Assessment of the long-term stability of pesticide residues under freezing conditions in brown rice and soybean certified reference materials.

The objective of this study was to assess the long-term stability of pesticide residues in brown rice and soybean. The long-term stability of pesticide residues in brown rice and soybean was assessed for 5415 days (over 14 years) and 1801 days (about 5 years), respectively. The samples-certified reference materials (CRMs) 7504-a (brown rice) and 7509-a (soybean) -were prepared by freeze-pulverization. Two target pesticides (etofenprox and fenitrothion) were selected for brown rice and four (chlorpyrifos, diazinon, fenitrothion, and permethrin) for soybean. Our analytical results for long-term stability based on highly reliable isotope dilution mass spectrometry were in the range of expanded uncertainty (k=2) for the certified values of each CRM. The concentration showed a decreasing trend in none of the target pesticides when the samples were stored at temperatures between -20 °C and -30 °C, which indicated that the target pesticides were stable for the tested long terms.

Coproduction of lipids and carotenoids by the novel green alga Coelastrella sp. depending on cultivation conditions.

A novel green alga Coelastrella sp. D3-1 was isolated, and its unique and significant lipid and carotenoid coproduction capability was characterised depending on cultivation conditions. The main component of produced lipids was triacylglycerol under nutrient depletion conditions, in which fatty-methyl-esters made up 20-44% of the dry cell weight (DCW) and consisted of abundant C16:0 and C18:1 fatty acids. The red (orange)-stage cells also produced a large portion of carotenoids (38.5% of the DCW) involving echinenone, canthaxanthin, and astaxanthin as major components accumulated over only 5-6 days under optimal conditions. Stress tests revealed resistance of the cells to pH 2-11, high temperatures (40-60 °C), ultraviolet irradiation, drought, and H2O2 treatment, thereby showing a robust nature. Both green- and red (orange)-stage cell extracts also showed antioxidant and anti-inflammatory abilities, implying that they have significant functions as useful biorefinery materials.

Internationally harmonized certified reference materials and proficiency testings for pesticide residue analysis.

Matrix certified reference materials (CRMs) and proficiency testings (PTs) are effective for evaluation of the quality of analytical results. The National Metrology Institute of Japan has developed five kinds of CRMs and has provided eight PTs so far for the quantification of pesticide residues in foods. Target pesticides were sprayed on growing crops, and the harvests were used for the preparation of CRMs and PT samples. In most cases, multiple analytical methods based on isotope dilution mass spectrometry were used to ensure the reliability of certified values (for CRMs) and reference values (for PT samples). These activities were carried out with corresponding to the international standards such as ISO 17034 and ISO/IEC 17043. An overview of the development of CRMs and the implementation of PTs is described, with some examples.

Applicability of Superheated Water Extraction for the Quantification of Pesticide Residues in Leafy Vegetables.

Superheated water extraction (SWE) of pesticide residues in a cabbage sample was demonstrated. The recovery yields of several relatively polar pesticides (log Pow < 3) by the spike-and-recovery method at 100°C were acceptable. Increasing the extraction temperature up to 150°C led to enhanced extraction efficiency except for pesticides that induced degradation. The recovery yields of some target pesticides having log Pow values of 3.5 - 4 were effectively enhanced by increasing the number of extraction cycles. The observed concentration of fenitrothion in a cabbage sample was comparable with those by the official Japanese analytical method. These results suggested SWE is potentially suitable for the extraction of different relatively high-to-medium polarity pesticides.

Proficiency testing by the National Metrology Institute of Japan for quantification of pesticide residues in grain samples from 2012 to 2018.

From 2012 to 2018, proficiency testings (PTs) were carried out to support the analytical skill development of participants. Grains were sprayed with various target pesticides and used as material samples for PTs. It was found that 27-56% and 10-43% of participants used the Japanese official multiresidue method and a solid-phase extraction technique with the Quick, Easy, Cheap, Effective, Rugged, and Safe (modified QuEChERS, known as "STQ" in Japan) method, respectively. Reported results were evaluated using two types of z-scores: one was based on consensus values calculated from the analytical results reported by the participants, and the other was based on values obtained by the National Metrology Institute of Japan (NMIJ). Acceptable z-scores based on the consensus values and NMIJ reference values were achieved by 78-95% and 67-94% of the participants, respectively. Many laboratories improved their z-scores by continuing participation in our PT.

Characterization of scallop midgut gland certified reference material for quantification of diarrhetic shellfish toxins.

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ±â€¯0.061) mg/kg for OA and (0.45 ±â€¯0.11) mg/kg for DTX1.

Interlaboratory comparison of liquid chromatography-tandem mass spectrometry quantification of diarrhetic shellfish toxins in scallop midgut glands.

An interlaboratory comparison (ILC) was organized as a measure of the analytical competency in the liquid chromatography-tandem mass spectrometry quantification of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in scallop midgut gland samples. The test sample was prepared using boiled midgut glands of naturally contaminated scallops with DTX1 and its esters by spiking with OA, and homogeneity and stability of this test sample was assessed to be appropriate. Twenty laboratories participated in the ILC based on the Japanese official testing method; they submitted two sets of analytical concentrations of target analytes along with the details of their analytical protocols. For assessing these data, assigned values were established from another ILC where ten participants quantified the target analytes by the standard addition method. The mean analytical results of the former ILC showed good agreement with the assigned values, and the corresponding relative reproducibility standard deviations met the criterion of CODEX STAN 292. Meanwhile, the results of more than half of the participants were out of the uncertainty range of the assigned values; these participants were encouraged to investigate their protocols to improve their analytical capability.

Comparison of Assigned Values from Participants' Results, Spiked Concentrations of Test Samples, and Isotope Dilution Mass Spectrometric Results in Proficiency Testing for Pesticide Residue Analysis.

The Hatano Research Institute (HRI) at the Food and Drug Safety Center has recently organized a series of proficiency-testing (PT) programs called the "External Quality Control for Food Hygiene" in order to evaluate the analytical capability of testing laboratories that inspect food samples in accordance with the Food Sanitation Act. In one of these programs, Pesticide I, consensus values calculated from the participants' analytical results were used as assigned values, and the spiked concentrations of the prepared test samples were used for evaluating variation among individual participants. In the present study, the values obtained in the 2013-2015 rounds have been assessed by comparing the analytical results of the target pesticides obtained by using two different isotope-dilution MS (IDMS) methods. These two IDMS methods are based on a combination of different pretreatment protocols and different GC separation columns. The weighted means of the observed analytical results were higher than the corresponding assigned values, but showed good agreement with the spiked concentrations. These results indicate that the spiking concentrations of the test sample from HRI are reliable, and therefore, these values can be used to evaluate the trueness of the participants' analytical method.

Difference between Consensus Value of Participants' Results and Isotope-Dilution Mass Spectrometric Results in Proficiency Testing for Pesticide Residues in Husked Wheat.

Proficiency testing was organized by the National Metrology Institute of Japan (NMIJ) as a measure of analytical competency in the quantification of pesticide residues in husked wheat powder. Seventy-one participants submitted analytical concentrations of the target pesticides (diazinon, fenitrothion, malathion, and etofenprox) along with details of the analytical method employed. Two types of assigned values were obtained for each target pesticide, i.e., the participants' analytical results and the results obtained by NMIJ based on isotope-dilution mass spectrometry (IDMS). The former values were lower than the latter due to the incomplete recovery yield of the target pesticides during the pretreatment process. The discrepancy between the two assigned values was particularly pronounced for malathion because of the longer duration of water-soaking used for the test samples. Two corresponding types of z-scores were then calculated to evaluate the analytical performance of the participants, where the z1-score indicates the performance of a participant relative to all participants, and the z2-score indicates the relative deviation of the analytical results of the participant from the IDMS value.

Evaluation of the impact of matrix effect on quantification of pesticides in foods by gas chromatography-mass spectrometry using isotope-labeled internal standards.

The impact of the matrix effect in GC-MS quantification of pesticides in food using the corresponding isotope-labeled internal standards was evaluated. A spike-and-recovery study of nine target pesticides was first conducted using paste samples of corn, green soybean, carrot, and pumpkin. The observed analytical values using isotope-labeled internal standards were more accurate for most target pesticides than that obtained using the external calibration method, but were still biased from the spiked concentrations when a matrix-free calibration solution was used for calibration. The respective calibration curves for each target pesticide were also prepared using matrix-free calibration solutions and matrix-matched calibration solutions with blank soybean extract. The intensity ratio of the peaks of most target pesticides to that of the corresponding isotope-labeled internal standards was influenced by the presence of the matrix in the calibration solution; therefore, the observed slope varied. The ratio was also influenced by the type of injection method (splitless or on-column). These results indicated that matrix-matching of the calibration solution is required for very accurate quantification, even if isotope-labeled internal standards were used for calibration.

Certification of reference materials for the determination of alkylphenols.

Certified reference materials (CRMs) are playing an increasingly important role in national and international standardizing activities. In Japan, primary standard solutions for analyses of endocrine disrupters are supplied under the national standards dissemination system named the Japan Calibration Service System (JCSS). For the traceability on reference materials used for preparation of the primary standard solutions based on the JCSS, the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed and certified high-purity reference materials of alkylphenols as NMIJ CRMs, such as 4-n-nonylphenol, 4-tert-octylphenol, 4-n-heptylphenol, 4-tert-butylphenol, and 2,4-dichlorophenol. Thereafter, it is essential to determine the alkylphenols by using these solutions based on the JCSS for environmental monitoring and risk assessments because analytical values obtained by using the solutions can ensure the reliability and traceability of the chemical analyses.

Proficiency testing for determination of pesticide residues in soybean: comparison of assigned values from participants' results and isotope-dilution mass spectrometric determination.

Proficiency testing (PT) for the determination of pesticide residues in soybean samples was organized by the National Metrology Institute of Japan (NMIJ). The candidate certified reference material, NMIJ CRM 7509-a, that was prepared from the raw soybeans containing target pesticides (diazinon, fenitrothion, chlorpyrifos, and permethrin) was used as the test sample. Forty participants submitted two sets of analytical results along with the details of the analytical method and conditions they applied. Two types of assigned values were established for each target pesticide: one was derived from the analytical results of the participants, and the other was provided from the analytical results by isotope-dilution mass spectrometry (IDMS). The latter values were 7.4-16% higher than the former values, plausibly because the analytical values from the IDMS measurements were not affected by the recovery ratio of the target pesticides during the analytical process. Thus, two kinds of z-scores were calculated for individual participants using the corresponding assigned values: one (z1-score) showed the relative performance score for the present PT and the other (z2-score) could be used for evaluation of the trueness of their analytical methods.

Evaluation of the performance of 57 Japanese participating laboratories by two types of z-scores in proficiency test for the quantification of pesticide residues in brown rice.

A proficiency test for the analysis of pesticide residues in brown rice was carried out to support upgrading in analytical skills of participant laboratories. Brown rice containing three target pesticides (etofenprox, fenitrothion, and isoprothiolane) was used as the test samples. The test samples were distributed to the 57 participants and analyzed by appropriate analytical methods chosen by each participant. It was shown that there was no significant difference among the reported values obtained by different types of analytical method. The analytical results obtained by National Metrology Institute of Japan (NMIJ) were 3 % to 10 % greater than those obtained by participants. The results reported by the participant were evaluated by using two types of z-scores, that is, one was the score based on the consensus values calculated from the analytical results of participants, and the other one was the score based on the reference values obtained by NMIJ with high reliability. Acceptable z-scores based on the consensus values and NMIJ reference values were achieved by 87 % to 89 % and 79 % to 94 % of the participants, respectively.

Development of soybean certified reference material for pesticide residue analysis.

A soybean certified reference material for pesticide residue analysis was developed by the National Metrology Institute of Japan. Three organophosphorus (diazinon, fenitrothion, chlorphyrifos) and one pyrethroid (permethrin) pesticides were sprayed on soybeans three times before harvest. These soybeans were freeze pulverized, homogenized, bottled, and sterilized by γ-irradiation to prepare the candidate material. Three isotope-dilution mass spectrometric methods that varied in terms of the solvents used for extraction of the target pesticides, the clean-up procedure, and the injection techniques and columns used for quantification via gas chromatography/mass spectrometry were applied to the characterization. Each target pesticide was quantified by two of these analytical methods, and the results were in good agreement. Homogeneity and stability assessment of the material demonstrated that the relative standard uncertainties due to the inhomogeneity and the instability for an expiry date of 55 months were 1.89-4.00% and 6.65-11.5%, respectively. The certified pesticide concentrations with expanded uncertainties (coverage factor k=2, approximate 95% confidence interval) calculated using the results of the characterization and the homogeneity and stability assessment were 21.7 ± 3.2 µg/kg for diazinon, 88 ± 21 µg/kg for fenitrothion, 11.1 ± 3.2 µg/kg for chlorpyrifos, and 20.1 ± 4.3 µg/kg for permethrin (as the sum of the constituent isomers).

Variation in concentration of perfluorooctanoic acid in methanol solutions during storage.

Perfluoroalkyl carboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) have been widely recognized as persistent environmental contaminants. For accurate quantification of PFCAs, their stability in calibration solutions is important because they are criteria of quantification. To examine stability of PFCAs in methanol, we monitored PFOA and its related compounds around 4 years. Interestingly, perfluorooctanoate was varied randomly, and methyl perfluorooctanoate (MePFOA) and methyl formate were observed when perfluorooctanoate decreased. Moreover, no detection of both methyl esters was in methanol solutions immediately after preparation. In each of prepared methanol solution of perfluorohexanoic, perfluoroheptanoic, and perfluorononanoic acids, their corresponding methyl esters and methyl formate were observed. Furthermore, MePFOA was observed even in the solutions stored around 4 months and thereafter MePFOA increased with increase in methyl formate. Therefore, PFCAs including PFOA should be used immediately after preparation when methanol is used as a solvent.

Development of apple certified reference material for quantification of organophosphorus and pyrethroid pesticides.

An apple certified reference material for the analysis of pesticide residues was issued by the National Metrology Institute of Japan. Organophosphorus and pyrethroid pesticides were sprayed on apples, and these were used as raw materials of certified reference material. The harvested apples were cut into small pieces, freeze-dried, pulverized, sieved, placed into 200 brown glass bottles (3g each), and sterilized by γ-irradiation. Stability and homogeneity assessment was performed, and the relative uncertainties due to instability (for an expiry date of 32 months) and inhomogeneity were 10.3-25.0% and 4.0-6.8%, respectively. The characterization was carried out using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides were obtained by isotope dilution mass spectrometry. Certified values were 2.28 ± 0.82 mg/kg for diazinon, 3.14 ± 0.79 mg/kg for fenitrothion, 1.55 ± 0.81 mg/kg for cypermethrin, and 2.81 ± 0.70 mg/kg for permethrin.

Separation of parabens on a zirconia-based stationary phase in superheated water chromatography.

A superheated water chromatography (SWC) method for the separation of alkyl esters of 4-hydroxybenzoic acid (parabens) using a zirconia-based stationary phase was developed and applied to real sample analysis. First, the SWC system was optimized in terms of the proper length of the preheating coil for establishing thermal equilibration of the mobile phase entering the column at the oven temperature. Next, the effect of the column temperature on the retention was investigated at 100-180°C. The elution time for all parabens decreased with increasing column temperature, and linear relationships between ln k and 1/T were obtained. At higher column temperatures, the elution time was further shortened because of the increased mobile-phase flow rate. Nevertheless, the loss of column efficiency at the higher flow rates was not significant. The application of the present method to the analysis of commercial lotions was then demonstrated. The quantification results obtained from SWC showed good agreement with those from a conventional HPLC method.

Evaluation of pressurized liquid extraction for determination of organophosphorus and pyrethroid pesticides in soybean.

The purpose of this study was to develop an analytical method for the determination of organophosphorus and pyrethroid pesticides in soybean by pressurized liquid extraction (PLE). Two organic solvents, acetone and acetonitrile, were evaluated as extraction solvents. In both cases, the amount of extract was enhanced with increasing extraction temperature. The extracts obtained using acetonitrile were measured by gas chromatography/mass spectrometry after a cleanup process based on the analytical method for the Japanese Positive List System for Agricultural Chemicals Remaining in Foods. The effect of extraction temperature (range: 40- 130°C) on extraction efficiency was evaluated by a recovery study using 21 organophosphorus pesticides and 10 pyrethroid pesticides as target analytes and acetonitrile as the solvent. The results indicated that at 130°C, some organophosphorus pesticides might be degraded, whereas extraction temperatures between 70°C and 100°C were optimal. Next, a prepared sample containing fenitrothion and permethrin was analyzed. Although the sample was not soaked in water prior to analysis, PLE provided analytical results comparable to those obtained by solvent extraction with homogenization. Therefore, PLE is considered a simple and alternative technique for the extraction of organophosphorus and pyrethroid pesticides in soybean.

Evaluation of the thermal effect on separation selectivity in anion-exchange processes using superheated water ion-exchange chromatography.

The thermal effect on retention and separation selectivity of inorganic anions and aromatic sulfonate ions in anion-exchange chromatography is studied on a quaternized styrene-divinylbenzene copolymer anion-exchange column in the temperature range of 40-120 °C using superheated water chromatography. The selectivity coefficient for a pair of identically charged anions approaches unity as temperature increases provided the ions have the same effective size, such that the retention of an analyte ion decreases with an increase in temperature when the analyte ion has stronger affinity for the ion-exchanger than that of the eluent counterion, whereas it increases when it has weaker affinity. The change in anion-exchange selectivity with temperature observed with superheated water chromatography has been discussed on the basis of the effect of temperature on hydration of the ions. At elevated temperatures, especially in superheated water, the electrostatic interaction or association of the ions with the fixed ion in the resin phase becomes a predominant factor resulting in a different separation selectivity from that obtained at ambient temperature.

Evaluation of microwave-assisted extraction for the analysis of organophosphorus and pyrethroid pesticides in green onions.

The aim of this study was to evaluate the efficiency of microwave-assisted extraction (MAE) for the analysis of organophosphorus (OP) and pyrethroid (PYR) pesticides in green onions by GC/MS. We optimized MAE extraction solvent, temperature, and time by using a certified reference material. As a result, the concentrations of two OP and two PYR target pesticides obtained by MAE with acetonitrile at 110 degrees C for 10 min were in good agreement with certified concentrations and comparable to the results by homogenization used as reference extraction technique. When the recovery test, performed by spiking the target pesticides into blank samples (5.0 g), was carried out with our optimized MAE conditions, mean recoveries of 16 OP and 10 PYR pesticides were 72-108% for a 1.0 pg spiking level and 70-119% for a 0.2 microg level. These results were acceptable according to the validation guideline for testing method of agricultural chemicals in food by Ministry of Health, Labour, and Welfare in Japan. The results suggested that MAE can be used for the analysis of OP and PYR pesticides in green onions.