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Rajvi Mehta - Analytical Problem - DEHP Leaching from PVC into Contents of Medical Devices

The use of plastics for the development and fabrication of medical devices has increased recently, the most used plastic being poly(vinyl chloride) (PVC). A phthalate ester plasticizer that is used to make PVC soft and flexible, di-2-ethylhexyl phthalate (DEHP), has been leaching from medical tubing and blood bags and into the contents of the tubing and/or bags. For example, some patients have received transfusions of stored blood containing detectable amounts of the plasticizers, and even some that migrated from medical tubing used for dialysis may have been associated with hepatitis-like symptoms that were seen in many dialysis patients.

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. The EPA has classified vinyl chloride as a Group A human carcinogen.
Manufacturers of these blood bags seem to adhere to the USP requirements for plastics testing, but these tests seem to have overlooked the presence of or migration of the DEHP from the plastics into the contents, which seems to indicate that a method should be used with a lower Limit of Detection.

My hypothesis is that with continued exposure to DEHP, many more adverse health effects could manifest and be detrimental to the human body. By analyzing samples of blood and plasma (the matrices in which the analyte can be found), the amount of DEHP (the analyte in question) that leaches from the PVC into the contents can be ascertained, and perhaps an alternative can be found. (1)

UV-Vis Absorption Spectrometry

The maximum wavelength of absorption for my analyte, DEHP, is 270 nm. The solvent used for the determination of this wavelength was hexane. (2) I was unable to find a value for the molar absorptivity of DEHP. I was going to attempt to calculate it, based on information given in another paper (3), but unfortunately, they did not give a path length used, and because of this, I was unable to calculate a value.


-Similar Analytical Problem (in case it doesn't show up in the comments):

The analytical problem that I think most resembles mine is the one with PFOA and Teflon. In this problem, PFOA is a surfactant that persists after it leaches out of Teflon, which can be compared with the DEHP plasticizer that leaches out of PVC. PFOA is also a carcinogenic, and DEHP has adverse effects that can be equally harmful to the human body.

Similar Analytical Problem

The analytical problem that is the most similar to mine is the one involving Teflon and PFOA. Megan Hartmann is the student that is presenting this topic. Her problem consists of PFOA leaching from the Teflon used to make pots and pans, and PFOA is carcinogenic and is therefore detrimental to human health. Megan's analyte is PFOA and her matrix is the air around Teflon pots and pans after they've been scratched, to see if any PFOA is released.

I think the studies that need to be done for our problems will be quite different. I am going to be measuring DEHP accumulation over time and so I will need to collect samples of blood and plasma that have been stored in medical devices made with PVC for varying periods of time, to show the relationship between accumulation over time. Megan will be testing to see if PFOA is released from Teflon; I don't think her analytical problem has any time dependence, and so she will not need to take samples over time. I think she only will be taking samples from different pots and pans made of Teflon. For my analytical problem, a negative control would be a sample from a medical device that is not made with PVC, and a positive control would be a sample from a medical device that is made with PVC. Megan's controls would be slightly similar; her negative control would be a sample from cookware that is not made with Teflon, while her positive control would be a sample from cookware that is made with Teflon.


Studies Needed to Investigate the Analytical Problem

Hypothesis: DEHP accumulates over time and can cause many adverse health effects that can be detrimental to the human body, such as liver cancer.

Studies: (A) Identify medical devices fabricated with PVC. (B) Measure DEHP accumulation levels in blood and plasma stored in said devices over time. (C) Measure DEHP accumulation levels in blood and plasma 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.

Analyte Levels in the Matrix: In the range of 24 to 72 hours, the DEHP levels in platelet concentrates ranged from 76-491 µg/mL, the levels in platelet rich plasma ranged from 34-181 µg/mL, and the levels in platelet poor plasma ranged from 52-285 µg/mL. (4)


Fluorescence Techniques

Even after checking various handbooks and sifting through the literature, I was unable to find maximum excitation and emission wavelengths for DEHP. From the molecular features of the compound, however, I would speculate that DEHP fluoresces naturally. It has an aromatic group in conjugation with two carbonyl groups, and so the pi electrons can easily move around, creating resonance. To determine the maximum excitation wavelength, I would pick any emission wavelength, and then scan all the excitation wavelengths until the maximum was found. After this, the maximum excitation wavelength will be fixed, and the emission wavelengths will be scanned until that maximum is found.

I would choose to use a spectrofluorometer for the analysis of my analyte. I choose this over a fluorometer because I will need to scan multiple wavelengths to determine my maximum excitation and emission wavelengths. Also, I will only be analyzing small samples of my analyte at a time, so using a standard instrument would be okay.


Chemical Structure and Standards

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The DEHP standard that I will need to use can be purchased from Sigma-Aldrich. The catalogue number is 36735, and it is available only in 1 g quantities, which cost $27.90.

Atomic and Mass Spectrometries

Atomic spectrometries cannot be used for the detection of my analyte because it is not a chemical element.

Mass spectrometry can definitely be used for the detection of my compound. My compound is di(2-ethylhexyl) phthalate, or DEHP. Its nominal mass is 391 Da and its exact mass is 390.277 Da. One of the methods I could use for mass spectrometry would include ESI as the ionization source and single quadropole for the mass analyzer. The literature source doesn't directly say that this mass analyzer is the one, but it was determined after searching for the instrument they used (5). The second method that I could use would use API as the ionization source and QTrap for the mass analyzer (6).

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Mass Spectrum of DEHP (6)

Sample Preparation

This sample preparation procedure is for use in LC:
(1) Centrifugation: The whole blood was centrifuged at 4,200g for 10 minutes, making PPP (platelet-poor plasma.
(2) Extraction: 1mL of PPP was removed from PVC bags and extracted with 3mL of acetonitrile, 1mL of NaOH (1 N), and 100 µL of an internal standard solution.
(3) Centrifugation II: Shake for 5 minutes, and then centrifuge at 4,000g for 10 minutes.
(4) The supernatant is then injected onto the column. (7)

Chromatographic Techniques

From the types of chromatography covered in class, I think that RPLC, HILIC, and GC would be the only viable options. My analyte, DEHP, doesn't carry a net charge, which means that ion-exchange will not work; it is too small for SEC; it won't have a specific affinity for anything in particular, which means that affinity chromatography will not work; and I will not need to separate it from its diastereomers, which means that chiral chromatography will not work.

RPLC would be my first choice to separate my analyte from other matrix components due to the nonpolar nature of the column used.

A commercial column used was purchased from VWR International. It is a C18 LICHROSPHER column, with a particle size of 5µm, a length of 125mm, and an inner diameter of 4mm. The catalogue number is 48219-354, and the price is $712.13. (8)

The mobile phase used for a separation utilizing the previous column was a 15:70:15 mixture of water:acetonitrile:THF. (8)

The detector suggested was a UV detector, but I think a fluorescence detector would be a better idea. My analyte is naturally fluorescent, and so would be easier to detect. Fluorescence is more sensitive than UV-Vis, and so it might be easier to differentiate between DEHP and its monoester metabolite, MEHP. Since fluorescence is more selective, it will remove other interferences from the matrices, blood and plasma.

Capillary Electrophoresis Techniques

Out of all the CE techniques that we covered in class, I think MEKC and CGE would be suitable to separate my analyte from other matrix components. Since DEHP is neutral, in MEKC it would separate from the other neutral components. CGE, which is a separation based on size, would separate DEHP from the other components in the blood and plasma matrices, including proteins of various sizes. CZE would not be suitable, because CZE is a separation based on charge and size, and as my molecule is neutral, it will not separate and will just stay with the EOF. cIEF is a separation based on different pIs. The pI of a molecule is the pH at which the molecule has a net zero charge, which could also mean that the positive and negative charges are balanced. Because DEHP is neutral and has no ionizable groups, it won't have a pI. When the voltage is applied, I think it would simply diffuse throughout the separation media rather than focusing.

MEKC would be my first choice for the CE technique that is most suitable for the separation of DEHP from other matrix components. The addition of a surfactant to the buffer in MEKC aids the formation of micelles, which have both hydrophobic and hydrophilic components. Thus, the hydrophobicity of the analyte plays a large role. Since DEHP is neutral and hydrophobic, it can partition into and out of the micelle and be separated from other neutral components, since while it is in the micelle, it has the same electrophoretic mobility as the micelle. It is also spending a different amount of time in the micelle compared to other neutral components due to varying affinities.

In the first paper I found, the buffer composition was SDS dissolved in a mixture of 0.02M sodium dihydrogen phosphate solution and 0.02M sodium tetraborate solution adjusted to a pH of 9.0. An electric field of 5-30kV was applied between the platinum electrodes that were in the carrier solution. The capillary used was fused silica that was 720mm long, with an inner diameter of 50 μm. On-column detection was used by UV Absorption at 210nm. The detector was placed 500mm from the positive end of the capillary. Samples were injected by vacuum injection (5 in. of Hg for 0.2s) and the injection volume was 1.5nL. (9)

In the second paper I found, the buffer composition was 100mM sodium cholate (SC), 50mM borate, and 15% methanol. The pH was 8.5. The applied electric field was 20kV. The capillary used was a fused silica capillary, with a total length of 60cm, an inner diameter of 75μm, and an outer diameter of 375μm. On-column detection was used by UV Absorption at 214nm. The detector was placed 50cm from the beginning of the capillary. Samples were injected by using a pressure of 3.5kPa (0.5 psi) for 3s. In this paper, they added PEG-400 to modify the micellar phase. When PEG-400 was increased from 0-5% in the same buffer, an increased resolution of DEHP was observed. When 2% PEG-400 was used, baseline separation of all the phthalates in the study was observed. Methanol and acetonitrile were also used to optimize the separation of the phthalates. A baseline separation was achieved when 15% methanol or 30% acetonitrile was added to the original buffer. (10)

In another paper found, the concentrations of DEHP in whole blood was, on average, 0.0238mg/mL (11). The limit of detection for MEKC in the second paper found was 0.054mg/L (10). This LOD is far lower than the concentrations that are observed in samples containing DEHP, and so I think that the on-column UV Absorption detectors used in both of these papers are sufficient for the analysis at hand.

Analytical Electrochemistry

It has been shown that aliphatic phthalate esters are reducible at highly negative potentials (about -1700mV). This reduction was shown at a hanging drop mercury electrode to have a two-step mechanism, corresponding to each of the current-voltage curves observed when using Normal Pulse Polarography (NPP) (12). This reduction is not reversible (12,13).

Each phthalate ester was shown to have a unique E1/2 value compared to the S.C.E. when using NPP, therefore if a standard of DEHP were tested, its E1/2 could be used for identification. Typical E1/2 range is -1600mV to -1800mV (12,13). In another paper, phthalate esters were analyzed using Differential Pulse Voltammetry (DPV) with a hanging drop mercury minielectrode, showing unique peak currents at respective E1/2 values. Again, DEHP standards can be used to distinguish DEHP from other phthalates (14).

Both NPP (13) and DPV (14) can be used for quantitation, since wave height in NPP and peak currents in DPV are proportional to the analyte concentration. Standard curves can be readily constructed showing strong linearity (14) and sensitivity in the low to sub μM range (better than HPLC-UV) and can be further improved to the nM range when preconcentration with SPE is done.

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. Aignasse, M.F. ; Prognon, P. ; Stachowicz, M. ; Gheyouche, R. ; Pradeau, D. A New Simple and Rapid HPLC Method for Determination of DEHP in PVC Packaging and Release Studies. Int. J. Pharm. 1995, 113, 241-246.

3. Yu, B.Y. ; Chung, J.W. ; Kwak, S. Reduced Migration from Flexible Poly(vinyl chloride) of a Plasticizer Containing β-Cyclodextrin Derivative. Environ. Sci. Technol. 2008, 42, 7522-7527.

4. Rock, G. ; Labow, R.S. ; Tocchi, M. Distribution of Di(2-ethylhexyl) Phthalate and Products in Blood and Blood Components. Environ. Health Perspect. 1986, 65, 309-316.

5. Inoue, K. ; Higuchi, T. ; Okada, F. ; Iguchi, H. ; Yoshimura, Y. ; Sato, A. ; Nakazawa, H. The Validation of Column-Switching LC/MS as a High-Throughput Approach for Direct Analysis of Di(2-ethylhexyl) Phthalate Released From PVC Medical Devices in Intravenous Solution. J. Pharm. Biomed. Anal. 2003, 31, 1145-1152.

6. Ito, R. ; Seshimo, F. ; Miura, N. ; Kawaguchi, M. ; Saito, K. ; Nakazawa, H. High-Throughput Determination of Mono- and Di(2-ethylhexyl) Phthalate Migration from PVC Tubing to Drugs Using Liquid Chromatography-Tandem Mass Spectrometry. J. Pharm. Biomed. Anal. 2005, 39, 1036-1041.

7. 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.

8. Bourdeaux, D. ; Sautou-Miranda, V. ; Bagel-Boithias, S. ; Boyer, A. ; Chopineau, J. Analysis by Liquid Chromatography and Infrared Spectrometry of Di(2-ethylhexyl) Phthalate Released by Multilayer Infusion Tubing. J. Pharm. Biomed. Anal. 2004, 35, 57-64.

9. 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.

10. 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.

11. 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.

12. Whitnack, G.C. ; Reinhart, J. ; Gantz, E.S.C. Polarographic Behavior of Some Alkyl Phthalate Esters. Anal. Chem. 1955, 27, 359-362.

13. Whitnack, G.C. ; Gantz, E.S.C. Polarographic Determination of Phthalate Esters in Plastics. Anal. Chem. 1953, 25, 553-556.

14. Qureshi, M.S. ; Fischer, J. ; Barek, J. ; Sirajuddin ; Bhanger, M.I. Voltammetric Determination of Aliphatic Phthalate Esters at a Hanging Mercury Drop Minielectrode and a Meniscus Modified Silver Solid Amalgam Electrode. Electroanal. 2010, 22, 1957-1962.

Comments

Blog 13. Great answers.

Blogs 9, 10, 11. Good answers.

Problems Using Similar Techniques

The preferred technique to separate my analyte from the matrix components would be RPLC, and the best detection method would be fluorescence. The RPLC column’s stationary phase would be similar to my analyte, which is something that you want in choosing a column. My analyte is naturally fluorescent, so it would not have to be derivatized before detection, which is beneficial. Fluorescence is a sensitive technique, so it might be easier to differentiate between DEHP and its monoester metabolite, MEHP. Also, since fluorescence is selective, it will remove other interferences from the blood and plasma matrices.

There were two analytical problems that mentioned using similar techniques to the ones I’ve chosen, specifically, RPLC and fluorescence. These are Taurine in Energy Drinks and Aflatoxin in the Oil.

Good answers for blog 8. However, it was hard to find because it was not posted as a comment.

Blogs 6 and 7. Good answers!

Blog 4. Good answers. The levels of DEHP that you found seem high. You may need to provide more context information.

Blog 5. Good answers.

My analytical problem is siloxanes in cosmetics.

Our problems are similar because we are both trying to determine if the analyte is accumlating in the body. There are many health concerns and the manufactuers of the analytes are not to concerned with their detrimental affects of their products.

My analytical problem : Butanol Production from Clostridia fermentation

Our problems are similar in that we both will need to track the change in concnetration of our analyte over time.

Great job!