DEVELOPMENT AND INDEPENDENT LABORATORY VALIDATION OF AN ANALYTICAL METHOD FOR THE DETERMINATION OF OXYACETANILIDE RESIDUE AND ITS METABOLITES IN TRITICUM AESTIVUM (GRAIN & STRAW) BY LC-MS/MS TANDEM MASS SPECTROMETRY

A novel and robust high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of residue of oxyacetanilide as its metabolitesnamely the N-fluorophenyl-N-isopropyl in Triticum Aestivum (Grain & Straw). The performance characteristics included linearity, specificity (matrix interferences), LOQ, reproducibility (%RSD) and trueness (%recovery). The method is, therefore, compliant with the definition of the residue for oxyacetanilide containing the N fluorophenyl-N-isopropyl as oxyacetanilide equivalent. This design specifically evaluates the effects of sample extraction interferences.


ISSN: 2320-5407
Int. J. Adv. Res. 8(02), 738-750 739 serious food shortages despite a rapid population increase.As reported earlier that 45% of the annual food construction is depleted by pest infestations 2 , effective pest organisation seems one of the main strategies to increase crop output for a rapidly growing population, that requires application of a wide variety of pesticides in agricultural fields to contest pests.
Pesticides have been broadly used all over the world since the20th century. These chemicals belong to various classes. Organochlorine, pyrethroid, herbicide, fungicide, nicotinoid and organophosphorus compounds, are the most vital groups. The Phys-chem characteristics of insecticides may differ significantly. Some chemicals contain halogens, others phosphorous, sulphur, chlorine or nitrogen. Herbicides play an important role in modern agriculture.Several compounds are very volatile and unstable, but several do not vanish at all. This assortment causes serious difficulties in the expansion of a worldwide residue analytical method, which should have the broadestpossibility possible. TheseMRM methods are instantlydesirable. Possibly, no other use of chemicals is regulated more extensively than that of pesticides. MRLrecognized for pesticides in crops and drinking water in utmost countries to avoid any antagonistic impact on public strength.Insecticide residues in surface water may cause adverse effects on marinecreatures. For these reasons,many laboratories are involved in the investigation of MRL or in the identification and quantification of these residues in ecologicalmedia. In this context, the use of several single-residue methods is usually too expensive. It must be noted that eachcorporation which applies for registering of a new insecticidemust provide residue analytical information. Contingent on the purpose, determination of pesticide residues may be target analysis or non-target analysis. These different chemicals act on a surprisingly low number of molecular targets in the plant. 3,4 An example of target analysis is the inspection of MRLs in food. The relevant analytes are fixed by the residue definition given in the MRL regulation. These residue definitions may include relevant metabolites or degradation products of the pesticides.
Wheat (Triticum aestivum) is leading food grain crop being a staple diet and prime importance in the realms of food crops in the world. It is foremost among cereals and as a main source of carbohydrates and protein for both human beings and animals; contains starch (60-90%), protein (11)(12)(13)(14)(15)(16).5%), fat (1.5-2%), inorganic ions (1.2-2%) and vitamins (B-complex and vitamin E). 5,6 In the past decades, many hypotheses have been made concerning the primary target sites of these herbicides. In general, these herbicides are more active on monocotyledonous plants than thedicotyledonous plants. The accumulation of pesticides in food commodities may have serious cascading effects on human health and ecosystems throughfood chains.Therefore, it is of great importance to investigate the uptake and accumulation behaviours of pesticides. The total area of the world under wheat is around 212.99 million ha with a grain yield of 596.20 million tons. 7 The most important species of wheat is Triticum aestivum occupying 85% of the total area under wheat cultivation. Weeds infestation is one of the major threats to crop growth and yield. Weeds compete with crop plants for nutrients, solar radiation, water, carbon dioxide, space, and many other growth factors. The fast-growing population of the country makes it imperative to achieve matching increases in the rate of food production.
Oxyacetanilide {N-(4-fluorophenyl)-N-(1-methyl-ethyl)-2-[5-(trifluoromethyl)-1, 3, 4-thiadiazol 2-yl] oxy] acetamide}, a selective herbicide to control grassy weeds in a wide range of crops disinclining cereals. 8,9 The objectives of this study were to develop a robust and reliable analytical method for the extraction, separation and identification of N-fluorophenyl-N-isopropyl in grain and straw extracts; for the purpose of accurate quantification using oxyacetanilide. The work was to independently validate for the determination of residue of oxyacetanilide in Wheat is the principal staple food crop for rural societies in Gujarat. This research work provides the framework for efficient, reproducible, specific, linear, accurate, high throughput, and costeffective analytical approach for the analysis of oxyacetanilide and its metabolite in Grain & Straw for residue analysis. Analytical method validation isapillar for standardising reference methods for its intended purpose. Laboratory studies provide reliable data forregulatory requirements, science, and quality control requirements.

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Sample collection:-Triticum Aestivum (Wheat grain and straw) specimens originating from the agricultural region of Saurashtra Gujarat, India and homogenized using a cutting mill for straw and a knife mill for grain samples, and stored in the deep freezer until extraction.
The individual stock solutions of oxyacetanilide(?) and 4-Fluoro-N-Isopropylaniline at 1 mg/mL were prepared in methanol. These stock solutions were used to prepare working standard solutions at 0.00008, 0.0001, 0.0004, 0.002, 0.004, 0.008 and 0.01 mg/L. The working standard solution prepared in 0.1 % formic acid in milli Q water (80%) and 0.1 % formic acid in methanol (20%). The stock, intermediate and working solutions were maintained at refrigerator condition (2-8°C).
Blank grain and straw samples were chosen for the preparation of matrix-interferences and recovery studies. Specificity was measured by analyzing blank wheat straw and grain sample evaluate potential interferences from endogenous components. Also, a comparison was made of the tR of standard solutions of compounds. In the validation study, the concentration of oxyacetanilide standard spiked-wheat (straw and grain) sample were selected to be at around 0.05 and 0.01 mg/L respectively as LOQ level. However, for 10 x LOQ level,this concentration level wasselected to be at around 0.5 and 0.1 mg/L respectively. For each concentration, the standard solution mixture of oxyacetanilide was spiked into wheat (straw and grain) (n=5).
For stock solution stability the stock solutions of oxyacetanilide and 4-fluoro-N-isopropylaniline were prepared and stored in refrigerator condition (2-8 °C) and compared with the freshly stock solutions of 4-fluoro-N-isopropylaniline and oxyacetanilideafter 21 days and 26 days respectively. For linearity solution stability, a 4-fluoro-N-isopropylaniline solutionwas prepared and stored in refrigerator condition (2-8 °C) and subsequently compared with the freshly prepared 4fluoro-N-isopropylanilinesolution after 31 days. For extract solution stability, wheat grain and straw at LOQ and 10 x LOQ level were extracted and analysed on the day 0 and stored in refrigerated condition (2-8 °C). After 27 days for wheat straw and 21 days for wheat grain, the stored extract solutions were analysed. (n = 3 for each).

Calculation:-
The below-mentionedequation was used to calculate the 4-fluoro-N-isopropylaniline residue R in samples for the transition 154 m/z -> 112/95 m/z: = C End x Multiplier M x DF Where: R: Residue found, in mg/kg C End : Final concentration of the analyte in the extract, in ng/mL V Ex : Water extract volume: 250 mL VR1: Aliquot of water extract: 10 mL. V End : Final volume: 5.0 mL (wheat grain), 25 mL (wheat straw) W: Specimen weight used: 5.0 g DF: Dilution factor Results and Discussion:-Optimisation of Extraction and Clean-up Procedure:-An amount of 5.0 g sample was taken into a 500 mL round bottom flask (RBF) and add water (40 mL for grain and 80 mL for straw). RBF was placed on magnetic stirring and the mixture was stirred for 1 h at room temperature. Then 5 mL of aqueous 1 N H 2 SO 4 was added and stirred for 2 minutes. An amount of 1 g KMnO 4 was added and the mixture was stirred for 5 min. The colour of the entire mixture appeared purple. Afterwards, 1 g of sodium bisulfite-sodium bisulfate-mixture was added to the flask and the mixture lost whole purple colour. A reflux condenser was attached on to the round bottom flask and 25 mL concentrated H 2 SO 4 was added through the reflux condenser into the flask. The mixture in the flask was heated and boil under reflux for at least 20 h. The mixture was allowed to cool for approximately 30 minutes and 100 mL of water 741 would be added through the reflux condenser. The reflux condenser was removed and the mixture filtered through a folded filter into a 250 mL graduated measuring cylinder. The cylinder was filled up to 250 mL with water. 10 mL of the extract was taken into a separating funnel containing 90 mL of water, the pH value wasin the range of 0.7 to 1. A volume of 20 mL of dichloromethane was added to the separating funnel, the funnel was shaken for 1 minute vigorously by hand and the organic (lower) phase was disposed of. This rinsingstep was repeated once again. 5 mL of 50 % aqueous sodium hydroxide solution (in an ice bath during addition) was added into the aq. phase, the mixture was stirred with a glass rod and pH was checked and the value of the mixture in the separation funnel wasin the range of 12.2 to 12.4.
A volume of 20 mL of dichloromethane was added and the separation funnel, shaken for 1 min. The lower phase was filtered through a glass funnel with anhydrous sodium sulphate into a round bottom flask. The sodium sulphate was wetted with dichloromethane before filtration. This step was repeated twice. 0.10 % formic acid in water was added to the extract (20 mL for wheat straw and 4 mL for wheat grain) as the keeper. The filtrate in the round bottom flask was evaporated in a rotary vacuum evaporator to the volume of the keeper. Water was added gravimetrically to the target water content (20 mL for wheat straw and 4 mL for wheat grain) and methanol was added (5 mL for wheat straw and 1 mL for wheat grain) and mixed. The mixture was ultra-sonicated for about 30 seconds.   The total analysis time was 9 min for the developed semi-automated method based on LC-MS/MS coupled to quadrupoles high-resolution mass spectrometry.4-fluoro-N-isopropylaniline was analysed with 154 m/z quantification along with two confirmation m/z ion 112 and m/z ion 95 and oxyacetanilide was analysed with 364 m/z quantification along with two confirmation m/z ion 152.2 and m/z ion 194. Moreover, the use of a semiautomated method minimizes sample handling, 742 which reduces interferences that may occur in the extraction stage.In this work, the influence of co-extracted compounds on analytical signals was assessed by injecting mobile phase, methanol, blank matrix extracts of wheat straw and wheat grain, a standard solution of 4-fluoro-N-isopropylaniline and oxyacetanilide. In separation selectivity testing, all blanks showed no interfering peaks at the tR of the target analytes, as can be seen from the chromatograms in Figures 3 and 4. Also, with the aim of studying the specificity of the method, a comparison was made of the tR of reference standard solutions sample as well as blank matrix extracts of wheat straw and wheat grain samples. As can be seen in Figures 3 and 4, the tR values were not significantly influenced by other interferences from the matrix, the method was specific for the analyte. The matrix of wheat straw and wheat grain was residue-free.
The slopes achieved insolvent 0.1 % formic acid in milli Q water (80%) and 0.1 % formic acid in methanol (20%). Next, the linearity and correlation coefficients were obtained in the range of 0.994 -0.999 from the calibration curves at concentrations ranging viz., 0.00008, 0.0001, 0.0004, 0.002, 0.004, 0.008 and 0.01 mg/L in Figures 1 and 2, from the blank levels and were found to be sufficient. .As far as we know, this is the first study focused on the development of a semiautomated method based on LC-MS/MS quadrupole for the determination of 4-fluoro-N-isopropylaniline, expressed as oxyacetanilide in wheat (straw and grain). Finally, the proposed methodology can be implemented in analytical laboratories for the determination of target compounds due to the simplicity of the procedure.

Conclusion:-
In-house inter-laboratory validation studies, using wheat straw and wheat grain samples containing N-fluorophenyl-Nisopropyl residues of oxyacetanilide demonstrated that the method is quick, rugged, selective, and sensitive enough to determine residue. This method is highly robust and suitable for cost-effective routine analysis of these herbicides, achieving acceptable recoveries for all the spike concentration, good sensitivity (LOQ, 0.01 mgkg −1 for wheat grain and 0.05 mgkg -1 for wheat straw) and acceptable % RSDs. The proposed LC-MS/MS method is rapid, sensitive, and successfully applicable for the simultaneous analysis of oxyacetanilide in their metabolite form N-fluorophenyl-N-isopropyl, in wheat straw and wheat grain. An analytical gradient elution developed in this method improved the peak shape and retention of the analytes over gradient elution. Positive mode ion-spray with MS/MS dimension gives admirable sensitivity and selectivity that produce distinct chromatographic peaks with slight nosiness. The present method allows the simultaneous determination of the presence and quantification with high reliability.