1. Assessment and Method Validation of Chlormequat
Chloride (CCC) in Food (Fruit, Vegetable & Grain) by
Liquid Chromatography Mass Spectrometry
By Virendra Vora*, Mukesh Kumar Raikwar and Vikas Bhardvaj
January 2012
The authors are Specialists at the Department of Pesticide Residue, Reliable Analytical
Laboratories Pvt Ltd, in Bhiwandi, Thane, India *Corresponding Author → See also:
«Study of Contamination Level of Pesticide Residues in Grapes (Vitis vinifera) in
Maharashtra»
◦
Abstract
A fast, sensitive and specific method for routine determination of Chlormequat
chloride residue from a variety of different food matrices was developed. It requires
no cleaning procedure and very easy to extract without any interference in liquid
chromatography (electrospray ionization) tandem mass spectrometry with using a
stable isotopically labeled internal standard. Mass spectral acquisition was done in
selected reaction monitoring mode (MRM), selecting the transitions from both the
35
Cl and the 37
Cl isotope of chlormequat. Recoveries after extraction was
determined with radio-labeled chlormequat and averaged over the spiking range
(0.010 – 0.050 mg/kg) in four different commodities were within 83-96%, with a
coefficient of variation batter than 2%. The method can be applied to grape, juice
concentrates, vegetables, fruits and cereal products, with typical limits of detection
for chlormequat estimated at 0.001 – 0.002 mg/kg. A survey of different food
commodities especially of grape revealed that chlormequat was detectable at very
low level and it is also a very stable compound in many of the food samples
analyzed, with the highest concentration recorded in grape (0.328 mg/kg, 100%
contamination) and okra (1.0 mg/kg) purchased from Maharashtra region.
Measurements were also conducted on two LC – MS instruments, also between
international and national labs and demonstrate the versatility and robustness of
the method and its applicability to instruments.
Introduction
Chlormequat chloride (IUPAC name 2-chloroethyltrimethyl ammonium chloride) is a highly stable
gibberellins biosynthesis inhibitor used as a plant growth regulator in many fruits and vegetables.
Chlormequat chloride (CCC) inhibits cell elongation, hence shortening and strengthening stems and
producing sturdier plants. It’s main use in agriculture is on cereal crops, such as wheat, barley and
oats, to increase resistance to lodging by limiting stem growth and to improve yields. However, it has
also been widely used to improve fruit setting, prevent premature fruit drop, increase fruit size and
reduce the need for tree pruning in top fruit. Due to the CCC properties with the relatively strongly
bound Cl- (Blinn, 1967) suggested that CCC residue could be accumulated in plant organs as well as
the products. However, other workers have subsequently reported that CCC is completely
metabolised since CCC residues could not be detected in animal tissues (Dekhuijzen et al., 1973;
Dekhuijzen et al., 1974). To protect animals and humans from consumption of CCC in food, the
European Community has set up the maximum CCC residual limits 0.05 for Fruits and Grains.
Several methods have been described for the determination of chlormequat. The first methods
developed were nonspecific spectrometric (Moony et al., 1967) and thin-layer chromatographic
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2. procedures (Stijve, 1980). Despite extensive cleanup, interferences were present, blank values were
too high, and reproducibility was poor. A method that is currently used in many laboratories for
chlormequat determination was developed by Greve and Hogendoorn (1996). In their method,
chlormequat is extracted from pears with methanol. After cleanup, using ionexchange
chromatography and further cleanup over alumina, chlormequat is converted into acetylene by
heating in alkaline medium at 215°C. The acetylene is measured in the head space by gas
chromatography (GC) with flame ionization detection. Although acceptable recoveries (68 – 92%)
and blanks were obtained, the method is still very labor intensive and unsuitable for routine analysis.
Direct determination of chlormequat has been described for the analysis of formulations. Capillary
electrophoresis was used with mass spectrometric detection (Moyano et. all, 1996). Mass
spectrometry (MS) enables detection of chlormequat, which lacks a chromophoric group, and makes
capillary electrophoresis or liquid chromatography (LC) promising alternatives to the classical
methods for residue analysis outlined above. In fact, very recently, an LC/MS procedure was
reported for the determination of chlormequat in Grapes (Banerjee et. all, 2007).
The purpose of the work described in this paper was to develop a common method for chlormequat
residue analysis in food, cereals, fruits and vegetables that is more suitable than the
Greve/Hogendoorn method for routine analysis. For method development reverse phase was used.
The extraction procedure was very easy, reproducible and robust, which require no cleanup
procedure. After complete validation, results were compared with international and national labs. The
method was validated and implemented for routine analysis (up to 5 ng/ml) on LCMS/MS.
Materials and Methods
Chemicals
Following chemicals were used in the project are methanol & water (J T Backer) as a mobile phase,
formic acid as a acidifying agent for extraction solvent, ammonium formate as a buffer for mobile
phase. All these reagents were purchased from Merk. PTFE (Polytetrafluoroethylene) filter of 0.22
µm was used to filter the sample. Standard chloramequat chloride and internal standard
Chlormequat Chloride C13
were purchased from “Accu std.”
Standard Stock solution of 1 mg/ml (1000 ppm) was prepared by dissolving 10 mg of CCC in
Methanol. The standard working range from 10 to 300 ng/ml was prepared by serial dilution with
methanol and std stock solutions were stored in the deep freezer at 4 °C when they were not in use.
Food Samples for Analysis
The fruits (Grape & Mango), Grain (Basmati Rice) and vegetable (Okra) from different region of
Maharashtra were collected to carry out the project on contamination of CCC. Grapes & Rice were
collected in winter season total number were 862 and 5 consequently, Mango (in fruit or juice form)
was collected in summer season in 10 number and Okra was collected in 12 number from the market
and farmer field in fresh condition and samples were stored at -18°C as a control sample and to
check the stability of CCC in sample (after few days and a month).
Sample Preparation and Extraction
Homogenize matrix require for efficient and precise extraction of CCC. To prepare the matrix,
sample was plucked (Grapes) or cut into small pieces (Mango, Okra) depending upon the physical
structure of sample. Cut or plucked sample was collected in prewashed & dried 1000 ml beaker then
sample was ground in a mixer for 10 min, obtained matrix fluid was blended with blender for 10 min
at 5000 rpm to homogenize thoroughly. Rice was ground to coarse/fine powder and homogenized
mechanically to obtained uniform matrix.
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3. The determination of chlormequat residues in plant material is difficult owing to the necessary
separation of chlormequat from naturally occurring choline. If separation is not quantitative high
blank values are found, which may lead to false positive results but due to its stability and polarity in
acidic methanol chlormequat get extracted very easily, so that the following simple method was used
for the extraction of CCC.
Weigh 10 gm of homogenized matrix in 50 ml plastic centrifuge tube (for recovery, spikes the sample
with specified amount of Standard and then vortex for 2 min). Add 20 ml of 0.1 % Formic acid in
methanol. Vortex for 5 min, then centrifuge at 10,000 rpm for 10 min. Pass the supernatant through
0.2 µ filter and Collect the supernatant in 2 ml vial for LC/MS.
LC/MS Conditions and Parameters
For sample analysis and method validation highly sensitive Agilent 6460 (Triple Quadruple) Mass
spectrometer was used. Standard of CCC and IS were tuned at 1 mg/kg dissolved in methanol and
following MRM (Multi reaction mode) and conditions were set for MS.
Compound
Name
Precursor
Ion
Product
Ion
Fragmentor
Collision
Energy
Polarity
CCC 122 58.1 130 35 Positive
CCC 122 62.9 130 35 Positive
CCC 124 58.1 130 35 Positive
CCC 124 65 130 35 Positive
CCC IS 128 58 120 35 Positive
CCC IS 128 69 120 20 Positive
CCC IS 126 58 120 35 Positive
CCC IS 126 67 120 20 Positive
Multi Reaction Mode (MRM)
Jet Stream ESI
Gas temp 330 °C
Gas flow 6.0 l/min
Sheath gas flow 8.0 l/min
Sheath gas temp 310 °C
Neb Pressure 30 psi
Capillary Voltage 3500
Nozzle Voltage 500
MS Conditions
HPLC Condition: Due to the high polarity of CCC, HILIC Atlantis silica column of Waters was used
with bellow mentioned mobile phase proportion. The column particle size of 5 µ, id 4.6 mm and
length 150 mm was selected to perform samples in large quantity as well as for better peak shape.
Samples were run at the flow rate of 0.5 ml/min.
Mobile Phase
A
8:2 Water : methanol with 5 mM Ammonium formate buffer
B
9:1 Methanol : Water with 5 mM Ammonium formate buffer
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4. Result and Discussion
Method Validation
Method validation is a documentary procedure to make sure that obtained result is precise, accurate
and consistent. Following procedure was followed for extraction and analysis method validation.
Blank 20 extraction solvents were run before the Linearity. Straight base line was seen in
chromatogram of all 20 runs which indicated that none ghost peak or interference peaks coming at
the same retention time. Linearity was plotted in solvent (Methanol) from 5 to 300 ng/ml (6 points).
0.99 Coefficient variation (CV) was obtained in both the solvent and all matrixes which say that the
response is equal to the concentration (No quenching at higher concentration) in the range from 5
ng/ml to 300 ng/ml. For recovery, spiking was done in each food matrix at known three different
concentrations (10, 20 and 50 µg/kg) with three replicates. Internal standard was spiked at same
concentration in all series to negate the error occur during the extraction.
Fig-1: MRM chromatogram of Standard (10 µg/kg) and IS (25 µg/kg) in Methanol
Fig-2: Chromatogram of spiked IS (@ 25 µg/kg) and CCC (@ 5 µg/kg) in Grape sample
CCC was run through C18 and HILIC column with the same above mobile phase and RSD was
calculated for both. HILIC column gave better elution, good peak shape and lowest RSD compare to
C18 column. Other mobile phases Water and Acetonitrile: Methanol (60:40) with 0.1 % formic acid
was also tried but best elution at 1 ng/ml was obtained only in Methanol: water. Repeatability with <
2% RSD was obtained between 6 points at three different (10, 20 and 50 µg/kg) concentrations. To
check reproducibility between instruments and chemists, validation was again performed on API
2000 Bio sciex LC-MS triple quadruple instrument and better than 2% RSD was found.
Pesticide CCC
Matrix Grape Mango Okra Rice
Rt 12.3 min. 12.4 min. 12.5 min 12.4 min
Calibration Range
0.005 – 0.3
(mg/kg)
0.005 – 0.3
(mg/kg)
0.005 – 0.3
(mg/kg)
0.005 – 0.3
(mg/kg)
Regression
Equation
y=0.0261x y=0.0234x y=0.0236x y=0.0245x
Linearity (R) 0.9993 0.9989 0.9976 0.9989
LOD (µg/kg) 0.001 (mg/kg) 0.001 (mg/kg) 0.001 (mg/kg) 0.001 (mg/kg)
LOQ (µg/kg) 0.005 (mg/kg) 0.005 (mg/kg) 0.005 (mg/kg) 0.005 (mg/kg)
Recovery (in %) 91.70 95.46 89.67 85.73
% RSD 1.27 1.76 0.85 2.0
Table 2 · Validation of Analytical Methods for CCC in Fruits & Vegetables
Samples Mango (µg/kg)
Conc. (µg/kg) R1 R2 R3 Mean % Recovery
0.01 0.0094 0.0096 0.0096 0.0095 95.33
0.02 0.0190 0.0192 0.0188 0.019 95.00
0.05 0.0480 0.0476 0.0485 0.0480 96.46
Samples Grape (µg/kg)
Conc. (µg/kg) R1 R2 R3 Mean % Recovery
0.01 0.0092 0.0086 0.009 0.0089 89.33
Table 3 · Percentage Recovery of CCC in Food
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5. 0.02 0.0198 0.0182 0.017 0.018 92.50
0.05 0.047 0.045 0.046 0.046 93.26
Samples Okra (µg/kg)
Conc. (µg/kg) R1 R2 R3 Mean % Recovery
0.01 0.0094 0.0082 0.0086 0.0087 87.33
0.02 0.019 0.0185 0.018 0.018 92.50
0.05 0.041 0.046 0.045 0.044 89.20
Samples Rice (µg/kg)
Conc. (µg/kg) R1 R2 R3 Mean % Recovery
0.01 0.0084 0.0081 0.0085 0.0083 83.33
0.02 0.016 0.016 0.018 0.017 86.00
0.05 0.043 0.046 0.041 0.043 87.86
Inter Laboratory and International Laboratory Checking
4 Grape samples were sent to the Europe Lab for testing where analysis was performed by another
method and same samples were analyzed by the above method. Then the both results were
compared to validate the repeatability of method. Deviation in results was not more than ± 2 % in
grape samples.
Stability Testing of CCC in Grapes
CCC is lacking in chromophore, is very polar and has low volatility (Baker et al., 1992) and since
does not degrade in the animal body (Calsamiglia et al., 1996, Reynolds et al., 2004). Around 29
Grapes samples were re-extracted after a month, which had been stored at -18 °C in deep freezer.
Obtained results were matched with the previous results, not more than ± 5 % variation between
results was found. This says that CCC is very stable compound and it even does not degrade in
food.
Conclusion
Nº
Name of
Sample
LOD
(mg/kg)
Total
Samples
Range
(mg/kg)
Samples
Contamination
%
Contamination
EU MRL
(mg/kg)
Samples
> MRL
1 Grapes 0.001 862
0.01-
0.328
862.00 100.00 0.05 206
2 Mango 0.001 10 Nil ND - 0.05 Nil
3 Okra 0.001 12 1.0 1 8.33 0.05 1
4 Rice 0.001 5 Nil ND - 0.05 Nil
Table 4 · Level of contamination of CCC in Fruits and Vegetables
From extraction and analysis method we can conclude that the method is very easy and simple to
perform the analysis on regular basis, large quantity of samples can be analyzed in a day with less
men power and cost. Validation of method by comparing data nationally (inter-laboratory checking)
and internationally say that method is very precise, robust, reproducible and accurate even
extraction method can be used almost for all foods with better than 85 % recovery. Stability testing
has proved that CCC is very stable compound in fruits, vegetables and grains.
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