Determination of glyphosate, maleic hydrazide, fosetyl aluminum, and

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Determination of glyphosate, maleic hydrazide, fosetyl aluminum, and ethephon in grapes by liquid chromatography/tandem mass spectrometry narong chamkasem J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02419 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Journal of Agricultural and Food Chemistry

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Determination of glyphosate, maleic hydrazide, fosetyl aluminum, and ethephon in grapes by liquid chromatography/tandem mass spectrometry

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Narong Chamkasem ([email protected])

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Southeast Food and Feed Laboratory (SFFL), U.S. Food and Drug Administration, 60 Eighth Street, N.E., Atlanta, GA 30309, United States

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Abstract A simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS-

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MS) method was developed for the determination of maleic hydrazide, glyphosate, fosetyl

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aluminum and ethephon in grapes using a reversed-phase column with weak anion-exchange and

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cation-exchange mixed-mode. A 5 g test portion was shaken with 50 mM HOAc and 10 mM

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Na2EDTA in 1/3 (v/v) MeOH/H2O for 10 min. After centrifugation, the extract was passed thru an

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Oasis HLB cartridge to retain suspended particulates and non-polar interferences. The final

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solution was injected and directly analyzed in 17 min by LC-MS-MS. Two MS-MS transitions

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were monitored in the method for each target compound to achieve true positive identification.

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Four isotopically-labeled internal standards corresponding to each analyte were used to correct

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for matrix suppression effects and/or instrument signal drift. The linearity of the detector

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response was demonstrated in the range from 10 to 1000 ng/mL. for each analyte with a

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coefficient of determination (R2) of ≥ 0.995. The average recovery for all analytes at 100, 500,

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and 2000 ng/g (n = 5) ranged from 87 to 111%, with a relative standard deviation of less than

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17%. The estimated LOQs for maleic hydrazide, glyphosate, fosetyl-Al, and ethephon were 20,

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19, 38, and 56 ng/g, respectively.

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ACS Paragon Plus Environment


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Keyword

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Liquid-chromatography-tandem mass spectrometry

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Glyphosate

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Maleic hydrazide

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Fosetyl aluminum

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Ethephon

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Introduction

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Glyphosate (N-phosphonomethyl glycine) is a non-selective post emergence herbicide used for

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the control of a broad spectrum of grasses and broad-leaf weed species in agricultural and

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industrial fields. It was used in the vineyard as postemergence herbicide to control weed between

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grape vine rows. There was a report that glyphosate was detected in ten wine samples from

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California as high as 19 ng/g using an enzyme-linked immunosorbent assay (ELISA) method (1).

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This method is quick, inexpensive, and sensitive; however, it does not have the ability to confirm

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the identity of glyphosate to prevent false positive results. According to the electronic code of

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federal regulations, the tolerance level for glyphosate in grapes is 0.2 µg/g (2). Glyphosate is too

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polar to be retained by a reversed-phase C18 column. The lack of chromophore and and the polar

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in nature of glyphosate molucule also necessitates the use of derivatization techniques for the

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determination by liquid chromatography and gas chromatography (3-5). Vreeken and co-workers

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developed an analytical method to analyze glyphosate, AMPA and glufosinate in water samples

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using a reversed-phase liquid chromatography separation after pre-column derivatization with 9-

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fluorenylmethyl chloroformate (FMOC-Cl) with LC-MS-MS (6). An LC-MS-MS method using

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a mixed-mode HPLC column (Acclaim Trinity Q1) was developed to directly determine

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glyphosate (without derivatization) in soybean, egg, milk and honey (7-9). This method should

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be applicable for grapes as well.

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Maleic hydrazide (MH) is a plant growth regulator with some herbicidal activity whose mode of

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action is to inhibit cell division. For example, it has been applied to control vine growth by



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limiting excessive vegetative growth and improve grapes quality in intensive vineyards (10). The

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early analytical method for this small molecule (MW = 112) was colorimetric method which is

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nonspecific and susceptible to interference (11). To increase sensitivity a labor intensive GC-

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ECD method was developed for MH, which involved a Diels-Alder condensation to first make a

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derivative (12). Newsome determined MH levels in potato, beets and carrots by ion-exchange

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liquid chromatography with a UV detector (13). Kubilius and Bushway developed capillary

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electrophoresis technique to determine MH in potato and onion at 2 to 20 µg/g level (14).

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Moreno et al. (2012) developed an HPLC-UV method and confirmation with MS using

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atmospheric pressure chemical ionization (APCI) interface to determine MH residue in garlic

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bulbs (15). According to the 40CFR180.175, MH has tolerance only in potato and onion at 50

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and 15 µg/g and no tolerance in grapes (16).

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Ethephon is a synthetic plant growth regulator used to improve fruit abscission for mechanical

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harvest, to promote or inhibit flowering, and to enhance sugar content (17). Ethephon analysis is

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mandatory for several foodstuffs especially table grapes in the coordinated community control

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program laid down in Commission Regulation (EC) No. 788/2012 (18). The tolerance level of

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ethephon in the US (40CFR180.300) for grapes is 2 µg/g (19). It is a very polar compound

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containing a phosphonic acid group (pKa = 2.5 and 7.2) and is stable in aqueous solution below

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pH 4 (20). An indirect gas chromatographic technique was developed by measuring the release

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of ethylene under alkaline condition using head-space analysis (21). Other gas chromatographic

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methods also involved a long derivatization step before injection on GC-MS (22,23). Since the

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compound is not retained well on a reversed-phase column, Anastassiates et al (24) used ion-


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chromatography as an alternative technique. Hydrophilic interaction liquid chromatography

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(HILIC) was introduced by Alpert to retain very polar analytes (25). Hanot et al. combined

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HILIC technique with tandem mass spectrometry to determine ethephon in grapes with a limit of

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quantification of 0.05 µg/g (26). This method used a mobile phase with a pH of 9.3 in order to

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minimize band broadening. This pH is outside the optimum pH limit from the manufacturer,

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therefore, column shelf life may be shorter than optimum.

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Fosetyl aluminum (fosetyl-Al) is used on grapevines to control downy mildew and fungal

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disease (Esca) as a replacement of the banned compound, sodium arenite (27,28). This

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compound is too polar to be retained by a reversed-phase HPLC column and it lacks UV

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absorption. Therefore, ion chromatography (29) and gas chromatography after derivatization (30)

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were used as alternate techniques. To improve retention of the analyte on a reversed-phase

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column, ion-pairing reagent was used in the mobile phase with electrospray mass spectrometer to

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determine fosetyl-Al in lettuce (31). This method has a relatively high limit of quantification of

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0.2 µg/g because the sample must be diluted up to five-fold to minimize severe matrix

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suppression. The tolerance for fosetyl-Al (listed as Aluminum tris (O-ethylphosphonate) in the

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US (40CFR180.415) is 10 µg/g (32).

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It is time-consuming and not practical to analyze a sample for all four analytes using four

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different methods. Anastasiades et al. developed an LC/MS multiresidue method that would

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determine these analytes in a single run (24). The sample was extracted with acidified methanol,



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without partition or cleanup, so the extract often contained large amounts of co-extractives which

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can impact the robustness of the method. This method employed a Hypercarb column, porous

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graphitic carbon, with acidified methanol/water mobile phase in a negative electro-spray mode

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for a run time of 30 min. The column requires a special priming/reconditioning procedure and

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shows significant peak tailing of glyphosate. Currently, the pesticide screening program of the

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Food and Drug Administration (FDA) does not have a multiresidue method that is quick,

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accurate, and sensitive for determining these compounds in a single run to support regulatory

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actions. The purpose of this study is to develop an LC-MS-MS method using a negative ion-

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spray ionization mode for the direct determination of glyphosate, MH, fosetyl-Al and ethephon

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in grapes. It also explains an extraction method that requires small sample size, non-toxic

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solvent, and an effective sample cleanup procedure to ensure method ruggedness, sensitivity, and

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selectivity.

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Experimental

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Chemical and reagents

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Pesticide standards (≥ 99% purity) were purchased from LGC Standards (Manchester,

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NH) consisting of glyphosate, MH, fosetyl-Al, ethephon, glyphosate 13C215N, MH D2, fosetyl-

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Al D15, and ethephon D4 . Methanol and water of HPLC grade were obtained from Fisher

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Scientific (Pittsburgh, PA). Formic acid was obtained as 98% solution for mass spectrometry

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from Fluka (Buchs, Switzerland.). Acetic acid, ammonium formate and

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ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were purchased from Fisher Scientific

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(Pittsburgh, PA). Oasis HLB (60 mg) solid phase extraction cartridges were obtained from

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Waters (Milford, MA). EDP 3 electronic pipettes at different capacities (0-10 µL, 10-100 µL,


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and 100-1000 µL) were purchased from Rainin Instrument LLC (Oakland, CA) and were used

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for standard fortification. A solution of acid/Na2EDTA solution (50 mM acetic acid/10 mM

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Na2EDTA) was prepared by mixing 572 µL of acetic acid and 0.74 g of Na2EDTA in 200-mL of

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purified water. The extracting solvent was prepared by mixing 750 mL of Na2EDTA solution

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with 250 mL of methanol.

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A solution of 500 mM ammonium formate/formic acid (pH 2.9) was prepared as follows: 15.76

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g of ammonium formate was dissolved in approximately 300 mL of HPLC water and adjusted

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with 98% formic acid (approx. 28.3 mL) until the pH reached 2.9 (using pH meter), and the

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solution was diluted to 500 mL with HPLC water. The HPLC mobile phase A was HPLC grade

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water and mobile phase B was prepared by mixing 100 mL of the 500 mM buffer solution with

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900 mL of purified water (final concentration was 50 mM).

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Standard Preparation

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Individual stock solutions of glyphosate and fosetyl-Al at 1 mg/mL were prepared in water. A

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stock solution of MH at 1 mg/mL was prepared in dimethyl sulfoxide (MH has poor solubility in

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both water and methanol). A stock solution of ethephon (1 mg/mL) was prepared in methanol.

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These stock solutions were used to prepare standard mix solutions in water at 100, 10, and 1

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ng/µL. The solutions were maintained at 4 °C in polypropylene tubes to avoid adsorption to

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glass. The internal standard (IS) solution of MH D2, fosetyl-Al D15, and ethephon D4, at 50

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ng/µL and glyphosate 13C215N at 10 ng/uL was prepared by dissolving the stock standard in

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water and stored in a polypropylene tube. The calibration standards were prepared in the

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extracting solvent with IS solutions for the calibration curves as described in Table 1.



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Sample Preparation and Extraction Procedure

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Two seedless grape samples (green and purple) were obtained from a local market. The samples

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were minced with a food processor until they had a smoothie-like texture. The representative portion was

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weighed at 5 ± 0.1 g each in 50-mL polypropylene centrifuge tubes (Fisher Scientific, Pittsburgh, PA) and

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fortified with native standard solutions at 100, 500, and 2000 ng/g (five replicates). The samples were

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vortexed briefly and stored in a freezer overnight. Two non-fortified grape samples were also prepared

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and used for matrix matched standard. On the extraction day, the spiked samples were thawed to room

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temperature. The extracting solvent (16 mL) was added to each tube using an automatic pipette. The final

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liquid volume of 20 mL was used for residue concentration calculation (assuming that grape has 80%

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moisture). The tubes were capped tightly and shaken for 10 min on a SPEX 2000 Geno grinder (SPEX

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Sample Prep LLC, Metuchen, NJ) at 2000 stroke/min then centrifuged at 4,130 rpm (3,000 x g) for 5 min

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using a Q-Sep 3000 centrifuge (Restek, Bellefonte, PA). Three milliliters of the extract were passed

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through an Oasis HLB cartridge, and the last milliliter of the extract was collected into a 2-mL plastic

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centrifuge tube. The sample extract (495 μL) was mixed with 5 μL of 50 ng/μL of IS solution in a new 1-

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mL plastic autosampler vial before analysis by LC- MS-MS.

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Instrumentation

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LC-MS-MS analysis was performed by using a Shimadzu HPLC system. The instrument was

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equipped with two LC-20AD pumps, a Sil-20AC autosampler, and a CTO-20AC column oven

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(Shimadzu, Kyoto, Japan), coupled with a 6500 Q-TRAP mass spectrometer from AB SCIEX

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(Foster City, CA). The Analyst software (version 1.6) was used for instrument control and data

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acquisition. Nitrogen and air from TriGas Generator (Parker Hannifin Co., Haverhill, MA) were

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used for nebulizer and collision gas in LC-MS-MS. An Acclaim™ Trinity™ Q1 (3 µm, 100 x

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3 mm) analytical column from Thermo Scientific (Sunnyvale, CA) and a C18 SecurityGuard


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guard column (4 x 3 mm) from Phenomenex (Torrance, CA) were used for HPLC separation at

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35 °C with sample injection volume of 5 µL. The mobile phase was 100% A (water) for 30 s at a

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flow rate of 0.5 mL/min then immediately ramped up to 100% B (ammonium formate/formic

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acid buffer) for 13 min to elute the analytes and strongly retain interfering compounds. The

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column was equilibrated with 100% A at a flow rate of 0.7 mL/min for 4 min for a total run time

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of approximately 17 min. A diverter valve connected between the HPLC column and the MS

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interface was used to direct the LC eluent to waste from 0 to 2 min and after 5 min. The MS

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determination was performed in negative electrospray mode with monitoring of the two MS-MS

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transitions using a scheduled MRM program of 80 s for each analyte. Analyte-specific MS-MS

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conditions and LC retention times for the analytes are shown in Table 2. The MS source

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conditions were as follows: curtain gas (CUR) of 30 psi, ion spray voltage (ISV) of -4500 volts,

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collisionally activated dissociation gas (CAD) is high, nebulizer gas (GS1) of 60 psi, heater gas

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(GS2) of 60 psi, source temperature (TEM) of 600 ºC.

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Results and discussion

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Chromatography Optimization

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including reversed-phase, anion-exchange, and cation exchange. It is a very versatile column

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that is suitable for the analysis of neutral and ionic compounds. It was previously used for

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glyphosate analysis in soybean and milk samples with an isocratic mobile phase of 50 mM

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ammonium formate (7 and 8). In order to improve the retention of glyphosate and the other polar

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pesticides in this study, a step gradient elution (9) was evaluated. At the beginning of the run, the

The Acclaim Q1 is a mixed-phased mode column possessing multiple retention mechanism,



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mobile phase A (water) was used at a flow rate of 0.5 mL/min for 0.5 min to elute polar non-

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ionic compounds such as sugar while the analytes were still retained at the head of the column.

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To elute the analytes, the mobile phase was switched to 100% mobile phase B (50 mM

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ammonium formate, pH 2.9) immediately. The retention times of MH, glyphosate, fosetyl-Al,

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and ethephon are 2.7, 3.0, 4.3, and 4.4 min, respectively. It was necessary to continue pumping

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the mobile phase B for approximately 13 min to elute compounds from grape matrix that may

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interfere with fosetyl-Al IS (mass 114/82). The higher salt concentration (70 mM) will elute the

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analytes faster but will result in signal reduction due to ion-suppression at the MS interface. The

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injection volume of 5 µL was chosen to obtain adequate sensitivity for this study. Larger

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injection volumes (10 to 20 uL) caused a significant signal reduction of MH and fosetyl-Al.

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MH has four prominent MS-MS transitions at 111/82, 111/83, 111/55, and 111/42 with the Sciex

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6500 MS system. However, grape samples tested in this study contain compounds having the

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MS-MS transitions at 111/83 and 111/55 eluted near the MH peak. Therefore, for MH, the MS-

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MS transition at 111/82 was used for quantification and the MS-MS transition at 111/42 was

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used for identification of the compound. There were no MS interference issues for glyphosate,

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fosetyl-Al and ethephon.

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Optimization of Sample Extraction Procedure

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Glyphosate is a chelator that can bind with metal ions in soil samples (33). A solution of acetic

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acid/EDTA was previously used to extract glyphosate, AMPA, and glufosinate in food samples

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with excellent results (7-9). Acetic acid lowers the pH of the sample to precipitate protein, and

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Na2EDTA prevents glyphosate from forming a chelation complex with metal ion (34). A mixed


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standard prepared in this solution was injected on the LC-MS-MS. All analytes exhibited good

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separation with good peak shape. Aqueous methanol solution with hydrochloric acid has also

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been used to extract polar pesticides from food matrices (24). This approach was also evaluated.

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A standard solution prepared in a solution of MeOH/aqueous HCl 0.1M (1:1) gave a non-

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symmetrical peak shape of fosetyl-Al (peak fronting). Therefore, this aqueous methanol solution

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was not used in this experiment.

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The Acclaim Q1 column has a reversed-phase retention and may retain any non-polar

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compounds under 100% aqueous mobile phase. The Oasis HLB cartridge was used for sample

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cleanup step to retain non-polar interference from the aqueous extract. This prevented the

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analytical column from being fouled with non-polar compounds. MH has a slight retention in

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reversed-phase mode (15) and it may be partially retained on the Oasis HLB cartridge during the

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cleanup step with acid/Na2EDTA solution. The elution profile of each standard was tested by

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passing a standard mix solution in acetic/Na2EDTA solution thru an Oasis HLB cartridge and

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determining the recovery for each compound. Approximately 30% of MH was retained on the

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SPE while the rest of the analytes passed thru the cartridge at nearly 100%. A much stronger

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solvent would be needed to recover MH during the cleanup step. Therefore, methanol was added

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to the extracting solvent ranging from 10 to 50% and a similar test was performed. It was found

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that 25% methanol in the acid/Na2EDTA was the optimum strength to quantitatively elute MH,

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and yet it was weak enough not to elute other sample matrix. To demonstrate that this solution

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was optimum, 5 g of purple grape were shaken with 16 mL of 25% methanol in acid/Na2EDTA

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solution on a Geno grinder at 2000 stroke/min for 10 min and then centrifuged at 3000 x g to



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obtain a clear extract. The extract was spiked with a standard mix at 500 ng/mL and 3 mL of this

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extract was passed thru the Oasis HLB cartridge. A purple color layer from the grapes extract

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was formed as a tight band on the top of the cartridge and the final extract was colorless. The

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cleaned extract was diluted 10 times with water and injected to the LC-MS-MS to obtain >90%

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recovery for all analytes. To further improve the recovery of ethephon during the sample

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cleanup, it is recommended to discard the first mL of the extract before collecting it. The Oasis

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HLB cartridge may have some active sites that need to be saturated with the sample extract in

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order to work properly.

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Evaluation of Matrix Effects

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To evaluate the degree of matrix effect (suppression or enhancement), calibration standard

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solutions of the analytes in solvent and in both organic grapes blank samples (after sample

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cleanup) were injected to the LC-MS-MS. The calibration curves were plotted between the peak

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response and analyte concentration (from 10 to 1000 ng/mL) using a linear regression curve fit.

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The matrix effect (% ME) was calculated as the slope of calibration curve of the analyte in the

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sample matrix is divided by the slop of calibration curve of the analyte in solvent and multiplied

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by 100. A value of 100% means that no matrix effect is present; if the value is less than100%, it

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means that there is matrix suppression, and if the value is more than 100%, it means that there is

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matrix enhancement. Table 3 shows the %ME of all analytes in two organic grape extracts.

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MH, fosetyl-Al, and ethephon demonstrated severe matrix suppression (7-32%), while only

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glyphosate had minimum matrix suppression (89 to 99%). Both grape varieties demonstrated

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similar degrees of matrix suppression. Based on this data, internal standards and/or matrix-

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matched standards are needed for accurate quantification of these analytes.


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Method Performance

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The proposed method was performed using two organic seedless purple and green grape samples

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collected from a local market. The calibration standard solutions at concentrations from 10 to

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1000 ng/mL were prepared in the extracting solvent and in two organic grape extracts with the

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addition of IS. These standard solutions were injected along with the fortified samples and

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sample blank. For comparison purposes, three different quantification methods were used to

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determine the accuracy and precision of the recovery results. They were a) standard in matrix

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with internal standard calibration method, b) standard in solvent with internal standard

264

calibration method and c) standard in matrix with external calibration method (Table 4). The

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calibration curves were linear fit with 1/x weighing and they all showed satisfactory linearity

266

with coefficient of determination (R2) of more than 0.995. The accuracy and precision of the

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method was evaluated via recovery experiment on two blank grape samples spiked at 100, 500,

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and 2000 ng/g. The selectivity of the method was evaluated by analyzing reagent blank, blank

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sample, and blank sample spiked at the lowest fortification level. No relevant signal (above 20%

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of the 100 ng/g sample) was observed at any of the transitions selected in the blank samples. A

271

reagent blank was injected immediately after the 1000 ng/mL standard and no carry-over was

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observed above 10% of the 10 ng/mL standard.

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Figure 1 shows the chromatograms of all analytes in blank purple organic grape spiked at 100

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ng/g with the signal/noise ratios of 50, 54, 26, and 18 for MH, glyphosate, fosetyl-Al, and

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ethephon, respectively. The sensitivity, expressed in terms of limit of detection (LOD), was

277

estimated as the concentration of analyte in matrix that generates signal of 3 times the baseline



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noise. Therefore, the estimate LOD for MH, glyphosate, fosetyl-Al, and ethephon are 6, 6, 12,

279

and 17 ng/g, respectively. The limit of quantification (LOQ) was estimated as the concentration

280

of analyte in matrix that generates signal 10 times the baseline noise. Therefore, the estimated

281

LOQ for MH, glyphosate, fosetyl-Al, and ethephon are 20, 19, 38, and 56 ng/g, respectively.

282

These LOQ levels are much lower than the tolerance level; therefore, the method is suitable for

283

regulatory work. Method linearity was determined for each target compound using a linear

284

regression curve fit (1/x weighing). The coefficients of determination (R2) are better than 0.995

285

for all analytes.

286 287

The accuracy (recovery) and precision (relative standard deviation or RSD) for the two organic

288

grape samples (purple and green) determined in two different days by using three different

289

calibration curves are shown in Table 5. The overall average recoveries were 76-103% with an

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RSD of ≤ 17 % for MH, 83-100% with an RSD of ≤ 6% for glyphosate, 82-117% with an RSD

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of ≤ 8% for fosetyl-Al, 78-109% with an RSD of ≤ 9% for ethephon. All three calibration

292

methods provided similar results. Since the matrix blank may vary in nature and is time-

293

consuming to prepare, the calibration standard in solvent with IS should be used in routine

294

analysis to save time and simplify the procedure.

295

This work describes a ten-minute extraction with methanolic aqueous solution of acetic acid and

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Na2EDTA for rapid and direct determination of MH, glyphosate, fosetyl-Al and ethephon residue

297

in grape samples. After centrifugation and cleanup with an Oasis HLB, the sample extract was

298

injected directly on the LC-MS-MS system. The mixed-mode Acclaim™ Trinity™ Q1 HPLC

299

column retains the analytes in the ion-exchange mode. The step gradient elution developed in

300

this method improved the peak shape and retention of the analytes. Non-ionic polar interferences


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such as sugar were eluted much earlier and diverted to waste to keep the ion-source clean.

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Negative mode ion-spray with MS-MS measurement gives excellent sensitivity and selectivity

303

that produces distinct chromatographic peaks with minimal interference. The use of internal

304

standard for each analyte minimized the matrix effect and provides accurate quantification. This

305

eliminates the need to use matrix-matched calibration standards. The method has good

306

sensitivity/selectivity and is suitable for the regulatory purposes

307 308



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Alferness, P.L.; Iwata, Y. Determination of glyphosate and (aminomethyl) phosphonic acid in soil, plant and animal matrixes, and water by capillary gas chromatography with mass-selective detection. J Agric Food Chem.1994;42:2751-2759.

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Qian, K,; Tang T,; Shi T,; Li P,; Li, Y,; Cao, Y. Solid‐phase extraction and residue determination of glyphosate in apple by ion‐pairing reverse‐phase liquid chromatography with pre‐column derivatization. Journal of separation science 2009; 32: 2394-2400.

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Ibáñez, M.; Pozo, Ó.J.; Sancho, J.V.: López, F.J.; Hernández, F. Residue determination of glyphosate, glufosinate and aminomethylphosphonic acid in water and soil samples by liquid chromatography coupled to electrospray tandem mass spectrometry. J Chromatogr A 2005;1081: 145-155.

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Vreeken, R.J.; Speksnijder, P.; Bobeldijk-Pastorova, I.; Noij. Th.H.M. Selective analysis of the herbicides glyphosate and amonimethylphosphonic acid in water by on-line solidphase extraction-high-performance liquid chromatography electrosprayionization mass spectrometry. J Chromatogr A. 1998;794 :187-199

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Chamkasem, N., Harmon, T. Direct determination of glyphosate, glufosinate, and AMPA in soybean and corn by liquid chromatography/tandem mass spectrometer. Anal Bioanal Chem. 408 (2016) 4995-5004

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Chamkasem, N.; Vargo, J.D. Development and independent laboratory validation of an analytical method for the direct determination of glyphosate, glufosinate, and aminomethylphosphonic acid in honey by liquid chromatography/tandem mass spectrometry Laboratory Information Bulletin U.S. Food and Drug Administration, Office of Regulatory Affairs, Rockville, MD (LIB 4613) June 2016.

10)

Lavee, S. Usefulness of growth regulators for controlling vine growth and improving grape quality in intensive vineyards. Acta Hortic. 1987; 206 :89-108

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AOAC 963.24. Maleic Hydrazide Pesticide Residues Spectrophotometric Method. 10.6.22. In: Official Methods of Analysis of AOAC International. 16th ed. Gaithesburg, MD, USA., 1998

12)

King, R.R. Gas chromatography determination of maleic hydrazide residues in potato tubers. J. AOAC. 1983;66:1327-1329.

13)

Newsome, W.H. A method for the determination of maleic hydrazide and its b-DGlucoside in food by high-pressure anion-exchange liquid chromatography J.Agric. Food Chem. 1980;28:270-272

14)

Kubilius, D.T.; Bushway, R.J. Determination of maleic hydrazide in potatoes and onions by capillary electrophoresis. J. Agri Food Chem. 1988;46:4224-4227

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Moreno, C.M.; Stadler, T.; da Silva, A.A.; Barbosa, L.C.A.; de Queiroz, M.E.L.R. Determination of maleic hydrazide residue in garlic bulbs by HPLC. Talanta. 2012;89; 369-376

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http://www.ecfr.gov/cgi-bin/textidx?SID=c30d88a8176fe20021d927e53e9422c7&mc=true&node=se40.26.180_1175&rg n=div8 (accessed August 1, 2016)

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Pierik, R.; Tholen, D.; Poorter, H.; Visser. E.J.; Voesenek, L.A. The Janus face of ethylene: growth inhibition and stimulation. Trends Plant Sci 2006;11:176–183

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The Commission of the European Communities (2012) Commission Implementing Regulation (EU) No 788/2012 of 31 August 2012 concerning a coordinated multiannual control programed of the Union for 2013, 2014 and 2015 to ensure compliance with maximum residue levels of pesticides and to assess the consumer exposure to pesticide residues in and on food of plant and animal origin.

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https://www.fsai.ie/uploadedFiles/Legislation/Food_Legisation_Links/Pesticides_Residues_in_f ood/Reg788_2012.pdf (accessed August 1, 2016)

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Marin, J.M.; Pozo, O.J.; Beltran, J.; Hernandez, F. An ion-pairing liquid chromatography/tandem mass spectrometric method for determination of ethephon residues in vegetables. Rapid Commun Mass Spectrom 2006;20:419–426

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Krautz, S.; Hanika, G. Eine einfache und schnelle gaschromatographische Bestimmung von Ethephon in Obste, Gemüse and Getreide mittels Head-space-Analyse. J Mol Nutr Food Res 1990;34:569–570

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Royer, A.; Laporte, F.; Bouchonnet, S,; Communal, P.Y. Determination of ethephon residues in water by gas chromatography with cubic mass spectrometry after ionexchange purification and derivatization with N-(tert-butyldimethylsilyl)-Nmethyltrifluoroacetamide. J Chromatogr A. 2006;1108:129–135

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Takenaka, S. New method for ethephon (2-chloroethylphosphonic acid) residue analysis, and detection of residual levels in the fruit and vegetables of Western Japan. J Agric Food Chem. 2002;50:7515–7519

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Anastassiades, M.; Kolberg, D.I.; Mack, D.; Wildgrube, C.; Sigalov, I.; Dörk, D.; Barth, A. (2015) Quick method for the analysis of residues of numerous highly polar pesticides in foods of plant origin involving simultaneous extraction with methanol and LC-MS/MS determination (QuPPe-Method). http://www.crlpesticides.eu/library/docs/srm/meth_QuPPe.pdf

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Alpert, A.J. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr A 1990;499:177–196

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Hanot, V.; Joly, L.; Bonnechere, A.; Van Loco, J. Rapid determination of ethephon in grapes by hydrophilic interaction chromatography tandem mass spectrometry. Food Anal. Methods. 2015;8:524-530.

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Di Marco, S.; Osti, F.; Calzarano, F.; Roberti, R.; Veronesi, A.; Amalfitano, C. Effects of grapevine application of foseyl-aluminum formulations for downy mildew control on “esca” and associated fungi. Phytopahtoal. Mediterr 2011;50 (supplement): S285-S299

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Magarey, P.A.; Wachtel, M.F.; Newton, M.R. Evaluation of phosphonate, fosetyl-Al and several phenylamide fungicides for post-infection control of grapevine downy mildew caused by Phasmorara viticola, Australasian Plant Pathology 1991;20:34-40

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Ouimette, D.G.; Coffey, M.D. Quantitative analysis of organic phosphonates, phosphonate and other inorganic anions in plants and soil using high-performance ion chromatography. Phytopathology 1988;78:1150-1155.

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Pelegri, R., Gamon, M.; Coscoll, R.; Beltrán, V.; Cunat. P. The metabolism of fosetylaluminum and the evolution of residue levels in orange and tangerines Pestic. Sci. 1993;39:319–323

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Hernández, F,; Sancho, J.V.; Pozo, O.J.; Villaplana, C.; Ibáñez, M.; Grimalt, S. Rapid determination of fosetyl-aluminum residues in lettuce by liquid chromatography/electrospray tandem mass spectrometry. J. AOAC 2003;86:832-838

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Duke, S.O.; Lydon, J.; Koskinen, W.C.; Moorman, T.B. Chaney, R.L.; Hammerschmidt,R. Glyphosate effects on plant mineral nutrition, crop rhizosphere microbiota, and plant disease in glyphosate-resistant crops. J. Ag Food Chem 2012;60:10375-10379

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Bruno, F.; Curini, R.; Di Corcia, A.; Nazzari, M.; Pallagrosi, M. An original approach to determining traces of tetracycline antibiotics in milk and eggs by solid-phase extraction and liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2002;16 : 1365-1376

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457 458 459 460 461

Figure 1

Chromatogram of green organic grape blank fortifed at 100 ng/g of four anlaytes with signal/noise ratio (blue trace is quantification transition, red trace is confirmation transition)

462 463

464

Note: This figure should be in color.



Page 22 of 28

22

465

Table 1.

Stock std

Preparation of calibration standard solutions.

µL used

ng/µL 1 1 1 10 10 10

5 10 25 5 25 50

IS (50 ng/µL)

Solvent/extract

µL used

µL used

5 5 5 5 5 5

490 485 470 490 470 445

ng total

466 467


(ng/mL) in liquid

5 10 25 50 250 500

10 20 50 100 500 1000

(ng/g) in 5 g sample 50 100 250 500 2500 5000

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23

468

Table 2.

Retention time and MRM conditions for LC-MS-MS analysis a.

469 470 Analyte

Precursor

Product

Retention

DP

EP

CE

CXP

Ion (m/z)

Ion (m/z)

Time (min)

Maleic hydrazide.1

111

82

2.59

-45

-10

-23

-10

Maleic hydrazide.2

111

42

2.59

-45

-10

-55

-10

Maleic Hydrazide D2 (IS)

113

42

2.59

-45

-10

-55

-10

Glyphosate.1

168

63

2.7

-50

-10

-27

-10

Glyphosate.2

168

79

2.7

-50

-10

-50

-10

Glyphosate 13C215N (IS)

171

63

2.7

-50

-10

-30

-10

Fosetyl aluminum.1

109

81

4.36

-50

-10

-14

-10

Fosetyl aluminum.2

109

63

4.36

-50

-10

-19

-10

Fosetyl aluminum D15 (IS)

114

82

4.36

-60

-10

-16

-10

Ethephon.1

143

107

4.44

-30

-10

-12

-10

Ethephon.2

143

79

4.44

-30

-10

-24

-10

Ethephon D4 (IS)

147

111

4.44

-30

-10

-11

-10

471 472 473 474 475

a. Compound dependent parameters: DP = declustering potential, CE = collision energy, EP = entrance potential, CXP = collision cell exit potential



Page 24 of 28

24

476 477 478

Table 3.

Matrix effect (%ME) evaluation (peak area of standard in solvent vs. in matrix). Slope

Sample

Analytes

solvent

matrix

% ME

Purple grape Maleic Hydrazide Glyphosate Fosetyl-Al Ethephon

210 239 3972 888

59 214 296 103

28 89 7 12

Green grape

195 254 3041 888

62 252 387 103

32 99 13 12

Maleic Hydrazide Glyphosate Fosetyl-Al Ethephon

479


Page 25 of 28


25

480

Table 4.

Average Recovery (%) and RSD (%) data obtained in the experiments (5 replicates at each level).

481 482 Spike level

Purple Grape (day 1) standard in standard in standard in matrix (IS) solvent (IS) matrix (ext) Recovery RSD Recovery RSD Recovery RSD (%) (%) (%) (%) (%) (%) 87 17 76 16 83 12 103 11 86 11 86 7 103 6 86 6 89 2

Green Grape (day 2) standard in standard in standard in matrix (IS) solvent (IS) matrix (ext) Recovery RSD Recovery RSD Recovery RSD (%) (%) (%) (%) (%) (%) 100 9 91 11 100 9 92 5 89 5 91 9 83 7 82 7 86 4

Analyte

(ng/g)

Maleic Hydrazide

100 500 2000

Glyphosate

100 500 2000

93 96 98

6 4 6

85 84 86

6 4 6

97 88 90

5 2 2

93 91 83

5 5 3

94 94 86

5 5 3

100 94 93

5 4 6

Fosetyl Aluminum

100 500 2000

97 110 113

8 1 3

117 110 113

8 1 3

95 83 86

6 3 4

92 88 87

1 2 4

115 109 107

1 2 4

100 95 82

4 3 3

Ethephon

100 500 2000

102 95 102

6 7 3

91 88 96

6 6 3

101 88 85

5 7 3

93 88 86

9 4 6

109 102 100

1 2 4

100 95 78

5 7 5



26

TOC


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27

Disclaimer The views expressed are those of the authors and should not be construed to represent the views or policies of the U.S. Food and Drug Administration. Any reference to a specific commercial product, manufacturer, or otherwise, is for the information and convenience of the public and does not constitute an endorsement, recommendation or favoring by the U.S. Food and Drug Administration.



254x190mm (96 x 96 DPI)


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Determination of glyphosate, maleic hydrazide, fosetyl aluminum, and

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