Application Note #1823788Evaluation of Matrix Effects for Dilute-and-shoot LC/MS-MS Analysis of Carbendazim in Orange Juice and Wine

AbstractA quantitative Multiple Reaction Monitoring (MRM) method for car-

bendazim analysis in orange juice and wine matrices was developed

for the Bruker EVOQ Elite LC-MS/MS system with a calibration range

from 0.005 up to 50 ppb. Matrix effects were evaluated by compar-

ing matrix effect% against dilution factors. Results indicate that or-

ange juice and wine matrices exhibit different matrix effects and pro-

vide the scientific reference to determine appropriate dilution factors.

Good instrument sensitivity and robustness were achieved during

method development.

Introduction - Residues in Food Analysis Carbendazim is a broad spectrum fungicide used for the production

and storage of arable crops. However carbendazim is also a known

endocrine disruptor with potentially harmful effects on human health

following consumption or exposure.1 Together with the pesticide’s

well documented environmental toxicity, this has resulted in the in-

creased regulation of carbendazim over the past few decades, culmi-

nating with a complete ban in the US. Carbendazim may pose a risk

to public health due to its residual presence in imported fruit

products. In the US in 2011 carbendazim was detected in imported

Brazilian orange juice. The following year a significant concentration

was detected within Chinese brand wines. In response the US Food

and Drug Administration (FDA) has limited the concentration of car-

bendazim in imports to no more than 10 ppb.2

MethodLiquid chromatography coupled with triple quadrupole mass spec-

trometry (LC-MS/MS) is widely considered the industry standard

technique for pesticide screening. Sample preparation for pesticide

analysis in juice generally employs two strategies: solid phase ex-

traction (SPE) or ‘dilute-and-shoot’. Of the two ‘dilute-and-shoot’ is

the preferred method due to its operational simplicity. Solutions are

commonly diluted prior to analysis in order to minimize the matrix

effects resulting from high concentrations of endogenous material.

However, few studies have been carried out to determine the appro-

priate dilution factors or to evaluate dilution-factor-caused matrix in-

terference for carbendazim analysis. In the current study, a quantita-

tive MRM method for carbendazim analysis in orange juice and wine

matrices was developed using a Bruker EVOQ LC-MS/MS system

with a calibration range from 0.005 to 50 ppb.3

Experimental

Sample PreparationOrange juice samples A and B were purchased from a local grocery

shop. Three wine samples were also purchased: red wine A, white

wine A, and red wine B. The orange juice and wine samples were

prepared for the ‘dilute-and-shoot’ analysis by diluting with water us-

ing 5X, 20X, 100X, 200X, 500X and 1000X dilution factors. 1 ppb of

carbendazim was spiked into each diluted samples. Nine carbendaz-

im calibration standards were also prepared at 0.005, 0.01, 0.05, 0.1,

5, 10 and 50 ppb levels.

Chromatography (Advance UHPLC) • Column: YMC C18-Hydro, 50 mm x 2mm x 3µm

• Injection volume 5µL

• Flow rate: 400 µL/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 (EVOQTM Elite) • VIP-Heated ESI – positive mode

• Spray voltage: 4000 V

• Nebulizer gas flow: 50 units

• Heated probe gas flow: 45 units

• Heated probe temperature: 450oC

• Cone gas flow: 15 units

• Cone temperature: 300oC

Results and Discussion

Sample PreparationFigure 2 shows the percentage of matrix effects against dilution fac-

tor plotted for the diluted orange juice and wine samples. For orange

juice above 100-fold dilution matrix effects are reduced to below

20%. For wine samples, at least 200-fold dilution is required to re-

duce matrix effect to below 20%.

Figure 3 illustrates the excellent linearity with four orders of magni-

tude from 0.005 to 50 ppb, achieving an R2 value of 0.995.

Figure 1: Molecular structure of carbendazim, a banned pesticide detected in imported orange juice and wine.

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

Chromatography (Advance UHPLC)

Conclusions An LC-MS/MS based MRM method was developed for carbendazim

quantitation in domestic and imported orange juice and wine using

the EVOQ Elite triple quadrupole LC-MS/MS. 100-fold diluted orange

juice and 200-fold diluted wine samples were found to be suitable di-

lution factors for the ‘dilute-and-shoot’ method.

The quantitation results from the EVOQ Elite LC-MS/MS analysis of

carbendazim in diluted blank matrices reveal that orange juice sample

A and B, white wine A and red wine B all exhibit trace concentrations

of carbendazim, albeit well below the 10 ppb permitted by the FDA.

The carbendazim level for 200-fold dilutedred wine A is detected at

3740 ppb, as illustrated by Figure 5, over 300x the acceptable level.

AuthorsHelen (Qingyu) Sun, Zicheng Yang; Kefei Wang

References(1) PAN Europe factsheet on Carbendazim, Brussels, 2nd March 2011

(2) Orange Juice Products and Carbendazim: Addendum to FDA Let-

ter to the Juice Products Association (January 9, 2012)

(3) Evaluataion of matrix effects for Dilute-and-Shoot LC-MS/MS anal-

ysis of carbendazim in orange juice and wine – poster presented by

Bruker Chemical and Applied Markets at North America Chemical

Residue Workshop, 21-24 July 2013

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EVOQ Sensitivity and ReproducibilityFigure 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.

Overlain Ion Chromatograms

Quantitation The EVOQ sensitivity and reproducibility evaluation set the limit of quantitation of carbendazim at 0.005 ppb. The RSD% of 10 replicate injec-

tion of 0.01 ppb carbendazim is 3.4%, demonstrating excellent instrument sensitivity and robustness.

Figure 3: Calibration curve from 0.005 ppb up to 50 ppb. Figure 4: EVOQ sensitivity and reproducibility evaluation. RSD% of 3.4% for 10 replicate injections indicates great sensitivity and robustness.

Calibration curve from 0.005 to 50 ppb EVOQ Sensitivity and Reproducibility

For research use only. Not for use in diagnostic procedures.

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