1. Rajvi Mehta Chem 4101, Fall 2011 December 9, 2011 DEHP Leaching from PVC into Contents of Medical Devices

2. Analyte: DEHP Di-2-ethylhexyl Phthalate (DEHP) is a phthalate ester plasticizer that is used to make PVC medical devices soft and flexible. It was shown that with a longer storage time, there was a larger accumulation of DEHP in the contents of the medical devices. Large amounts of DEHP can cause a variety of symptoms and diseases, including birth defects, decreased fetal weight, miscarriages, and liver cancer, and it can also act as an endocrine disruptor. 1,2 Photo taken from http://t0.gstatic.com/images?q=tbn:ANd9GcRx32nvoGEdRB5aeXu3O0yQipGQxxo9ug9BaI8fdXjXQbHK-W2d

3. Analytical Problem and Hypothesis Analytical Problem o The use of plastics for the development and fabrication of medical devices has increased recently, the most used plastic being poly(vinyl chloride) (PVC). DEHP has been leaching from the medical devices and into their contents, such as plasma and blood. 1 Hypothesis o With continued exposure to DEHP, many more adverse health effects could manifest and be detrimental to the human body.

4. Studies Needed Identify medical devices that are fabricated with PVC. Measure DEHP accumulation levels in blood stored in said medical devices over time. Measure DEHP accumulation levels in blood stored in medical devices that are (a) known to be made using DEHP, as a positive control, and (b) not made using DEHP, as a negative control. Controls and Standards DNNP can be used as an internal standard. A standard stock solution of DEHP in acetonitrile can be prepared and from this stock solution, working solutions can be prepared by suitable dilutions with acetonitrile. A calibration curve can then be obtained by analyzing a 1mL plasma sample that is spiked with 100µL of dilutions to obtain µg DEHP/mL plasma. 3

5. Method of Choice The method of choice for this analytical problem is HPLC with a fluorescence detector. HPLC was chosen as a separation method due to shorter analysis times and better resolution, as compared with electrophoretic methods. 6,7 HPLC will also be more efficient in separating the plasma matrix components from the analyte. Fluorescence was chosen as a detection method, even though it was not explicitly used in the literature, due to high selectivity and sensitivity. The concentration of DEHP in whole blood is, on average, 0.0238mg/mL. 4 The limit of detection for the fluorescence detector, which is 1.3 fg, is therefore sufficient for the detection of DEHP. 5

6. 1260 Infinity Quaternary LC System HPLC schematic taken from http://media.americanlaboratory.com/m/20/Article/504-fig1.jpg Pressure range up to 600 bar Injection range: 0.1-100 µL Flow range: 0.2-10 mL/min ZORBAX Extend-C18 Column 20mm x 2.1mm i.d. 1.8 micron particle size 8

7. 1260 Infinity Fluorescence Detector Fluorescence schematic taken from http://s4jkuch.edu.glogster.com/fluorescence-spectroscopy/ Multi-wavelength detection to improve sensitivity and selectivity Excitation and Emission wavelength ranges: 200-1200nm Source: Xenon flash lamp LOD: 1.3 fg S/N: >3,000 5

8. Sample Preparation Whole blood sampleCentrifuge (4,200g for 10 min)Platelet-poor plasma (PPP) Extraction (1mL PPP, extracted w/ acetonitrile, NaOH, and an internal standard solution) Centrifuge (Shake for 5 min, then 4,000g for 10 min) 3

9. Hypothetical Results These are HPLC chromatograms of the PPP sample: (A) blank plasma sample; (B) blank plasma sample spiked with 112.50 μ g/mL DEHP; (C) supernatant of PPP stored in bag for 14 days containing 512.55 μ g/mL DEHP. 3 DNNP was used as an internal standard. For DEHP quantification, the peak ratio (DEHP peak area/DNNP peak area) was calculated for each PPP sample, and the amount of DEHP was then determined by use of the calibration curve. 3

10. Other Methods

11. Conclusions The method of analysis, HPLC with a fluorescence detector, was chosen for its high sensitivity and selectivity. The LOD of the detector (1.3 fg), was many orders of magnitude lower than the average concentration of DEHP in a plasma sample (0.0238mg/mL), indicating that the detector would be able to detect the DEHP. 4,5

12. References 1. Jaeger, R.J.; Rubin, R.J. Migration of a Phthalate Ester Plasticizer from Polyvinyl Chloride Blood Bags into Stored Human Blood and its Localization in Human Tissues. New Engl. J. Med. 1972, 287, 1114-1118. 2. Mitani, K. ; Narimatsu, S. ; Izushi, F. ; Kataoka, H. Simple and Rapid Analysis of Endocrine Disruptors in Liquid Medicines and Intravenous Injection Solutions by Automated In-Tube Solid-Phase Microextraction/High Performance Liquid Chromatography. J. Pharm. Biomed. Anal. 2003, 32, 469-478. 3. Dine, T. ; Luyckx, M. ; Cazin, M. ; Brunet, C. ; Cazin, J.C. ; Goudaliez, F. Rapid Determination by High Performance Liquid Chromatography of Di-2-ethylhexyl Phthalate in Plasma Stored in Plastic Bags. Biomed. Chromatogr. 1991, 5, 94-97. 4. Valeri, C.R. ; Contreras, T.J. ; Feingold, H. ; Sheibley, R.H. ; Jaeger, R.J. Accumulation of Di-2-ethylhexyl Phthalate (DEHP) in Whole Blood, Platelet Concentrates, and Platelet-Poor Plasma: 1. Effect of DEHP on Platelet Survival and Function. Environ. Health Perspect. 1973, 103-118. 5. Agilent 1260 Infinity Fluorescence Detector User Manual. http://www.chem.agilent.com/Library/usermanuals/Public/G1321-90012_FLD-AB_USR_EN.pdf 6. Guo, B. ; Wen, B. ; Shan, X. ; Zhang, S. ; Lin, J. Separation and Determination of Phthalates by Micellar Electrokinetic Chromatography. J. Chromatogr. A. 2005, 1095, 189-192. 7. Takeda, S. ; Wakida, S. ; Yamane, M. ; Kawahara, A. ; Higashi, K. Migration Behavior of Phthalate Esters in Micellar Electrokinetic Chromatography With or Without Added Methanol. Anal Chem. 1993, 65, 2489-2492. 8. Agilent 1260 Infinity Quaternary LC VL System Manual and Quick Reference. http://www.chem.agilent.com/Library/usermanuals/Public/G1311-90310_QuatLC_VL_ebook.pdf