. Recovery was in the range 82-93%. Two combination of drugs, viz. amlodipine+ramipril and amlodipine+enalapril, were separated by the three modes of liquid chromatography. The percentage recovery was in the range 80-92% by open column.     

Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection

Ultrasound-assisted extraction was used for the determination of phenolic compounds present in strawberries. The optimization study of the extraction was carried out using spiked samples (100 mg/kg). The sample immersed in an aqueous solution containing hydrochloric acid (0.4 M) was sonicated for 2 min (duty cycle 0.2 s, output amplitude 20% of the nominal amplitude of the converter, applied power 100 W with the probe placed 1cm from the bottom of the water bath and 5 cm from the walls of the precipitate glass). Subsequent separation was carried out by liquid chromatography (LC) with photodiode array UV detection. Calibration curves using the standard addition in green strawberries typically gave linear dynamic ranges of 2-300 mg/l for all analytes; R(2) values exceeded 0.996 in all cases. The method was applied to two types of strawberries to demonstrate the applicability of the proposed method, which is much faster and produces less analyte degradation than methods as solid-liquid, subcritical water and microwave-assisted extraction.     

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.     

Some characteristics of an intensified photodiode array spectrometer system for use in plasma emission spectrometry

A 1024-element silicon photodiode array with a microchannel plate image intensifier was coupled to a Czerny-Turner spectrometer and basic characteristics of the spectrometer system were studied using an Hg lamp, a hollow cathode lamp and a d.c. argon plasma as emission sources. The intensifier proved to be useful to enhance signals without increasing the electronic background. The signal to dark current ratio of the cooled photodiode array was larger than that of a photomultiplier (HTV R457) in the wavelength region above 380 nm. Effects of entrance slit width, integration time per scan and intensifier gain on signal to background ratios and signal to noise ratio are presented and optimum conditions for emission measurements using the system are described.     

Extraction and separation of cationic surfactants from river sediments: application to a spectrophotometric determination of cationic surfactant in an aquatic environment using membrane filters.

The quantitative extraction of cationic surfactant (CS+) in river sediments was studied. Further, the developed method was applied to the spectrophotometric determination of CS+ in urban river sediment samples by solid-phase extraction with membranes. A mixture of methanol and hydrochloric acid was proposed as an eluent. Dried sediment was digested in the eluent under ultrasonic irradiation. After elution, the eluent was evaporated to almost dryness. The residue was dissolved in a small volume of methanol and diluted to a certain volume with water. The pH of the solution was adjusted to 4-5 to separate iron and some other metals as precipitates of hydroxides. The solution was passed through two-piled membranes: first glass-fiber and then polytetrafluoroethylene (PTFE) membranes. A small volume of methanol was passed through the membranes to elute any CS+ retaining on the membranes. After passing the methanol solution through a cationic exchange resin column, the retained CS+ was eluted with methanol containing a high concentration of sodium chloride. Water, Bromophenol Blue (BPB) and hydrochloric acid were added to the solution. The solution was passed through a mixed cellulose ester membrane filter to retain an ion associate of CS+.BPB-. The retained ion associate was dissolved in a small volume of N,N-dimethylformamide together with the membrane filter, followed by the addition of triethanolamine to make the solution alkaline. The absorbance due to BPB2- was measured at 603 nm against a reagent blank. This method was applied to the determination of CS+ in river water and sediment. A cationic surfactant in sediments at 10(-5) mol kg-1 levels was detected with satisfactory precision. It was found that CS+ was about 500-fold enriched in the sediment from water at the place where domestic wastewater was discharged.     

Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.     

Normal-phase high-performance liquid chromatographic separations using ethoxynonafluorobutane as hexane alternative. II. Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry applications with methanol gradients

We have reported recently that high-speed normal-phase (NP) HPLC separations of a broad range of organic compounds can be performed on cyano columns using gradients of methanol in hexane-like solvent-ethoxynonafluorobutane (ENFB), available commercially. In this communication, we demonstrate that atmospheric pressure chemical ionization (APCI) in combination with mass spectrometry (MS) can be effectively used for detection in such separations. The efficiency of APCI under conditions studied has also been compared to the efficiency of traditional electrospray ionization (ESI) in combination with MS for reversed-phase (RP) HPLC of the same compounds. The compounds included in this study were steroids, benzodiazepines, and other central nervous system-active substances, nonsteroidal anti-inflammatory drugs, tricyclic antidepressants, and beta-adrenergic blocking agents. Non-polar compounds were found to respond stronger when APCI-MS technique was used, whereas APCI and ESI ionization efficiencies were comparable when polar substances were studied. The combination of normal-phase HPLC separation conditions with mass spectral detection may expand the range of LC-MS applications traditionally associated with reversed-phase HPLC and ESI-MS detection.     

Advances in the gas chromatographic determination of persistent organic pollutants in the aquatic environment

Environmental chemists have been challenged for over 30 years to analyse complex mixtures of halogenated organic pollutants like polychlorinated biphenyls (PCBs), polychlorinated alkanes (PCAs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins and polychlorinated furans (PCDD/Fs). Gas chromatography (GC) often proved to be the method of choice because of its high resolution. The recent developments in the field of comprehensive two-dimensional GC (GCxGC) show that this technique can provide much more information than conventional (single-column) GC. Large volume injection (e.g. by programmed temperature vaporiser, or on-column injection) can be employed for the injection of tens of microliters of sample extract, in that way substantially improving the detection limits. Electron-capture detection (ECD) is a sensitive detection method but unambiguous identification is not possible and misidentification easily occurs. Mass spectrometric (MS) detection substantially improves the identification and the better the resolution (as with MS/MS, time-of-flight (TOF) MS and high-resolution (HR)MS), the lower the chances of misidentification are. Unfortunately, this comes only with substantially higher investments and maintenance costs. Co-extracted lipids, sulphur and other interferences can disturb the GC separation and detection leading to unreliable results. Extraction, and more so, sample clean-up and fractionation, are crucial steps prior to the GC analysis of these pollutants. Recent developments in sample extraction and clean-up show that selective pressurised liquid extraction (PLE) is an effective and efficient extraction and clean-up technique that enables processing of multiple samples in less than 1h. Quality assurance tools such as interlaboratory studies and reference materials are very well established for PCDD/Fs and PCBs but the improvement of that infrastructure is needed for brominated flame retardants, PCAs and toxaphene.     

Liquid chromatographic retention and separation of phenols and related aromatic compounds on reversed phase columns

Summary Isocratic column liquid chromatographic systems with UV absorbance detection at 280 nm have been developed for the separation of 29 phenolics and related compounds.The selectivity was investigated on silica-, carbon- and polymer-based separation columns for the separation of phenolic type of components. The effects of various acetonitrile/buffer mixtures, and pH of the mobile phase, and their impact on the retention of the phenols was assessed. Tables of retention times on the four columns for the 29 phenols with two different acetonitrile/buffer mixtures, together with the retention times at three pHs from 6.5 to 2.3 with varying levels of organic modifier on the LiChrospher RP 18 column are presented.As an application, the analysis of real river water samples from the Ebro river is described using a solid phase extraction step prior to injection into the chromatographic system.     

Rapid separation and determination of process-related substances of paracetamol using reversed-phase HPLC with photo diode array as a detector.

A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.     

Liquid chromatographic separation of enantiomers of beta-amino acids using a chiral stationary phase

The enantiomers of both alpha-substituted beta-alanines and beta-substituted beta-alanines may be chromatographically separated using silica-bonded chiral stationary phases derived from N-acetylated alpha-arylalkylamines. The amino acids are chromatographed as alkyl esters of N-3,5-dinitrobenzoyl derivatives; separability factors range from 1.11 to 1.65 for nine alpha-substituted beta-alanines and from 1.08 to 1.20 for nine beta-substituted beta-alanines. The enantiomers of beta-aminoisobutyrate and beta-leucine, chiral beta-amino acids occurring in animal tissues and physiological fluids, are among those resolved. The enantiomers of R,S-beta-aminoisobutyrate and several related alpha-alkyl-beta-alanines were prepared by chromatographic resolution of diastereomeric dipeptides.     

Liquid chromatographic determination of imazosulfuron in drinking water and in soil using ultraviolet detection.

Reversed-phase liquid chromatography (LC) is used to determine a relatively new sulfonylureic herbicide, imazosulfuron, 1-(2-chloroimidazo-[1,2-a] pyridin-3-ylsulfonyl)-3-(4,6-dimethoxy-2-pyrimidinyl)-urea (TH-913), in drinking water and in soil. TH-913 is extracted from water using solid-phase extraction on C18 bonded silica. Soil samples (20 g) are extracted with 300 ml of methanol-water (50:50) and the acidified extracts are transferred onto Sep-Pak C18 and processed as described for water samples. Off-line desorption is done with 20 ml of methanol-water (50:50). The eluate is evaporated to dryness, the residue dissolved in acetonitrile and analysed by LC with UV detection at 238 nm. The recoveries of TH-913 from water were over 95% (at 0.05 microg/l level) and from soil over 90% (at 0.005 mg/kg level).