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.     

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.     

Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and alpha-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with beta-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartridge which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wave-length 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma and urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 micrograms/kg body weight. Zearalenone was rapidly metabolized to alpha-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and alpha-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.     

Optimum conditions for formation of aflatoxin M1-trifluoroacetic acid derivative

Because thin-layer chromatographic (TLC) confirmation of identity and reverse-phase liquid chromatographic (LC) determination with fluorescence detection of aflatoxin M1 both require the derivative formed in the reaction of M1 and trifluoroacetic acid (TFA), various reaction conditions were studied to obtain complete derivative formation. Of the various organic solvents tested, the reaction between M1 and TFA proceeded best in the nonpolar solvents hexane and isooctane. Other parameters investigated were reaction temperature and time, aflatoxin M1 concentration, and solvent volume. The following procedure is considered optimum: 200 microL each of hexane and trifluoroacetic acid are mixed with M1 standard in a silylated glass vial or with milk residue in a regular glass vial with a Teflon-lined screw cap and heated 10 min at 40 degrees C. The mixture is evaporated to dryness under N2, and the derivative is saved for TLC or LC. No unreacted aflatoxin M1 was detected by reverse-phase LC after this procedure was incorporated for analysis of milk samples.     

Liquid chromatographic determination of cephapirin residues in milk

A liquid chromatographic (LC) method was developed for quantitative determination of cephapirin residues in milk that also resolved cephapirin from ampicillin, cloxacillin, and penicillin G. Diluted milk was passed through a C18 cartridge on which the cephapirin was adsorbed; then, interfering material was removed by washing with water and methylene chloride and cephapirin residues were eluted with methanol-acetonitrile (25 + 75). After drying, residues were dissolved in the mobile phase for injection. The LC system had an ultrasphere-ODS column with RP-18 Spheri-10 guard column and a UV detector with a 254 nm filter. The mobile phase was 85% sodium acetate (0.01M) and 15% methanol-acetonitrile (25 + 75) with a flow rate of 1 mL/min. Sensitivity was 20 ppb or less with a recovery of 61-80% in the range studied. Other beta-lactam antibiotics tested did not interfere with detection of cephapirin. Analysis of 30 samples of commercial homogenized milk obtained for a survey of antibiotics in consumer milk in Canada revealed no detectable cephapirin residues.     

Chromatographic methods for determination of macrolide antibiotic residues in tissues and milk of food-producing animals

Tylosin, an antibiotic developed specifically for agricultural use, and erythromycin are the main macrolide antibiotics used in animal production. Two-dimensional thin layer chromatography has been used for detection of tylosin in poultry meat, eggs, and milk and for erythromycin in poultry meat. Detection limits reported are, for tylosin, 0.1 ppm in poultry meat, 0.05 ppm in egg, and 0.01 ppm in milk, and for erythromycin, 0.25 ppm in poultry meat. Liquid chromatography (LC) has also been used for determination of tylosin in milk, blood, and tissues of animals. Samples (milk, blood serum, or tissue homogenates in water or pH 2.2 buffer) were deproteinized with acetonitrile, tylosin was partitioned into methylene chloride, and the extracts were concentrated and dissolved in acetonitrile. Chromatography was done on a reverse phase end-capped C18 column using 0.002-0.005 M ammonium dihydrogen phosphate-acetonitrile-methanol (10 + 60 + 30-5 + 80 + 15). Solvent composition was varied with the type of sample analyzed. The method will detect 0.1 ppm tylosin in tissues and less in milk and blood serum. The LC method was more sensitive than microbiological assays for detection of tylosin in tissues of treated swine; recoveries of tylosin by the LC method were frequently several-fold higher.     

高寒地区燕麦 (Avena sativa L.) 人工草地生物量分配对施肥和混播措施的响应

【目的】了解高寒地区燕麦人工草地生物量分配格局对燕麦品种、施肥措施和混播水平的响应机制,为高寒地区人工草地建植提供依据。 【方法】采用“三因素四水平”正交试验设计,包括 4 个燕麦品种,分别为青燕 1 号、林纳、青海 444、青海甜燕麦;4 种施肥措施,分别为不施任何肥料 (CK₀)、尿素 75 kg/hm2 + 磷酸二铵 150 kg/hm2 (NP)、有机肥 1500 kg/hm2 (OM) 和尿素 37.5 kg/hm2 + 磷酸二铵 75 kg/hm2 + 有机肥 750 kg/hm2 (NP + OM);4 个箭筈豌豆混播水平,分别为 0、45、60、75 kg/hm2,在燕麦拔节期、抽穗期、开花期和乳熟期分别对各处理燕麦和箭筈豌豆各器官生物量进行了测定。 【结果】燕麦人工草地地上生物量以青燕 1 号 (85.57%)、尿素 + 磷酸二铵 + 有机肥处理 (84.91%) 和 0 kg/hm2 混播水平 (85.81%) 时分配比例最高;地下生物量以林纳 (18.04%)、不施肥 (16.02%) 和 30 kg/hm2 混播水平 (16.29%) 时分配比例最高。茎、叶、穗和根生物量分配比例分别为 44%～48%、19%～24%、17%～22% 和 14%～18%。茎、叶、穗和地下生物量分配随季节分别呈增加、先降后增、增加和下降的变化趋势;在各因素影响下,燕麦生长前期以叶生长为主,中、后期以茎生长为主,而箭筈豌豆在生长前期以根生长为主,中、后期以叶生长为主。燕麦生物量分配在整个生育期处于优势地位,地上生物量分配始终高于地下,表现为异速生长关系。茎和地下生物量分配在生长前期和后期分别受施肥措施和品种的影响;叶生物量分配在整个生育期受混播水平的影响;燕麦穗生物量分配主要受品种和混播水平的影响。 【结论】品种、施肥措施和箭筈豌豆混播水平均对高寒地区燕麦人工草地生物量分配具有显著影响,选用青燕 1 号品种,单播,配施尿素 37.5 kg/hm2、磷酸二铵 75 kg/hm2、有机肥 750 kg/hm2 时地上生物量分配最高;选用林纳,并混播 30 kg/hm2 的箭筈豌豆,采用不施肥措施时地下生物量分配最高。     

Analysis of free and esterified ergosterol in tomato products

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.     

Liquid chromatographic determination of aflatoxin M1 in milk

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.     

Liquid chromatographic determination of desfuroylceftiofur metabolite of ceftiofur as residue in cattle plasma

A liquid chromatographic (LC) method has been developed for the determination of the desfuroylceftiofur metabolite of ceftiofur as a residue in the plasma of animals. Plasma sample in 0.1M pH 8.7 phosphate buffer containing dithioerythritol is incubated under nitrogen for 15 min at 50 degrees C. The sample is centrifuged, charged to a C18 cartridge, and washed with 0.1M ammonium acetate. The desfuroylceftiofur residue on the cartridge is derivatized by adding 0.1M ammonium acetate containing iodoacetamide and letting the cartridge stand in the dark for 30 min. The cartridge is then drained and rinsed, and the desfuroylceftiofur acetamide is eluted with methanol. The mixture is evaporated to dryness, dissolved in pH 10.6 sodium hydroxide, and charged to a SAX cartridge. The derivative is eluted with 2% acetic acid, reduced in volume, and dissolved in mobile phase for liquid chromatography. The LC system includes a C8 column and guard cartridge with UV detection at 254 nm. The gradient mobile phase (flow rate 1 mL/min) is 0.01M pH 5 ammonium acetate programmed to 29% methanol-water (60 + 40) in 25 min. Recoveries were 90-100% with a sensitivity of 0.1 ppm or less. The procedure has been applied to the plasma of cattle, rats, horses, pigs, and dogs.     

Effects of injection solvent and mobile phase on efficiency in reverse-phase liquid chromatographic determination of aflatoxin M1

The effects of injection solvent and mobile phase composition on the reverse-phase liquid chromatographic determination of aflatoxin M1 (M1) were examined. M1 was converted to the more highly fluorescent derivative aflatoxin M2a (M2a). Using a C-18 column and a mobile phase of H2O-MeCN-MeOH (60 + 20 + 20) (MP-A), M2a was dissolved in various ratios of MeCN-H2O prior to injection. Chromatographic efficiency for the M2a peak varied from ca 2000 theoretical plates when injected in 30% aqueous MeCN to ca 9000 plates when injected in water alone. However, using the same C-18 column but with a mobile phase of H2O-IPA-MeCN (80 + 12 + 8) (MP-B), the M2a peak exhibited 25,000 plates when injected in 30% aqueous MeCN and 10,000 plates when injected in water alone.     

Liquid chromatographic determination of vitamins D2 and D3 in fortified milk and infant formulas

A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of tetracycline antibiotics in beef and pork tissues using ion-paired liquid chromatography

A simplified procedure was developed for determination of tetracycline antibiotics in tissues which improved stability of these compounds in sample extracts and eliminated the need for troublesome cleanup procedures. Tissues were homogenized in water. Acetonitrile (16 mL) and then 1 mL of 0.1 M H(3)PO(4) were added to 4 mL of homogenate and the clear supernatant was filtered. The filtrate was mixed with hexane and dichloromethane and the resulting water layer was collected, evaporated to 1-2 mL, and filtered into autosampler vials. Ion-pairing liquid chromatography was used to separate tetracyclines from interferences in sample extracts, eliminating the need for further cleanup. Analysis was isocratic using a Phenomonex Prodigy ODS(3) column with a mobile phase of 4 mM oxalic acid, 4 mM sodium oxalate, 10 mM sodium decanesulfonate-acetonitrile (70 + 30 for oxytetracycline and tetracycline; 66 + 34 for chlortetracycline). Recoveries were generally in the 90-100% range with limits of quantitation of 0. 05-0.1 ppm. The procedure was evaluated with beef and pork muscle, liver, and kidney.     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

Characterization of major anthocyanins and the color of red-fleshed Budd Blood orange (Citrus sinensis)

High-performance liquid chromatography (HPLC) with photodiode array detection was applied for the characterization of anthocyanins in red-fleshed Budd Blood (Citrus sinensis) orange. More than seven anthocyanin pigments were separated within 30 min by using a binary gradient (0.1% H(3)P0(4) in water and 0.1% H(3)PO(4) in acetonitrile) elution on a Prodigy ODS column. Separations by reversed-phase HPLC and semipreparative HPLC on a Prodigy 10-microm ODS Prep column, and acid and alkali hydrolyses were used for identification of anthocyanins. The primary anthocyanins in Budd Blood orange grown in Florida were cyanidin-3-(6"-malonylglucoside) (44.8%) followed by cyanidin-3-glucoside (33.6%). Two other minor pigments were also acylated with malonic acid. Malonated anthocyanins represented the major proportion (>51%) of anthocyanins in Budd Blood orange. Total anthocyanin contents and juice color parameters (CIE L,a,b) were compared with six other Florida-grown blood oranges.     

Thin layer chromatographic confirmation of aflatoxin M1 extracted from milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on M1 after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110 degrees C for 10 min and then developed in hexane-acetone-chloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p-Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.     

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.     

Liquid chromatographic determination of oxfendazole in swine feeds

A relatively simple analytical method is presented for determination of oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinyl-benzimidazole) at levels as low as 0.012% in swine feeds, using cation exchange liquid chromatography (LC). The sample was extracted with a solvent mixture of methanol-glacial acetic acid (90 + 10) at 45 degrees C, using a gyrorotory shaker. Plant pigments and other feed excipients were removed using zinc acetate treatment and pH-controlled extraction. Oxfendazole was further separated from the remaining interferences and quantitatively determined by LC on a Partisil SCX column with acetonitrile-0.01M phosphate buffer as mobile phase. The method is stability-specific, linear, precise, and accurate at 80-120% labeled strength (relative standard deviation 0.9-1.7 with mean recovery of 98-99%). Supporting data at a level of 0.0135% oxfendazole in swine feed indicated that this method is capable of complete recovery of oxfendazole from medicated swine feeds.     

Liquid chromatographic method for analysis of elemental sulfur in pesticide formulations

A nonaqueous reverse-phase liquid chromatographic (LC) method has been developed to determine elemental sulfur in pesticide formulations. Samples were extracted in 50 mL of stabilized tetrahydrofuran (THF) by gentle swirling while sonicating for 1 min. A 5 microL aliquot was injected into the LC instrument equipped with a Vydac 218 TP 54 column. The mobile phase was methanol-acetonitrile-stabilized tetrahydrofuran (58.5 + 40 + 1.5). Sulfur was monitored at 280 nm. Retention time was approximately 5 min with total analysis time of 7 min. For 6 different products analyzed 12 times each, the coefficients of variations were all less than 3.5%. Purity of each sulfur peak was checked by using a photodiode array detector in the spectrum and absorbance ratio modes. No impurities were observed at the monitoring wavelength.     

Liquid chromatographic determination of protein acid hydrolysate content in blended soy sauce

Five methods were investigated for the determination of levulinic acid in soy sauce to determine the addition of protein hydrolysate, mainly acid hydrolysate of defatted soybeans. Best results were obtained by using liquid chromatography (LC) with 0.004 M HClO4 as the mobile phase and bromcresol purple as a post-column reagent. An innovative LC method with 0.1% H3PO4 as eluant was developed for determination of levulinic acid at 280 nm in soy sauce. This was the most time-saving method.