The Experiment

Materials & Methods

HPLC Conditions:

Obtaining A Representative Sample
- Stir and obtain 3 different samples of a single brand of orange juice
-Then mix the 3 samples together and stir to make it homogeneous

Sample Preparation:
- Spike 0.5 mL of Orange Juice with Triphenyl Phosphate (as an Internal Standard) at 50 ppb.
- Dilute 3:1 with HPLC Grade Water & vortex
- Centrifuge 15-20 min at 6000 rpm to remove the sediments
- Collect supernatant which the carbendazim dissolves in
- Run HPLC/MC analysis

Internal Standard Calibration Method:
- Prepare 5 known standards of Carbendazim of different concentrations, with Triphenyl Phosphate as the internal standard.
- Inject each standard and obtain the detector response at each concentration
- Draw a calibration graph between normalized peak areas and concentration of standard solutions to obtain the Calibration Graph

Sample Quantification:
- Inject prepared samples into HPLC and obtain the detector response
- Calculate the concentration of carbendazim from the Calibration Graph

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If you are wondering what is Triphenyl Phosphate...

Triphenyl phosphate is used as the internal standard because it is available in high purity (>99% from Sigma-Aldrich), and chemically similar and does not react with Carbendazim and the other fungicides of interest in the study. In addition, it does not cause any chemical or matrix interference in the chromatographic process, and its physico-chemical properties are similar to carbendazim.

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Results & Discussion

Here are the MRM extracted ion chromatographs for Carbendazim (spiked at 2 ppb) & Triphenyl phosphate (spiked at 50 ppb) in orange juice extract from the study.

Carbendazim was spiked in the orange juice extract to simulate a scenario of orange juice contaminated with the fungicide carbendazim.

This is the calibration graph for the concentration of carbendazim in orange juice extract, from 2.0 - 500 ppb concentration levels.

References:
n.d. 2012. LC/MS/MS of Fungicides and Metabolites in Orange Juice with Agilent Bond Elut Plexa and Poroshell 120. [ONLINE] Available at: http://www.chem.agilent.com/Library/applications/5991-0051EN.pdf. [Accessed 27 January 2013].

n.d. 2012. The Determination of Benomyl and Carbendazim in Municipal and Industrial Wastewater. [ONLINE] Available at: http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007_11_06_methods_method_631.pdf. [Accessed 17 January 2013].

Detection of Carbendazim

In this project, we will be looking at a study by Agilent Technogies, Inc. that describes the detection of carbendazim and 3 other fungicide residues in Orange Juice, and HPLC coupled to Mass Spectrometry (MC) was the detection method of choice.

In this study, the detector used in this image would be a Mass Spectrometer.

How HPLC works:

In our case, what we need for our detection would be a Normal Phase HPLC

In a normal phase HPLC, we use a NON-POLAR mobile phase and a POLAR stationary phase. Firstly, we have 2 types of mobile phase 1) 0.1% formic acid in WATER and 2) 0.1% formic acid in METHANOL. 

So in our solvent  reservoir, we need 0.1% formic acid, water and methanol.

During the actual run of the analysis, the solvent are run through a vacuum degasser, where the solvent are removed of dissolved gas that cause inconsistent flowrate which could affect the results, into the quarternary pump where the different solvents are mixed  to give the desired mobile phase that would pass through into the column. At this point the autosampler, the injection system, would introduce 5µm of our samples or standards into the column together with our mobile phase which is a packed column of C18 on 2.7µm spherical silica particles. At the end, we will use a mass spectometry detection method.

How Mass Spectrometry works:

The molecule is first ionised to give it a positive charge and then accelerated to have a constant kinetic energy for all molecules. Along the path which the molecules are travelling, they are deflected with a magnetic field. The strength of this deflection depends on the size of the molecules and also the charge that it obtained when it was earlier ionised, so the most deflected molecules are those with a large molecular weight and those with more charge. The detection of the ions is done by detecting the neutralised electric current.

Image credits: 

HPLC/MC - http://www.waters.com/webassets/cms/category/media/other_images/primer_e_lcsystem.jpg

Isolation of Carbendazim

Given that carbendazim decomposes at 250°C, we cannot use Gas Chromatography (GC) to isolate carbendazim in our samples. Thankfully, we could use High Performance Liquid Chromatography (HPLC)! HPLC analysis does not require high temperatures as there is no flash vaporization technique involved. Instead, samples are analysed at room temperature and therefore there is no risk of carbendazim decomposing during the analysis! Hurray for HPLC!

 

However, we should not celebrate too quickly and keep in mind that carbendazim slowly decomposes at alkaline pH, hence we will need to either acidify the sample, or maintain it at a neutral pH as we run it for analysis. 

Image credits:
HPLC - http://www.knauer.net/typo3temp/pics/aa01b4668f.jpg

Carbendazim - The Good & The Bad (Really Bad)

The Good

- A commonly used fungicide
- Used for many Fungal Diseases in plants. Mould, blackspot, blight, mildew, etc. (add pictures)
- Carbendazim works by inhibiting the development of fungi by inhibiting tubulin formation at mitosis.
(Carbendazim inhibits fungal mitotic microtubule formation)

The Bad REALLY Bad: Health effects

- Mutagenic:
  - Potent aneugen even at LOW exposures
  - Inhibits polymerization of tubilin= aneuploidy

- Reproductive:
  - Damage mammal womb development
  - Deformities like lack of eyes and hydrocephalus <--- "brain juice overload"

- Carcinogenic:
  - A study on mice fed with carbendazim for 2 weeks showed a tendency of the development of benign and malignant tumors 

More about Carbendazim

IUPAC name: Methyl benzimidazol-2-ylcarbamate

Molecular formula: C₉H₉N₃O₂

Relative molecular mass: 191.2

Physical characteristics:
Pure carbendazim is a colourless, white crystalline, odourless solid which decomposes on melting at approx. 250°C.

Solubility:
(at 20°C)
8mg/litre water (at pH 7)
68mg/litre dichloromethane
100mg/litre chloroform
300mg/litre acetone
5g/litre dimethylformamide
300g/litre ethanol

Stability:
Stable in storage at 35-50°C. Slowly decomposes in alkaline solution. Stable in acid, forming water soluble salts.

Chemical structure for Carbendazim:


How the fungicide looks like:


For more info about Carbendazim, check out this webpage we found!
http://www.inchem.org/documents/pds/pds/pest89_e.htm

Image credits: Chemical Structure - http://www.sigmaaldrich.com/medium/structureimages/90/mfcd00055390.png
Carbendazim fungicide powder - http://i00.i.aliimg.com/photo/107523050/Carbendazim.jpg