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Ian Ronningen- Pesticides and Toxins in fragrances and natural flavors

The focus of my analytical problem is the presence of residual pesticides, or natural toxins in natural flavors and fragrances. The raw materials for natural flavors and perfumes are frequently sourced from fruits, spices, herbs, flowers, microbial fermentation and a number of other agricultural sources (1). Extraction is commonly done via steam distillation, expression, tincturing, distillation, and a number solvent extraction techniques. These are then commonly concentrated for a number of reasons, including added stability and ease of transportation (1). Any toxins, pesticides compounds present would also be concentrated, which would eventually reach the consumer (3). Produce is generally cleaned and treated before the fragrance or flavor is extracted but treatments are kept mild to preserve the character of the raw material. Another aspect to this is flavor and fragrance companies sourcing rare and unique raw materials from remote and undeveloped countries that may have sup-par quality control measures, accidental inclusion of heavy metals from groundwater, or use of banned pesticides still remain and issue. Part of my interest in this topic is consumer perception that "naturally" flavored products are healthier than artificial flavors.

The hypothesis is that these toxins and pesticides are concentrated in the raw materials for naturally flavors and fragrances. These could have an acute or chronic health impact on human health. Additional financial impact could stem from levels of contaminants below a level that could pose harm to the consumer.

Target analytes include common pesticides, naturally produced plant toxins, heavy metals.

These matrixes will commonly be alcohol, powdered, in oil emulsions, and occasionally in water. Matrices will be complex, and methodology will require high throughput, high sensitivity, and rapid multi-residue detection for application (2).

(1) Reineccius, Gary. Flavor Chemistry and Technology. Boca Raton : Taylor & Francis, c2006.
(2) Seiber, J N. "Contributions of Pesticide Residue Chemistry to Improving Food and Environmental Safety: Past and Present Accomplishments and Future Challenges." Journal of agricultural and food chemistry 59.14 (2011):7536-7543.
(3) Culliney, T W. "Pesticides and natural toxicants in foods." Agriculture, ecosystems & environment 41.3-4 (1992):297-320.

UV-Vis Absorption Spectrometry-

Driss et al (1993) has shown application of UV-Vis absorption in water at a wavelength of 254 nm in water, for simultaneous detection of parathion-methyl, parathion, fenitrothion, diazinon, azinphos-methyl, azinphos methyl, phosmet, carbaryl. Additionally another study reported detection of a mixture of seven insecticides using a wave length of 220 nm in a 80:20 or 70:30 methanol to water solution (Farran, 1988). A wave length of 290 nm was selected by Sultatos et al, however in this publication they did note that the maximum variation of each compound did vary somewhat, their detection included: Parathion, Paraxon, p-Nitrophenol, Chlorpyrifos, Chlorpyrifos Oxon, Trichloro-2-pyridinol, Methyl Chlorpyrifos, Methyl Chlorpyrifos Oxon, Azinphos Methyl, Coumaphos. Two of these papers also took time to analyze the oxon form of these toxins as oxons are converted into compounds with higher toxicity, and fat solubility in the human liver than their native form (Davies, 1975). All three of these methods use a separation method before detection, in many methods this is either gas or liquid chromatography. The structural differences between the various pesticides is limited and as such distinction between them using UV-Vis without a separation technique would be extremely challenging, if not impossible.

Molar absorptivity has been a challenge to find for all potentially applied pesticides, however research has been done to investigate molar absorptivity on some organophosphates, and some chloroacetanilides. These include: ε = 466 ± 41 for alachlor, ε = 3558 ± 323 for diazinon, ε = 5.5 ± 0.5 for dichlorvos, in units M-1cm-1 (Feigenbrugel, 2005). Although some literature values were identified, extensive literature was not found relating to the molar absorptivity of potential contaminants, this could stem from the application of mass spectroscopy for complex food matrices, do note this is speculative.

With identified analytes UV-vis absorption is expected, especially considering many of the organophosphates have a phenolic ring, or a nitrogen containing ring such as pyridine, pyrazine, pyrimidine. These compounds alone can be detected using UV-Vis spectroscopy, and additional ring substitutions would most likely enhance the likelihood of detection.

Please see comment on fruit proteins allergen post.

BLOG 6. Chemical structure and standards

Selected Structures:

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Analytical Standard Sources-
Atrazine-Fluka 99% Pure- 250mg for $35.20. Product # 45330

Alachlor- Fluka 99% Pure- 250 mg for $20.70. Product # 45316 (C13 labeled 10mg for $282.50, #34086)

Diazinon- Fluka 99% Pure- 250 mg for $30.90. Product# 45428 (Deterium- 10mg for $691.10, Fluka Product# 492175)

Warfarin- Fluka 99% Pure- 250 mg for $34.50. Product#45706

Standard Preparation- Prepare samples through dilution of sample to analytical range. Using equally spaced concentrations prepared through serial dilution, analyzed in triplicate (more depending on time). Standard curve should cover potential range of analyte concentration, as an example if the expected concnetration is 50 ppm, a calibration curve should include many points (5-100 ppm) and be linear in that region. Additionally standard addition of a selected (potentially a not present pesticide which is spiked at a certain level) could be used in this case.

Mass Spectra for Atrazine (ESI Positive Mode):

Source is (4) Schreiber, A. "Application of spectral libraries for high-performance liquid chromatography-atmospheric pressure ionisation mass spectrometry to the analysis of pesticide and explosive residues in environmental samples." Journal of chromatography 869.1-2 (2000):411.
Atrazine Spectra: For Blog 7:
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Davies J, Barquet A, Vaclavek C, et al. Human Pesticide Poisonings by a Fat-Soluble Organophosphate Insecticide. Archives of Environmental Health. December 1975;30(12):608.

Driss, M R. "Determination of carbaryl and some organophosphorus pesticides in drinking water using on-line liquid chromatographic pre-concentration techniques." Journal of chromatography 639.2 (1993):352-358.

Farran, A. "Identification of organophosphorus insecticides and their hydrolysis products by liquid chromatography in combination with UV and thermospray-mass spectrometric detection." Journal of chromatography 455(1988):163.

Feigenbrugel, V. "Near-UV molar absorptivities of acetone, alachlor, metolachlor, diazinon and dichlorvos in aqueous solution." Journal of photochemistry and photobiology. A, Chemistry 174.1 (2005):76

Sultatos, L G. "Determination of organophosphorus insecticides, their oxygen analogs and metabolites by high pressure liquid chromatography." Chromatographia 15.10 (1982):669.


Blog 13? - 1 pt

Blogs 9, 10, 11. Great answers.

Blog 11.

There have been application reported using CZE, MEKC, cIEF and CGE (2). However it seems like for my analysis the best option would be MEKC since it is applicable to a wide number of analytes in my matrix and has excellent resolving power, and when coupled with effective SPE methods would allow for effective analysis by MS(1,2, 3). Although addition of ionic additives might be required to resolve compounds with similar structure. CGE however would not be as effective as the other techniques, since my analytes are small molecular weight the impact of the gel would be limited.

A 75 mM cholic acid with 6mM sodium tetraborate decahydrate in deionized water. An uncoated capillary was selected (50 cm x 75 um I.D.) and the separation was carried out using a 30 kV votage. A final pH of 9.2 was selected, all conditions were modeled after Pico et al (2).

Mass Spectrometry is the best selection for compound identification, and elucidation. At the given concentration this analyzer should be fine, especially considering concentration using SPE.

(1). HernándezBorges, J. "Highly sensitive analysis of multiple pesticides in foods combining solid‐phase microextraction, capillary electrophoresis‐mass spectrometry, and chemometrics." Electrophoresis 25.13 (2004):2065.
(2) Pico, Y. "Capillary electrophoresis for the determination of pesticide residues." TrAC. Trends in analytical chemistry 22.3 (2003):133.
(3) Rodríguez, R. "Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits." Analytical chemistry 75.3 (2003):452.

Blog 10. (Please note Blog 9 was caught by spam filter)

My analytical problem would best be analyzed by RP-HPLC-MS (Triple Quad). This method allows for rapid analysis, high resolution and high sample throughput with simple and rapid sample prep.

The problems that were similar to mine, although they did not specifically us RP-HPLC coupled to a MS as an method of analysis. RP-HPLC was used for analysis of taurine in energy drinks, and for analysis of aflatoxin in cooking oil. Although both problems used a different detector, either could be adjusted to use MS.

BLOG 9. Chromatographic techniques

The types of chromatography that are applicable to my analytical problem include reversed phase liquid chromatography, and gas chromatography either directly or with derivatization. The other types of chromatography would not be able to separate my analytes. Size exclusion would not be feasible due to the small differences in molecular weight, meaning poor separation is likely. Affinity chromatography would limit number of analytes analyzed, which is a negative due to the number of analytes present. HILIC is a possibility, however pesticide analysis sees a wider application of reversed phase separation methods.

Although GC is an adequate method, most analysis has moved to HPLC, which is what I will also use for my separation technique.

Columns: All from sigma aldrich.
-Ascentis Express RP-Amide HPLC Column 53913-U -2.7 μm particle size, L × I.D. 10 cm × 2.1 mm
-Ascentis Express C18 HPLC Column 53823-U -2.7 μm particle size, L × I.D. 10 cm × 2.1 mm
-Ascentis Express Phenyl-Hexyl HPLC Column 53336-U-2.7 μm particle size, L × I.D. 10 cm × 2.1 mm

All of these columns would be adequate for separation of my analytes, a traditional C18 column allows for good separation of pesticides, but would struggle with polar compounds. If the analysis did involve a high number of polar compounds a C18 chain with a internal polar group, like the RPAmide column, would also be a good choice, and would still provide good separation of all other compounds. If a majority of the compounds were polar aromatics the phenyl-Hexyl column would be great, especially since many of the analytes are heterocyclic rings.

All of the selected columns can be used for either HPLC or for UHPLC, are able to be run at high pressure, and are stable over a broad pH range. A 10cm column would allow for effective separation, while still maintaining a reasonable back pressure, and could be run even on a traditional HPLC system. Run conditions would most likely start at 5-10% water, with either formic acid or ammonium formate adjusted to pH 3-3.5. A gradient elution would be required for rapid analysis and adequate peak resolution. The HPLC system would be coupled to a triple quadrupole mass spectrometer, potentially operating in MS/MS. This will give an adequate limit of detection, is very selective for analytes, especially when running in MS/MS mode. It also has the possibility of comparing to extensive pesticide residues and would allow easier identification of unknown analytes.
Gong, A. "Analysis of trace atrazine and simazine in environmental samples by liquid chromatography-fluorescence detection with pre-column derivatization reaction." Journal of chromatography 827.1 (1998):57.

Krause, R T. "Determination of fluorescent pesticides and metabolites by reversed-phase high-performance liquid chromatography." Journal of chromatography 255(1983):497.

 Picó, Y. "Environmental and food applications of LC–tandem mass spectrometry in pesticide‐residue analysis: An overview." Mass spectrometry reviews 23.1 (2003):45.

Schreiber, A. "Application of spectral libraries for high-performance liquid chromatography-atmospheric pressure ionisation mass spectrometry to the analysis of pesticide and explosive residues in environmental samples." Journal of chromatography 869.1-2 (2000):411.

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Excellent answer. Check spelling.

BLOG. 8- Sample Preparation and clean up.

I am lucky in the fact that there are commercially available SPE cartridges that are designed to clean up and produce effective sample clean up in food products that are being analyzed for pesticides.

The product is sold and distributed by Restek, Catalog#26125. The product is also part of an AOAC (1, 3) method for pesticide analysis.

Although this is a great tool some sample prep is still required. This includes (2,4):

Step One- Weighing sample and solvent extraction. 1 mL of acetonitrile per 1 g of sample, add internal standard. Shake for one minute.

Step 2- Using annhydrous salts (magnesium sulfate, sodium chloride, trisodium citrate dihydrate, disodium hydrogencitrate sesquihydrate) dry sample, weights are 4 g, 1g, 1g, 0.5 g respectively. Shake for one minute.

Step 3. Centrifuge at 3,000 U/min for 5 minutes.

Step. 4. Transfer to dispersive SPE (dSPE) tube. shake tube, and centrifuge for 5 minutes at 3,000 U/min. After elution clean up pH may be adjusted with formis acid. I will analyze both before and after pH adjustment to ensure maximum detectable pesticides.

After this preparation these samples are ready for GC or HPLC analysis.

These products are called Q-sep QuEChERS dSPE tubes. They come in a variety of formulations which can select for various contaminants. They can contain: magensium sulfate for partitioning of water away from organic solvent, primary and secondary amine exchange (PSA) material for removal of sugars and fatty acids, graphitized carbon for removal pigments and sterols, and C18 packing for removal of nonpolar compounds.

For my use I would want a dSPE cartridge with magnesium sulfate, PSA, graphitized carbon, and potentially C18. These ensure reduction of noise and will help with low concentration analytes.


1. M. Anastassiades, S.J. Lehotay, D. Stajnbaher, F.J. Schenck, J. AOAC International 86, p. 412-431 (2003).
2. QuEChERS-A Mini-Multiresidue Method for the Analysis of Pesticide Residues in Low-Fat Products.
3. AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with
Magnesium Sulfate.
4. EN 15662, Foods of Plant Origin—Determination of Pesticide Residues Using GC-MS and/or LC-MS/MS
Following Acetonitrile Extraction/Partitioning and Clean-up by Dispersive SPE—QuEChERS method

Excellent answers for Blogs 6 and 7.

BLOG 7. Atomic and mass spectrometries

In my analysis I will not be able to use any type of atomic spectrometries.

Application of Mass Spectroscopy is widely used in the field of pesticide analysis. In the case of my analysis MS/MS methods are important to use to help with identity confirmation based from the fragmentation of the analyte. This is important due to the difficulties with seperation and co-elution, through application of MS/MS specific analytes of interest can be selected for and quantified (2).

Selected Analytes of interest- Note citation 1,2, and 3 provide tables of general pesticide structure and mass and fragmentation data.
Guthion- 317.005 g/mol
Potosan- 328.32 g/mol
Warfarin- 308.33 g/mol
Piperonyl butoxide- 338.438 g/mol
n-Propyl isome- 362.4168 g/mol
Carbofuran- 221.25 g/mol
Naptalam- 291.30 g/mol
propham-179.22 g/mol
Atrazine- 215.68 g/mol

Lehotay uses GC as a separation technique coupled to an ion trap mass spec using electron impact ionization(1). And although this was effective in identification and quantification I think application of a LC system employing a either a Q-tof or a triple quad mass spec would be best. Although a Q-tof is a high resolution analyzer the triple quad would have an improved dynamic range, Pico et al. discusses the LOD and the quantification ability of each instrument as shown in a literature review, it seems that triple quad mass analyzers are the most used, and are run in single ion monitoring mode to increase sensitivity. It is further discussed that positive ionization is used for basic analytes such as: triazines, carbamates, and organophosphorus pesticides, ammonium quaternary herbicides, and phenylureas, where negative ionization is used for acidic pesticides like phenoxy acid and sulphonylureas (2). Electrospray ionization has a slight majority of use over APCI in this literature review (2), and so would be a good starting point for my analysis, I also believe that application of a triple quad mass analyzer would be a good selection for analysis. Thurman et al. discusses the increased sensitivity of APCI-Positive in analysis of neutral and basic fragments, and ESI-negative for anionic and cationic analytes (4).

See Spectra attached in blog posting.

(1) Lehotay, S. J., & Eller, K. I. Development of a method of analysis for 46 pesticides in fruits and vegetables by supercritical fluid extraction and gas chromatography/ion trap mass spectrometry. Journal of AOAC International 78 (1995): 3.

(2) Picó, Y. "Environmental and food applications of LC–tandem mass spectrometry in pesticide‐residue analysis: An overview." Mass spectrometry reviews 23.1 (2003):45.

(3)Schreiber, A. "Application of spectral libraries for high-performance liquid chromatography-atmospheric pressure ionisation mass spectrometry to the analysis of pesticide and explosive residues in environmental samples." Journal of chromatography 869.1-2 (2000):411.

(4)Thurman, E M. "Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides." Analytical chemistry 73.22 (2001):5441.

Blog 4. Great studies, but they are not directly addressing the hypothesis. You either need to revisit your hypothesis or modify the studies. (-0.5 pt).

Blog 5. Great answers. Fluorescent analysis of some pesticides is more sensitive if these are derivatized.

Blog 5. Fluorescence-

Pesticides being a rather general term, there are a number of different analytes that could be selected.

Some identified analytes with fluorescent characteristics are as follows:

-Guthion- in water pH 11 after hydrolysis- Excitation 312 nm, emission 380 nm.

-Potasan- in methanol- 320 nm excitation, 385 nm emission

-Warfarin- in methanol- 320 nm excitation, 385 nm emission

-Piperonyl butoxide - in methanol- 292, 248 nm excitation, 318 nm emission

-n-Propyl isome- in water, pH 7- 285 nm excitation, 326 nm emission

All above from source (2).

Below data was gained from (3) Using a acetonitrile water gradient

Aniline- 245 nm excitation- 340 nm emission
Biphenyl- 258 nm excitation- 316 nm emission
carbofuran- 278 nm excitation- 306 nm emission
para-chloroaniline- 292 nm excitation- 343 nm emission
naphthalene acetamide- 286 nm excitation- 335 nm emission
naptalam- 328 nm excitation- 427 nm emission
propham- 242 nm excitation - 306 nm emission

Atrazine, a widely used and an increasingly used pesticide, can be derivitized using 4-(2-Phthalimidyl) benzoyl chloride (PIB-Cl) for detection, using 312 nm excitation, and 420 nm for emission (1).

(1) Gong, A. "Analysis of trace atrazine and simazine in environmental samples by liquid chromatography-fluorescence detection with pre-column derivatization reaction." Journal of chromatography 827.1 (1998):57.

(2)Hornstein, I. "Pesticide Residues, Spectrophotofluorometry for Pesticide Determinations." Journal of agricultural and food chemistry 6.1 (1958):32.

(3)Krause, R T. "Determination of fluorescent pesticides and metabolites by reversed-phase high-performance liquid chromatography." Journal of chromatography 255(1983):497.

There are a few key studies that I have identified.

1. Investigation of commercial raw material cleaning processes. The goal of this study is to identify the effectiveness of toxin or pesticide removal using a commercial washing method. In many cases the washing method is a fairly light treatment, this is to preserve all flavor and aroma impact characteristics. Some materials, like citrus, may have more severe treatments, however raw materials like flowers (lavender) would have a light washing process. There would be two studies that I would like to do here. First, identify key incoming materials and analyze them pre-washing, and post washing for toxic compounds. This would establish the efficiency of the process. Secondly, doping compounds of varying nature (hydrophobicity, solubility) and identifying which compounds are most efficiently removed by the process, this would help industry establish which pesticides might be a best choice to use on the raw materials. It would also be worthwhile to analyze and quantify the concentration of various pesticides and toxins in the wash solution, since if this concentration is high you are not washing but contaminating your raw materials.

Further a second study to identify various extraction methods. I would like to investigate pressing of citrus, steam distillation and solvent extraction, the final two are commonly used for spice extractions, and more delicate extractions. Identifying the extraction efficiency of the analytes versus the flavor and aroma compounds. This is an important experiment, if compounds are present after the wash, but are not recovered during the flavor or aroma extraction process then the importance of the toxin is minimal. However if an analyte is extracted with a higher efficiency than flavor and aroma compounds then washing practices might need to be adjusted, or incoming quality measures adjusted to limit this toxin. I would like to conduct experiments that quantify toxin amount before extraction, and quantify toxin present in the resulting extract.

Identifying amounts of analyte on products is a difficult task to do. The USDA sets upper limits of pesticide residues on commodities, in the case for oranges the ranges for regulated compounds is 50 ppm to ppb range. This is for a commodity product. The flavor and fragrance industry are currently sourcing rare raw materials and flavor and aroma compounds from non-commodity products which may not be regulated, however the tolerances for most products are ppm-ppb range.

Source: USDA International Maximum Residue Limit Database (no ACS citation available.) Online access and limits set for regulated pesticides in food products.

My problem is similar to this one. We are talking about the organic toxin substance is food-degree stuff. The interference are both complex for our analysis.

Answers to BLOG 3? (-1 Pt)

Great posting!