Quantitative Multiple Reaction Monitoring of Carbendazim in Orange Juice and Wine Using the EVOQ Elite LC-MS/MS System

Carbendazim, a broad spectrum fungicide, is employed in the production and storage of arable crops. Nevertheless, it is recognized as an endocrine disruptor and potentially affects human health following exposure or consumption. In addition, the well documented environmental toxicity of this pesticide has led to the increasingly stringent regulation of carbendazim over the last few decades, eventually ending in a complete ban in the United States.

The residual presence of carbendazim in imported fruit products may cause potentially harmful effects on public health. Brazilian orange juice imported to the United States in 2011 was found to contain carbendazim. In the following year, imported Chinese brand wines were found to contain significant concentration of carbendazim. Hence, the FDA has restricted the carbendazim content in imports to no more than 10ppb.

Liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) is widely recognized as the industry standard analysis method for pesticide screening. This article discusses the development of a quantitative MRM method for the analysis of carbendazim in orange juice and wine matrices utilizing a Bruker EVOQ LC-MS/MS system with a calibration range from 0.005ppb to 50ppb.

EVOQ Elite LC-MS/MS System

The EVOQ triple quadrupole for liquid chromatography (LC-TQ) (Figure 1) was intended for a singular purpose to perform reliable quantification of many real samples at the fastest sample-to-report time possible. It offers unprecedented sensitivity, linearity, accuracy, precision and an extensive dynamic range for multiple reaction monitoring (MRM) assays. Advances in atmospheric pressure ionization (API) technology and software make it a revolutionary technique for routine high-sensitivity, quantitative analysis.

The EVOQ Elite LC-MS/MS system.

Figure 1. The EVOQ Elite LC-MS/MS system.

With the lowest system delay-volume possible, the Advance LC pump technology provides unprecedented ballistic gradient reproducibility at analytical flow rates. This design-for-purpose feature reduces gradient delay and ascertains highly precise retention times, resulting in shorter run-to-run cycle times with the reproducibility essential for quantitative LC-MS/MS analyses.

The LC-TQ performance is extended by the OLE module by facilitating method-driven, on-line clean-up or sample pre-concentration. The inclusion of a third pump in the same module saves bench space and simplifies configuration under full software control, while delivering the flexibility of quick method development. The following are the key benefits of the EVOQ Elite LC-MS/MS system:

  • Ultra-high sensitivity for biomolecules and tiny molecules can be easily obtained due to the novel interlaced quadrupole (IQ) dual ion funnel.
  • Reliably run matrix-rich samples on the powerful orifice plate-based API interface.
  • Thermally labile molecules can be efficiently analyzed at high flow rates with the innovative vacuum insulated probe (VIP) heated electrospray probe.
  • Exception-based data-review software saves time by conveniently highlighting chromatograms that do not satisfy preset method criteria.

Sample Preparation and Experimental Set-up

Sample preparation for analyzing pesticides in juices typically applies two strategies: solid phase extraction (SPE) or ‘dilute-and-shoot’, of which the ‘dilute-and-shoot’ is the suitable technique owing to its operational simplicity. The dilution of the solutions is typically performed before the analysis to reduce the matrix effects caused by high concentrations of endogenous material. Nevertheless, few analyses have been performed to identify the appropriate dilution factors or to assess dilution-factor-caused matrix interference for carbendazim analysis.

Here, the samples analyzed included orange juice samples A and B, and wine samples of red wine A, white wine A, and red wine B. The samples were diluted with water for the ‘dilute-and-shoot’ analysis, using 5X, 20X, 100X, 200X, 500X and 1000X dilution factors, followed by spiking with 1ppb of carbendazim. In addition, nine carbendazim calibration standards were made at 0.005, 0.01, 0.05, 0.1, 5, 10 and 50ppb levels.

Chromatography (Advance UHPLC)

  • Column: YMC C18-Hydro, 50mm x 2mm x 3µm
  • Injection volume 5µL
  • Flow rate: 400µL/min
  • Solvent A: Water + 0.1% Formic acid + 10mM Ammonium Formate
  • Solvent B: MeOH, + 0.1% Formic acid + 10mM Ammonium Formate
  • Gradient conditions:
    0.0min 30% B
    0.2min 30% B
    2.0min 60% B
    2.1min 95% B
    3.1min 95% B
    3.2min 30% B
    5.2min 30% B

Mass Spectrometry (EVOQ Elite)

  • VIP-Heated ESI – positive mode
  • Spray voltage: 4000V
  • Nebulizer gas flow: 50 units
  • Heated probe gas flow: 45 units
  • Heated probe temperature: 450°C
  • Cone gas flow: 15 units
  • Cone temperature: 300°C

Experimental Results

The percentage of matrix effects plotted against dilution factor for the diluted orange juice and wine samples are delineated in Figure 2, showing the reduction of matrix effects to below 20% for orange juice with above 100-fold dilution. For wine samples, at least 200-fold dilution is needed to decrease the matrix effect to less than 20%.

Matrix effect% against dilution factor reveals appropriate dilution factors of 100-fold for orange juice and 200-fold for wine.

Figure 2. Matrix effect% against dilution factor reveals appropriate dilution factors of 100-fold for orange juice and 200-fold for wine.

The limit of quantitation of carbendazim set by the EVOQ sensitivity and reproducibility evaluation is at 0.005ppb (Figure 3). The RSD% of 10 replicate injection of 0.01ppb carbendazim is 3.4%, showing outstanding sensitivity and robustness of the EVOQ Elite LC-MS/MS system (Figure 4).

Calibration curve from 0.005 ppb up to 50 ppb.

Figure 3. Calibration curve from 0.005 ppb up to 50 ppb.

EVOQ sensitivity and reproducibility evaluation. RSD% of 3.4% for 10 replicate injections indicates great sensitivity and robustness.

Figure 4. EVOQ sensitivity and reproducibility evaluation. RSD% of 3.4% for 10 replicate injections indicates great sensitivity and robustness.

The quantitation results (Table 1) from the analysis of carbendazim in diluted blank matrices using the EVOQ Elite LC-MS/MS system show that all samples have trace concentrations of carbendazim, but well below the 10ppb level accepted by the FDA. However, the carbendazim concentration for 200-fold diluted red wine A was found to be 3740ppb, which is more than 300x the acceptable level (Figure 5).

Overlaid ion chromatograms of 200-fold diluted red wine B wine and 200-fold diluted red wine A red wine.

Figure 5. Overlaid ion chromatograms of 200-fold diluted red wine B wine and 200-fold diluted red wine A red wine.

Table 1. EVOQ LC-MS/MS Analysis of Carbendazim in Diluted Blank Matrices.

Blank Matrix Dilution factor Measured Conc. (ppb) Calculated Conc. (ppb)
Orange juice A 100 0.012 1.2
Orange juice B 100 N/A < 0.5
White wine A 200 N/A < 0.5
Red wine A 200 0.007 1.4
Red wine B 200 18.7 3740

Conclusion

The results clearly demonstrate the ability of the EVOQ Elite triple quadrupole LC-MS/MS to develop an LC-MS/MS based MRM method suitable for carbendazim quantitation.

This information has been sourced, reviewed and adapted from materials provided by Bruker - Chemical And Applied Markets.

For more information on this source, please visit Bruker Daltonics.

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