Determination of depletion and statistical distribution of morantel-related residues in bovine milk following administration of morantel tartrate to dairy cows

Residue depletion studies were conducted in dairy cattle to monitor morantel-related residues in milk following oral administration of morantel tartrate (Rumate. Eleven lactating cows of various ages, periods of lactation, and known milk production were orally dosed with the bolus formulation of morantel tartrate with an actual dose range of 8.4-9.8 mg/kg body weight. Representative samples of milk were collected at 10-14 h intervals post-dose, and subsamples were assayed for the major and minor hydrolysis products of morantel-related residues, MAPA and CP-20,107. Residues assayed as precursors of MAPA peaked at the second milking (24 h post-dose) and were below 25 ppb (range: less than 12-24 ppb). Precursors of CP-20,107, which confirm the identity of morantel, also peaked at 24 h post-dose (range: 2.1-3.3 ppb) and declined rapidly thereafter. A statistical model was used to project the level of residues at the upper limit of 99% of the total target animal (i.e., dairy cattle) population with 95% confidence. The calculated peak levels from this model were 50 and 5.0 ppb for morantel-related residues convertible to MAPA and CP-20,107, respectively.     

Determination of morantel-related residues in bovine milk by electron capture gas chromatography

A gas chromatographic assay was developed to determine major residues of morantel in bovine milk over a range that is suitable for monitoring residues of the drug. The method is based on hydrolysis of the N-methyl-tetrahydropyrimidine portion of morantel and its metabolites to N-methyl-1,3-propanediamine, and converting the diamine to an N,N-bis-(2-nitro-4-trifluoromethylphenyl) derivative. The addition of an internal standard, the N-desmethyl-N-ethyl homolog of pyrantel, to the milk sample circumvents any potential problem that could arise from variable reaction yields, and eliminates the true recovery as a factor affecting the accuracy and precision of the procedure. The concentrations of the derivatives are determined by pulsed electron capture gas chromatography over a linear dynamic range that is equivalent to 12.5-50 ppb morantel. The method was evaluated at the 0, 12.5, 25, and 50 ppb levels in fortified bovine milk, and in a withdrawal sample containing physiologically incurred morantel residues. Mean values of 14 +/- 1.7, 24 +/- 3.7, and 47 +/- 6.9 were found for the fortified samples, approximately 3 ppb for control milk, and 16 +/- 1.7 ppb for the withdrawal sample.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.     

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.     

Liquid chromatographic determination of morantel tartrate in cattle feed

A liquid chromatographic (LC) method is proposed for measuring 0.485-0.970% morantel tartrate in cattle feeds. The drug is leached from feed, diluted, separated from interfering substances on a silica column, and measured in the effluent stream by 313 nm spectrophotometric detection. Two potential degradation products, i.e., cis-isomer of morantel tartrate and N-(3-methylaminopropyl)-trans-3-(3-methyl-2-thienyl)acrylamide, and a related anthelmintic, i.e., pyrantel tartrate, do not interfere. Average recovery of drug from liquid spiked samples and laboratory blends was 98-100% with a maximum coefficient of variation (CV) of 2.3%. Results for pelleted and crumbled commercial scale feeds ranged from 94 to 102% of label claim, with a maximum CV of 1.5%.     

Liquid chromatographic determination of sulfamethazine in milk

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.     

Direct competitive enzyme-linked immunosorbent assay for sulfamethazine residues in milk

A direct competitive enzyme-linked immunosorbent assay (ELISA) is described for the detection and estimation of sulfamethazine residues in milk. Samples are cleaned up rapidly by acidifying and centrifuging the milk, adjusting the supernatant liquid to pH 7.0, and centrifuging again. The supernate is then assayed using set points to estimate sulfamethazine levels in the sample in the range of 1 ppb to 1 ppm. Multiple samples of milk can be screened in 1.5-2 h by this ELISA method.     

Rapid reverse phase liquid chromatographic determination of aflatoxin M1 in milk

A rapid, economical, and reliable liquid chromatographic (LC) method is described for determination of aflatoxin M1 in milk. The method includes an improved AOAC extraction procedure, cleanup of the extract on a silica cartridge, and LC quantitation. Alternatively, a rapid column cleanup procedure can be used. Milk artificially spiked with aflatoxin M1 at 0.05, 0.1, and 0.5 ppb was analyzed using both new approaches as well as an AOAC method coupled with LC for quantitation of the toxin. Recovery of aflatoxin M1 by the first approach of the new method ranged between 93.4 and 99.1%, and for the alternative procedure between 92.4 and 96.8%. The AOAC method gave lower recovery (85.6-90.7%) of toxin, but the results from this method had a somewhat smaller standard deviation for replicate analyses than did results of the new method.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

Liquid chromatographic determination of carbadox, desoxycarbadox, and nitrofurazones in pork tissues

A liquid chromatographic (LC) method has been developed for the determination of carbadox, desoxycarbadox, and nitrofurazones in the 10-40 ppb range in pork muscle, liver, and kidney tissues. Tissues were homogenized in absolute ethanol, and the homogenates were treated with metaphosphoric acid and reduced in volume by rotovaporization. Hexane was added to the concentrates, which were then centrifuged to remove fat. After addition of KH2PO4 to the aqueous phase and extraction with ethyl acetate, the extracts were passed through alumina columns before analysis by reverse phase LC. Overall average recoveries (10-40 ppb range) for carbadox and desoxycarbadox from spiked tissues were 53% +/- 13.6 and 61% +/- 7.2, respectively; overall average recoveries for nitrofurazone and furazolidone were 43% +/- 7.3 and 77% +/- 10.9, respectively. Before these optimum determinations, degradation by even minimal incandescent light was found to reduce recovery especially of desoxycarbadox. The results of this photochemical degradation are reported and briefly discussed.     

Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

Immunochromatography of fusarochromanone mycotoxins

The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).     

Flunixin residues in milk after intravenous treatment of dairy cattle with (14)C-flunixin

Flunixin meglumine is used in veterinary medicine as an alternative to narcotic analgesics and as an antiinflammatory agent. Eight Holstein dairy cows were dosed intravenously once daily on three consecutive days with (14)C-flunixin meglumine at approximately 2.2 mg of flunixin free acid/kg of body weight. Milk was collected twice daily to determine the decline of the total radioactive residues (TRR) in milk and to identify or characterize residue components. TRR in milk declined rapidly and averaged 66, 20, and 14 ppb, respectively, for the first, second, and third milkings after administration of the last dose. Milk was extracted, and the extracts were examined for radioactive residues. Mean extractability of milk TRR was always greater than 80%. Flunixin and 5-hydroxyflunixin were identified by coelution with analytical standards using reverse phase HPLC. These two residues were the main radioactive residues found in milk and together accounted for 64, 37, and 44% of the extractable residues, for the first, second, and third milkings, respectively, after administration of the last dose. The presence of 5-OH flunixin in milk was confirmed by HPLC/MS/MS.     

Liquid chromatography-tandem mass spectrometry for the determination of protein-bound residues in shrimp dosed with nitrofurans

An analytical method was developed for the determination of bound residues of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin with a sensitivity of 1 ppb in shrimp. In this procedure, shrimp tissue is prewashed with solvents followed by overnight acid hydrolysis, during which the side chains of the bound residues are released and simultaneously derivatized with 2-nitrobenzaldehyde. After liquid-liquid extraction cleanup, the derivatives are detected and quantitated using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with an atmospheric pressure chemical ionization interface. The method was validated using control shrimp fortified with each side-chain analyte at 1, 2, and 4 ppb. Method accuracies were >80% with coefficients of variation of <20% for all four analytes. Tissues from dosed shrimp were assayed to demonstrate the effectiveness of the method for recovering bound residues of nitrofurans. In shrimp dosed with nitrofurans, nitrofurantoin exhibited the lowest level of bound residues.     

Quantitation of the nitrosamine 2-ethylhexyl-4-(N-methyl-N-nitrosoamino) benzoate (NPABAO) in sunscreen products.

We have devised a method to quantitate the nitrosamine, 2-ethylhexyl-4-(N-methyl-N-nitrosoamino) benzoate (NPABAO), in commercial products containing the sunscreen ingredient, Padimate O. The method involves a minimum of cleanup steps to afford a nonaqueous extract from product emulsions suitable for analysis by a liquid chromatograph interfaced to a thermal energy analyzer (LC/TEA). The method is applicable to lotions, creams, and gels. Oils are normally soluble in the mobile phase and can be analyzed directly on the LC/TEA without additional cleanup procedures. The method has a minimum detectable limit of about 30 ppb and yields greater than 80% recovery. It is highly reproducible and generates no NPABAO artifactually prior to quantitation on the LC/TEA. Application of the method to 22 different commercial product formulas disclosed that the level of NPABAO in each of the products is below 250 ppb, with 18 of the products containing less than 100 ppb. Of interest was the observation that musk ketone, a common fragrance constituent, produces a false-positive TEA response that can interfere with accurate analysis of NPABAO content in typical commercial products.     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Sulfamethazine (sulfadimidine) residues in Canadian consumer milk

A survey on the presence of sulfamethazine (sulfadimidine) residues in consumer milk has been conducted in 10 cities across Canada. In each city, homogenized milk was purchased at 3 different retail outlets, each supplied by different processing plants. A total of 30 samples was analyzed by a liquid chromatographic method. The limit of quantitation was 5 ppb. In addition to automatic integration, visual inspection of the chromatograms was required to distinguish between low concentrations of sulfamethazine and 2 unknown interfering peaks. Two samples, from different cities, contained 11.4 and 5.24 ppb of the drug. Drug identity was confirmed by mass spectrometry. All other samples appeared to be free of the drug.     

Determination of N-nitrosodimethylamine in nonfat dry milk: collaborative study

Ten laboratories participated in a collaborative study of a method for the determination of N-nitrosodimethylamine (NDMA) in nonfat dry milk. NDMA is eluted with dichloromethane from a mixture of Celite, acidic sulfamic acid, and nonfat dry milk (all packed in a chromatography column), concentrated in a Kuderna -Danish concentrator, and finally analyzed by a GC-thermal energy analyzer technique. Ten samples were studied: 6 were naturally contaminated (NDMA levels 0.38-3.56 ppb) and 4 were spiked with known levels (0.96 and 3.2 ppb) of NDMA. The coefficients of variation (CV) of the complete data for the naturally contaminated samples (excluding the 2 samples containing the lowest levels) were 8.5% and 22.5% for repeatability and reproducibility, respectively. The corresponding CVs for the spiked samples were 14.4% and 20.4%, respectively. The percent recoveries of the added NDMA in the spiked samples (at the 2 levels indicated above) were 101.6 +/- 3.2 (omitting 1 outlier) and 95 +/- 2.1, respectively. The method has been adopted official first action.     

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%.