Method for the determination of bas 510 f in plant

Technical Procedure:
Method for the Determination of
Cypermethrin Isomers in Plant Matrices
BASF Method Number 567/0
Test Facility
Number of Pages
This document contains manufacturing and trade secrets of BASF. It is the property of BASF and may be used only for that purpose for which it was intended by BASF. Every other or additional use, exploitation, reproduction, publication or submission to other parties requires the written permission of BASF, with the exception of regulatory agencies acting within the limits of their administrative authority. BASF Method No. 567/0 was developed and adapted for the analysis of Alphacypermethrin
and Cypermethrin in various plant matrices.
Data and information contained in this technical protocol were not necessarily produced
under GLP regulations nor was this report audited by QAU. The report as published here is
intended as a laboratory and reference guide for the use of BASF method No. 567/0.
This method was developed by a laboratory-team consisting of the following persons:
M. Seiferlein, P. Agterhoff, U. Gröschel, J. Stewart, J. Varner
Abstract
567 was developed for the determination of Alphacypermethrin and This report includes the analytical procedure of BASF method No. 567. BAS 310 I (Alphacypermethrin) or BAS 311 I (Cypermethrin) is extracted with a 70:25:5 methanol/water/hydrochloric acid mixture. After centrifugation, an aliquot of the extract is partitioned into cyclohexane. Sillica Gel SPE column is used for further purification, if required. Final determination is performed by LC/MS/MS using the ammonium adduct of cypermethrin. The limit of quantitation (LOQ) of the method for Cypermethrin or Alphacypermethrin is 0.01 mg/kg. Table of Contents
Introduction . 5
Materials. 5
2.2 Test and Reference Substances. 5 2.3 Equipment for Extraction and Sample Clean-up . 7 2.4 Reagents. 8 2.4.1 Chemicals . 8 2.4.2 Solutions and Solvent Mixtures. 8 2.4.3 Standard Solutions of BAS 310 I . 8 2.4.4 Standard Solutions of BAS 31I I . 9 Analytical Procedure . 10
3.1 Spiking of Samples for Recovery Experiments . 10 3.2 Extraction of the Sample Material . 10 3.3 Clean-up. 10 3.3.1 Liquid/liquid Partition with Cyclohexane . 10 3.3.2 SPE-Sillica Gel Column Clean up (optional: may be required for some matrice) . 10 3.3.3 Preparation of the Final Volume . 11 3.4 Final Determination by LC/MS/MS . 12 3.4.1 Instrumentation and Conditions . 12 3.4.2 Calibration Procedures . 13 3.4.3 Limit of Quantitation . 13 Calculation of Results . 14
Flowchart of Method 567/0 . 15
Method Management and Time Requirement. 16
Confirmatory Technique. 16
1 Introduction
Alphacypermetrin (BAS 310 I) is an insecticide of the pyrethroide group, which is used against several diseases in various crops and which is a racemate of two enantiomers. These two enantiomers are also constituents of the insecticide Cypermethrin, which consists in total of eight stereoisomers The method has a limit of detection of 0.01 mg/kg for Alphacypermethrin alone and 0.01 mg/kg for total Cypermethrin. 2 Materials
List of Abbreviations
High Pressure Liquid Chromatography with mass spectrometry Test and Reference Substances
BAS 310 I
Racemate comprising (S)-cyano-3-phenoxybenzyl-(1R,3R)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate and (R)-cyano-3-phenoxybenzyl-(1S,3S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate ISO Common Name: Alpha-Cypermethrin CAS Registry No.: BAS 311 I
(RS)-a-cyano-3-phenoxybenzyl(1RS,3RS;1RS,3SR)-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate ISO Common Name: Cypermethrin CAS Registry No.: Equipment for Extraction and Sample Clean-up
Equipment
Size, Description
Manufacturer
Catalog No.
NOTE: Equivalent equipment from other suppliers may be substituted.
2.4 Reagents
2.4.1 Chemicals
Chemical Manufacturer/Supplier
Note: Equivalent reagents and chemicals from other suppliers may be substituted.
2.4.2 Solutions and Solvent Mixtures
Methanol / water / 2N hydrochloric acid, 70/25/5, v/v/v Sample Solvent for prep. of final volume 4mM ammonium formate + 0.1% formic acid in water: Water / formic acid / ammonium formate 0.25 g ammonium formate in 999 mL water + 1 mL formic acid 4mM ammonium formate + 0.1% formic acid in methanol: Methanol / formic acid/ ammonium formate 0.25 g ammonium formate in 999 mL methanol + 1 mL formic acid 2.4.3 Standard Solutions of BAS 310 I
Stock Solutions
Prepare a stock solution containing 1 mg/ml of BAS 310 I in acetone. This can be done by
weighing e.g. 10 mg of BAS 310 I and diluting with 10 ml of acetone. The weight should be
corrected for purity.
Prepare a standard solution containing 10 µg/ml of BAS 310 I in acetone. This can be done
by mixing e.g. 1 ml of the 1 mg/ml solution in a 100 ml volumetric flask and diluting to the
mark with acetone.
Examples for Preparation of Fortification Solutions
Take solution
Dilute with methanol to a final
Concentration
(µg/ml)
volume of (ml)
(µg/ml)
Please note: the fortification solutions are prepared in methanol since the samples are
extracted with methanol/water/hydrochloric acid.
Standard Solutions for LC/MS/MS
Prepare a standard solution containing 0.1 µg/ml (100 ng/ml) of BAS 310 I by taking 1 ml
of the 10 µg/ml solution in a 100 ml volumetric flask and diluting to the mark with 80:20
methanol:water.
Examples for Preparation of Standard Solutions for Calibration
Take solution
Dilute with 80:20
Concentration
methanol:water to a final
volume of (ml)
It is recommended to store the standards in a refrigerator and renew the stock standard solution after 90 days and the other standard solutions after 30 days. 2.4.4 Standard Solutions of BAS 31I I
Prepare standard solutions of Cypermethrin in the same manner as described for the BAS 310 I standards. 3 Analytical
Procedure
Spiking of Samples for Recovery Experiments
5 g of plant material are weighed into a 250 ml wide neck powder bottle. The spiking solution containing BAS 310 I or BAS 311 I is added according to the following table: Concentration of
Volume of
spiking solution
spiking solution
Fortification
Extraction of the Sample Material
Weigh 5 g of prepared* sample material into a 250 ml wide neck powder bottle and add exactly 100 ml of extraction solvent (see chapter 2.4.2). If the sample is very finely ground, place on a shaker table and shake for approximately 15 minutes. If sample is coarsely ground, homogenise using a Polytron for about 3 minutes at 7000 rpm. Take an approx. 10 ml portion (definite volume not necessary) of the extract and centrifuge for about 5 min at approximately 4000 rpm. *Note: In order to work with a sample weight of 5 g, it is essential to have excellent sample homogeneity and
small sample particles. This can be achieved using dry ice during the initial preparation (grinding/homogenisation). If this prerequisite is not met, larger amounts of sample should be used (e.g. 25 g of sample and 3.3 Clean-up
Liquid/liquid Partition with Cyclohexane
Transfer exactly 1 ml (1% aliquot) of the supernatant into a 10 ml culture tube containing 1 ml of reagent-grade water. Add exactly 5 ml of cyclohexane to the culture tube and shake well. If the SPE clean-up is required, proceed directly to Step 3.3.2. Many plant matrices do not require the optional SPE clean-up step. In this case, remove a 3 mL aliquot (60%, overall aliquot 0.6%) of cyclohexane phase and transfer to a 10 mL culture tube. Then proceed directly to Step 3.3.3 – Preparation of Final Volume. SPE-Sillica Gel Column Clean up (optional: may be required for some matrice)
Condition a 3 ml 100 mg SPE Sillica Gel Column by washing it with 3 ml of ethyl acetate followed by 3 ml cyclohexane. Remove a 3 mL aliquot (60%, overall aliquot 0.6%) of cyclohexane phase and transfer to the SPE column. Wash the column with 2 ml of cyclohexane. Set a 10 ml culture tube into the vacuum box and elute the analyte with 3 ml elution solution (cyclohexane/ethyl acetate 80/20 v/v). Collect the eluate in the 10 ml culture tube. Do all these steps including the column-conditioning step under low vacuum with a flow rate of about 0.5 to 1.0 ml/minute. 3.3.3 Preparation of the Final Volume
Evaporate the aliquot from Step 3.3.1 or the eluant from Step 3.3.2. to dryness at approx. 40°C using a gentle stream of nitrogen. Dissolve the residue in 1.0 mL of 80:20 methanol:water and transfer the solution into an HPLC vial. In case of high residues, an appropriate dilution may be necessary to remain in the linear range of the calibration curve. Final Determination by LC/MS/MS
3.4.1 Instrumentation and Conditions
Sciex API 3000 LC/MS/MS Mass Spectrometer Phenomenex Columbus C18, 2.0 mm X 100 mm, 5 μ particle size Solution A: 4mM ammonium formate + 0.1% formic acid in water Solution B: 4mM ammonium formate + 0.1% formic acid in methanol m/z 433 → m/z191 Analysis is based on the ammonium m/z 435 → m/z 193 adduct of Cypermethrin Note: The equipment listed above may be substituted by instruments with similar specifications.
The instrument conditions, injection volume, column and gradient steps may be modified, but any changes must be recorded in the raw data. 3.4.2 Calibration
Procedures
Calculation of results is based on peak intensity measurements (peak area or peak height) using a calibration curve. The standard curve is obtained by direct injection of the BAS 310 I or BAS 311 I standards into the LC/MS/MS in the range of 0.25 ng/ml to 2.5 ng/ml. In a given injection run, the same volume is used for all samples and standards. The calibration curves are obtained by plotting peak area or height versus the concentration of BAS 310 I or BAS 311. Evaluation is usually based on the most intense daughter ion, which in this case was the transition m/z 433 → m/z 191. Establish the stability of the detection response by injecting several concentrations of standards. Each injection set should begin and end with standard injections, and each standard level should be injected at least in duplicate. 3.4.3 Limit of Quantitation
The limit of quantitation is defined as the lowest fortification level successfully tested. For all matrices, the limit of quantitation is 0.01 mg/kg for BAS 310 I and 0.01 mg/kg for BAS 311 I. Calculation of Results
4.1 Principle
Calculation of results is based on peak area (or height) measurements and are calculated from the calibration curve. Residues in treated samples are reported as total Cypermethrin. 4.2 Calculation
The individual concentrations of Cypermethrin or BAS 310 I in mg/kg are calculated as
shown in equation I:
I. Residue
Final volume of the sample after all dilution steps [mL] Concentration of analyte as read from the calibration curve [ng/ml] Factor remaining after all unit conversions The recoveries of spiked compounds are calculated according to equation II: Recovery % = Residue in fortified sample – Residue in Control x 100
Flowchart of Method 567/0
Method Management and Time Requirement
The analysis of one series of samples (= 26 unknown samples, 2 fortified samples for recovery experiments, 2 blank samples) requires 8 working hours. This time includes the calculation of the results, the preparation of the equipment as well as the reporting of all raw data under GLP. 7 Confirmatory
Technique
The LC/MS/MS final determination for BAS 310 I and BAS 311 I is a highly selective detection. Two separate transitions are monitored; therefore, no confirmatory technique is required. 8 Ruggedness
The following recovery results were obtained during pre-validation ruggedness testing. This data was not collected under GLP. SPE Clean-up
Fort Level
Recovery %
(yes / No)

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