Separation of kafirins on surface porous reversed-phase high-performance liquid chromatography columns

Surface porous high-performance liquid chromatography (HPLC) columns were investigated for the separation of kafirins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8, and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed and were slightly faster than those achieved on the surface porous C18 stationary phase. However, the resolution was higher on the latter column. Using an ammonium hydroxide/acetonitrile mobile phase, separations were performed on a novel, alkaline stable surface porous C18 stationary phase. The resolution at alkaline pH was not as high, however, as with the traditional acidic acetonitrile mobile phases. In comparison to fully porous stationary phases, the surface porous phases provided higher resolution with much lower separation times (17 versus 40 min). Total peak areas were correlated to total protein content of sorghum (r(2) = 0.96; n = 10), and a method to measure in vitro pepsin digestibility using reversed-phase (RP)-HPLC peak areas showed good correlation to the traditional nitrogen combustion method (r(2) = 0.82; n = 20). Thus, the surface porous stationary phases could be used not only for more rapid separations but also to provide simultaneous information on total protein content and digestibility.     

Determination of bisphenol a and bisphenol B residues in canned peeled tomatoes by reversed-phase liquid chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.     

Environmental analysis by on-line immunoextraction and reversed-phase liquid chromatography: optimization of the immunoextraction/RPLC interface

The use of antibodies in HPLC columns for on-line immunoextraction combined with reversed-phase liquid chromatography (RPLC) is of growing interest in environmental and agricultural analysis. This technique is typically performed by using a small RPLC precolumn to capture and concentrate analytes as they elute from the immunoextraction column; however, there is little information on the conditions required for optimizing this interface. This study examined the behavior of this interface by using 2,4-dichlorophenoxyacetic acid (2,4-D) and related herbicides as model analytes. It was found that analyte dissociation from immunoextraction columns followed first-order decay and that the elution of these analytes through the immunoextraction/RPLC interface gave an exponentially modified Gaussian profile. Computer simulations were used to see how analyte elution through the interface changed with different dissociation and retention conditions. Several guidelines were developed from this work that could be used for developing and optimizing on-line immunoextraction/RPLC systems for other chemicals of environmental or agricultural interest.     

Analysis of pesticides in nuts by online reversed-phase liquid chromatography-gas chromatography using the through-oven transfer adsorption/desorption interface

A new method to determine pesticide residues in nuts is presented, in which the pesticides are extracted from samples with a small amount of ethyl acetate and anhydrous sodium sulfate. No additional cleanup or concentration steps are necessary. The extract is directly injected into the high-pressure liquid chromatograph, where preseparation of the pesticide residues from other components coextracted from the nuts is carried out using methanol/water as the eluent. The selected liquid chromatography fraction containing the pesticides is automatically transferred to the gas chromatograph using the through-oven transfer adsorption/desorption interface. The calculated limits of detection for each pesticide varied from 0.1 to 61.3 microg/kg. The repeatabilities of the analysis and the overall procedure (extraction and analysis) were satisfactory. No variations in the retention time were observed. The method was applied to the analysis of pistachio nut, peanut, walnut, hazelnut, and sunflower seed.     

Organic solvent-free reversed-phase ion-pairing liquid chromatography coupled to atomic fluorescence spectrometry for organoarsenic species determination in several matrices

A novel method has been developed to determine As-containing animal feed additives including roxarsone (ROX), p-arsanilic acid (p-ASA) and nitarsone (NIT), as well as other organic As species (dimethylarsonic acid (DMAA) and monomethylarsonic acid (MMAA)) by ion-pairing high-performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry (IP-HPLC-HG-AFS). A simple isocratic reversed-phase (RP) HPLC method with a mobile phase containing citric acid and sodium hexanesulfonate (pH 2.0) was developed using a C(18) column. The use of an organic solvent free mobile phase turns this methodology into an environmentally friendly alternative. Several ion pair forming agents, such as sodium hexanesulfonate, tetrabutylammonium bisulfate and perfluoroheptanoic acid, were studied. The limits of detection for As species were calculated in standard solution and resulted to be 0.2, 0.5, 0.6, 1.6, and 1.6 μg As L(-1) for MMAA, DMAA, p-ASA, ROX and NIT, respectively. This method exhibited convenient operation, high sensitivity and good repeatability. It was applied to As speciation in different samples including arugula, dog food, dog urine and chicken liver.     

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.     

Purification of cyclopiazonic acid by liquid chromatography

A purification procedure for cyclopiazonic acid has been developed, using sequential preparative and semi-preparative liquid chromatography. Crude cyclopiazonic acid (324 mg) was extracted from a 1 L fermentation medium with chloroform-methanol (80 + 20), dried, dissolved in chloroform, and chromatographed on an oxalic acid/silica preparative column with chloroform-methanol (99 + 1) as the eluant. A semi-preparative oxalic acid/silica column and chloroform-methanol (99.5 + 0.5) were then used for rechromatography of the partially purified cyclopiazonic acid. This second chromatographic treatment yielded fractions from which cyclopiazonic acid was readily crystallized (106.7 mg; 33% recovery). Analytical chromatography was developed using an amino column in an ion-exchange mode, with a methanol-phosphate buffer eluant. Response was linear from 10 to 800 micrograms/injection of standard solutions. Cyclopiazonic acid chemically binds sodium from soda-lime vials.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).     

Analysis of unsaponifiable compounds of edible oils by automated on-line coupling reversed-phase liquid chromatography-gas chromatography using the through oven transfer adsorption desorption interface

An automated method for analysis of unsaponifiable compounds in edible oils is presented. The method involves the on-line coupling of reversed-phase liquid chromatography and gas chromatography (LC-GC) using the through oven transfer adsorption desorption (TOTAD) interface. The oil is injected directly with no sample pretreatment step other than filtration. It may also be considered to dilute the oil sample. In the LC step, a short C4 column using a methanol/water eluent separates analytes from the other components of the oils, which are made up of mainly triglycerides. A LC fraction of up to 1.6 mL containing the analytes is transferred to GC at a flow rate of 0.1-2 mL/min. The TOTAD interface allows solvent venting and the introduction of the analytes into the GC column. The proposed fully automated method allows the analysis of different groups of compounds (free sterols, tocopherols, squalene, and erythrodiol and uvaol) in one chromatographic run or the analysis of these compounds in different groups. Sensitivity is more than necessary, and repeatability is good, the CV ranging from 3 to 12% for the full analysis.     

Determination of nicarbazin in chicken tissues by liquid chromatography and confirmation of identity by thermospray liquid chromatography/mass spectrometry

A liquid chromatographic method is described for the quantitative measurement of nicarbazin in chicken liver, fat, muscle, and skin tissues. The 4,4'-dinitrocarbanilide (DNC) portion of nicarbazin is extracted from tissues with ethyl acetate. After filtration and evaporation, the extract is purified by liquid-liquid partitioning with acetonitrile-hexane and alumina cartridge chromatography. DNC is separated and measured by reverse-phase liquid chromatography (RP-LC) with an octadecylsilyl (ODS) column and a UV detector set at 340 nm. The overall average recovery of DNC added to tissues was 83.4 +/- 3.1%. The lowest level validated in tissues by this procedure was 0.10 ppm. The limit of detection was estimated to be 0.020 ppm. This method provides a sensitive, selective, rapid, and reproducible alternative to existing purification, separation, and detection techniques, such as differential pulse polarography and colorimetry, for determination of nicarbazin in chicken tissues. Identity of DNC is confirmed by subjecting the purified extracts to thermospray-LC/mass spectrometric analysis using negative-ion detection and selected ion monitoring. Three structural-indicating ions at m/z 302, 272, and 164 are monitored in the thermospray-mass spectrum which are characteristic of the DNC molecule.     

Dimetridazole residues in pork tissue. I. Assay by liquid chromatography with electrochemical detector

A liquid chromatographic (LC) method with electrochemical detection in the reductive mode was developed for the quantitative determination of dimetridazole (DMZ) and its major metabolite (HMMNI) at residue levels in pork tissue. For blood plasma, a sample is precipitated with 2 volumes of acetonitrile and centrifuged, and a diluted aliquot of the supernatant liquid is chromatographed. For muscle, a 10 g sample is extracted 3 times with dichloromethane. After evaporation of the combined extracts, the residue is redissolved in a mixture of hexane and mobile phase (0.3% TEA in 0.6M ammonium acetate pH 5.0 and acetonitrile, 85 + 15) and centrifuged, and an aliquot of the lower phase is chromatographed. Chromatography is accomplished using valve switching with 2 liquid circuits, employing the same mobile phase for both. The sample is deaerated by sparging with helium under slight positive pressure to prevent rediffusion of the oxygen. The sample is first loaded into a deoxygenator and the flow is stopped for complete deoxygenation. The flow is then resumed to transfer the sample into the first, low back-pressure column (ODS, 10 microns, 4.6 x 200 mm). Switching the valve at this point removes the deoxygenator from the circuit and connects the first column to a second one (ODS, 5 microns, 4.6 x 150 mm) in tandem. After the effluent is passed through a second deoxygenator to reduce the residual oxygen in the mobile phase, it is monitored by an electrochemical detector with a screened wall jet cell and a gold mercury electrode, set at -1.2 V.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of thiamphenicol in bovine plasma by liquid chromatography

A liquid chromatographic method is described for the measurement of thiamphenicol in bovine plasma. The plasma (1 mL) is extracted with ethyl acetate. After the solvent is evaporated under a stream of nitrogen, the residue is reconstituted in methanol-water and analyzed by reverse-phase liquid chromatography with UV detection at 224 nm. The intra-day recoveries for bovine plasma spiked with 5 and 50 micrograms/mL of thiamphenicol were 102 and 101%, respectively, with coefficients of variation of 2.40 and 0.28%, respectively. The interday recoveries for the 5 and 50 micrograms/mL samples were 103 and 101%, respectively, with coefficients of variation of 3.40 and 0.94%, respectively. The sensitivity of the method allows quantitation to at least the 100 ng/mL level.     

Determination of sulfite in foods by headspace liquid chromatography

Sulfite was determined in a variety of foods by liquid chromatography (LC) after the samples were mixed with a solution containing mannitol, FeSO4, and Na2HPO4, adjusted to pH 11, and left to stand for 15 min at room temperature. An aliquot of the mixture was placed in a headspace vial and mixed with 50% H3PO4. After 15 min, a portion of the headspace was removed with a syringe containing LC mobile phase without acetonitrile. The syringe was shaken and an aliquot of the solution was analyzed on an anion exchange column with a mobile phase of 0.03M methane sulfonate (pH 10.8) containing 5% acetonitrile. Sulfite was detected amperometrically (glassy carbon electrode) at +0.7 V. The method was successfully compared to the FDA-modified Monier-Williams procedure for a variety of foods. Minimum detectable levels were about 1 microgram/g, based on a 15 g sample.     

Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography

Successful enantiomeric separation of 10 chiral pesticides by high-performance liquid chromatography (HPLC) using cellulose-tris(3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was performed. The mobile phase was n-hexane modified by ethanol, propanol, 2-propanol (IPA), butanol, or isobutanol. The effects of mobile phase composition and column temperature on the separation were investigated. Baseline separation was obtained with ethofumesate, fluroxypyr-meptyl, malathion, benalaxyl, diclofop-methyl, methamidophos, vinclozolin, and lactofen, whereas near baseline separation was obtained with profenofos and acetochlor. Butanol was the best modifier for benalaxyl; isobutanol was the best modifier for lactofen, malathion, diclofop-methyl, and ethofumesate; and IPA was the best modifier for the other five. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector. The quantitative analysis methods for the enantiomers of ethofumesate, benalaxyl, and diclofop-methyl were established. Validation parameters include linearity, precision, and limit of detection (LOD). The enantiomeric residual analysis procedures in soil and water samples were also developed using acetone extraction and C(18) solid phase extraction. The methods were reliable for residual analysis of the enantiomers.     

Monitoring of changes in substrate characteristics during mushroom compost production

Substrates from three mushroom compost facilities in Northern Ireland, employing similar production technologies, were examined to assess the quality of the compost produced. Biochemical investigation highlighted changes in substrates through each step of the production cycle. Thermogravimetric analysis (TGA) provided useful information on fiber fraction content and extent of substrate breakdown. A comparison of productivity, chemical, and thermal data permitted assessment of the degree of bioconversion that had occurred in the decomposition from raw materials to finished substrate for each composter. One of the composters consistently produced substrate of inferior quality compared to the other two, indicating production inefficiencies during composting. Results demonstrated that allied to chemical analyses, TGA is a useful tool, providing valuable information on substrate quality and, in particular, for studying the bioconversion of lignocellulosic materials in mushroom compost.     

Quantitative determination of amines in wine by liquid chromatography

A liquid chromatographic (LC) procedure is described for the determination by dansylation of the following 16 kinds of biogenic amines found in wine: monomethylamine (MM), ethylamine (EM), iso- and n-propylamine (Pr), iso- and n-butylamine (Bu), iso- and n-amylamine (Am), pyrrolidine (PY), 2-phenethylamine (PH), tryptamine (TR), putrescine (PU), cadaverine (CA), histamine (HI), tyramine (TY), and spermidine (SP). The amines in white and red wine were applied to a column of Amberlite CG-50 type I resin (Na-form) after the column had been washed with water and eluted with 1N hydrochloric acid. This eluate was evaporated to dryness under reduced pressure and derivatized with dansyl chloride (DNS). LC separations were performed on Finepak SIL C18S and LiChrosorb RP-8 columns with an acetonitrile-water elution gradient. In the survey of commercial wines by this method, most of the samples were found to contain 12 amines, including iso-Am, CA, PU, TY, and others. The highest levels of these amines were 4.84 micrograms PU/mL in red wine, and 5.11 micrograms iso-Am/mL in white wine. The total levels of amines in red wine were comparatively higher than in white wine.     

Determination of N-methylcarbamate pesticides in liver by liquid chromatography

A liquid chromatographic (LC) method using a 2-step purification technique for the simultaneous determination of 10 carbamates in bovine, swine, and duck livers has been developed. Carbamates are extracted from liver samples with methylene chloride. After evaporation, the residues from the extract are dissolved in methylene chloride-cyclohexane (1 + 1) and cleaned up by gel permeation chromatography. The eluate containing carbamate residues is evaporated to dryness, reconstituted in methylene chloride, further purified by passing it through an aminopropyl Bond Elut extraction cartridge, and analyzed by liquid chromatography using post-column derivatization with orthophthalaldehyde and fluorescence detection. Excitation and emission are set at 340 and 418 nm, respectively. Liver samples for beef, pork, and duck were fortified with 5, 10, and 20 ppb of mixed carbamate standards. The average of 10 recoveries of 10 carbamates at all 3 levels of fortification was greater than 80% with coefficients of variation less than 17%.     

Determination of caffeine, theobromine, and theophylline in standard reference material 2384, baking chocolate, using reversed-phase liquid chromatography

A rapid and selective isocratic reversed-phase liquid chromatographic method has been developed at the National Institute of Standards and Technology to simultaneously measure caffeine, theobromine, and theophylline in a food-matrix standard reference material (SRM) 2384, Baking Chocolate. The method uses isocratic elution with a mobile phase composition (volume fractions) of 10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min with ultraviolet absorbance detection (274 nm). Total elution time for these analytes is less than 15 min. Concentration levels of caffeine, theobromine, and theophylline were measured in single 1-g samples taken from each of eight bars of chocolate over an eight-day period. Samples were defatted with hexane, and beta-hydroxyethyltheophylline was added as the internal standard. The repeatability for the caffeine, theobromine, and theophylline measurements was 5.1, 2.3, and 1.9%, respectively. The limit of quantitation for all analytes was <100 ng/mL. The measurements from this method were used in the value-assignment of caffeine, theobromine, and theophylline in SRM 2384.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.