Vinh-Tran-Analytical-Problem-Triclocarban in Human Urine
There are many antimicrobial agents found in soaps. One of the most widely used is Triclocarban (TCC,3,4,4'-trichlorocarbanilide). Toxicological studies in the 1970s concluded that TCC was safe to use in personal care products but recent findings reveal significant biological effects. Studies show that at high concentrations, TCC may act as an agonist to testosterone and other steroids which are also known as endocrine disrupting substances (EDS). Because of its widespread use, studies revealed the presence of TCC in surface water in large concentrations (g/L) and in other biological organisms such as algae and snails. Moreover, US EPA reports detected TCC in all 84 sewage samples with concentrations as high as 0.44 g/kg. These studies reveal the high persistency of TCC in the environment, which may be likely due to the use of antimicrobial soaps and detergents by humans.
While studies have shown the presence the TCC in the environment, the proposed analytical problem would be to detect TCC in human serum, mainly urine samples. This is important since exposure to TCC could influence human health. Animal studies have shown that EDS and such chemicals linked to a variety of problems including cancer, reproductive failure and developmental anomalies. These problems may translate into the human biological system and the FDA has begun its investigation. The hypothesis is that with continued exposure to TCC through daily activities such as showering and washing hands that TCC enters the human biological system. By analyzing urine samples, human exposure to TCC will be monitored and determined.
1. Schebb, N. H.; Inceoglu, B.; Ahn, K. C.; Morisseau, C.; Gee, S. J.; Hammock, B. D. Investigation of Human Exposure to Triclocarban after Showering and Preliminary Evaluation of its Biological Effects. Environ. Sci. Technol. 2011, 45, 3109-3115.
2. ScienceDaily. Antibacterial Chemical Disrupts Hormone Activities, Study Finds. http://www.sciencedaily.com /releases/2007/12/071207150713.htm (accessed September 19, 2011).
UV-Vis Absorption Spectrometry
Jungerman and Beck determined germicide mixtures in soaps and detergents by preparing acidic and basic alcohol mixtures of TCC to be analyzed by UV-Vis spectrophotometry. The acidic alcohol sample was prepared with 95% ethanol (96mL) with glacial acetic acid (4mL) while the basic alcohol sample was prepared with 95% ethanol (96mL) and concentrated ammonium hydroxide (4mL). These samples were then analyzed for absorbance between 220 nm and 400 nm. The spectrum revealed that TCC absorbed the most between 200 and 280 nm with the two highest points of absorbance at about 210 nm and 265 nm. An extinction maxima was determined to be 265 nm for both the acidic and basic samples.
The researchers also used the same methods to detect other trichloro compounds that are also used as bacteriostatic additives to soaps of which have very similar organic structure to TCC. Their experiments revealed that these trichloro compounds do absorb the highest at wavelengths of about 260 to 300 nm. Therefore if a more modern experimental procedure were to be performed, the same solvent mixture would be used and the wavelength range above would be used to analyze the presence of TCC in urine samples. Of course these urine samples must first be purified and TCC extracted to a pure sample in order to reduce noise and other contaminant signals. I have yet to research and find an appropriate method to the purification of TCC.
3. Jungerman, E.; Beck, E.C. Determination of Germicide Mixtures in Soaps and Detergents. Journal of the American Oil Chemist's Society. 1961, 38, 513-515
Similar Analytical Problem: My analytical problem is similar to John Raia's Detection of Analytical Surfactants in Water since my compound is also used in large scales and are manufactured in different soaps and detergent products. For a more in depth explanation, please view John Raia's blog postings.
Similar Analytical Problem(s)
One similar analytical problem is Rajva Mehta's dealing with DEHP leaching from PVC into contents of medical devices. Our analytes include chlorine compounds that may cause human biological effects and are considered carcinogens. She will be testing blood and plasma samples and will have to follow multiple purification steps such as in my analytical problem to isolate the desired analyte. Her hypothesis is that with continued exposure to DEHP, many more adverse health effects could manifest and be detrimental to the human body. Another similar problem is Perfluorooctanoic acid levels in human blood by Andrew Szeliga. While his analyte contains Fluorine, our problems are similar that our compounds contain halogens and would need to test human bodily fluid tests. He hypothesizes that people living or working in close proximity to sources of PFOA will have dangerously elevated levels of PFOA in their bloodstream. The studies that we both will have to conduct is to compare samples against a control. These experiments will allow us to conclude the presence of our analytes in the human body.
Hypothesis: Continued exposure to TCC through daily activities such as showering and washing hands with soaps and detergents containing the antimicrobial will cause TCC to enter into the human biological system.
Studies: (1) Get a representative sample group of people for the study (i.e. those who live in the cities, suburbs, and rural areas) because the use of soaps may depend on the location of individuals. (2) Divide the group into two main focus areas: Shower study (focus on TCC and personal hygiene) and Dish study (focus on TCC and ingestion by utensils, etc.). The shower study will be given shampoo/body wash containing TCC and given dish washing soap without TCC and vice versa for the dish study. (3) Create a detailed plan entitling each individual a set time of the day to either shower/wash dishes with TCC containing soaps/detergents (each individual will follow these same instructions) (4) Prior to beginning the experiment, the participants will be given shampoo/body wash and dish washing detergent not containing TCC as to set a sample standard and measure the levels of TCC as the experiment progresses. (5) Start the experiment - Group 1: control (non TCC Dish and Shower), Group 2: Dish Study (TCC dish washing detergent and non TCC shampoo), Group 3: Shower Study (TCC shampoo and non TCC dish washing detergent), Group 4: Dish and Shower (both contains TCC) (6) Measure levels of TCC accumulation in the human biological system by collecting urine samples weekly in each individual of every group.
Alternative Studies: After collecting data for a month, the experiment can eventually be extended to three months. After those three months of TCC accumulation, we can set up a study to observe how fast the rate of disappearance of TCC will be in each case study and individual. This will be done by the same process and methods by collecting urine samples and measuring the levels of TCC present.
Chemical Structure and Standards
Cambridge Isotope Laboratories, Inc.
Description: Isotopically labeled Triclocarban (4'-CHLOROPHENYL-13C6, 99%), 100 ug/mL in CH3CN
Catalog Number: CLM-7286-1.2
Quantity: 1.2 mL
http://www.isotope.com/cil/products/displayproduct.cfm?prod_id=9241 (Date Accessed Oct. 25, 2011)
Atomic and Mass Spectrometries
Atomic spectrometry cannot be used to quantify Triclocarban. However, two types of mass spectrometry, ESI-Quad and ESI-linear trap-FT-ICR, can be used to quantify Triclocarban (MW = 315.58 g/mol). Electrospray ionization will be used as the ionization source and triple quadrupole and an ion trap as the mass analyzers. In the research article I found, the conditions used to obtain the mass spectrum were the flow injection of 10 ng of analyte with the scanning in the m/z range of 275-400. In order to detect the base ion and its acetic acid adduct ion, an eluent mixture containing acetonitrile (70%), water (30%), and acetic acid (10mM) was used because the method was LC MS with liquid chromatography preceding the injection of analyte into the mass spectrum. This is to purify sample from other possible contaminants. The analyte mass spectrum was compared to an internal standard mass spectrum of radiolabeled Triclocarban (13C6).
Halden, R. U.; Paull, D. H.; Analysis of Triclocarban in Aquatic Samples by Liquid Chromatography Electrospray Ionization Mass Spectrometry. Environ. Sci. Technol. 2004, 38, 4849-4855
Sample Preparation Procedures
Direct Urine Analysis
(1) Centrifugation: 50-100 uL aliquots of urine will be mixed with an internal standard (IS) solution 1/1 (v/v), vortexed, followed by centrifugation (16,000 g) at 4 C for 5 min.
(2) Anaylsis: The supernatant will be directly injected into an LCMS.
Urine Analysis after Hydrolysis
(1) HCl (100 uL) will be added to 500 uL of sample urine to produce a final acid concentration of 1 M in a 3 mL glass vial.
(2) The mixtures are vortexed and heated (20 min) to 100 C, cooled down on ice, and will be neutralized with 6 M aq NaOH (90 uL)
(3) An aliquot of resulting solution will be mixed with IS solution 1:4 (v/v) and analyzed by LCMS.Source: View Source 1 above
From different research articles and from class lectures, reverse phased HPLC would be most suitable to separate my analyte from other matrix components because my analyte TCC is polar and would elute readily as compared to other techniques. My compound is not chiral and therefore chiral chromatography is not suitable and size exclusion has too low of a resolution for my analysis. Gas chromatography might not be ideal since my compound is known not to be that volatile. However, ion exchange chromatography may also be suitable since it separates compounds based off polarity and my analyte is quite polar. My first choice would be reversed phase HPLC because it suits ideally to separate my polar compound from a possible compound in similarity, Triclosan, from the mixture and is much faster as well as more cost efficient if I were to perform the experiment on a large scale.
A suitable commercial column can be purchased from Waters (Product Number: 176000863): ACQUITY UPLC BEH C18 Column, 2.1 x 50 mm, 1.7 µm, 3/pk. The trifunctionally bonded BEH particle gives a very wide usable pH range (pH 1-12), superior low pH stability, and ultra-low column bleed for high sensitivity MS applications.
The mobile phase will consist of a buffer solution (A) and run on a gradient with acetonitrile (B) as follows by Guo et. al.:0 min, 50% A/50% B with a flow rate of 0.4mLmin−1, then 40% A/60% B with a flow rate of 0.5mLmin−1 at 1.0min, finally, reconditioning the column with 50% A/50% B after washing column with 90% B at the rate of 0.3mLmin−1 for 1.5 min.
I would use a MS detector because it is most sensitive to detect trace levels of my analyte mixture (up to pico grams).
http://www.waters.com/waters/partDetail.htm?partNumber=176000863 (Date Accessed: Nov 10, 2011)
(source will follow this blog posting)
Capillary Electrophoresis Techniques
Capillary Zone Electrophoresis (CZE) and Micellar Electrokinetic Chromatography (MEKC) are the two types of capillary electrophoresis that would be suitable to separate my analyte. CZE would work best because TCC may exist as an anionic species and with an applied voltage, the ionic analyte may be separated due to its size and charge from other contaminants especially Triclosan (TCS) that has a similar charge but is smaller in size and therefore would help to detect TCC more easily. MEKC would be suitable since TCC is hydrophobic in nature and would therefore distribute itself between the hydrophobic interior of the micelles and the hydrophilic exterior buffer solution. Capillary Gel Electrophoresis (CGE) and cIEF would not be suitable because they both are usually performed to separate proteins or biological molecules.
My first choice would be CZE because it will allow for an efficient separation of my charged analyte mixture and does not require a micellar mixture which may impact cost efficiency if this were to be performed on a large scale. It is also the preferred technique to separate small ions such as TCC.
Since TCC is expected to exist as an anionic mixture, a coated capillary tube is ideal. My buffer would be acetonitrile at a basic pH (~8-9) in order to separate my anionic species.
I would perform this separation in tandem with a mass spectrometer because it would be able to detect minute concentrations of my analyte. A MS detector is also chosen because it is one of the few techniques that is suitable for detection of my analyte TCC because other analytical techniques do not work such as AES and Fluorescence Spectroscopy. It has a very low limit of detection at 1-0.01 attomoles as compared to other techniques and is suitable for my hypothesis because it will allow for an accurate detection of TCC in urine.
Triclocarban is electroactive because it contains a carbonyl group that bridges the two chlorinated benzene rings. However, TCC has an extremely low water solubility (<2 mgL-1) which prevents the study of its electrochemical degradation in an aqueous medium. As noted in Sires et. al.Triclocarban, including a possible contaminant in my analytical project, Triclosan, both of which are antimicrobial agents, can be oxidized to give stable intermediates by Electro-Fenton degradation. The main oxidant as described in this system is the hydroxyl radical that is produced on both the anode surface and from water oxidation and in the medium by Fenton's Reaction. This reaction takes place between electrogenerated H2O2 and Fe2+ coming from the cathodic reduction of O2 and Fe3+.
TCC can produce four stable intermediates: two hydroxylated derivatives (hydroquinone and chlorohydroquinone) and two nitroderivatives (1-chloro-4-nitrobenzene and 1,2-dichloro-4-nitrobenzene).
In order to identify TCC using electrochemistry, a Pt/O2 diffusion cell will be used and samples of TCC (standards at different known molar concentrations as well as the patient urine samples) will be prepared with 0.05M Na2SO4 as the background electrolyte and 0.20mM Fe3+ as catalyst at pH 3.0. The electro-Fenton system will be operated at 60 mA and at room temperature.
The samples will be added to a solution of acetonitrile/water (v/v) and subject to electrolysis (~10 min) where the mixture will be analyzed by GC/MS to identify the hydroxylated derivatives and the nitroderivatives. Sires et. al. provided a table of the retention times as well as the molecular peak of the intermediates which may be used in comparison for the identification of TCC in my analytical problem if this technique were to be used.
In order to quantify my unknown concentration of TCC in human urine, I first will have to make a standard plot of the concentration of TCC versus time in known molar amounts in different acetonitrile/water (v/v) systems. After the decay of TCC by electrolysis, reversed-phase HPLC will follow in order to quantify the amount of generated carboxylic acids from the degradation by ion-exclusion chromatography. This amount will be a direct correlation to the amount of TCC in the solution and therefore a standard plot can be produced. Unknown samples will be subject to degradation and LC will provide quantitative results of generated carboxylic acids which is then compared to these standard curves for quantitation.
Source: Sires, I.; Oturan, N.; Oturan, M. A.; Rodriguez, R. M.; Garrido, J. A.; Brillas, E. Electro-Fenton Degradation of Antimicrobials Triclosan and Triclocarban. Electrochimica Acta. 2007, 52, 5493-5503