Evaluation of isoflavone aglycon and glycoside distribution in soy plants and soybeans by fast column high-performance liquid chromatography coupled with a diode-array detector

An ultrafast HPLC/UV-vis DAD method working at 254 nm was applied for the determination of isoflavone aglycons and glycosides (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin, and biochanin A) in roots, stems, leaves, and soy pods of soy plants and in soybeans of five varieties (Korada, Quito, Rita, OAC Erin, and OAC Vison). An Atlantis dC18 ultrafast RP chromatographic column (20 mm x 2.1 mm, 3 microm particle size) was applied for separation of the isoflavone aglycons and glycosides. A flow rate of the mobile phase (0.1% (v/v) acetic acid, pH 3.75-solvent A and methanol-solvent B) was 0.35 mL min(-1), and the column temperature was 36 degrees C. A linear gradient profile from 13 up to 22% B (v/v) from zero to 2.5 min, up to 30% B to 3.21 min, up to 35% B to 4 min, up to 40% B to 4.5 min, up to 50% B to 5.14 min, and followed by negative gradient up to 13% B to 7.71 min was used. The absolute limits of detection per sample injection (5 microL) were the highest for biochanin A (166.2 fmol) and the lowest for genistin (17.0 fmol), respectively. An accelerated solvent extraction (ASE) in combination with sonication was applied for isolation of biologically active compounds. A solid-phase extraction procedure was used to purify the extracts in the case of analysis of soy plants parts. The recoveries of 96-106% were obtained for the different concentrations of the isoflavone aglycons and glycosides and the different matrixes (overall RSDs 2-9%). The highest isoflavone concentrations were found in roots (12.5 microg g(-1) dry weight), while the amounts were about 3-1100 microg g(-1) fresh weight in different varieties of soybeans.     

Determination of ergosterol in ganoderma spore lipid from the germinating spores of Ganoderma lucidum by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and determination of free ergosterol in ganoderma spore lipid (GSL) extracted from the sporoderm-broken germinating spores of Ganoderma lucidum. Sodium hydroxide in methanol was added for the hydrolysis of ergosteryl esters to determine the total content of ergosterol in GSL by HPLC. A 0.04 M concentration of sodium hydroxide in reaction mixtures was appropriate for the complete hydrolysis of ergosteryl esters without a significant loss of ergosterol during saponification. In addition, the ergosterol content in four commercial GSL softgel supplements from four different firms was determined. The results showed that the ergosterol content in these samples had significant differences. Ergosterol content may be a suitable marker for evaluating the quality of GSL products.     

Determination of ETU in tomatoes and tomato products by HPLC-PDA: evaluation of cleanup procedures

An HPLC-PDA method for the determination of ethylenethiourea (ETU), the main degradation product of the organic fungicides ethylene bis(dithiocarbamate)s (EBDCs), in tomatoes and tomato products is reported. Solid-matrix liquid-liquid (l-l) partitioning and separatory funnel l-l partitioning for the cleanup were examined. The effect of salt addition, pH, and phase ratio on analyte recovery at the cleanup step was studied. It was found that solid-matrix l-l partitioning afforded higher precision and more selective separation of the analyte. According to the method proposed, the samples were extracted with methanol/water (3:1, v/v) and cleaned up on an Extrelut 20 column. ETU was eluted with dichloromethane and separated on a reversed phase HPLC column. For tomato products with degrees Brix > 20 further purification through silica cartridge was adopted. The method was validated over the following ranges of concentrations: 0.01-0.5 mg/kg for tomatoes, 0.01-0.1 mg/kg for tomato juice, and 0.05-0.25 mg/kg for tomato paste. The accuracy (recoveries > 70%) and the precision obtained (%RSD < 10%) were satisfactory.     

Determination of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography

The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans-astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis-astaxanthin and 13-cis-astaxanthin.     

Liquid chromatographic method for quantitative determination of free fatty acids in butter

A liquid chromatographic method has been developed for the determination of free fatty acids in butter. The fatty acids are converted to the p-bromophenacyl esters, via a crown ether-catalyzed reaction, without separation from the other butter components. The esters are separated on a C18-bonded silica column by using an acetonitrile-water solvent gradient and quantitated using the ester of heptadecanoic acid as internal standard. C16 and C18:1 co-elute in the acetonitrile-water system but are separated using an isocratic methanol-acetonitrile-water system. Limits of detection range from 7 ng for butyric acid to 45 ng for linoleic acid. The average coefficient of variation (n = 10) for 10 free fatty acids from a butter was 5.83%.     

Determination of levamisole in animal tissues using liquid chromatography with ultraviolet detection.

An efficient and sensitive liquid chromatographic method is described for the determination of the anthelminthic drug levamisole, in muscle, liver, kidney and fat of sheep, pigs and poultry, using thiabendazole as internal standard. Samples were extracted by homogenizing with chloroform, and were applied to Supelco Si solid-phase extraction columns and eluted with methanol. Chromatographic analysis was performed on a LiChrospher 60 RP-Select B column using methanol/ammonium acetate buffer 0.05 M (55/65, v/v) as mobile phase and reading at 220 nm. The quantification limit for the assay was 4 ng/g. Mean recoveries were about 84% for liver, 85% for kidney, 89% for muscle and 84% for fat. The assay has been used for statutory testing purposes.     

Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures

The usefulness of the solvent mixture dichloromethane/methanol for lipid extraction and the determination of lipid classes and fatty acids in samples of different natures was conducted. Two different extraction methods were compared, one containing chloroform/methanol, another containing dichloromethane/methanol. Total lipid extraction showed some minor differences but no variation in the lipid classes. Regarding the fatty acid profile, in Echium virescens seeds, 17 major fatty acids could be identified and quantified, and all were equally extracted when either solvent system was employed. In Echium acanthocarpum hairy roots, 17 major fatty acids were quantified, showing some statistical differences for one cell line in favor of chloroform. The data obtained from the liquid nutrient medium were also comparable. The cod roe sample showed 31 major fatty acids, showing no statistical differences between the two solvent systems. Contrarily, the CH 2Cl 2 method was able to extract 31 main fatty acids found in European seabass dorsal muscle more efficiently than the CHCl 3 method. The results indicate that, for lipid extraction and fatty acid assessment, dichloromethane/methanol can readily replace the commonly employed chloroform/methanol, thus avoiding the major health, security, and regulatory problems associated with the use of chloroform.     

Determination of ascorbic acid and carotenoids in food commodities by liquid chromatography with mass spectrometry detection

Two methods, one to determine ascorbic acid and one to determine lycopene and beta-carotene, in vegetables and fruits by liquid chromatography coupled with mass spectrometry (LC-MS) have been established. The chromatographic separation of the studied compounds and their MS parameters were optimized to improve selectivity and sensitivity. In both methods, separation was carried out with two coupled columns, first a C(18) and then a dC(18), using as mobile phase 70% methanol (0.005% acetic acid) and 30% acetic acid 0.05% for ascorbic acid determination and a mixture of methanol, tetrahydrofuran, and acetonitrile (60:30:10 v/v/v) for carotenoid analysis in isocratic mode. The molecular ion was selected for the quantification in selective ion monitoring (SIM) mode. Ascorbic acid was detected with electrospray ionization probe (ESI) in negative mode, while chemical ionization atmospheric pressure (APCI) in positive mode was used for the target carotenoids. The methodology for ascorbic acid analysis is based on an extraction with polytron using methanol and a mixture of methaphosphoric acid and acetic acid. Extraction of the carotenoids was carried out with tetrahydrofuran/methanol (1:1) (v/v). The proposed methods were applied, after their corresponding validations, to the analysis of four varieties of tomatoes, tomato in tin enriched and dried tomato, and to the analysis of mango and kiwi fruits, to compare the content in these compounds. Moreover, the influence of the process of freezing and the effect that the manipulation/preservation has in the content of ascorbic acid in tomato have also been studied.     

HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization

Precolumn derivatization applying o-phthaldialdehyde (OPA) was used to analyze free lysine, histidine, and ornithine, precursors of the respective biogenic amines cadaverine, histamine, and putrescine, which are considered indicators of fish quality and safety. This method uses 75% methanol to eliminate the use of strong acids as the extraction solution. Each analysis took 35 min, was reproducible, and allowed separation of primary amino acids in fish samples. A binary solvent delivery system coupled with a fluorescence detector and an Ultrasphere ODS column were utilized for HPLC separation. Linearity of the calibration curves was very good (r(2) = 0.99) for the amino acids of interest. Minimum concentrations of detection were 40 pmol/mL for histidine and lysine and 70 pmol/mL for ornithine. Average recoveries were 72% for lysine, 93% for histidine, and 98% for ornithine. This method used solvent gradient elution to study the levels of these analytes in mahi-mahi, bigeye tuna, and flounder.     

Optimizing conditions for the extraction of pigments in cochineals (Dactylopius coccus Costa) using response surface methodology

A simple method was developed for the extraction and determination of color pigments in cochineals (Dactylopius coccus Costa). The procedure was based on the solvent extraction of pigments in insect samples using methanol:water (65:35, v:v) as extractant. Two-level factorial design was used in order to optimize the solvent extraction parameters: temperature, time, methanol concentration in the extractant mixture, and the number of extractions. The results suggest that the number of extractions is statistically the most significant factor. The separation and determination of the pigments was carried out by high-performance liquid chromatography with UV-visible detection. Because the absorption spectra of different pigments are different in the visible region, it is convenient to use a diode array detector to obtain chromatographic profiles that allow for the characterization of the extracted pigments.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

Determination of oxytetracycline in premixes and veterinary products by liquid chromatography

A liquid chromatographic method for the assay of oxytetracycline in premixes and veterinary products is described. Premix samples are extracted with acidified methanol, diluted with mobile phase, and filtered before chromatography on a C-8, reverse phase column. The assay method separates oxytetracycline from epioxytetracycline, tetracycline, and chlortetracycline. Total elution time for oxytetracycline is less than 5 min at 1.5 mL/min. Five spiked premix samples each of 2 and 50 g/lb had a coefficient of variation of 3.5 and 4.5% and a mean recovery of 99 and 104%, respectively. The results of premixes and veterinary products assayed by this method compared closely with those of the same assayed by the official AOAC microbiological method.     

Comparison of extraction buffers for the detection of fumonisin B(1) in corn by immunoassay and high-performance liquid chromatography

The Associatian of Official Analytical Chemists approved method for quantification of fumonisin B(1) (FB(1)) in corn meal or corn-based food products includes extraction into methanol (MeOH)/water (3:1, v/v). Disposal of the extraction medium can pose safety and environmental problems. To secure a rapid and inexpensive screen for FB(1) contamination, a sensitive competitive ELISA using a rabbit polyclonal antibody was developed. This assay was used in a comparative study measuring the extraction efficiency of FB(1) in aqueous or organic solvent buffers using 16 field corn samples. An aqueous phosphate buffer was found to be suitable for extracting FB(1), thus eliminating the need for organic solvents. HPLC and ELISA determinations compared well in fortified samples at known concentrations between 1 and 50 microg/mL of extract. Overestimation at levels >50 microg/mL were common. The characteristics and application of the ELISA for screening purposes are discussed.     

Improved liquid chromatographic determination of riboflavin in milk and dairy products

Reported here is a simple liquid chromatographic (LC) method for the determination of riboflavin in milk (liquid, evaporated, and dry), yogurt, and cheese. The method involves passing liquid samples or filtrates of semisolid and solid samples through a C18 cartridge. Retained riboflavin is then eluted with an aliquot of 50% methanol in 0.02M acetate buffer of pH 4. A volume of the eluate is injected into the LC system consisting of a C18 column, a solvent of water-methanol-acetic acid (65 + 35 + 0.1, v/v) with a flow rate of 1 mL/min, and a UV detector set at 270 nm. The method is precise and accurate and compares favorably with the present AOAC method. Moreover, it involves fewer sample preparation steps and has a total analysis time of less than 1 h.     

Simplified cleanup and liquid chromatographic determination of oxamyl residues in potato tubers

A simple and efficient method is presented for the extraction, cleanup, and liquid chromatographic (LC) determination of oxamyl residues in potato tubers. Samples are extracted with methanol, partitioned into dichloromethane, and cleaned up using Sep-Pak Florisil cartridges. LC determination is performed using a Zorbax PSM 60 size exclusion column with an acetonitrile-water (1 + 9) mobile phase and UV detection at 254 nm. Recovery of oxamyl from spiked control tubers averaged 94.1 and 85.9% at fortification levels of 0.4 and 0.08 micrograms oxamyl/g tuber, respectively. The minimum detectable concentration of oxamyl by this method is 0.01 micrograms/g.     

Solid Phase Extraction Method for Selective Determination of Phthalate Esters in the Aquatic Environment

The use of solid phase extraction and capillary GLC provides thebasis for the selective determination of phthalate ester plasticizers in rivers and marine water samples. Of the severalsolvent ratios (methanol in dichloromethane) used for selective elution of phthalate esters from the C18 solid phase glass catridge, the 50/50 ratio, CH₃OH in CH₂Cl₂ (v/v)gave the best result. The method was tested on river and marinewater samples that receive effluent from industries that use phthalate esters. The rivers and marine water samples were grossly polluted as several phthalate esters, for example, dimethyl (DMP), diethyl (DEP), dibutyl (DBP) and diethylhexyl (DEHP)were present at 0.03–2306±9.4 μg L^-1. A study on uncontaminated water was done to establish background levels.     

Rapid assay for analyzing biogenic amines in cheese: matrix solid-phase dispersion followed by liquid chromatography-electrospray-tandem mass spectrometry

A new rapid and sensitive method based on matrix solid-phase dispersion (MSPD) followed by liquid chromatography-electrospray-tandem mass spectrometry was devised for the determination of biogenic amines at trace levels in cheese samples. The method required 0.25 g of sample, CN-bonded silica as a dispersant sorbent, and a formic acid aqueous solution/methanol mixture as an eluting solvent. Extraction recoveries from soft cheese products were calculated in the 98 +/- 4-110 +/- 6% range. A procedure based on solid-phase extraction was also evaluated for the extraction of these compounds in cheese. Chromatographic separation was performed using a C18 column with an aqueous ammonium acetate/methanol mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision, and trueness. Results in the 0.05-0.25 mg kg(-1) range were obtained for the LOD of histamine, tyramine, and beta-phenylethylamine in soft cheese samples. Linearity was established over 2 orders of magnitude. Excellent precision in terms of intra-day repeatability was calculated (RSD% < 5). The applicability of the method to the determination of biogenic amines in cheese products was demonstrated.     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Extraction and characterization of water–repellent materials from Australian soils

Organic materials responsible for water–repellency in some Australian soils were extracted with an amphiphilic mixture of iso -propanol/15.7 m ammonia (7:3, v:v) in a Soxhlet apparatus, after which the water–repellent soils were rendered wettable. The successful extraction by an organic solvent system indicates that the bulk of hydrophobicity in these soils is not covalently linked to the surface of the sand. The extracted materials restored hydrophobicity on acid washed sands or ignited sands at levels comparable to the original soils.
Spectroscopic and chromatographic examination of the extracted materials indicated that both free and esterified long–chain, 16–32 carbon atom, fatty acids were present with a bimodal distribution showing maxima at C₁₆ and C₂₂. The ¹³C–NMR and infrared spectra of the most hydrophobic extract suggest that hydrophobicity is caused by molecules with extensive polymethylene chains. Calculations with model compounds indicate that at least a close packed monolayer is required before measurable hydrophobicity can be detected with the molarity of ethanol droplet penetration test.     

Preparation, isolation, and characterization of cutin monomers and oligomers from tomato peels

Cutin in tomato peels was depolymerized in methanolic base to yield cutin monomers or a mixture of cutin oligomers. These products were isolated by typical solvent extraction methods or by precipitation, and the isolates were characterized by chromatographic and spectroscopic analyses. It was determined that the compositions of the isolates from both isolation procedures were similar, although solvent extraction gave higher yields. However, the precipitation method, which is easy to carry out and avoids the use of undesirable organic solvents, may be preferable in commercial processes for recovering these compounds.