Multi-residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/Tandem Mass Spectrometry.

Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points in sample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initially extracted with Na2EDTA-McIlvaine's buffer (pH 7.0). Medium polar compounds were then extracted from the same samples with acetonitrile containing 0.1% formic acid. Secondly, cleanup was performed using a single SPE polymer cartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from 0.1 to 5 µg/kg. Residues were detected in 24 out of 110 samples and analyzed using the validated method.

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry.

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.

Rapid determination of fumagillin residues in honey by liquid chromatography-tandem mass spectrometry using the QuEChERS method.

A new, rapid, and efficient method for determining the fumagillin residues in honey was developed. The samples extracted were analyzed using LC/MS/MS. Chromatographic separation of fumagillin was performed in gradient mode on a C8 column (100 x 2.0 mm, 5 microm) at 40 degrees C. The mobile phase consisted of a mixture of 2 mM ammonium formate-0.01% formic acid solution and methanol; the flow rate was set to 0.2 mL/min. Under these conditions, it was possible to measure fumagillin and its isomers as a single peak. The sample preparation procedure used is based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which is fast (approximately 30 min) and uses less organic solvent. The fumagillin was extracted with acetonitrile containing 0.1% formic acid, then purified using a solid-phase extraction method with an Oasis mixed-mode weak anion-exchange cartridge. The overall recovery of fumagillin ranged from 88.1 to 99.4%; the intra- and interassay CVs were <4.5% and <4.9%, respectively. The LOQ was 0.1 microg/kg. LC/MS/MS coupled with the QuEChERS method showed strong potential as a method for determining fumagillin residues in honey.

Development of a highly sensitive liquid chromatography with tandem mass spectrometry method for the qualitative and quantitative analysis of high-intensity sweeteners in processed foods.

A new method for the simultaneous determination of two sweeteners (Advantame and Neotame) in processed foods using liquid chromatography (LC) with tandem mass spectrometry(MS/MS) was developed herein. Chromatographic separations were performed using an ACQUITY UPLC CSH C18 column at 40 °C via a mobile phase comprising 10-mmol/L ammonium formate and methanol. Samples were prepared via rapid dialysis using 30% methanol solution in a thermostatic shaker set at 160 rpm and 50 °C for 1 h. The matrix in the test solution had no effect on the identification and quantification of the compound without a clean-up step using solid-phase extraction (SPE). This method satisfied all validation criteria with a limit of quantification (LOQ) of 0.01 µg/g for all samples. Using this method, the amounts of Advantame and Neotame in 24 processed foods were subsequently investigated, with the results indicating their detection beyond the lower LOQ. Moreover, a multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) method was developed and described to further enhance product-identification ability.

[Determination of hymexazol in agricultural products by GC-NPD].

A method for the determination of hymexazol in agricultural produdcts by gas chromatography with a highly sensitive nitrogen-phosphorus detector (GC-NPD) was investigated. Hymexazol was extracted with acetonitrile, and the acetonitrile layer was separated by salting-out. The water layer was loaded onto a Chem-Elut column. Hymexazol in the water layer was adsorbed on the column, and eluted with ethyl acetate. The acetonitrile layer and the eluate were mixed and evaporated. The residue was dissolved in ethyl acetate, and the sample solution was cleaned up on a C18 column. Hymexazol in the eluate was analyzed by GC-NPD with a high-polarity capillary column (DB-FFAP) and highly deactivated inlet liner. Recoveries of hymexazol spiked in agricultural products (tomato, lemon, soybean and other samples) at the level of 0.1 mug/g ranged from 65.0 to 84.7%. The limit of detection was 0.02microg/g.

[Survey of pesticide residues in imported cereal products (1994.4 approximately 2006.3)].

A survey of pesticide residues in 490 imported cereal products on the Tokyo market from April 1994 to March 2006 was carried out. Eight kinds of organophosphorus pesticides (chlorpyrifos, chlorpyrifos-methyl, DDVP, diazinon, etrimfos, malathion, MEP and pirimiphos-methyl) were detected at levels between Tr (below 0.01 ppm) and 0.82 ppm from 91 samples. In our investigations, chlorpyrifos-methyl and malathion tended to be detected in samples from America, pirimiphos-methyl in those from Europe, and MEP in those from Oceania. Thus, pesticide residues seemed to be different in produce from different areas. Residue levels of these pesticides were calculated as between 0.08 and 13.2% of their ADI values according to the daily intake of cereal products. Therefore, these cereal products should be safe for normal usage.

Clean-up method of forchlorfenuron in agricultural products for HPLC analysis.

A simplified method for the determination of forchlorfenuron in agricultural products by HPLC with UV detection was investigated. A chopped sample homogenate from agricultural products was extracted with acetone. The extract was filtrated and concentrated. The residues was loaded onto a Chem Elut column and extracted with ethyl acetate. The crude extract was purified on Oasis HLB and Bond Elut PSA mini-columns using a mixture of methanol and ethyl acetate. Forchlorfenuron was analyzed by HPLC with UV detection (263 nm). HPLC separation was performed on an ODS column with methanol-water as the mobile phase. Recoveries of forchlorfenuron from several agricultural products fortified at the level of 0.1 microg/g were in the range of 87.6-99.5%. The limit of detection (S/N=3) was 0.005 microg/g in the sample.

[Survey of pesticide residues in baby foods (1999.4-2005.6)].

A survey of pesticide residues in 316 baby foods on the Tokyo market from April 1999 to June 2005 was carried out. Fifteen kinds of pesticides, including organophosphorus, organochlorine, carbamate, pyrethroid and others, were detected between at levels Tr (below 0.01 ppm) and 0.55 ppm from 28 samples. The baby foods in which residual pesticides were detected were produced from flour, leaf vegetable and fruits. The pesticides were detected in baby foods produced with minimal processing, such as straining and squeezing. For the baby foods containing pesticides, the amounts of intake of the pesticides were calculated from the recommended servings, and compared with the ADI values. Residual pesticide levels in baby food were between 0.06 and 16.6% of the ADI. Therefore, these foods should be safe in normal usage.

[Survey of pesticide residues in rice (April, 1995-March, 2005)].

Pesictide residues in 343 samples of domestic rice and 32 samples of imported rice purchased on the Tokyo market from April 1995 to March 2005 were investigated. Residues of eleven kinds of pesticides (including organophosphorus, organochlorine, carbamate, pyrethroid and organonitrogen pesticides and bromide) were detected at levels between trace and 1 ppm in 47 domestic rice samples. DDVP and bromide residues were detected at levels between 0.01 ppm and 5 ppm in 18 imported rice samples. For rice samples that were found to containing pesticides, the amounts of pesticide intake were calculated according to the daily intake of rice, and they were compared with the ADI values. Since residual pesticide levels were 17/10,000-2/5 of ADI, all the rice samples were considered to be safe for human consumption.

Simultaneous determination of multi-class veterinary drugs in chicken processed foods and muscle using solid-supported liquid extraction clean-up.

We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography-mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70-105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0µg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.

Simultaneous determination of amantadine, rimantadine, and memantine in processed products, chicken tissues, and eggs by liquid chromatography with tandem mass spectrometry.

A simultaneous determination of amantadine, rimantadine, and memantine in processed products (deep-fried chicken, fried chicken, fried quail egg, and grilled chicken) with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This new method was also applicable for chicken tissue (muscle, liver, and gizzard) and eggs. The chromatographic separation was performed on a Kinetex® XB-C18 core-shell technology column using a mobile phase of acetonitrile and 0.1% formic acid in a 10mmol/L ammonium formate solution, resulting in the complete separation of isomers (rimantadine and memantine) and any other obstructive peaks from the sample matrices. Sample preparation was performed by a modified QuEChERS method using acetonitrile and a 0.1% acetic acid extraction solution and cleaned using an Oasis® MCX cartridge. The sample matrix had no effect on the identification of the compounds. For quantification, an external solvent calibration curve was used. This new method exhibited good accuracy ranging from 79.9% to 91.5%. The relative standard deviation of repeatability (RSDr) ranged from 1.2% to 3.6% and the relative standard deviation of within-laboratory reproducibility (RSDWR) ranged from 1.3% to 6.0%. These standard deviations satisfied the criteria for Japanese validation guidelines. The limit of quantification (LOQ) was 1.0µg/kg for all samples. Analyte residues were not detected in 55 samples using the validated method.

Determination of Fluopicolide in Livestock Products and Seafood by LC-MS/MS.

An analytical method for the determination of fluopicolide in livestock products and seafood was developed using LC-MS/MS. Sodium chloride was added to livestock products and seafood samples and fluopicolide was extracted twice with acetone after acidification with formic acid. The fat from the crude extract was removed using a macroporous diatomaceous earth column, followed by purification with a combination of mini-columns of GC (graphite carbon) and PSA (ethylenediamine-N-propyl silylation silica gel). The average recovery (n=5) of fluopicolide from 10 types of livestock products and seafood (cattle fat, cattle liver, cattle muscle, chicken, eel, egg, freshwater clam, honey, milk and salmon) spiked at the MRLs or at the uniform limit (0.01 ppm) was 96-100%, with a relative standard deviation of 2.3-6.2%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

Rapid Multi-Residue Analysis of Pesticides in Pulses by LC-MS/MS.

Rapid multi-residue analysis of pesticides in pulses was developed using LC-MS/MS. Pesticide residues in 5 g of homogenized pulses were extracted with 30 mL of acetonitrile and salted out with 4 g of anhydrous magnesium sulfate and 2 g of sodium chloride in the presence of citrate buffer in a disposable tube. The resulting residues were extracted with 30 mL of acetonitrile, and co-extractives were removed on a handmade four-layer column, consisting of a layer of Z-Sep/C18 (20 mg/50 mg) dry particles on top of a three-layer, custom-made (pre-packed) column (lower bed: 60 mg of PSA, middle bed: 30 mg of GC, and top bed: 60 mg of C18) packed in a 10 mm internal diameter polypropylene column (3 mL). The developed method showed good recoveries of pesticides in soybean, lentil, white kidney bean and garbanzo. According to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan, recovery tests were conducted in soybeans fortified with 107 kinds of pesticides at the levels of 0.01 and 0.1 µg/g, respectively. At each concentration 2 samples were extracted on 5 separate days. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. As regards the trueness of this method for 107 pesticides in soybeans, 97 pesticides were in the range of 70-120% with satisfactory repeatability and within-run reproducibility. This new method is expected to be applicable for routine examination of pesticide residues in soybeans.

Determination and surveillance of hydrocortisone and progesterone in livestock products by liquid chromatography-tandem mass spectrometry.

A simple analytical method for the determination of hydrocortisone and progesterone in bovine, swine, and chicken muscle and eggs was developed. Hydrocortisone and progesterone were extracted with acetonitrile and subsequently cleaned-up using an Oasis HLB mini-cartridge. The method was validated in accordance with Japanese guidelines and exhibited trueness from 86.6% to 104.3% and precision (relative standard deviations (RSDs) of repeatability and within reproducibility were under 8.7% and 11.7%, respectively). The method was applied to 103 bovine muscle, 137 swine muscle, 69 chicken muscle and 52 egg samples that were commercially available in Tokyo, Japan. The hydrocortisone concentration was 0.9-41.2 µg kg(-1) in all bovine muscle samples, with an average of 7.7 µg kg(-1) and a median of 6.2 µg kg(-1). The progesterone concentration in 50 samples exceeded the limit of quantification (LOQ) and reached a maximum of 95.4 µg kg(-1). Hydrocortisone was also detected in all swine muscle samples at concentrations of 2.0-56.0 µg kg(-1). Its average and median concentrations amounted to 13.1 and 11.3 µg kg(-1), respectively. Twenty-three samples contained progesterone levels surpassing the LOQ, with a maximum concentration of 107.0 µg kg(-1). No chicken muscle samples contained any of the analytes. The progesterone concentration was 15.5-200.0 µg kg(-1) in all egg samples, with an average of 95.4 µg kg(-1) and a median of 90.5 µg kg(-1).

[Single-laboratory validation study of simple and simultaneous determination method for pesticide residues in meat by LC-MS/MS].

We performed a single-laboratory validation study of a simple and simultaneous determination method for pesticide residues in meat using LC-MS/MS. Water was added to the sample and the mixture was homogenized. Next, pesticides were extracted with acetonitrile containing 1 vol% formic acid using a homogenizer, and salted out with magnesium sulfate, trisodium citrate and sodium chloride. After centrifugation, the acetonitrile layer was made up to standard volume and analyzed by LC-MS/MS. This method was assessed by performing recovery tests in retail bovine, swine and chicken muscle samples spiked with the 132 pesticides at the levels of 0.01 and 0.04 µg/g. Among them, 125 pesticides satisfied the Japanese method validation guideline criteria in bovine, 120 in swine and 127 in chicken.

Determination and surveillance of nine acaricides and one metabolite in honey by liquid chromatography-tandem mass spectrometry.

A simple and accurate analytical method for the determination of acaricides in honey was developed and validated in accordance with Japanese validation guidelines. Analytes - amitraz, N-2,4-dimethylphenyl-N-methylformamidine (DMPF), etoxazole, fenpyroximate, fipronil, hexythiazox, propargite, pyridaben and spirodiclofen - were extracted with ethyl acetate under basic conditions and subsequently cleaned up using an InertSep(®) MA-1 polymer-based anion-exchange column. The method was validated by fortified recovery tests at three different concentrations (1, 5 and 10 µg kg(-1)) performed with three samples daily on five different days. The method exhibited recoveries of 77-116% and precision (relative standard deviations - RSDs) of repeatability and within-laboratory reproducibility ranged from 2% to 22% and from 3% to 23%, respectively. The sample solution was successfully cleaned up to enable quantification using external solvent calibration curves. The limits of quantification (LOQs) were estimated to be 1 µg kg(-1) for all analytes. The method was applied to honey samples commercially available in Tokyo, Japan. Analysis of 250 honey samples indicated that amitraz was present in 127 samples, and that its residual concentration was less than 20 µg kg(-1). Propargite was detected in 23 samples at concentrations less than 1 µg kg(-1).

[Determination of Amantadine in Poultry Tissues and Egg by LC-MS/MS].

An accurate and selective analytical method for amantadine, which is used as antiviral drug to treat influenza A virus infection, was developed using LC-MS/MS. Residual amantadine was extracted from 4 kinds of food sample (poultry muscle, liver, gizzard and egg) with acetonitrile-pH 3.0 McIlvaine buffer (7 : 3), then cleaned up with an Oasis® MCX mini-cartridge. An external standard calibration curve was used for quantification, after sample purification by the combination of a reverse-phase strong cation exchange mixed mode cartridge for cleanup and a HILIC column for HPLC. The method was validated by performing recovery tests in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Recovery ranged from 79.3% to 91.7%, RSDs of repeatability were under 3.3%, and RSDs of within-laboratory reproducibility were under 8.4%. This new method was applied to samples of poultry and egg purehased in Tokyo, but residual amantadine was not detected at all.

Simple method for assaying serum oxytocin and changes of serum oxytocin level during parturition in cynomolgus monkeys.

A novel and simple assay system using a 96-well ELISA plate was established for measuring serum oxytocin in cynomolgus monkeys. This method omits the centrifuge for B/F separation because the second anti-rabbit IgG antibody-coated ELISA plate can easily separate the first anti-oxytocin rabbit antibody-bound radiolabeled oxytocin. Since this method has the advantage of omitting B/F separation, it becomes possible to measure a large number of samples with simple steps. In addition, accurate and reproducible results could be obtained by this method. The optimal reaction condition made it possible to measure more than 8 pg/ml of serum oxytocin. The changes of serum oxytocin level in relation to the first delivery was determined in a total of 11 female monkeys who were divided into two groups, infant-accepting mothers (4 monkeys) and infant-rejecting ones (7 monkeys). The serum oxytocin levels of pre-delivery (one to 4 days before delivery) and post-delivery (within 12 hr after delivery) in infant-accepting mothers were 33.6 +/- 4.57 and 43.5 +/- 16.4 pg/ml, respectively. Those in infant-rejecting mothers were 39.0 +/- 9.6 and 31.4 +/- 7.0 pg/ml. Two-way ANOVA (accepting/rejecting x pre/post) revealed a significant interaction of two factors (F (1, 9) = 5.39, p < 0.05). This result implies the possibility of a different pattern of oxytocin secretion between infant-accepting and infant-rejecting mothers during parturition.

[Determination of sucralose in foods by HPLC using pre-column derivatization].

The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.

[Analysis of imazalil and its major metabolite in citrus fruits by GC-FTD].

A simplified simultaneous analytical method of imazalil (IZ) and its major metabolite, alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (IZM), in citrus fruits was developed, and commodities samples were investigated. A homogenate of citrus fruits was extracted with ethyl acetate under basic conditions. The crude extract was partitioned between 0.025 mol/L of sulfuric acid and ethyl acetate. The analytes were extracted from the aqueous fraction under basic conditions with ethyl acetate. The extract solution was purified with an ENVI-Carb cartridge, and then analyzed by GC-FTD and GC/MS. Recoveries of IZ and IZM added to grapefruit at the level of 0.05 microgram/g were 90.0 and 108.7%, and those in the case of lemon were 100.4 and 93.0%, respectively. The detection limits were 0.01 microgram/g in samples. By this method, IZ and IZM were analyzed in 46 citrus fruits on the market and were detected simultaneously in some samples.