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From: Food Quality & Safety magazine, April/May 2012

Analyze organophosphorus pesticides in the apple matrix by GC/MS/FPD using an Agilent J&W DB-35ms Ultra Inert GC Column

by Doris Smith and Ken Lynam

Agilent J&W DB-35ms Ultra Inert GC Column

Organophosphorus pesticides are widely used in the agricultural industry for crop protection. Human toxicities for this class of molecules have shown acute and chronic effects from pesticide poisoning. OP pesticides affect the ner­vous system of insects and mammals by inhibiting an enzyme, acetylcholinesterase, that is important in helping regulate nerve impulses.1

Children are considered more susceptible to organophosphate toxicity because their pesticide dose per body weight is larger compared to that of adults.2 Children also have lower levels of detoxifying enzymes that deactivate OP pesticides, con­tributing to their vulnerability to pesticide exposure.3,4 Recent studies have shown a correlation between OP pesti­cide exposure and an increased risk for attention deficit hyperactivity disorder and other neurodevelopmental deficits in children.5-7 Because the main source of exposure for children is through consumption of food contain­ing OP pesticide residues, analytical testing capable of determining residual pesticides in food samples is critical.8

The multiresidue determination of pesticides in fruits and veg­etables usually involves an organic extraction of the pesti­cides from the plant matrix, followed by a cleanup procedure to remove co-extractives and other interferences. Anastassiades and colleagues developed the QuEChERS [quick, easy, cheap, effective, rugged, and safe] method for the analysis of pesticide residues in produce.9 This approach simplifies the traditional, labor-intensive extraction and cleanup procedure, while providing a fast, robust, and cost-effective method suitable for extracting pesticide residues.

Flow chart of the Agilent QuECHERS extraction procedure for apple samples
Figure 1: Flow chart of the Agilent QuECHERS extraction procedure for apple samples

Chromatographically active compounds such as OP pesticides can adsorb onto active sites in the sample flow path, particularly at trace levels, compromising an ana­lyte’s response. These pesticides tend to show peak tailing through interaction with active sites in a chromatographic system. This makes analysis challenging, particularly in diffi­cult sample matrices. Minimizing activity in the gas chromatography column is essential to ensure accurate quantitation. Agilent’s J&W DB-35ms Ultra Inert column minimizes column activity so difficult and active analytes can be consistently analyzed at trace levels. The use of the mid-polarity DB-35ms UI phase also offers additional selectivity over a non-polar phase, which can assist in resolving potentially co-eluting peaks, or shift a peak of interest away from matrix interferences.

Chromatographic Conditions
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Table 1: Chromatographic Conditions
Table 2: Flow Path Supplies
Table 3: Correlation Coefficients for the OP Pesticides Calibration Standards Analyzed by GC/MS-SIM and FPD in Phosphorus Mode with a Split Ratio for MSD:FPD = 3:1

A gas chromatographic system capable of multisignal detec­tion can provide complementary data for identification, confirmation, and quantitation of target analytes from a single injection. This method provides simultaneous detection of OP pesticides by gas chromatography/ mass spectrometry/selected ion monitoring and flame photometric detector in phosphorus mode by splitting the column effluent between the mass selective detector and FPD. The approach chosen here uses a GC/MSD/FPD system to identify and confirm the order of elu­tion for peaks of interest. Once the elution order is estab­lished, the chromatographic parameters can easily be trans­ferred to a GC/FPD system. The use of FPD detection without flow splitting is expected to increase sensitivity threefold, fur­ther improving lower level detection.

The GC/MS system was also equipped with backflush capa­bility, which shortens instrument cycle time by backflushing late-eluting matrix components through the inlet purge valve. Long bakeout times between injections are avoided by using this technique. Backflushing has the additional benefit of increasing the time intervals for source cleaning by effectively clearing deleterious matrix components from the system.10


An Agilent 7890A GC, combined with an Agilent 5975C GC/MSD equipped with a flame photometric detector and an Agilent 7683B auto­matic liquid sampler, were used for this series of experiments. A purged two-way capillary flow technology device was used to split the effluent 3:1 to the MSD:FPD. The CFT device also allowed for post-column backflush. Table 1 lists the chro­matographic conditions used for these analyses. Table 2 lists the flow path consumable supplies used in these experi­ments.

Reagents and Chemicals

All reagents and solvents were high-performance liquid chromatography or Ultra Resi grade. Acetonitrile from Honeywell (Muskegon, Mich.), toluene from Honeywell Burdick & Jackson, and acetone from JT Baker were purchased through VWR International (Radnor, Pa.). The 12-component custom pesticide standard was prepared by Ultra Scientific (N. Kingstown, R.I.).

Solutions and Standards

The OP pesticide stock standard solution (100 μg/mL of 12 OP pesticides) was diluted in acetone to yield spiking solutions of one and 10 g/mL. A surrogate standard, triphenyl phosphate, was prepared at concentrations of one, 15, and 100 g/mL in toluene. The spiking solutions were used to prepare the calibration curves in the matrix blank extract by appropriate dilution.

Sample Preparation

An organic apple sample was purchased from a local grocery store. The apple was chopped into small cubes and frozen at minus 80 degrees C overnight. The samples were then comminuted thoroughly to achieve sample homogeneity. The sample extraction method used the QuEChERS method. Figure 1 illustrates the sample preparation procedure graphically in a flow chart.

Chromatographic conditions
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Figure 2: GC/MS-SIM and FPD chromatograms of a matrix matched OP pesticides standard analyzed on an Agilent J&W DB-35ms UI 20 m × 0.18 mm × 0.18 µm capillary GC column (Agilent p/n 121-3822UI). Chromatographic conditions are listed in Table 1. The effluent split ratio is MSD:FPD = 3:1.

Samples containing 15 (± 0.1) grams of apple were weighed into centrifuge tubes. Quality control samples were spiked with appropriate amounts of spiking solutions to yield QC samples with quantitative concentrations relative to the 3:1 split ratio of 150, 300, and 750 ng/mL levels for GC/MS-SIM determination, and 50, 100, and 250 ng/mL by flame photometric detection. Each sample received a 15-mL aliquot of ACN. Two ceramic bars (Agilent p/n 5982-9313) were added to each sample to aid in sample extraction. The samples were vortexed for one minute. An Agilent original QuEChERS extraction salt packet (Agilent p/n 5982-5555) containing 6 grams of MgSO4 and 1.5 grams sodium chloride was added to each centrifuge tube. The capped tubes were shaken on a Geno/Grinder at 1500 rpm for one minute. The samples were centrifuged at 4000 rpm for five minutes.

An 8 mL aliquot of the upper layer was transferred to an Agilent QuEChERS dispersive SPE 15-mL tube for general fruits and vegetables (Agilent p/n 5982-5058). The dSPE tube was vortexed for one minute and then centrifuged at 4000 rpm for three minutes to complete the sample extraction. The extract from the dSPE tube was transferred to a GC vial and analyzed by SIM GC/MS and GC/FPD using the chromatographic conditions listed in Table 1.

Extractions of water and acetonitrile aliquots were prepared in the same manner as the samples and served as reagent blanks.

Discussion of Results

The OP pesticides were resolved on an Agilent J&W DB-35ms UI 20 m × 0.18 mm × 0.18 μm analysis column in about 30 minutes. The 12-component pesticide matrix-matched standard shown in Figure 2 shows good peak shapes for the pesticides in the GC/MS-SIM and FPD chromatograms. OP pesticides, particularly those that are more polar, can be problematic, often yielding broad peak shapes or excessive tailing and making reliable quantitation at low levels difficult. The high level of inertness of the DB-35ms UI column results in better peak shape and decreased sample adsorption, allowing lower detection limits. Figure 3 depicts the excellent peak shape seen for four of the more polar OP pesticides—oxydemeton-methyl, methamidophos, mevinphos, and acephate—with the DB-35ms UI column.

Enlarged section of GC/MS-SIM and FPD
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Figure 3: Enlarged section of GC/MS-SIM and FPD chromatograms of the more problematic polar pesticides analyzed on an Agilent J&W DB-35ms UI capillary column (Agilent p/n 121-3822UI). Chromatographic conditions are listed in Table 1. The effluent split ratio is MSD:FPD = 3:1.

The performance of the DB-35ms UI high efficiency column yielded excellent linearity and recovery over the calibration range of this study. The linearity of the column as defined by the r2 values of the pesticide standard curve was ≥0.992 for all the pesticides using both detectors. The individual OP pesticide analyte values are shown in Table 3.

The GC/MS-SIM analysis was able to detect down to the 15-20 ng/mL range for most of the pesticides. A higher SIM signal is necessary to quantify the more volatile pesticides below the 30 ng/mL range, mainly because of matrix interferences. Because flame photometric detection in phosphorus mode is selective only for analytes containing phosphorus, it is able to detect low levels of OP pesticides in complex matrices without the matrix interferences. The FPD was able to detect the OP pesticides down to 15 ng/mL, with the exception of naled, which could only be detected at higher levels (>25 ng/mL). Naled can undergo debromination, which can affect detection, especially at trace levels. The detection levels for the targeted OP pesticides were well below the U.S. maximum residue levels in an apple matrix, except in the case of chlorpyrifos, which has an MRL of 10 ppb for apples and grapes.11 Analysis by GC/FPD without flow splitting offers increased sensitivity to monitor the lower levels of detection needed for chlorpyrifos.

Recovery and Repeatability of OP Pesticides
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Table 4: Recovery and Repeatability of OP Pesticides in Spiked Apple Matrix with an Agilent J&W DB-35ms UI Column (Agilent p/n 121-3822UI)

The extraction process, using the QuEChERS method, was effective in retaining the OP pesticides in the spiked apple sample and providing sufficient cleanup of the sample matrix for GC/MS analysis. Figure 4 shows the OP pesticide mix spiked into an apple matrix sample. The matrix was prepared using a QuEChERS sample preparation approach that included extraction/partitioning and dispersive SPE. A GC/MS-SIM blank matrix trace is shown below the analyte trace to indicate the level of potential matrix interference with the analytes of interest. Peak shapes for the OP pesticides are still quite sharp and well resolved, indicating excellent performance on the DB-35ms UI column in a fruit matrix.

Recoveries were determined by GC/MS-SIM at the 150, 300, and 750 ng/mL levels, and 50, 100, and 250 ng/mL using the FPD in phosphorus mode. The recoveries for most of the pesticides were greater than 75%, with average RSDs below 10%. Recoveries for the individual OP pesticides are listed in Table 4. Lower recoveries were noted for the more polar pesticides: oxydemeton-methyl, methamidophos, and acephate. One possible explanation is that these polar, highly water-soluble pesticides may have been partially lost through incomplete partitioning into the aqueous layer during the extraction step.12


This application note successfully shows a quick and efficient analytical method to monitor low and trace level OP pesticide residue in apple samples. Splitting the column effluent to an MSD and FPD facilitated selectivity, identification, and confirmation of OP pesticides from a single injection, thereby increasing laboratory productivity. Using GC/MS in full scan mode enabled identification of specific pesticides, while SIM mode offered selectivity and sensitivity for quantitation of the pesticides at trace levels. Confirmation and further specificity were achieved by FPD in phosphorus mode. FPD detection was effective at minimizing matrix interferences, enabling lower detection.

The Agilent QuEChERS method for general fruits and vegetables provided enough sample cleanup to minimize matrix interferences while still maintaining low-level analyte detection. The simple QuEChERS extraction method allows for faster sample prep, facilitating higher sample throughput. Residual sample matrix carryover is removed through use of backflush, which eliminates the need for a bakeout cycle, significantly reducing analytical run times.

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Figure 4: GC/MS-SIM chromatogram of the apple extract blank and a 150 ng/mL spiked apple extract analyzed on an Agilent J&W DB-35ms UI capillary column (Agilent p/n 121-3822UI). Chromatographic conditions are listed in Table 1.

The Agilent J&W DB-35ms UI capillary column resolves the targeted OP pesticides and provides excellent peak shapes for the polar pesticides, allowing for more reliable quantitation at low levels. Detection levels for the OP pesticides were at or below the U.S. maximum residue levels for various fruits. Matrix-matched calibration standards yielded regression coefficients r2 ≥ 0.992, and recoveries from fortification studies were greater than 75% with an average RSD <10% for both GC/MS-SIM and FPD, further demonstrating the effectiveness of using an Agilent J&W DB-35ms UI column for residual pesticide determination.

Smith and Lynam are employed at Agilent Technologies Little Falls site in Wilmington, Del. Smith is an applications chemist for Volt Management, Inc., and provides application support for Agilent’s line of GC capillary columns, including the latest line of J&W Ultra Inert columns. Lynam is a senior applications chemist in the Chemistries and Supplies Division of the Chemical Analysis Group. His experiences in industrial chemical, pharmaceutical, and environmental settings over more than 20 years have provided him the opportunity to develop application solutions using GC, GC/MS, HPLC, and SFC techniques.


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