Performance of a proposed determinative method for p-TSA in rainbow trout fillet tissue and bridging the proposed method with a method for total chloramine-T residues in rainbow trout fillet tissue

Chloramine-T is an effective drug for controlling fish mortality caused by bacterial gill disease. As part of the data required for approval of chloramine-T use in aquaculture, depletion of the chloramine-T marker residue (para-toluenesulfonamide; p-TSA) from edible fillet tissue of fish must be characterized. Declaration of p-TSA as the marker residue for chloramine-T in rainbow trout was based on total residue depletion studies using a method that used time consuming and cumbersome techniques. A simple and robust method recently developed is being proposed as a determinative method for p-TSA in fish fillet tissue. The proposed determinative method was evaluated by comparing accuracy and precision data with U.S. Food and Drug Administration criteria and by bridging the method to the former method for chloramine-T residues. The method accuracy and precision fulfilled the criteria for determinative methods; accuracy was 92.6, 93.4, and 94.6% with samples fortified at 0.5X, 1X, and 2X the expected 1000 ng/g tolerance limit for p-TSA, respectively. Method precision with tissue containing incurred p-TSA at a nominal concentration of 1000 ng/g ranged from 0.80 to 8.4%. The proposed determinative method was successfully bridged with the former method. The concentrations of p-TSA developed with the proposed method were not statistically different at p < 0.05 from p-TSA concentrations developed with the former method.     

Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish.

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.     

Determination of ciprofloxacin and enrofloxacin in edible animal tissues by terbium-sensitized luminescence.

A terbium-sensitized luminescence method is described for the determination of the sum of residues of enrofloxacin and its major metabolite ciprofloxacin in edible animal tissues. Several parameters affecting both detection and extraction were studied. Analytes were extracted from spiked samples of chicken and trout tissues with pH 7.4 buffer-dichloromethane. The organic extract was evaporated and the residue dissolved in aqueous nitric acid and defatted with hexane. Determination was carried out directly in the aqueous phase (in a micellar medium). The calibration curves were linear up to 75 micrograms l-1. The detection limit was 3.5 micrograms kg-1 (for a 5 g sample) and the repeatability was 7.0% (n = 7). The sensitivity was similar for both quinolones and therefore calibration can be carried out with either ciprofloxacin or enrofloxacin. In any case, the differences were < 10%.     

Determination of flunixin residues in bovine muscle tissue by liquid chromatography with UV detection.

A new and sensitive liquid chromatography-ultra violet method with a detection limit of 6 ng/g (ppb) and a limit of quantification of 15 ng/g was developed for the determination of flunixin residues in bovine muscle tissue. Flunixin in homogenized animal tissue was extracted with acetonitrile after enzyme digestion. The tissue digest (extract) was then cleaned up on a solid-phase extraction cartridge and eluted with acidified hexane. After the eluate was evaporated to dryness under nitrogen at 55 degrees C, the residue was reconstituted in 1 mL mobile phase solution and analyzed by reversed-phase gradient chromatography with UV detection at 285 nm. The method was then applied in a survey study of slaughter animals to determine whether flunixin is being used in an off-label manner for veal and beef production in Canada.     

Determination of chlorotoluron, isoproturon and metoxuron in soil by GLC-NPD and confirmation using GLC-MS

Summary The analysis of several phenyl urea herbicides in soil by GLC-NPD, directly or after alkylation, and the confirmation of residues by GLC-ITD is reported. Soil was extracted with methanol, the organic solvent evaporated, the residue dissolved in hexane and then analyzed by gas chromatography on a 3% OV-17 glass packed column. An aliquot of the extract was ethylated overnight with EtI, NaH and Me₂SO as solvent. The mixture was hydrolyzed, the ethylated compounds extracted with hexane and determinated by GLC-NPD on a BP-5 fused silica capillary column. Values obtained with the direct GLC analysis were reproducible and similar to those obtained after ethylation. Recovery of each herbicide was higher than 80% and the limit of detection was 0.01 ppm. These compounds were also analyzed by GLC-ITD. The sensitivity in the SIM mode was near 0.1 ng and the residues can be confirmed with this technique down to 0.01 ppm.     

Simultaneous Determination of 13 Phenoxy Acid Herbicide Residues in Soybean by GC‐ECD

Abstract

In this study, a gas chromatography method for the multiresidue determination of 13 phenoxy acid herbicide residues (4‐CPA, 4‐CPP, phenoxy butyric acid, dicamba, MCPA, MCPP, MCPB, 2,4‐D, 3,4‐D, 2,4,5‐T, 2,4,5‐TP, 2,4‐DP, and 2,4‐DB) in soybean was developed. After the removal of fat using n‐hexane, the sample was extracted with acetonitrile‐50 mM HCl (v/v 7:3), and then followed by liquid‐liquid partition with n‐hexane (saturated by acetonitrile). The soybean extract was further cleaned up by anion exchange column. Then the residues were derived with pentafluorobenzyl bromide and the resulting pentafluorobenzyl (PFB) esters were analyzed by a gas chromatograph equipped with an electron capture detector (ECD), quantified by the external standard method. Recoveries of spiked samples at two fortified levels (0.01 and 0.1 mg/kg) are all above 70%. And the relative standard deviation was less than 20%. It shows that the limits of determination (LOD) of this method (S/N≥3) can meet the requirement of maximum residue analysis in import/export inspection for soybean.     

Spectrophotometric determination of pentaerythritol tetranitrate (PETN) in waste water from lead styphnate primer plants

An accurate spectrophotometric method is proposed for the determination of pentaerythritol tetranitrate (PETN) in waste water from lead styphnate primer plants by use of phenoldisulphonic acid. The waste water is filtered through a sintered glass crucible and the PETN is determined in the filtrate and the residue. In the determination of PETN in the filtrate, sodium hydroxide is added and the PETN is extracted with methylene chloride (in alkaline solution, styphnate and TNT are not extracted). The methylene chloride solution is then evaporated to dryness, the residue is treated with a solution of phenoldisulphonic acid in sulphuric acid, water and ammonia are added, and the yellow colour is measured. In the determination of PETN in the residue, the PETN is dissolved in acetone, an aliquot of the acetone solution is treated with water and sodium hydroxide, the PETN is extracted with methylene chloride and the colour is developed as above. Various factors affecting the determination were investigated. The solubility of PETN in water was studied.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Screening and quantitative confirmatory method for the analysis of glucocorticoids in bovine milk using liquid chromatography-tandem mass spectrometry

An LC/MS/MS method was developed and validated for the simultaneous identification, confirmation, and quantification of 12 glucocorticoids in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. The developed method can detect and confirm the presence of dexamethasone, betamethasone, prednisolone, flumethasone, 6alpha-methylprednisolone, fluorometholone, triamcinolone acetonide, prednisone, cortisone, hydrocortisone, clobetasol propionate, and clobetasol butyrate in bovine milk. Milk samples are extracted with acetonitrile; sodium chloride is subsequently added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is evaporated to dryness and reconstituted in a water-acetonitrile mixture, and determination is carried out by LC/MS/MS. The method permits analysis of up to 30 samples in 1 day.     

Development of a rapid,multi-class method for the confirmatory analysis of anti-inflammatory drugs in bovine milk using liquid chromatography tandem mass spectrometry

A rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of seven licensed anti-inflammatory drugs (AIDs) in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. Two classes of AIDs were investigated, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). The developed method is capable of detecting and confirming dexamethasone (DXM), betamethasone (BTM), prednisolone (PRED), tolfenamic acid (TLF), 5-hydroxy flunixin (5-OH-FLU), meloxicam (MLX) and 4-methyl amino antipyrine (4-MAA) at their associated maximum residue limits (MRLs). These compounds represent all the corticosteroids and NSAIDs licensed for use in bovine animals producing milk for human consumption. These compounds have never been analysed before in the same method and also 4-methyl amino antipyrine has never been analysed with the other licensed NSAIDs. The method can be considered rapid as permits the analysis of up to 30 samples in one day. Milk samples are extracted with acetonitrile; sodium chloride is added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid–liquid purification by the addition of hexane. The purified extract is finally evaporated to dryness and reconstituted in a water/acetonitrile mixture and determination is carried out by LC–MS/MS. Decision limit (CCα) values and detection capability (CCβ) values have been established for each compound.     

水产品中螺旋霉素、替米考星、泰乐菌素与北里霉素残留量的超高效液相色谱-紫外检测法同时测定

建立了水产品肌肉组织中螺旋霉素、替米考星、泰乐菌素、北里霉素同时测定的超高效液相色谱-紫外检测(UPLC-TUV)方法。样品经乙腈提取后,浓缩至近干,用4%NaCl溶解残渣,正己烷除脂,经固相萃取小柱净化,乙腈洗脱;以乙腈-25 mmol/L磷酸二氢铵(pH2.5,含10%乙腈)为流动相,以ACQUITYUPLC BEHC18为分离柱,柱温为45℃,流速为0.3 mL/min,紫外检测。方法在0.100~20.0 mg/L范围内呈线性相关,螺旋霉素、替米考星、泰乐菌素和北里霉素的相关系数分别为0.998 7、0.999 3、0.999 4和0.998 0。平均回收率为70%~102%,相对标准偏差为2.9%~11.2%,螺旋霉素、替米考星、泰乐菌素和北里霉素的检出限分别为25、25、50、75μg/kg。方法满足水产品肌肉组织中螺旋霉素、替米考星、泰乐菌素和北里霉素的残留量测定。     

Determination of soluble styphnate and nitrate in waste-water from lead styphnate primer plants

Methods are proposed for the determination of soluble styphnate and nitrate in waste-water from lead styphnate primer plants. The styphnate is extracted from the waste-water with methylene chloride and is determined by measurement of the absorbance of the methylene chloride solution at 273 nm. The nitrate is determined in the aqueous solution left after the methylene extraction by measurement of the absorbance at 220 nm. For complete extraction of the styphnate by the methylene chloride, the solution must be moderately acidic (about 6% perchloric acid). The acidity for the determination of the nitrate is not critical. Before the determination of the nitrate in the aqueous extract, it is necessary to boil the solution to eliminate residual methylene chloride which would interfere with the determination of nitrate. PETN is extracted by the methylene chloride but does not interfere with the determination of the styphnate, since it shows no interfering peaks. Chloride, sulphate, phosphate, fluoride and carbonate do not interfere with the determination of styphnate or nitrate.     

液相色谱-串联质谱法测定食用植物油中的棉酚

建立了食用植物油中棉酚的液相色谱-串联质谱（LC-MS/MS）分析方法。待测物经无水乙醇涡旋振荡提取,C18色谱柱分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,LC-MS/MS测定,外标法定量。方法的测定低限（S/N>10）为1 mg/kg;在添加浓度为1、2和200 mg/kg水平下,棉酚的加标回收率为87.4%~100%,相对标准偏差为3.9%~12.2%。结果表明,本方法灵敏度高,测定结果准确,回收率稳定,可用于食用植物油中棉酚残留的确证检测。     

Confirmation of phenylbutazone residues in bovine kidney by liquid chromatography/mass spectrometry

A confirmatory method is described for phenylbutazone (PB) residues in bovine kidney tissue. Ground kidney tissue is diluted with water, and the mixture is made basic with 25% ammonium hydroxide in water; the lipids are extracted with ethyl and petroleum ethers. The ether layer is discarded, and the tissue is acidified with 6N HCl. PB residues are extracted with tetrahydrofuranhexane (1 + 4). The extract is passed through a silica solid-phase extraction column, and the eluate is evaporated to dryness. The residue is dissolved in acidified acetonitrile-water-acetic acid (50 + 49.4 + 0.6). A single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface is used to confirm the identity of the PB residues in the kidney extract. Negative-ion detection with selected-ion monitoring of 4 ions is used. Sets of control and fortified-control kidney tissues (at 50, 100, and 200 ppb PB) and several kidney tissue field samples were analyzed for method validation. The method was tested further during the course of a survey to determine the incidence of PB residues in bovine kidney samples obtained from slaughterhouses across the country. In addition, the method was tested for use with an ion-trap mass spectrometer coupled to a liquid chromatograph, which allowed confirmation of PB at lower levels (5-10 ppb) in kidney tissue.     

Determination of thiamphenicol residues in chicken muscles by column liquid chromatography

A column liquid chromatographic (CLC) method for the determination of thiamphenicol residues in chicken muscles was developed. The drug is extracted from minced muscles with ethyl acetate and the extract is evaporated to dryness. The residue is dissolved in 10% sodium chloride solution and partitioned with n-hexane. Thiamphenicol is extracted with ethyl acetate and, after evaporation of the solvent, the residue is cleaned up by alumina column chromatography. CLC analysis is carried out on a Nucleosil C18 column with ultraviolet detection of thiamphenicol at 230 nm. The average recoveries of thiamphenicol added to muscles at 0.2 and 0.1 ppm were 92.8 and 90.0%, respectively. The detection limit was 5 ng for thiamphenicol standard, which corresponds to 0.05 ppm in muscles.     

固相萃取-高效液相色谱法测定蘑菇中咪鲜胺的残留量

建立了蘑菇中咪鲜胺残留的固相萃取一高效液相色谱（SPE-HPLC）检测方法。对提取条件、固相萃取条件及仪器参数等进行了优化。提取液经C18和PSA固相萃取柱净化、浓缩后HPLC分离,紫外225nm检测。方法的检出限为0.038mg／kg,咪鲜胺在蘑菇样品中的添加回收率为82.6%-100.3%,变异系数是1.8％-9.2%,符合农药残留检测要求。     

Peer validation of a method to confirm chloramphenicol in honey by liquid chromatography-tandem mass spectrometry

Chloramphenicol (CAP) is extracted from an aqueous dilution of honey using ethyl acetate. The extracts are evaporated and redissolved in water. CAP is then extracted from the aqueous solutions using reversed-phase solid-phase extraction cartridges. CAP is eluted from the reversed-phase cartridges with acetonitrile-water and re-extracted into ethyl acetate. The ethyl acetate is evaporated, and the residue is reconstituted in an aqueous solution. Extracts are chromatographed using a reversed-phase column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions of precursors m/z 321 or 323 are monitored. The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 2 laboratories.     

Determination of DiazaCon in quail feed and quail serum by ion pair reversed-phase chromatography.

Liquid chromatographic (LC) methods were developed for quantitating the potential avian contraceptive DiazaCon in quail feed and serum. DiazaCon was extracted from ground quail feed with basic n-butyl chloride. The n-butyl chloride extract was evaporated to dryness. The DiazaCon residues were dissolved in an aqueous methanolic ion pairing solution and quantitated by LC at 206 nm. Avian sera was combined with an equal volume of a pH 4 aqueous solution of ion pairing reagent and filtered to remove interfering proteins. DiazaCon was quantitated by LC. Mean recoveries for 500 and 2000 ppm fortified feed were 89.1 and 91.0%, respectively. The mean recovery for sera fortified at 5 levels ranging from 35 to 2000 ppm was 84.9%. Method limits of detection were approximately 14 and 13 ppm for feed and sera, respectively.     

Determination and confirmation of malachite green and leucomalachite green residues in salmon using liquid chromatography/mass spectrometry with no-discharge atmospheric pressure chemical ionization

A liquid chromatography/mass spectrometry (LC/MS) method was developed to quantitate and confirm residues of leucomalachite green (LMG) in salmon tissue after their conversion to chromic malachite green (MG) in the extraction process. The method uses no-discharge atmospheric pressure chemical ionization (APCI) in conjunction with an ion-trap instrument to generate product-ion spectra. In the sample preparation procedure, salmon tissue is extracted with acetonitrile/buffer, the LMG residue is partitioned into methylene chloride, the LMG is converted to MG using an organic oxidizing agent, and the MG is isolated on alumina/propylsulfonic acid solid-phase extraction cartridges. The method was validated by fortifying salmon with different levels of LMG, and then detecting the residue as MG The LC/MS conditions, including a comparison of electrospray and no-discharge APCI, were evaluated and optimized. MG was not confirmed in any of the control tissue extracts, and all fortified samples analyzed during validation met the confirmation criteria as described. In addition to providing confirmatory data, this method can provide an alternative method for quantitation of MG in salmon. The recoveries of LMG measured as MG by this LC/MS method, at fortification levels of 1-10 ng/g were very high (86-109%), with low relative standard deviation(RSD) values (6.4-13%). The results agreed very closely with those obtained for the same extracts using an LCNIS procedure, indicating that matrix suppression was not an issue. The presence of LMG in salmon tissue samples fortified at 0.25 ng/g was confirmed by this method, with an average recovery of 70.1% and an RSD of 12.0%. Sample extracts from fish exposed to MG were also analyzed.     

High-pressure liquid chromatographic analysis of carbofuran and two non-conjugated metabolites in crops as fluorescent dansyl derivatives

Carbofuran, and non-conjugated 3-hydroxycarbofuran and 3-ketocarbofuran were extracted from carrots, corn and potatoes with acetone and partitioned into hexane-methylene chloride. The organic extract was evaporated to a small volume for clean-up on a 2% deactivated Florisil column. All three carbamates were eluted with 15% acetone in hexane. The pesticide residues were hydrolysed to their corresponding phenols with 0.1 M sodium carbonate followed by derivatization with dansyl chloride in acetone. The derivatives were extracted and analysed by high-pressure liquid chromatography with fluorescence detection (excitation, 360 nm; emission, greater than 400 nm). Absolute recoveries for all three compounds were between 50 and 65% for spiked samples by the extraction method used. Detection limits approached 0.01 ppm in the foods studied.