Column liquid chromatographic determination of carbadox and olaquindox in feeds.

A column liquid chromatographic method for simultaneous determination of carbadox and olaquindox in swine feeds is described. The drugs were extracted from feeds with carbon tetrachloride-dimethylformamide (80:20) at 60 degrees C for 30 min. The extract was mixed with water (25:45). After centrifugation the aqueous layer was chromatographed on a reversed-phase column using gradient elution and ultraviolet detection at wavelengths of 305 and 262 nm. Recoveries from samples fortified at levels of 20-50 ppm were 92 +/- 9% for carbadox and 93 +/- 6% for olaquindox (means +/- standard deviations, n = 71).     

Liquid chromatographic determination of pyrantel tartrate in medicated formulations

The validation of a novel liquid chromatographic (LC) method for the determination of pyrantel tartrate in feed is presented. The method provides a significant improvement over the efficiency and precision of AOAC Official Method 978.30. The method was shown to be accurate, precise, linear, and robust for medicated articles. Unlike the official method, the LC method was shown to be a superior stability-indicating method. After the method was validated by using laboratory blends, the effectiveness of the method was demonstrated with marketed product as well.     

Determination of decoquinate in animal feeds by liquid chromatography: collaborative study

The performance characteristics of a liquid chromatographic (LC) method for the analysis of decoquinate (DEC) in supplements, premixes, and complete animal feeds at medicating and trace levels were collaboratively studied. DEC is extracted from ground feed samples with 1% calcium chloride-methanol solution using mechanical agitation for 90 min. After centrifugation for 5 min and dilution (if necessary), an aliquot of the extract is diluted with water. The diluted extracts are filtered and analyzed by reversed-phase LC with fluorescence detection. Suspect positive trace-level samples are confirmed by using an alternate excitation wavelength. Fourteen test samples of medicated feeds, supplement, and medicated premix, along with 8 test samples for trace-level analysis, were sent to 13 collaborators (one in Canada, 4 in Europe, and 8 in the United States). Test samples were analyzed as blind duplicates. Acceptable results were received from 12 laboratories for the medicated test samples and from 13 laboratories for the trace-level samples. Repeatability relative standard deviation estimates ranged from 1.3 to 5.6%. Reproducibility relative standard deviations estimates ranged from 2.8 to 6.1%, and HorRat values ranged from 0.22 to 0.74.     

High-performance liquid chromatographic determination of oxantel and pyrantel pamoate

A method is described for the determination of oxantel and pyrantel pamoate in proprietary broad spectrum anthelmintic tablets. Quantitation is performed by using an octyl Spherisorb column with a mobile phase consisting of acetonitrile and water modified with butylamine. Carbaryl-(1-naphthyl methylcarbamate) is used as internal standard.     

Reversed-phase liquid chromatographic determination of riboflavin in feeds

A method for determination of riboflavin in animal feeds using liquid chromatography (LC) was developed for feed samples fortified with riboflavin at 1 mg/lb or greater (up to 10,000 mg/lb). Feed samples were extracted in 0.1 N HCl with heating on a steam bath for 30 min, followed immediately by mechanical shaking for 30 min. Sample extracts were diluted to target volume with 2% acetic acid and filtered; riboflavin was determined by LC on a reversed-phase C18 column with 2% acetic acid-acetonitrile (85 + 15) mobile phase for separation and fluorescence detection with excitation at 460 nm and emission at 530 nm. The extraction was compared with that of the AOAC Official Method for riboflavin in food and feed premixes. The 2 method extractions were not significantly different from each other at the 95% confidence level. The developed method also had good linearity over 4 orders of magnitude, recovery of 95-99% from spiked feed samples, a limit of detection of riboflavin at 0.00034 microg/mL in solution, a limit of quantitation of 0.023 mg/lb in feed, and good ruggedness.     

Determination of lasalocid sodium in animal feeds and premixes by reversed-phase liquid chromatography: collaborative study

A liquid chromatographic (LC) method for the analysis of lasalocid sodium in premixes, complete animal feeds, and trace-level feeds was collaboratively studied. The method employs a 0.5% HCI acidified methanol extraction followed by 20 min sonication in a water bath heated to 40 degrees C. Samples are then shaken on a mechanical shaker for 1 h and stored overnight, followed by an additional 10 min shaking the following morning. Sample extracts are diluted if necessary with extractant, filtered, and injected onto an LC system. Determination of all lasalocid homologs is by reversed-phase LC with fluorescence detection at 314 nm excitation and 418 nm emission. Eight samples of drug premixes, medicated feeds, and mineral supplements, along with 2 samples for trace-level analysis were sent to 20 collaborators in the United States, Canada, and The Netherlands. Study data were returned by 17 laboratories. Two additional supplemental trace-level samples and a blank feed were provided to 15 of the collaborating laboratories, and test data were received from all 15 participants. For the drug premixes, medicated feeds, and mineral supplements, RSDr values (within-laboratory repeatability) ranged from 1.2 to 19.9%, RSDR values (among-laboratory reproducibility) ranged from 3.4 to 32.3%, and HorRat values ranged from 0.35 to 3.73. For the trace-level samples, only lasalocid A, the predominant homolog comprising > 90% of the sum of all homolog peak area, was quantified. All laboratories correctly identified the analyte. Although some instrument response was reported by a number of laboratories for the blank feed, all but one laboratory's results were well below the 1 mg/kg limit of quantification. RSDr values for the initial 2 trace-level samples were excessive, ranging from 51.6 to 64.4%. RSDR values ranged from 51.6 to 75.7%, and HorRat values ranged from 3.6 to 4.0. Data for the initial trace-level samples indicated that the test samples were improperly prepared to ensure homogeneity, and a new set of supplemental samples was provided to collaborators, with significantly improved results. RSDr values for the 2 supplemental trace-level samples ranged from 1.6 to 2.5%, RSDR values ranged from 5.6 to 9.2%, and HorRat values ranged from 0.43 to 0.62.     

Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination

A commercially available system is described for the fully automated clean-up and high-performance liquid chromatographic (HPLC) analysis of aflatoxins in foods and animal feeds. The system marketed primarily for handling solid-phase extraction columns has modified software to facilitate use with immunoaffinity columns. Sample extract clean-up followed by injection onto an HPLC column with post-column iodination and fluorescence detection is carried out completely unattended. A coefficient of variation of 5.1% for aflatoxin B1 analysis was obtained, and the accuracy of the system was demonstrated by the analysis of peanut butter certified reference material.     

Liquid chromatographic determination of nicarbazin in feeds

A new liquid chromatographic method has been developed for determination of nicarbazin in feeds. Approximately 40 g feed is extracted with 200 mL acetonitrile-water (80 + 20, v/v). An aliquot of the extract is filtered and assayed using a reversed-phase isocratic method that measures the 4,4'-dinitrocarbanilide moiety of nicarbazin at a wavelength of 340 nm. For medicated feeds, the method uses a standard linear range of 5 to 100 microg/mL. For lower levels, a linear range of 50 to 150 ng/mL can be used. The method has a limit of detection of 250 ng/g and a limit of quantitation of 500 ng/g in a 40 g feed sample. Recovery was 99.1%, with a range of 95.2 to 101.8%. In the typical U.S. dosing range of 27 to 113.5 g/ton, the precision of the method based on one analyst, one day, and 2 weighings ranged from 2.8% (113.5 g/ton) to 4.7% (27 g/ton).     

A sensitive high-pressure liquid chromatographic method for the determination of propranolol in microliter serum samples

Summary A high-pressure liquid chromatographic assay capable of quantitating subnanogram per milliliter concentrations of propranolol in microliter volume of serum was developed. Propranolol was extracted from serum with hexane at pH 9.7. Further sample clean up was achieved by back extraction. Separation of propranolol from its metabolites and normal serum constituents was performed on 10 m C₁₈ reversed-phase column packing. High sensitivity was attained with fluorescence detection using a low UV excitation wavelength. This assay is suitable for use in pharmacokinetic studies involving small laboratory animals.     

Liquid chromatographic determination of maleic hydrazide in technical and formulated products: collaborative study

Fourteen collaborating laboratories assayed maleic hydrazide (MH), 6-hydroxypyridazin-3(2H)-one, in technical and formulated products by reversed-phase liquid chromatography (LC) with sulfanilic acid as an internal standard. The active MH in the samples (6 lots) ranged from 16% (expressed as the potassium salt) to 98% (MH in the technical). A small amount of 1 M KOH was added to the technical MH and analytical standards to create the potassium salt of the analyte which is soluble in water. Test samples and standards were extracted with water containing the internal standard before analysis by LC on a C8 column with an ion-pairing eluting solution and UV detection at 254 nm. The concentration of MH was calculated by comparing the peak area response ratios of the analyte and the internal standard with those in the analytical standard solution. Eleven laboratories weighed each test sample twice with single analysis. Three laboratories weighed each sample once and made duplicate injections on the LC system. The data were analyzed using the 11 laboratories' results. A second data analysis was done including all laboratory results using a Youden pair approach, selecting one of 2 duplicate assay values randomly for each laboratory and sample. In the first data analysis, the repeatability standard deviation ranged from 0.07 to 1.39%; reproducibility standard deviation ranged from 0.22 to 1.39%. In the second data analysis (using all laboratory data), repeatability standard deviation ranged from 0.09 to 0.86%; reproducibility standard deviation ranged from 0.22 to 1.31%.     

Gas chromatographic determination of bifenthrin in technical and selected formulated products: collaborative study

A GC method for the analysis of technical and formulated bifenthrin samples was evaluated in a collaborative study. Bifenthrin is determined by using a 50% (trifluoropropyl)-methylpolysiloxane wide-bore capillary column and flame ionization detector. Ten samples, consisting of four formulations and a technical material were analyzed by 12 collaborators using Youden pairs. The four formulation types included in this study were microemulsion (ME), wettable powder (WP), suspension concentrate (SC), and emulsifiable concentrate (EC). Variability in the analysis of two of the formulation types, SC and EC, was later found to be due to the noncommercial containers used to hold the test samples. Because of this, valid data could not be obtained for the EC and SC. For the two formulations for which valid data could be obtained, ME and WP, and the technical chemical, accuracy and variability results are typical of large data sets. For the technical chemical and the two formulations for which valid data were obtained, Official First Action is recommended.     

Determination of monensin, narasin, and salinomycin in mineral premixes, supplements, and animal feeds by liquid chromatography and post-column derivatization: collaborative study

A liquid chromatographic (LC) method for the analysis of monensin, narasin, and salinomycin in mineral premixes, supplements, and complete animal feeds at medicating and trace levels was collaboratively studied. The method uses methanol-water (90 + 10) extraction with mechanical shaking for 1 h, filtration, and dilution if necessary. Determination of the 3 ionophores is by reversed-phase LC using post-column derivatization with vanillin and detection at 520 nm. Suspect positive trace-level products and medicated feeds containing unexpected ionophores are confirmed by hexane extraction or post-column derivatization with dimethylaminobenzaldehyde (DMAB). Twenty-five test samples of medicated feeds, supplements, and mineral and drug premixes, and 9 test samples for trace-level analysis were sent to 11 collaborators in Bulgaria, Czech Republic, Portugal, France, The Netherlands, United States, and Canada. Acceptable results were received from 10 laboratories. For the medicated complete feeds, supplements, and mineral premixes, RSDr values (within-laboratory repeatability) ranged from 2.5 to 5.2%, RSDR values (among-laboratory reproducibility) ranged from 2.7 to 6.8%, and HorRat values ranged from 0.31 to 1.30. For the drug premixes, the result variability was excessive and HorRat values ranged from 2.27 to 14.1. For the trace-level test samples, all laboratories correctly identified the analytes and did not report any false positives. RSDr values ranged from 1.3 to 9.5%, RSDR values ranged from 5.2 to 13.1%, and HorRat values ranged from 0.4 to 0.97.     

Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley: collaborative study

A collaborative study was conducted to evaluate a liquid chromatographic (LC) method with immunoaffinity column cleanup for determination of ochratoxin A. The method was tested at 3 concentration levels of ochratoxin A in barley, which represent possible future European regulatory limits. The test portion was extracted with acetonitrile-water by blending at high speed. The extract was filtered, diluted with phosphate-buffered saline (PBS), and applied to an ochratoxin A immunoaffinity column. The column was washed with water and the ochratoxin A eluted with methanol. The solvent was then evaporated and the residue redissolved in injection solvent. After injection of this solution onto reversed-phase LC column, ochratoxin A was measured by fluorescence detection. Eight samples of low level naturally contaminated barley and 2 samples of blank barley (ochratoxin A not found at the limit of detection of 0.2 microg/kg at the signal-to-noise ratio of 3 to 1) were sent, along with ampules of ochratoxin A, calibrant, and spiking solutions, to 15 laboratories in 13 different European countries. Test portions were spiked with ochratoxin A at levels of 4 ng/g, and recoveries ranged from 65 to 113%. Based on results for spiked samples (blind duplicates) and naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 4 to 24%, and the relative standard deviation for reproducibility (RSDR) ranged from 12 to 33%. The method showed acceptable within- and between-laboratory precision, as evidenced by HORRAT values, at the low level of determination for ochratoxin A in barley.     

Liquid chromatographic determination of residual nitrite/nitrate in foods: NMKL collaborative study

Nitrite and nitrate are used as additives in the food industry to provide color and taste and to control undesirable gas and flavor production by anaerobic bacteria by virtue of their antimicrobial properties. The analytical method that has been widely used to determine nitrite and nitrate involves the use of toxic cadmium. In response to a request from the Nordic Committee on Food Analysis, a study was performed to obtain an alternative chromatographic method to determine residual nitrite and nitrate in meat products. The study was done in 3 stages: (1) comparative evaluation of the performance of 3 liquid chromatographic methods, (2) internal validation of the selected ion chromatographic method, and (3) a collaborative study in which 17 laboratories from European countries participated. Furthermore, the applicability of the method to matrixes other than meat and meat products was demonstrated. The results of the collaborative study show that the European Prestandard prENV 12014-4 is well suited for the determination of nitrite and nitrate in different foods (e.g., meat products, vegetables, baby food, and cheese). The limits of detection for nitrite and nitrate ions are 1 and 10 mg/kg, respectively. Recoveries of residual nitrite/nitrate ranged from 96 to 108%. Repeatability and reproducibility were satisfactory.     

Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.     

Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up.

The analysis of the fungal mycotoxin ochratoxin A in cereals and animal products is described using an immunoaffinity column clean-up and high-performance liquid chromatographic determination. The clean-up can be carried out manually or using a commercially available automated sample preparation system. The method has been applied to cereals such as wheat, rye and barley, unprocessed breakfast cereals and animal products such as pigs' kidneys and blood sausages. Recoveries ranged from 70-80% for spiked samples (10 micrograms/kg) and the method had a relative standard deviation of 1.3% (n = 8) for the analysis of a wheat sample naturally contaminated at 13.7 micrograms/kg ochratoxin A and relative standard deviation of 3.0% (n = 8) for a pig kidney sample spiked at 10 micrograms/kg ochratoxin A. The immunoaffinity approach was significantly faster than methods employing conventional chromatographic clean-up, and extracts were freer of co-extractives giving a limit of detection of 0.2 micrograms/kg.     

Liquid chromatographic determination of deoxynivalenol in baby food and animal feed: interlaboratory study

An interlaboratory study was conducted for the determination of deoxynivalenol in baby food and animal feed by high-performance liquid chromatography with UV detection. The study included 14 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 89% (at 120 microg/kg) to 85% (at 240 microg/kg) for baby food and from 100% (at 200 microg/kg) to 93% (at 400 microg/kg) for animal feed. On the basis of the results for spiked samples (blind duplicates at 2 levels), as well as those for naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in analyses of baby food ranged from 6.4 to 14.0% and in analyses of animal feed, from 6.1 to 16.5%. The relative standard deviation for reproducibility (RSDR) in analyses of baby food ranged from 9.4 to 19.5% and in analyses of animal feed, from 10.5 to 25.2%. The HorRat values ranged from 0.4 to 1.0 and from 0.7 to 1.3, for baby food and animal feed, respectively. The method showed acceptable performance for within-laboratory and between-laboratory precision for each matrix, as required by European legislation.     

Development and validation of a liquid chromatographic/ tandem mass spectrometric method for determination of chlortetracycline, oxytetracycline, tetracycline, and doxycycline in animal feeds

A selective and accurate LC/MS/MS method for the simultaneous determination of chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), and doxycycline (DC) in animal feeds was developed. Samples were extracted with Na2EDTA-McIlvaine buffer and further purified with Oasis HLB SPE columns. The purified extract was separated on an Xbridge C18 column and detected by LC/MS/MS with positive electrospray ionization in the multiple reaction monitoring mode. This method provided average recoveries of 80.9 to 119.5%, with CVs of 1.7 to 9.8% in the range of 0.5 to 50 mg/kg CTC, OTC, TC, and DC in feeds, except the average recovery of CTC was 76.0%, with a CV of 14.6% in pig feed spiked with 0.5 mg/kg CTC. The linear ranges for the four TCs determined by LC/MS/MS ranged from 0.005 to 2.5 microg/mL with a linear correlation coefficient (R2) >0.99. The LOD and LOQ for CTC, OTC, TC, and DC in pig and poultry feeds ranged from 0.003 to 0.02 and 0.01 to 0.05 microg/g, respectively. The method was successfully applied for the analysis of 30 real feed samples, and no illegal use was detected.