1. ASHLEY COVELLI CHEM 4101 DECEMBER 8 TH, 2010 Absorption of Salicylic Acid in Skin After Repeated Usage of Facial Cleansers

2. What is Salicylic Acid? Compound found naturally in willow tree, “Salix alba,” then prepared using Kolbe-Schmidt process Purpose in cosmetic products: anti-acne agent, skin- conditioning agent, reported to enhance percutaneous penetration of other chemical agents in solution 1 Problem : Salicylic acid is believed to be harmful to humans/animals (if found above toxic threshold of 30 mg/100mL), causing problems such as: skin irritation, increased sun sensitivity, and reproductive and developmental toxicity at concentrations ≤3% in personal care products 1

3. Hypothesis Hypothesis: Salicylic acid is hazardous to human health upon repeated exposure. Most facial cleansers contain roughly 2% of salicylic acid. The concentration levels of this compound found below the skin in humans will be analyzed, the analyte concentration in serum should be below the toxic threshold of 30 mg/100mL using a proper analytical method. Importance of solving the analytical problem: By analyzing the amount of salicylic acid in products and comparing these concentrations to concentrations of salicylic acid present below the dermis, would help the FDA regulate hazardous cosmetic products. As a result of this study, cosmetic companies may wish to reformulate their products in order to provide a less hazardous product for consumers.

4. Summary of Other Analytical Techniques… MethodAdvantagesDisadvantages CZE with UV-Vis Detector 2 CZE enables a detection of a large variety of analytes to be analyzed, 2 μg/mL to be detected in solution using a simple buffer (with a pH around 8.8) The analyte must first be derivatized before analyzed using this method, as analytes separate based on charge once voltage is applied. UV-Vis Absorbtion 1 Absorbances of various solutions may be analyzed to determine concentration of analyte in cleanser (pure salicylic acid should absorb in 305-310 nm range), minimal preparation needed Interfering substances may be present in solution (i.e. water), small uncertainties may arise when obtaining absorbances due to the difficulty of reproducing the exact wavelength setting ESI-MS 3 High sensitivity, low LOD, low signal-to-noise ratio (0.1μL-1) Qualitative analysis, but not an efficient way to quantify salicylic acid in solution, expensive IR Good for qualitative analysis, fast method, multiple scans, distinct functional groups found in analyte may be seen in fingerprint region Presence of water and other analytes may be problematic, it may be hard to distinguish frequencies because of overlapping vibrational modes

5. Analytical Method of Choice: HPLC with UV-Vis detector Sensitive method which is needed when analyzing low concentrations Small sample volumes needed Quantitative determination of analyte concentration in solution Relatively low limit of detection (around 0.1 μg/mL for analyte) 1 System is reasonably priced ($1,425 for C8 column and UV detector) After analyzing, sample is not destroyed. Relatively good reproducibility

6. HPLC column (Ascentis® C8 column – 581444-U Supelco) 4 and UV detector (Agilent 325 Dual Wavelength UV-Vis HPLC Detector) 5 Reverse phase HPLC column (size=25 cm × 10.0 mm )4 : (approximately $425.00) Main advantage is the control of elution selectivity Particle size of salicylic acid is <7.4 μm 6 Mobile phase: combinations of methanol and water Stationary phase: C8 packing (octyl) on silica gel with added buffer (i.e. sodium phosphate) Flow rate: approximately 1 mL/min 6 UV Detector 5 (approximately $10,000): Excellent quantitative technique Internal Standard to obtain calibration curve Absorbances obtained at wavelength range: 305-310 nm due to salicylic acid 6 From Beer’s law plot, concentration of salicylic acid can be determined Rapid analysis Relatively high sensitivity ( around 0.1 μg/mL for analyte) 1

7. Experimental Design Prepare Internal Standard and standard solutions 1.SubcutaneousMicrodialysis  obt ain blood samples 2. Centrifuge the blood samples to obtain plasma 3. Inject samples into HPLC-UV vis instrument 4. Obtain an absorbance spectrum of solution, the wavelength absorbed in the range 305- 310 nm is due to salicylic acid. 1. Flow rate: 1 mL/min Mobile Phase: methanol/water Stationary Phase: C8

8. ANALYTIC CHARACTERISTICS USING SIMILAR METHODS: Figures of Merit for Analytical Method of Choice 1 : ParameterSalicylic Acid Retention Time5.5 minutes Limit of Detection0.1 μg/mL Concentration Range0.04-3.0 mol salicylic acid/mL Limit of Quantification0.3 μg/mL Reproducibility3% UV-vis Absorption305-310 nm Signal to noise ratio>3 Linearity CurveR 2 =0.9982

9. Proposed Analysis Retention time for Salicylic acid=5.5 minutes Quantify concentration of Salicylic acid in serum based on absorbance values obtained from UV detector and calibration plot Confirm concentrations of SA obtained from UV with areas of peaks obtained from HPLC Compare the levels of SA in serum to the concentration of the cosmetic product the consumer has been consecutively using Be sure to run controls using animal serum and various concentrations of analyte

10. Sample Spectrum 1,7

11. Conclusion Importance of usage of safe cosmetic products, detection of SA in consumers is important to monitor Practical analytical method as all compounds are able to be detected, thus multiple analyte concentrations may be compared Fast detection and moderate cost High levels of SA may cause serious long term health effects Salicylic acid is also an ingredient in asprin, how does consumption of this drug compare affect serum levels, is this also hazardous?

12. References 1. Francis; Taylor. Safety Assessment of Salicylic Acid, Butyloctyl Salicylate, Calcium Salicylate, C12–15 Alkyl Salicylate, Capryloyl Salicylic Acid, Hexyldodecyl Salicylate, Isocetyl Salicylate, Isodecyl Salicylate, Magnesium Salicylate, MEA Salicylate, Ethylhexyl Salicylate, Potassium Salicylate, Methyl Salicylate, Myristyl Salicylate, Sodium Salicylate, TEA Salicylate, andTridecyl Salicylate. Int. J. of Tox. 2003, 22, 1-108. 2. Makino, K.; Itoh, Y. Determination of nonsteroidal anti-inflammatory drugs in human specimens by capillary zone electrophoresis and micellar electrokinetic chromatography. Electrophoresis. 2004, 25, 1488–1495 3. Kasprzyk-Horden, B.; Dinsdale, R.; Guwy, A. Multiresidue methods for the analysis of pharmaceuticals, personal care products and illicit drugs in surface water and wastewater by solid-phase extraction and ultra performance liquid chromatography–electrospray tandem mass spectrometry. A. Bioanal Chem. 2008, 391,1293–1308. 4. http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=16706624 http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=16706624 5. http://www.chem.agilent.com/en-US/Products/Instruments/lc/analytical/systems/120 0seriesbinarylc/Pages/default.aspx http://www.chem.agilent.com/en-US/Products/Instruments/lc/analytical/systems/120 0seriesbinarylc/Pages/default.aspx 6. Sarveiya, Vikram. Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers. J. of Chrom. 2004, 803, 225-231. 7. Uges, D. An HPLC method for the determination of salicylic acid, phenacetin and paracetamol in serum, with indications; two case-reports of intoxication. Int. Jour. Of Clin. Pharm. 2001. 3, 1309-1315.