Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.     

Determination of melengestrol acetate in bovine tissue: collaborative study.

Seven laboratories collaboratively studied a method for the assay of melengestrol acetate at the 0, 10, and 20 ppb levels in bovine fat, liver, muscle, and kidney. The study included fortification of tissue by each laboratory and analysis of fat samples taken from treated heifers which had endogenous levels of 0, 10, and 20 ppb melengestrol acetate. The multistep cleanup procedure used included extraction, solvent partition, column chromatography, and electron capture gas-liquid chromatographic, determination. Results of the study for muscle, liver, kidney, and fat showed that the method gave satisfactory recoveries and accuracy. In fat, the most critical tissue, recovery was greater than 93%. A statistical comparison of the results reported for fat tissue from treated heifers demonstrated that 5 of the 7 laboratories obtained similar results. The results produced by the method can be expected to be repeatable within and among laboratories. On the basis of the collaborative results the method has been adopted as official first action.     

Residue depletion of nitrovin in chicken after oral administration

In this study, the residue depletion of nitrovin in chicken was studied after feeding the birds with dietary feeds containing 10 mg/kg of nitrovin for 7 consecutive days. Tissues (muscle, fat, kidney, and liver) and plasma were collected at different withdrawal periods and determined by a high-performance liquid chromatography-ultraviolet (HPLC-UV) method. The limit of detection for nitrovin in tissue and plasma samples was 0.1 ng/(g or mL), and the inter- and intrarecoveries from the blank fortified samples were in the range of 71.1-85.7%. At the withdrawal period of 0 days, the residue concentration of nitrovin in plasma was the highest (average of 84.98 ng/mL) compared to those in muscle, fat, liver, and kidney (average of 21.04, 61.18, 24.04, and 68.28 ng/g, respectively). At the withdrawal period of 28 days, the residue levels of nitrovin in muscle, fat, liver, and plasma were all higher than 1.0 ng/(g or mL) and the highest concentration was in liver (average of 5.8 ng/g). These data are in support of the ban of nitrovin as a feed additive in food-producing animals.     

Metabolism of 2,4-dichlorophenoxyacetic acid in laying hens and lactating goats

2,4-Dichlorophenoxyacetic acid (2,4-D) labeled with (14)C was found to be rapidly eliminated by laying hens and lactating goats dosed orally for 7 consecutive days at 18 mg/kg of food intake and for 3 consecutive days at 483 mg/kg of food intake, respectively. Excreta of hens and goats contained >90% of the total dose within 24 h after the final dose. Tissue residues were low and accounted for <0.1% of the dose in these animals. For hens, the residues in muscle, liver, and eggs (0.006-0.030 ppm) were lower than those found in fat and kidney (0.028-0.714 ppm), 2,4-D equivalents. The tissue with highest residue in goat was the kidney at 1.44 ppm, 2,4-D equivalents. Milk, liver, composite fat, and composite muscle had significantly lower residue levels of 0.202, 0.224, 0.088, and 0.037 ppm, respectively. The most abundant tissue residue was 2,4-D and acid/base releasable residues of 2,4-D. A minor metabolite was identified as 2,4-dichlorophenol.     

Residue depletion of cefquinome in swine tissues after intramuscular administration

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed for the detection of cefquinome (CEQ) residues in swine tissues. The limit of detection (LOD) of the method was 5 ng g(-1) for muscle and 10 ng g(-1) for fat, liver, and kidney. Mean recoveries of CEQ in all fortified samples at a concentration range of 20-500 ng g(-1) were 80.5-86.0% with coefficient of variation (CV) below 10.3%. Residue depletion study of CEQ in swine was conducted after five intramuscular injections at a dose of 2 mg kg(-1) of body weight with 24 h intervals. CEQ residue concentrations were detected in muscle, fat, liver, and kidney using the HPLC-UV method at 265 nm. The highest CEQ concentration was measured in kidney tissue during the study period, indicating that kidney was the target tissue for CEQ. CEQ concentrations in all examined tissues were below the accepted maximum residue limit (MRL) recommended by the Committee for Veterinary Medical Products of European Medical Evaluation Agency (EMEA) at 3 days post-treatment.     

High pressure liquid chromatographic determination of zearalenone in chicken tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.     

Comparison of peroxide value methods used for semihard cheeses

The objective was to evaluate alternatives to the peroxide value method of choice in the dairy industry, the method issued by the International Dairy Federation. Furthermore, the study evaluated the feasibility of alternative solvents for extracting lipids and subsequent peroxide value determinations. Packaged cheeses were stored under illuminated display at 4 degrees C to obtain samples with various peroxide contents but with uniform gross composition. The hydroperoxide contents were measured during 3 weeks of storage by applying two lipid extraction methods, Folch and Bureau of Dairy Industry (BDI) extractions, and three different hydroperoxide extraction solutions [chloroform/methanol (7:3, v/v), hexane/2-propanol/methanol (5:7:2, v/v/v), and methanol/decanol/hexane (3:2:1, v/v/v)], prior to standard colorimetric measurements. Extraction yields of fat from Havarti cheeses using the Folch and BDI extraction methods were approximately 109 and 61%, respectively, of the yields obtained by the International Dairy Federation gravimetric reference method. Although differences in fat extraction yields were compensated for, significantly higher peroxide values resulted from the Folch extraction method than from the BDI extraction method. The peroxide values obtained by the three methods were all in the same range, and pronounced linear correlations between peroxide contents determined using the three solutions were noted (r (2) in the range of 0.951-0.983). Peroxide value levels were not significantly different in samples stored in the dark or exposed to light.     

High-throughput method for the quantitation of total folate in whole blood using LC-MS/MS

A high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method for red blood cell (RBC) folate analysis was developed from a previously described manual (M) LC-MS/MS method. The HT LC-MS/MS method used 96-well plates in which RBC folates were hydrolyzed with concentrated HCl in the presence of the [13C6]pABA internal standard (IS). The pH of the hydrolysate was adjusted to 5.0 before cleanup using 96-well plate OASIS HLB SPE cartridges. The analyte and IS were eluted with ethyl acetate/hexane (95:5, v/v) and methylated with methanol and trimethylsilyldiazomethane. The methylated analyte and IS were quantified with LC-MS/MS as previously described. The HT LC-MS/MS method was validated by determining the recovery of six different folate vitamers, which were quantitatively recovered (84-105% with CV<9.0%). RBC folate concentrations in whole blood samples correlated between HT and M LC-MS/MS methods (r=0.922, p<0.0001 for n=43 samples) and between the HT LC-MS/MS method and a chemiluminescence assay (r=0.664, p<0.001 for n=325 samples). Comparison of the results between HT LC-MS/MS and chemiluminescence methods with Bland-Altman difference plots and by ROC curve analysis indicates that the chemiluminescence assay underreports RBC folate concentrations. The HT LC-MS/MS method allows for high-throughput sample preparation for the analysis of RBC folate.     

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry

A sensitive method has been developed for the simultaneous determination of residues of 25 β?-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS3). Nine β?-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.     

Improved microbiological assay for penicillin residues in tissues and stability of residues under cooking procedures

The microbiological assay for penicillin residues was improved by using centrifugation to remove physical barriers to diffusion, a small buffer/meat extraction ratio, and a more sensitive 2-layer assay system. Recoveries from muscle, kidney, and liver tissues ranged between 70.1 and 86.7% with measurable levels of 0.03--0.05 unit/g. By comparison, the Food and Drug-suggested methodology yielded recoveries of 45.9--54.0% and levels of detectability of 0.08--0.10 unit/g. Cooking of hamburger, steaks, and port chops indicated that procaine penicillin withstood cooking conditions, and significant levels of the original activity remained.     

Residue depletion of tilmicosin in chicken tissues

A high-performance liquid chromatography (HPLC) method with detection at 290 nm was modified and validated for the determination of tilmicosin residues in broiler chicken tissues. The limits of detection (LOD) of the method were 0.01 microg/g for muscle and 0.025 microg/g for liver and kidney. Average recoveries ranged from 80.4 to 88.3%. Relative standard deviation values ranged from 5.2 to 12.1%. Residue depletion of tilmicosin in broiler chickens was examined after dosing over a 5-day period by incorporation of the drug into drinking water at 37.5 and 75.0 mg/L. Tilmicosin concentrations in liver and kidney were highest on day 3 of medication and on day 5 in muscle, in both low- and high-dose groups. The residue levels in both groups were significantly higher in liver than in kidney or muscle. A minimum withdrawal time of 9 days was indicated for residue levels in muscle, liver, and kidney tissues below the maximum residue level (MRL).     

Liquid chromatographic determination of chloramphenicol in calf tissues: studies of stability in muscle, kidney, and liver

A liquid chromatographic method has been developed for the measurement of chloramphenicol (CAP) in muscle, liver, and kidney. The mean recovery levels were 82.6, 75.3, and 79.2% in muscle, liver, and kidney, respectively. The method was repeatable and reproducible for CAP measurement in muscle, with a detection limit of 1 microgram/kg. Investigation of CAP stability in muscle, liver, and kidney showed that CAP stability in muscle was good at -20 degrees C; for spiked liver and kidney, degradation of CAP was observed, and the use of piperonyl butoxide (PB) for metabolism inhibition was recommended for recovery and linearity studies. However, PB was unnecessary for preservation of treated animal tissues if samples were cut into cubes and cooled at -20 degrees C, just after slaughter, pending analysis. With these limitations, CAP can be measured in liver and kidney.     

Liquid chromatographic determination of sulfamoyldapsone in swine tissues

A liquid chromatographic (LC) method is described for the quantitative determination of sulfamoyldapsone (2-sulfamoyl-4,4'-diaminodiphenyl sulfone) in swine muscle, liver, kidney, and fat. Sulfamoyldapsone was extracted from tissues with acetonitrile saturated with n-hexane. The extract was washed with n-hexane saturated with acetonitrile, concentrated, and cleaned up by alumina column chromatography. Sulfamoyldapsone was separated on an ODS column by using acetonitrile-methanol-water (6 + 18 + 76) and was detected at 292 nm. Overall average recovery of sulfamoyldapsone added to tissues at levels of 0.1 and 0.5 microgram/g was 93.3% +/- 6.0. Detection limit was 0.02 microgram/g in these tissues.     

Endogenous estradiol-17beta in bovine tissues

The development of an assay for the estrogen estradiol-17beta in bovine muscle, kidney, liver, and uterine endometrium is described in this report. The tissue was homogenized, extracted, partitioned, and subjected to routine radioimmunoassay. Chromatography was not used. The first part of the report presents data on the accuracy, reproducibility, and sensitivity of the assay. The concentration of estradiol-17beta in the tissues compared with muscle was ranked as follows: uterine endometrium (10-fold higher), liver (3-fold), and kidney (3-fold). The estrogen concentration was not significantly different in the muscle between heifers, but was significantly greater (P less than 0.05) in the liver and kidney of heifers and steers. In heifers pre- and post-estrous phase endometrium (a target tissue of E2beta) contained significantly greater concentrations of estradiol-17beta than did luteal phase endometrium (P less than 0.05), but mean levels in the edible tissues were not significantly different (P greater than 0.05) between these 2 of the 4 stages of the estrous cycle.     

Development of an enzyme-linked immunosorbent assay for the detection of pentachloronitrobenzene residues in environmental samples

A competitive enzyme-linked immunosorbent assay (ELISA) for pentachloronitrobenzene (PCNB), a fungicide and chemical intermediate, was developed using a polyclonal antiserum produced against a hapten-protein conjugate of pentachlorophenoxypropionic acid-bovine serum albumin (BSA). An indirect competitive ELISA of PCNB showed an IC50 of 37 ng/mL and a limit of detection (LOD) of 7 ng/mL. The ELISA can tolerate up to 10% (v/v) methanol, 5% (v/v) acetonitrile, or 5% (v/v) acetone without significant fluctuation of Amax and IC50. The assay sensitivity showed little change in a range of pH from 6 to 8 and concentrations of 0.05-0.2 M NaCl in the assay buffer. Very low cross-reactivities were observed for some structurally related compounds except for hexachlorobenzene (12%). The average recoveries of PCNB from fortified well water, river water, and soil samples were in ranges of 88-94, 80-91, and 70-81%, respectively. The correlations between the gas chromatographic and ELISA results were excellent (r 2 >or= 0.97, slopes from 0.86 to 1.10) for those fortified samples. The ELISA is a good alternative tool for monitoring PCNB residues in environmental samples.     

Extraction methods for quantitation of gentamicin residues from tissues using fluorescence polarization immunoassay

Sodium hydroxide digestion of unhomogenized kidney and skeletal muscle for 20 min at 70 degrees C was a superior method for extracting gentamicin from tissues, compared with simple homogenization, trichloroacetic acid precipitation of homogenized tissue, and sodium hydroxide digestion of homogenized tissue. Fluorescence polarization immunoassay was used to quantitate gentamicin. Sodium hydroxide digestion of unhomogenized tissue allowed for the recovery of 90.0 +/- 5.9% (means +/- SD) from renal cortex and 79.9 +/- 3.5% from skeletal muscle. The limit of sensitivity was 17.4 ng/g kidney tissue, 15.8 ng/g digested muscle, and 39.0 ng/g digested heart. The within-assay coefficient of variation (CV) at 100 ng/g kidney was 9.2%; at 500 ng/g kidney, the CV was 2.5%; and at 2000 ng/g kidney, the CV was 1.5%. The between-assay coefficient of variation was less than 7.5% for all concentrations from kidney, and the 99% confidence interval at 100 ng/g kidney was 71.7-112.4 ng gentamicin/g kidney. The within-assay coefficient of variation (CV) at 100 ng/g muscle was 15%; at 500 ng/g muscle, the CV was 2.6%; and at 2000 ng/g muscle, the CV was 2.3%. The between-assay coefficient of variation was less than 15% for all concentrations from muscle, and the 99% confidence interval at 100 ng/g muscle was 72.5-136.8 ng gentamicin/g muscle. Gentamicin-free milk could be distinguished from milk containing gentamicin concentrations of 10 ng/mL milk with 95% confidence, and from milk containing concentrations of 30 ng gentamicin/mL milk with 99% confidence. Quantitative results at or below the tolerance level can be obtained within 90 min of sample acquisition using these extraction and assay methods.     

Total lipid extraction of homogenized and intact lean fish muscles using pressurized fluid extraction and batch extraction techniques

The reliability and efficiency of pressurized fluid extraction (PFE) technique for the extraction of total lipid content from cod and the effect of sample treatment on the extraction efficiency have been evaluated. The results were compared with two liquid-liquid extraction methods, traditional and modified methods according to Jensen. Optimum conditions were found to be with 2-propanol/n-hexane (65:35, v/v) as a first and n-hexane/diethyl ether (90:10, v/v) as a second solvent, 115 degrees C, and 10 min of static time. PFE extracts were cleaned up using the same procedure as in the methods according to Jensen. When total lipid yields obtained from homogenized cod muscle using PFE were compared yields obtained with original and modified Jensen methods, PFE gave significantly higher yields, approximately 10% higher (t test, P < 0.05). Infrared and NMR spectroscopy suggested that the additional material that inflates the gravimetric results is rather homogeneous and is primarily consists of phospholipid with headgroups of inositidic and/or glycosidic nature. The comparative study demonstrated that PFE is an alternative suitable technique to extract total lipid content from homogenized cod (lean fish) and herring (fat fish) muscle showing a precision comparable to that obtained with the traditional and modified Jensen methods. Despite the necessary cleanup step, PFE showed important advantages in the solvent consumption was cut by approximately 50% and automated extraction was possible.     

Determination of halogenated contaminants in human adipose tissue

A method has been developed for determination of organochlorine contaminants in human adipose tissue. After fat extraction from the tissue with acetone-hexane (15 + 85, v/v), organochlorines were fractionated from fat by gel permeation chromatography with methylene chloride-cyclohexane (1 + 1, v/v) as solvent. After Florisil column cleanup, the GPC extract was analyzed by capillary column gas chromatography using 2 columns of different polarity. Compound identity was confirmed by gas chromatography-mass spectrometry using selected ion monitoring. Recoveries for fortification levels of 10-500 ng/g were greater than 80% except for trichlorobenzene and hexachlorobutadiene (ca 60%).     

Nature of the residue of [(14)C]Cloransulam-methyl in lactating goats

Two lactating goats were given a daily oral dose of either [UL-aniline-(14)C; AN] or [triazolopyrimidine-7,9-(14)C; TP]cloransulam-methyl for 5 consecutive days. Each animal received a dietary equivalent of approximately 10 mg/kg of test material, approximately 2225 times the realistic maximum dietary exposure for a dairy animal. Milk, urine, and feces samples were collected in the morning and afternoon for each animal. Each goat was sacrificed within 23 h of receiving the last dose, and the liver, kidneys, samples of blood, fat, muscle, and gastrointestinal tract contents, and urine from the bladder were collected. All of these samples were analyzed for (14)C content. Cloransulam-methyl (CM) was rapidly excreted by the animals, with 99.9% of the recovered radioactivity appearing in the urine and feces. Radiochemical analysis showed very low residues, with the highest being in the kidneys at 0.122 and 0. 128 mg equiv of CM/kg (AN and TP labeled compounds, respectively). Radioactive residues were extracted and fractionated from kidney, liver, and milk. Analysis showed approximately 0.066 mg/kg CM in the kidney but <0.003 mg/kg in the liver. Only one metabolite, cloransulam, was identified (in liver, 9.5% of total radioactive residue; 0.005 mg/kg). All other metabolites were present at lower levels. Sulfonanilide bridge cleavage was not a significant degradation route for cloransulam-methyl in ruminants. These data indicated a very low bioaccumulation potential for cloransulam-methyl and its metabolites in ruminants. For a ruminant exposed to anticipated levels of cloransulam-methyl in its diet, parent and metabolites, in total, would not be expected to exceed 50 ng/kg in the kidney and liver.     

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.