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.

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.

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 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.

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).

[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.

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).

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.

[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.

[Studies on rapid multi-residue analysis of pesticides in agricultural products by LC-MS/MS].

Rapid multi-residue analysis of pesticides in agricultural products was studied by using LC-MS/MS. Pesticide residues in 10 g of homogenized agricultural products were extracted with 30 mL of acetonitrile and salted out with 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride in the presence of citrate salts for buffering in a disposable tube. Co-extractives were removed by use of our original triple layered column (C18/GC/PSA; 60/30/60 mg). According to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan, we conducted recovery tests in 5 kinds of agricultural products (brown rice, kiwi, cabbage, sweet potato and spinach) spiked with 60 pesticides at the level of 0.01 or 0.1 µg/g. Each concentration of pesticide spiked was extracted from 2 samples per day on 5 days. Pesticides in the test solution were determined by two types of LC-MS/MS using scheduled MRM. Using this method, 58 out of 60 pesticides satisfied the guideline criteria in brown rice, 59 in kiwi, 55 in cabbage, 55 in sweet potato and 56 in spinach. This method is applicable for routine examination of pesticide residues in agricultural products.

[Survey of pesticide residues in imported spices and herbs (1997.4-2011.3)].

A survey of pesticide residues in 313 samples of imported spices and herbs on the Tokyo market from April 1997 to March 2011 was carried out. Thirty-seven kinds of pesticides, including organophosphorus, organochlorine, pyrethroid, carbamate and others, were detected between levels of trace (below 0.01 ppm) and 3.3 ppm from 64 samples. The rate of detection was highest in peel (100%) followed by stem (66.7%), fruit (34.5%), bark (33.3%), flower (31.3%) and leaf (14.7%). No residues were detected in root, seed or whole grass. Organochlorine pesticides were detected in all plant parts. The insecticides were detected in products from all production areas, suggesting that their use is common. Residue levels of these pesticides were calculated as less than 1% of their ADI values, based on the daily intake of spices and herbs. Therefore, these spices and herbs should be safe when consumed in customary amounts.

Single-laboratory validation study of rapid analysis method for multi-class veterinary drugs in milk, fish and shellfish by LC-MS/MS.

A method of rapid analysis of multi-class residual veterinary drugs in milk, fish and shellfish was validated in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed in one injection. Analytes were extracted from samples with two kinds of solvent, acetonitrile containing 1 vol% formic acid and anhydrous acetonitrile, and salted out with 4.0 g of magnesium sulfate, 1.5 g of trisodium citrate and 2.0 g of sodium chloride. This method was assessed by performing recovery tests in retail milk and 4 kinds of fresh cultured fish and shellfish (salmon, tiger shrimp, red sea bream and bastard halibut) spiked with the 43 target analytes at the levels of 10 and 100 µg/kg. Using this method, 40 out of 43 drugs satisfied the guideline criteria in milk, 37 drugs in salmon, 42 drugs in tiger shrimp, 41 drugs in red sea bream and 39 drugs in bastard halibut.

[Survey of pesticide residues in imported tea (1992.4-2010.3)].

A survey of pesticide residues in 116 imported tea samples on the Tokyo market from April 1992 to March 2010 was carried out. Twenty-two kinds of pesticides, including organophosphorus, organochlorine, pyrethroid and others, were detected at levels between trace (below 0.01 ppm) and 4.0 ppm in 76 samples. The rate of detection was 90% in non-fermented tea, 89% in semi-fermented tea, and 49% in fermented tea. Organophosphorus pesticide was not detected in puer tea, which is fermented with bacteria. The pesticide residues tended to be decreased by a fermentation process. However, organochlorine and pyrethroid pesticides appeared to be chemically stable, so they were still detected in fermented teas. Residue levels of these pesticides were calculated as less than 1% of ADI, except for ethion (45% of ADI) based on the daily intake of tea. Therefore, these teas should be safe when drunk in customary amounts.

Application of carbonated apatite coating on a Ti substrate by aqueous spray method.

The fabrication and characterization of a carbonate-containing apatite film deposited on a Ti plate via an aqueous spray method is described. The mist of the spray solution emitted from a perpendicularly oriented airbrush was made to strike a warmed Ti substrate. The thicknesses of the sprayed film and those heat-treated at 400 °C-700 °C under Ar gas flow were in the range 1.21-1.40 µm. The results of elemental analyses and Fourier transform infrared spectroscopy of the powders that were mechanically collected from the surface of the sprayed film suggest that the film was Ca(10)(PO4)6(CO3) · 2CO2 · 3H2O. The presence of the carbonate ion and the lattice CO2 molecule was confirmed via the aforementioned analyses; the finding was also consistent with the X-ray diffraction patterns of the films and the chemical identity of the sprayed and heat-treated films that were measured using X-ray photoelectron spectroscopy. The sprayed film comprises a characteristic network structure, which contains round particles within the networks, as was observed by field-emission scanning electron microscopy. A scratch test indicated that the shear stress of the sprayed film (21 MPa) significantly improved to 40 and >133 MPa after heat-treatment at 600 °C and 700 °C, respectively, under Ar gas flow for 10 min.

Development and validation of rapid analysis method for multi-class veterinary drugs in livestock products by LC-MS/MS.

A method for rapid analysis of multi-class residual veterinary drugs in livestock products was developed and validated in accordance with the Japanese guideline for pesticides. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed simultaneously in only 18 minutes. The extraction process was developed by modifying the QuEChERS approach to provide faster and less expensive extraction. The samples were extracted by using two kinds of solvent, acetonitrile and acetonitrile including 1 vol% formic acid, and salted out with magnesium sulfate, trisodium citrate and sodium chloride. Using these two extractants, 40 out of 43 drugs satisfied the guideline criteria in bovine muscle and swine muscle, 39 drugs were found in chicken muscle, and 37 drugs were found in eggs. The limit of quantification was less than the MRL for all analytes.

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.

Screening assay of residual antibiotics in livestock samples by LC-MS/MS.

A LC-MS/MS screening assay of multi-class antibiotics was developed for 19 residual antibiotics in livestock samples. Sample preparation employed the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach using 0.5% formic acid in acetonitrile-methanol (8 : 2), with salting-out using magnesium sulfate, trisodium citrate and sodium chloride. Recovery values from 5 different livestock samples ranged from 45.5 to 121.6%, and the RSDs were under 18% at two concentration levels. The limit of quantification values of 19 analytes were under 10 µg/kg in all livestock samples, and the procedure can detect almost all analytes under the MRL. Screening capability was confirmed by employing spiked samples. This new screening assay for residual antibiotics in livestock samples is expected to be useful for routine laboratory tests.

[Study on rapid analysis method of pesticide contamination in processed foods by GC-MS and GC-FPD].

A simple and rapid method using GC-MS and GC-FPD for the determination of pesticide contamination in processed food has been developed. Pesticides were extracted from a sample with ethyl acetate in the presence of anhydrous sodium sulfate, then cleaned up with a combination of mini-columns, such as macroporous diatomaceous earth, C18, GCB (graphite carbon black) and PSA. Recovery tests of 57 pesticides (known to be toxic or harmful) from ten kinds of processed foods (butter, cheese, corned beef, dried shrimp, frozen Chinese dumplings, grilled eels, instant noodles, kimchi, retort-packed curry and wine) were performed, and the recovery rates were mostly between 70% and 120%. This method can be used to judge whether or not processed foods are contaminated with pesticides at potentially harmful levels.

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.