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A lack of published analytical method for determination of chlorfluazuron(CFZ) in human plasma has prompted concerns about their safety and toxicity . Since most of published methods describe the determination of CFZ in plant, soil, air and water not in plasma the importance of this paper is clear.
This method based on pressurized liquid chromatography and LC-MS/MS has been developed for determining chlorfluazuron in human plasma.
Chromatography was performed on a Luna 5 µm C18(2) (Phenomenex) 50-2.0mm.The mobile phase was a binary gradient water-acetonitrile with 0.1% formic acid at flow rate of 0.25 ml min−1. The LC separation and MS/MS optimization were studied to select the most appropriate operating conditions. The method developed has also been validated. The limits of quantification (LOQs) was from 20ng ml-1.The LC-MS/MS method developed is simple, rapid, accurate, sensitive, and reliable for the quantification and confirmation of(CFZ) in human plasma.CFZ was quantified in human plasma. Excellent linearity was achieved over a range of concentrations from 20ng to 2000ngml-1 with correlation coefficients0.995-0.999 (n=3).The combination of pressurized liquid chromatography and LC-MS/MS provides a sensitive and selective method for the determination of CFZ in human plasma .
Benzoylureas (BUs) constitute an important group of pesticides with herbicide, insecticide, or acaricide activity that act as insect growth regulators [1, 2]. These compounds were introduced in 1978 by Bayer of Germany; the first was triflumuron (Alsistyns). Others appearing since then are chlorfluazuron (Atabrons, Helixs), followed by teflubenzuron(Nomolts, Darts), hexaflumuron (Consults), flufenoxuron (Casades) and flucycloxuron (Andalins) [3, 4]. Their mode of action is the inhibition of chitin synthesis in the cuticle of the insect with rupture of malformed cuticle or death by starvation. The herbicide systemic activity of the BU fluometuron is the inhibition of the photosynthesis in crops of cottons, sugarcane, leguminous soy, and tomatoes . BUs began to be used in Central America in 1985, to control a severe, resistant leafworm complex (Spodoptera spp., Trichoplusia spp.) outbreak in cotton. Their greatest value is the control of caterpillars and beetle larvae [6-9].Although these pesticides show a low toxicity for mammals because the chitin is absent in plants and in vertebrates,their residues can often reach populations through the food chain causing chronic exposure and long-term toxicity effects [10-12].In laboratory animals, chlorfluazuron had very low acute toxicity if ingested, inhaled or exposed on the skin. It was slightly irritating to the eyes but did not cause skin irritation or allergic reactions when applied to the skin. Chlorfluazuron was absorbed only to a limited extent when swallowed and excreted mainly in the faeces.Short and long-term exposure to low concentrations of chlorfluazuron in the diet was without serious consequences in animal studies. The only consistent finding was an increase in cholesterol levels. Chlorfluazuron did not cause birth defects in laboratory animals.There is evidence to indicate that chlorfluazuron persists and bio-accumulates in the body. In rats, high levels of chlorfluazuron residues were deposited in fat and depletion was slow, with a depletion half-life of approximately 42 days. Goats and hens secreted chlorfluazuron into milk and egg yolk, respectively. If given in the diet of food-producing animals, there is a significant potential for accumulation and retention of residues in the fat and for excretion in milk.As chlorfluazuron is used in subterranean termite baiting stations, public exposure is unlikely to occur. However, due to its persistent and bio-accumulative nature, care needs to be taken not to misuse the chemical.Chlorfluazuron partitions primarily to the soil/sediment compartments where it adsorbs strongly to soil organic matter. It is moderately persistent in soil and movement to watercourses in runoff must be avoided. It is highly toxic to aquatic arthropods such as daphnia due to its chitin inhibiting properties, but is of low toxicity to mammals, birds, bees, fish, soil micro-organisms and the earthworms.Pesticide residue levels are generally regulated through various national and international standards, established as maximum residue limits (MRLs) .. The only tool to control the quantity of pesticides in food and to enforce tolerances is their analytical determination in samples [14, 15]. Although MRLs are legislated for many food types, control and screening in the literature as well as in the laboratories are really restricted to pesticide residue determination in fruits and vegetables [10, 16-19].There is a need to increase the applicability of analytical methods to nearly any food type currently in the market. Most used techniques to determine pesticide residues in fruits and vegetables are GC and LC [19-23]. Generally,because of their high polarity and low volatility, the BUs are analyzed by LC with UV [4, 7], fluorescence [9, 15], chemiluminescence or MS [4, 8, 10, 14] including those MS detectors capable of MS/MS, triple quadrupole (QqQ)[24-26] and IT [25, 26]. Several analytical studies concerned to the determination of not only BUs but also simultaneously with other types of pesticide have been recently reported. Zrostlikova et al. . Based on that self-poisoning with pesticides is a major public health problem across the Asia Pacific Region.  , the lack of published analytical method for determining CFZ in human plasma , It is very important for us to developed a method for determining CFZ in human plasma .
2.1. 2.2. Sample preparation
Materials and standards
Chlorfluazuron (CFZ) Fenfuracarb(FBC) and was supplied by Riedel-de-Haen-(Germany).
Individual stock solutions were prepared by dissolving 2.5mg of each compound in 10 ml of methanol in case of Chlorfluazuron (CFZ) were stored in glass-stopper bottles at 4 °C. Standard working solutions at various concentrations were daily prepared by appropriate dilution of aliquots of the stock solutions in aacetonitrle .
HPLC-grade solvents ( acetonitrile, methanol and isopropanol were supplied by Merck (Darmstadt, Germany), while Formic acid was supplied by Fluka analytical(Germany).
Deionized water (<8 MΩ cm resistivity) was obtained from the Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA). All the solvents and solutions were filtered through a 0.45-μm cellulose filter from Scharlau (Barcelona, Spain) before use.
2.2. Sample preparation:
Sample preparation was performed by protein precipitation with acetonitrle . One hundred microliter aliquots of plasma from calibration samples, quality control samples, or clinical plasma samples were transferred to 0.5 ml microcentrifuge tubes. Add 20 μL of the appropriate standard stock to the standards, 20 μL of buffer to the test samples and QCs ,20 μL of ISW to all tubes except the blank. Add 20 μL of buffer to the blank. Then add 200 µL of Acetonitrile to all samples. Vortex mix for ca. 5s. Centrifuge (5min at 1000g).Decant the supernatant into autosampler vials and cap then centrifuge the auto sampler vials (5min at 1000g).
2.3 Sample quantification
Concentrations of CFZ were determined based on the ratio of their peak area for its monitored mass transition and the peak area of the mass transition characteristic for the internal standard(FBC). A calibration curve covering the entire therapeutically used plasma concentration range was established for CFZ using quadratic regression analysis of the ratio of analyte peak area/internal standard peak area versus analyte concentration with a weighting factor of 1/x2 . Unknown CFZ concentrations were calculated from the calibration curve based on the measured peak area ratios for the CFZ monitored
2.4.LC/MS and LC/MS/MS analysis
Separation was achieved on a Luna 5 µm C18(2) (Phenomenex) 50-2.0mm preceded by a C18security guard cartridge Gemini 5 μm C18 (Phenomenex) 4 x 3 mm). The mobile phase mobile phase A is 0.1% formic in water, while mobile phase B is 0.1% formic acid in in 95:5 methanol : water,at a flow rate of 0.25 ml min−1. The elution was gradient at zero time. The % of B was 0% then the % of B will gradually increase till became 100% at 2 min and kept till 3.95 min then at 4 min the % of B was dropped to 50% and finally pump was stoped at 7 min.Detection was performed using a MDS Sciex API2000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) that was operated in positive ion mode with turbo electrospray ionization. All analyses were performed in the multiple reaction monitoring (MRM) mode. Instrument control and data acquisition was performed using the Analyst v1.4.2 software package (Applied Biosystems, Foster City, CA).Optimization of the detection conditions was performed by direct infusion of the analytes (1 µg/ml, dissolved in methanol) from a syringe pump into the mass spectrometer.
The auto tuning function of the Analyst software was used, and the optimized parameters were used for the detection of CFZ .The parameter settings were as follows:
turbo ionspray gas 40/min, nebulizer (nitrogen) gas 40.00 psi, curtain gas 20.00 psi, collision-activated dissociation gas 2.00 psi, ionspray voltage 5000 V, temperature 400 °C, focusing potential 200 V declustering potential 40 V, entrance potentials 9 V, collision energy 29 V, collision cell exit potential 8 v.The mass spectrometer was tuned, optimizing the ionization source parameters, voltages of the lenses and trap conditions in the Expert Tune mode of the using Analyst software.whilst infusing a standard solution (1 μg ml−1) via a syringe pump at a flow rate of 4 μl min−1, which was mixed with the mobile phase at 0.5 ml min−1 by means of a T-piece. Operating conditions of the source were the same.The mass spectrometer was run in full scan and MRM modes. Positive ions were detected using the standard scan at normal resolution (scan speed 10 300 m/z/s; peak with 0.6 FWHM/m/z).
3. Results and discussion
3.1. Liquid chromatography-mass spectrometry
Chemical structures and molecular weights of the CFz and FBC together with the main ions obtained by LC/ESI-MS operating in positive polarity, with their relative abundances and tentative assignation.
For each compound , the ions monitored in Multiple radical ion monitoring (MRM) were the base peak of the mass spectrum, corresponding to the protonated molecule [M + H]+. For (CFZ), we observed, in agreement with what is reported in the literature 
The base peak of the mass spectrum was subjected to the first stage of MS/MS for all studied compounds. The MS2 analysis of tested pesticides with the ESI interface in positive polarity provided ions for all of them.
Chemical structures and molecular weights of the studied compounds together with the main ions obtained by LC/ESI-MS operating in positive polarity, with their relative abundances and tentative assignation, are shown in tables (5,6).
Based on the chemical structures of the analytes, an electrospray ionization interface (ESI) was used for ion generation. A Q1 full scan of each analyte and IS were acquired in both positive and negative mode when tuned under constant infusion at 600 µl/h of a 1 µg/ml methanol solution of the analytes. The signal-to-noise ratio was used as the measure of sensitivity . The positive ion mode of the ESI was selected for all analytes and IS due to a greater sensitivity compared to the negative ion mode. The protonated form of the analyte molecules [M + H]+
A chromatogram acquired from a blank human plasma sample spiked with 1000 ng/ml FBC , CSN
and 2000n g/ml CFN is shown in Fig. 1 . All of these analyte were in the same mixture with EPX as internal standard.For all analytes, good linearity in the calibration curves was achieved with coefficients of determination ofR2 > 0.992. The calibration curve for CFZ the assay was allowed quantification in a range of 20 to 2000 ng/ml .
Fig(1) Fig(1): Chromatogram CFZ in presence of FBC(IS) at ULOQ
Method of validation
3.2.1. Linearity and LLOQ.
Linearity for CFZ was validated from the corrected peak areas of spiked control plasma samples, containing working solution aliquots of 20 - 0000ng mL−1 and a standard concentration of I.S. at 50 ng mL−1.Three sets of standard of mix containing CFZ and IS in plasma . The chromatographic method is applied and responses have been expressed as the compounds peak areas corrected to the I.S. area.
3.2.2. Matrix selectivity
Six different blank plasma named as PL001, PL002, PL003, PL004,PL005,PL006 and standard st#2 and st#n-1 were used to carry out the chromatographic analysis. They named as PL001, PL002, PL003, PL004, PL005, and PL006 .We found that the six blank plasma do not interfere with any peak of CFZ or even the IS.
3.2.3. Intra-assay precision and accuracy.
Three level of QC was prepared and named as (QCH, QCM and QCL) the repeatability of this method was done in same day and hopefully the QC passed the test.
3.2.4.Inter batch precision and accuracy.
The analysis of clinical samples involved the analysis of the three QC level. clinical samples were arranged into 10 batches each of them contain the standards, three QC level, blank and zero blank. examples of these batches were showed in table(1).
We measure the following stability
1) Bench stability: € 3 hours at room temperature on the lab bench.
2) Freeze/ thaw :€ 1 cycle (ie. freeze to -80C , thaw and the assay
3.2.6Extract stability inside auto sampler
This validation test was done to see the stability of acetonitrilic extract inside the auto sampler.
Summary of all the above validation is in table (1).
Table (1) :Summary of validation
Comments / references
n = 3
PAR = -8.76-08 [CFZ]2 + 0.00105[CFZ) +0.00327
n = 6
tested: PL001, PL002, PL003, PL004,PL005,PL006
Intra-assay precision and accuracy n = 8
Inter assay precision and accuracy n=5
24 hr on auto sampler
3.4. Clinical samples
We receive 42 clinical samples (plasma) for patients expose to pesticides poising.
We apply our method to determined the concentration of each analyte .
3 of these samples were out of concentration range, so it was diluted and re assayed.
The concentration range of these samples was (86.5.---2800) ng ml-1
The procedure described here was used to test the stability in matrix under any Conditions.
The proposed method was successfully developed and validated to provide reliable, sensitive and specific data for the biological monitoring of the most widely used methyl benzyl urea pesticides chlorfluazuron human plasma .This method could be employed to provide more accurate explanations for occupational or acute exposures to this pesticide from now on.