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Determination of chlormequat and mepiquat residues and their dissipation rates in tomato cultivation matrices by ultra-performance liquid chromatography-tandem mass spectrometry.

This study described the development and validation of a simple, rapid, specific and sensitive method for detecting chlormequat chloride (CQ) and mepiquat chloride (MQ) residues in tomato cultivation matrices covering soil, water, seedling samples. The dissipation rates of CQ and MQ in tomato cultivation matrices were also determined in this study. A Hydrophilic Interaction Liquid Chromatography (HILIC) column was used for chromatographic separation. A triple quadrupole mass spectrometer equipped with an electrospray ionisation source in positive ion mode by multiple reaction monitoring was used for detection. Soil samples were extracted with accelerated solvent extraction (ASE) and cleaned up with WCX phase extraction column; water samples were extracted with WCX phase extraction column; seedling samples were extracted with methanol-ammonium acetate solution. LODs and LOQs of CQ and MQ were 0.02μg/kg and 0.1μg/kg in soil samples, 0.005ng/mL and 0.02ng/mL in water samples, and 0.05μg/kg and 1.0μg/kg in seedling samples, respectively. The mean recovery rate of CQ in soil, water and seedling samples ranged from 76.98% to 111.60%. While the mean recovery rate of MQ in soil, water and seedling samples ranged from 96.90% to 105.40%. The fastest to the slowest metabolising rates of CQ and MQ were as follows: soil samples>seedling samples>water samples. In conclusion, this study provided a new potential method for detecting CQ and MQ in tomato cultivation matrices using ultra-performance liquid chromatography-tandem mass spectrometry.

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[HPLC-MS/MS determination of residual amount of 4 plant growth retardants in 6 dried root and Rhizome Herbs].

HPLC-MS/MS was applied to the determination of residual amount of plant growth retardant such as paclobutrazol, daminozide, chlormequat and mepiquat chloride in dried root and rhizome herbs. The sample was extracted twice with acetonitrile containing 0.1% formic acid. The separation was performed on a Waters Atlantis HILIC column with an elution system consisting of acetonitrile-5 mmol•L⁻¹ ammonium acetate solution with 0.1% formic acid, methanol and acetonitrile. The MS spectrum was acquired in positive mode with multiple reactions monitoring (MRM). The linear range was 6-1 500 μg•kg⁻¹, and the optimized method offered a good linear correlation (r>0.997 8), excellent precision (RSD<11%) and acceptable recovery (from 79.3% to 103.3%). Four kinds of plant growth retardant have detected in some ofhenise herbs like Ophiopogonis Radix, Angelicae Sinensis Radix, Achyranthis Bidentatae Radix, Alismatis Rhizoma, Chuanxiong Rhizama and Notoginseng Radix et Rhizama, is among the more severe cases, dwarf lilyturf, multi-effect azole detection quantity is 63.4~1 351.66 μg•kg⁻¹, and Daminozide was detected in Ophiopogonis Radix, Angelicae Sinensis Radix, Chuanxiong Rhizama, Alismatis Rhizoma.

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Fast analysis of quaternary ammonium pesticides in food and beverages using cation-exchange chromatography coupled with isotope-dilution high-resolution mass spectrometry.

A fast separation based on cation-exchange liquid chromatography coupled with high-resolution mass spectrometry is proposed for simultaneous determination of chlormequat, difenzoquat, diquat, mepiquat and paraquat in several food and beverage commodities. Solid samples were extracted using a mixture of water/methanol/formic acid (69.6:30:0.4, v/v/v), while liquid samples were ten times diluted with the same solution. Separation was carried out on an experimental length-modified IonPac CS17 column (2 × 15 mm) that allowed the use of formic acid and acetonitrile as mobile phase. Detection limits for food and beverage matrices were established at 1.5 μg/L for chlormequat, difenzoquat and mepiquat, and 3 μg/L for diquat and paraquat, while for drinking water a pre-analytical sample concentration allowed detection limits of 9 and 20 ng/L, respectively. Precision, as repeatability (RSD%), ranged from 0.2 to 24%, with a median value of 6%, and trueness, as recovery, ranged from 64 to 118%, with a median value of 96%. The method developed was successfully applied to investigate the presence of herbicide residues in commercial commodities (mineral water, orange juice, beer, tea, green coffee bean, toasted coffee powder, cocoa bean, white corn flour, rice and sugar samples).

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The skeletal developmental toxicity of chlormequat chloride and its underlying mechanisms.

Chlormequat Chloride (CCC), a widely used plant growth regulator, could decrease body weight in animals; however, the mechanism has not been well studied. This study was designed to evaluate the skeletal development toxicity of CCC on pubertal male Sprague-Dawley (SD) rats and to investigate whether CCC impacts the development of chondrocyte, osteoblast and osteoclast through growth hormone (GH) and insulin like growth factor 1 (IGF-I). Rats from 23 to 70 on postnatal days were exposed to CCC daily by gavage at doses of 0, 75, 150, and 300mg/kg bw/d. The results showed that the size of femurs and tibias, bone mineral density and biomechanical parameters were significantly decreased in the 300mg/kg bw/d group compared with the control group. The concentration of osteocalcin (OCN) and C-terminal telopeptide of type I collagen (CTX-I) in blood in the 150mg/kg bw/d group was also changed. The mRNA expression ratio of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in 150 and 300mg/kg bw/d group was increased. Histological analysis of proximal and distal epiphyseal plates of the right femurs showed that both the proliferative zone and hypertrophic zone narrowed in CCC-treated groups. The concentration of IGF-I in blood was reduced with an increase in exposure doses of CCC. The mRNA expression of growth hormone receptor (GHR) in tibia was decreased in the CCC-treated group. The results indicated that CCC might indirectly impact the formation and activation of chondrocytes, osteoblasts and osteoclasts because of the decline of GHR and IGF-I, leading to skeletal development damage.

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The effects of chlormequat chloride on the development of pubertal male rats.

Chlormequat Chloride (CCC) is a plant growth regulator that is widely applied in agriculture. Previous studies have shown that long-term exposure of CCC could decrease body weight in animals. However, the underlying mechanisms have not been studied. In this study, CCC was administered to rats daily by gavage on postnatal days 23-60 at doses of 0, 75, 150 and 300mg/kg bw/d. The results showed that body weight and the length of the right femur were significantly decreased in the 300mg/kg bw/d group. Histological analysis of proximal growth plates of the right femurs showed narrowed proliferative zones and hypertrophic zones in CCC-treated groups. The mRNA expression of growth hormone, growth hormone receptor and insulin like growth factor 1 were decreased in the CCC-treated group. The results indicated that CCC may affect the expression of growth hormone and insulin-like growth factor 1 and subsequently cause a decrease in body weight and bone length.

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Multi-residue analysis of pesticides, plant hormones, veterinary drugs and mycotoxins using HILIC chromatography - MS/MS in various food matrices.

One of the recent trends in Analytical Chemistry is the development of economic, quick and easy hyphenated methods to be used in a field that includes analytes of different classes and physicochemical properties. In this work a multi-residue method was developed for the simultaneous determination of 28 xenobiotics (polar and hydrophilic) using hydrophilic interaction liquid chromatography technique (HILIC) coupled with triple quadrupole mass spectrometry (LC-MS/MS) technology. The scope of the method includes plant growth regulators (chlormequat, daminozide, diquat, maleic hydrazide, mepiquat, paraquat), pesticides (cyromazine, the metabolite of the fungicide propineb PTU (propylenethiourea), amitrole), various multiclass antibiotics (tetracyclines, sulfonamides quinolones, kasugamycin and mycotoxins (aflatoxin B1, B2, fumonisin B1 and ochratoxin A). Isolation of the analytes from the matrix was achieved with a fast and effective technique. The validation of the multi-residue method was performed at the levels: 10 μg/kg and 100 μg/kg in the following representative substrates: fruits-vegetables (apples, apricots, lettuce and onions), cereals and pulses (flour and chickpeas), animal products (milk and meat) and cereal based baby foods. The method was validated taking into consideration EU guidelines and showed acceptable linearity (r ≥ 0.99), accuracy with recoveries between 70 and 120% and precision with RSD ≤ 20% for the majority of the analytes studied. For the analytes that presented accuracy and precision values outside the acceptable limits the method still is able to serve as a semi-quantitative method. The matrix effect, the limits of detection and quantification were also estimated and compared with the current EU MRLs (Maximum Residue Levels) and FAO/WHO MLs (Maximum Levels) or CXLs (Codex Maximum Residue Limits). The combined and expanded uncertainty of the method for each analyte per substrate, was also estimated.

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Fatal poisoning with plant growth regulator - chlormequat.


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Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model.

In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim, chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B>A) directed transport of [(14)C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates.

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Chlormequat chloride retards rat embryo growth in vitro.

Chlormequat chloride is the most widely used plant growth regulator in agriculture to promote sturdier growth of grain crops by avoidance of lodging. Therefore, human exposure to chlormequat chloride is very common, but its developmental toxicity has not been studied. Thus, we investigated the developmental toxicity of chlormequat chloride by applying rat whole embryo culture (WEC) model, limb bud micromass culture and 3T3 fibroblast cytotoxicity test. Chlormequat chloride at 150μg/ml (0.93mM) retarded the rat embryo growth without causing significant morphological malformations and at 500μg/ml (3.1mM) caused both retardation and morphological malformation of the embryos. However, the proliferation and differentiation of limb bud cells were not affected by chlormequat chloride at as high as up to 1000μg/ml (6.2mM) applied. This concentration of chlormequat chloride did not affect the cell viability as examined by 3T3 fibroblast cytotoxicity test either, suggesting that cellular toxicity may not play a role in chlormequat induced inhibition of rat embryo growth. Collectively, our results demonstrated that chlormequat chloride may affect embryo growth and development without inhibiting cell viability.

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Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus.

The plant-specific TCP transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. However, no comprehensive analysis of the TCP family in watermelon (Citrullus lanatus) has been undertaken previously.

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