DEHP Leaching from PVC into Contents of Medical Devices

DEHP Leaching from PVC into
Contents of Medical Devices
Rajvi Mehta
Chem 4101, Fall 2011
December 9, 2011
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
Photo taken from
Analytical Problem and Hypothesis
• Analytical Problem
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
With continued exposure to DEHP, many more adverse
health effects could manifest and be detrimental to the human
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
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
 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
1260 Infinity Quaternary LC System
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 size8
HPLC schematic taken from
1260 Infinity Fluorescence Detector
• Multi-wavelength detection to
improve sensitivity and
• Excitation and Emission
wavelength ranges: 200-1200nm
• Source: Xenon flash lamp
• LOD: 1.3 fg
• S/N: >3,0005
Fluorescence schematic taken from
Sample Preparation
Whole blood sample
Centrifuge (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
Hypothetical Results
• DNNP was used as an internal
• 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
These are HPLC chromatograms of the PPP sample: (A) blank plasma
sample; (B) blank plasma sample spiked with 112.50μg/mL DEHP; (C)
supernatant of PPP stored in bag for 14 days containing 512.55μg/mL
Other Methods
Micellar Electrokinetic
Chromatography (MEKC)
Can separate neutral
Longer analysis time, as
compared with HPLC
Gas Chromatography (GC)
Shorter analysis time
Destroys sample
Mass Spectrometry (MS)
Analyte is easily identifiable
Difficult for quantitation;
expensive; destroys sample
UV-VIS Absorption (UVVIS)
Simple methodology;
Lower selectivity and
 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
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.
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.
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,
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.
Agilent 1260 Infinity Fluorescence Detector User Manual.
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.
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.
Agilent 1260 Infinity Quaternary LC VL System Manual and Quick Reference.