of polyeteylene (uhmwpe) of ion · hawig an o.i.= 1.1 5 for di fferent temperature figure 4.8:...

OXLlDATION OF ULTRA HIGE MOLECULAR WEIGHT POLYETEYLENE

(UHMWPE) CONTAINING TRACES OF COBALT ION

A thesis submitted in conformity with the requirements for the degree of

MASTERS OF APPLED SCIENCE

Graduate Department of Chernical Engineering and Applied Chemistry

University of Toronto

O Copyright by Jie Yu, 2000

National Library Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington OttawaON K1AON4 Ottawa ON KI A ON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence al!owing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

OXIDATION OF ULTRA Bl[GH MOLECULAR WEIGEIT POLYETHYLENE

(UfIMWPE) CONT-G TRACES OF COBALT ION

MASTERS OF APPLIED SCIENCE, 2000

JIE(GE2ACE) W

UNIVERSITY OF TORONTO

ABSTRACT

Wear of metal and UHMWPE implant components generates sub-micron particles

contaminated with metal ions. The associated particles have been shown to be phagocytosed

by human macrophages. This thesis is focused on the study of üHMWPE particle

degradation products and the process by which these products may be generated under

sirnulated thermal and oxidative/hydrolytic pathways of the implant environment. Oxidation

of particles was measured using Fourier transform i n h e d spectroscopy (FTIR). It was

s h o w that metailic ion (i.e., cobalt) accumulation into the particles (at trace levels of 10

ppm) can dramatically accelerate their oxidative degradation at relatively low temperatures

(Le. 60°C). Degradation products were isolated using high performance liquid

chromatography (HPLC) and characte~zed using liquid chromatography/mass spectrometry

(LCMS), mass spectrometry (MS/MS) and attenuated total reflectance FTR (ATR-FTIR).

The analysis of the isolated products showed that the degradation of the UHMWPE could be

fiirther propagated by the particle interaction with macrophage associated oxidants, such as

H202. It is postulated that the generation of degradation products from phagocytosed

UHMWPE particles and their uptake intra-cellularly could mediate cellular responses. Such

considerations have not been previously investigated for implant derived UHMWPE

particles.

This thesis was the accumulation of the efforts of many individuals. Their wisdom and

assistance made the completion of the document possible.

I would like to thank Dr. I.P. Santerre and Dr. Enn Boynton, my supervisors, for al1 the time

and effort they invested in me. Their continued enthusiasm and encouragement gave me a

sense for the mahing of a bue researcher.

Other individuals who need to be mentioned for their kindness and expertise are: Dr. Linjie

Meng and Ms. Ling Li.

Individuals 1 would like to thank for their cooperation are: Angela Lee and Shaomo Xing.

Wiîh the exchange of their knowledge, the completion of the thesis was made possible.

The following individuals picked me up during the times when it would never end. Yi-wen

Tang, Mei-ling Yang, Yoav Finer as well as Jeannette Ho. They kept telling me that I could

do it.

Finally, I would like to thank my husband, Hong (Simon) Shi, my father, Xilin Zuo, and my

mother, Youlan Cheng. With their patience and encouragement, 1 was able to finish my

work.

LIST OF ABBREVIATIONS

AA

ATR-FTIR

FTIR

HPLC

MS

0.1.

U H r n E

LDPE

HDPE

PTFE

LC/MS

SEM

Atomic absorption spectrometry

Attenuated total reflectance FTIR

Fourier transfomi infiared spectroscop y

High performance liquid chromatography

Mass spectroscopy

Oxidation index

Ultra high molecular weight polyethylene

Low density polyethylene

High density polyethylene

Teilon@

Liquid chromatographyAdass spectroscopy

Scanning electron microscopy

TABLE OF CONTENTS ABSTRACT

ACKOWLEDGEMENTS

LIST OF ABREVIATION

TABLE OF CONCENTS

LIST OF TABLES

LIST FU FIGC'RES

LIST OF APPENDICES

1 .O INTRODUCTION

2.0 BACKGROUND AND LITERATURE REVIEW

2.1 History of the Implants and Wear Problern

2.2 Polyethylene Materials

2.2.1 Polyrnerization of UHMWP E

2.2.2 UHMWPE Processing and Result in Properties

2.3 Sterilization of UHMWPE

2.4 Degradation of UHMWPE

2.4.1 Oxidative Degradation of UHMWPE

2.4.1.1 Gamma Irradiation and Oxidative Degradation

2.4.1.2 In Vivo ChemicaI Oxidation

2.4.1.3 Shelf Storage Environment

2.4.1.4 Thermal Oxidation

2.4.2 Metal Ions and their Role as Catalyst in Oxidation

3.0 MATERLALS AND METHODS

3.1 Material Selection

3.2 Test Sample Preparation

3.2.1 Sterilization of Samples

3.2.2 Coating of UHMWPE Particles with Cobalt Chionde

3.2.3 Cobalt ion Concentration Test (Atornic Absorption

Spectroscopy)

3.3 Oxidation of UHMWPE Particles

. . 11

... 111

iv

v

viii

X

xiv

3.3.1 Oxidation b y Thermal Treatment

3.3.2 Chernical Oxidation of U H W E Particles

3.3.2.1 Determination of Hydrogen Peroxide Activity

3 A2.2 Half-life Study for Hydrogen Peroxide

3.3.3 Incubation Experiments

3.4 Charactenzation of UHMWPE Oxidation by Fourier

Transform Infrared Spectroscopy (FTIR)

3.4.1 Definition of Oxidation Index for UHMWPE Particles

3.4.2 Typical FTIR Spectra Diagrams Associated with the

Particles

3.4.3 ATR-FTIR Analysis of Isolated Degradation Products

3.5 Extraction of Degradation Products

3.6 High Performance Liquid Chromatography (HP LC)

3 -7 Identi& Biodegradation Products using L C M S? (Liquid

Chromatography-Mass Spectrometry)

3.8 Identification of Biodegradation Products using MSIMS

4.0 RESULTS

4.1 Particle Surfiace Characterization

4.2 Cobalt Ion Analysis

4.3 Oxidation of UHEvrWPE

4.3.1 Thermal Oxidation of CobaltWHMWPE Particles

4.3.2 Catalyzed Oxidation by Other Metal Ions

4.3.3 Oxidation Following Incubation in Aqueous and

Hz02 Solutions

4.4 Hydrolytic Degradation of Oxidized UHMWPE

4.4.1 HPLC Aaalysis of Incubation Solutions

4.4.2 LCMS Results for Second and Third Batches

4.4.3 Mass Spectroscopy Isolated Products (MS/MS)

4.4.4 Chernical Characterization of Isolated HPLC Fractions

by ATR-FTIR

5.0 DISCUSSION

5.1 Cobalt Treated Samples

5.2 Thermal and Irradiation Oxidation of UHMWPE Particles

5.2.1 Metal Ion Catalyst Function in the Thermal

Oxidation Process

5.2.2 A Mathematical Representation of UHMWPE Oxidation

5.3 Chernical Oxidation of UHM\NPE

5.4 Nature of Released UHMWPE Degradation Products

5.4.1 Product Isolation and Identification

5.4.2 Proposed Chernical Structures of Isolated

Degradation Products

6.0 SUMMARY AND CONCLUSION

7.0 RECOMMENDATIONS

8.0 REFERENCES

Appendices

LIST OF TABLES

Table 2.1: Properties of LDPE and HDPE

Table 2.2: Standard Properties of UHMWPE Powder

Table 2.3: Properties of UHMWPE Fabncated Form

Table 3.1: Material Properties for the Selected UHMWPE

Table 32: Experimental Heat-treatments Periods for UHMWPE Particles

with Cobalt Ion Samples

Table 33: Hydrogen Peroxide Released in Macrophages at Four

Different Contrasting States

Table 3.4: Chernicals for Hydrogen Peroxide Assay

Table 3.5: Chemical Treatments for UHMWPE Particles

Table 3.6: Summary of Oxidation Index Definition for UHMWPE

Table 3.7: Samples using ATR-FTiR Chromatogram A in Figure 4.10

Table 3.8: Gradient Program Run for HPLC

Table 4.1: Cobalt Concentration in UHMWPE Particles in Relation to the

UHMWPEICoCI:, ratio used in the Treatment Process

Table 4.2: Oxidation Index Values at Different heat Treatments

Table 43: Summary of Oxidation Indices of Metal-coated UHMWPE Particles

Table 4.4: Association between LC Separation and MS Analysis of UHMWPE

Derived Degradation Products

Table 5.1: Redox Potentials (Aqueous Solution)

Table 5.2: Oxidation Index (0.1.) during the Initiation Phase of Oxidation

at Different Temperature f?om Figure 4.3

Table 5.3: Mass Ions Associated with Peak Area fTom Chromatogram A

of Figure 4.10

Table 5.4: Probable Chemical Structure of Fragmented Ions related to

the m/Z 387 products in Figure 4.39




the m/Z 387 products in Figure 4.4 1

Table 5.7: Probable Chemicai Structure of Fragmented Ions related to

the d Z 670 products in Figure 4.42

Table 5.8: Probable Chemical Structure of Fragmented Ions related to t




LIST OF FIGURES

Figure 2.1: Chemicd Structure of UHMWPE

Figure 3.1: Typical FTIR Spectra of Non-Oxidized and Oxidized UHMWPE

Figure 3.2: Preparation of HPLC Sarnples

Figure 3.3: HPLC Block Diagram in this thesis work

Figure 3.4: Schernatic of Triple quadrupole Mass Spectrometer

Figure 4.1: Scanning Electron Microscopy of UHMWPE Resin Particles

Figure 4.2: Scanning Electron Microscopy of UHMWPE Resin Particles

after undenvent Incubation process

Figure 4.3: Oxidation of UHMWPE with Cobalt Particles, post gamma Irradiation

Figure 4.4: FTIR Analysis of Oxidized UHMWPE with Cobalt for

Various heating times at IOSOC

Figure 4.5: FTIR Analysis of Oxidized UHMWPE with Cobalt for

Various heating times of 13 houn

Figure 4.6: Experimental and Mathematical Relationship Between Time and

Temperature for oxidation index of 1.15 and 1.5

Figure 4.7: Linear Mathematical Representation for Thermal Treated sarnples

HaWig an O.I.= 1.1 5 for Di fferent Temperature

Figure 4.8: Oxidation index for Différent incubation Treatment

Figure 4.9: Cobalt coated Sarnples with DiEerent incubation Treatments (First batch)

Figure 4.10: Cobalt coated Sarnples with Different Incubation Treatments

(Second batch)

Figure 4.1 1 : Cobalt coated Samples with Different Incubation Treatments (Third batch) 53

Figure 4.12: (LC/MS) Mass ion spectnun showing Products found at Retention

time of 49 Minutes for Sample #40 57

Figure 4.13: (LC/MS) Mass ion spectnim showing Products found at Retention

time of 52.4 Minutes for Sampie #40 57

Figure 4.14: (LCMS) Mass ion s p e c t m showing Products found at Retention

time of 53.4 1 Minutes for Sample #40


time of 58.4 Minutes for Sample #40


time o f 62.4 Minutes for Sample #40



Figure 4.18: (LCMS) Mass ion spectnim showing Products found at Retention


Figure 4.19: (LC/MS) Mass ion s p e c t m showing Products found at Retention

tirne of 60.36 Minutes for Sample #45



Fipre 4.21: (LCIMS) Mass ion spectnim showing Products found at Retention

time of 64.15 minutes for sampie #45


tirne of 46.7 Minutes for Sample #13


time of 48.9 Minutes for Sarnple #13

Figure 4.24: (LCMS) Mass ion spec tm showing Products found at Retention

time of 49.08 Minutes for Sarnple # 13

Fipre 4.25: (LCMS) Mass ion spectnim showing Products found at Retention



time of 53 .O2 Minutes for Sample # 13

Figure 427: (LC/MS) Mass ion spectrum showing Products found at Retention

tirne of 55.65 Minutes for Sample # 13

Figure 4.28: (LCMS) Mass ion spectnim showing Products found at Retention

time of 60.1 1 Minutes for Sample #13



Figure 430: (LC/MS) Mass ion spectnim showing Products found at Retention

time of 64.0 1 Minutes for Sample # 13


time of 42 .O 1 Minutes for Sarnple #5 1

Figure 4.32: (LCJMS) Mass ion spectnim showing Products found at Retention

time of 46.65 Minutes for Sample #5 1


time of 56.3 1 Minutes for Sample #5 1

Figure 434: (LCMS) Mass ion spectnun showing Products found at Retention

time of 60.2 Minutes for Sarnple #5 1

Figure 4.35: (LCIMS) Mass ion s p e c t m showing Products found at Retention

time of 6 1.92 Minutes for Sample #5 1

Figure 4.36: (LCIMS) Mass ion spectrum showing Products found at Retention


Figure 4.37: (LC/MS) Mass ion spectrum showing Products found at Retention


Figure 438: (LCMS) Mass ion spectrum showing Products found at Retention


Figure 4.39: (MSIMS) Mass ion spectnim showing Fragments for the

Parent ion with a m/Z of 387, found at the Retention tirne of

49 Minutes for Sample #40

Figure 4.40: (MSMS) Mass ion spectrum showing Fragments for the

Parent ion with a m/Z of 358, found at the Retention tirne of

52 Minutes for Sarnple #40

Figure 4.41: (MSIMS) Mass ion spectnmi showing Fragments for the

Parent ion with a d Z of 387, found at the Retention tirne of


Figure 4.42: ( M S M S ) Mass ion spectnim showing Fragments for the

Parent ion with a rn/Z of 670, found at the Retention t h e of


Figure 4.43: (MSIMS) Mass ion spec tm showing Fragments for the

Parent ion with a m/Z of 330, found at the Retention time of

62.4 Minutes for Sample #40

Figure 4.44: (MSIMS) Mass ion spectnim showing Fragments for the

Parent ion with a d Z of 387, found at the Retention time of

49 Minutes for Sample # 1 3

Figure 4.45: (MSIMS) Mas ion spectnim showing Fragments for the

Parent ion with a rn/Z of 654, found at the Retention time of


Figure 4.46: ATR-FTTR of HPLC Isolate from Area 1

Figure 4.47: ATR-FTIR of HPLC Isolate fiom Area 2

Figure 4.48: ATR-FTIR of HPLC Isolate fiom Area 3

Figure 4.49: Corrected ATR-FTIR Spectrum for Area 1

Figure 4.50: Corrected ATR-FTIR Spectnun for Area 2

Figure 4.51: Corrected ATR-FTIR Spectrum for Area 3

Figure 5.1: FTIR Diagrams for Non-irradiated (virgin) and Post-irradiated

UHMWPE Particles Samples

LIST OF APPENDICES

Appendix A: Calculation of the Amount of UHMWPE

Particles needed in the Incubation Vial. 116

Appendix B: Estimation of the hydrogen peroxide concentration in macrophages 117

Appendix C: Calibration Curves for Hydrogen Peroxide Activity (KI, 99% pure) 119

Appendix D: LC/MS Data for Products Between 100 to 900 Atomic Mass Units 120

Appendk E: Statistical analysis data 126

1.0 INTRODUCTION

Ultra-high molecular weight polyethylene (UHMWPE) is the matenal of choice for

contemporary metal-on plastic total joint replacements. Since Sir John Charnley implanted

the first polyethylene acetabular component in 1962, total joint replacement has become a

standard treatment for severe degenerative joint diseases and post-trauma injuries of the hip

and knee. Today, total joint arthro pl asty is a rernarkabl y effective surgical procedure,

relievine pain and restoring patient mobility for the lifetime of a device. In North Amerka

alone, it was estimated that 400,000 total joint replacements were performed in 1993 [Li et

al., 19941. Joint replacements, when they are perfonned well, c m last up to 15 years with

success rates in excess of 90%, depending on the surgeon's skill, the patient's activity level,

as well as the implant's design. However, approximately 10% of these implants have been

shown to mechanically destabilize or loosen.

UHMWPE is widely used together with metal alloys (Cobalt/Chrome, Titanium or Stainless

Steel) in orthopeadic implants. While the former is used as the acetabular cup, the latter

comprises the bal1 configuration. A cornrnon problem associated with these devices is that

the Wear of metal and UHMWPE implant components has resulted in the formation of Wear

particulates. Both mechanical loading and the oxidation of the metallic and polyrner

materiais in the physiological environment contribute to this degradation process. The

generated micro-sized particles are taken up by white cells and are believed to stimulate or

play a direct role in the periprosthetic inflammation and bone resorption taking place around

the implants [Shanbhag et ai., 19971. hflammatory bone resorption, which extends dong the

implant-bone interface, plays a central role in component loosening. Furthemore, this

condition can progress away from the interface and lead to significant amounts of bone loss

or osteolysis. When osteolysis occurs, the mechanical integrity of the implant is lost.

Therefore, revision surgery is needed, even if the components are well k e d [Sacomen et al.,

19981.

Research in the past decade has investigated the innuence of sizdshape and type (i.e.,

UHMWPE, metal, etc.)bandry et al., 1999; Jacob et al., 19971 of Wear particle debns

generated, on celi function, however, the nature of degradation products generated by the

oxidative process of complexed metal ions derived fiom the metal components and

polyrneric Wear debns has been ignored. The role of the metal ions and their influence on the

oxidation of UHMWPE is a p ~ c i p l e focus of this study. It has been hypothesized that:

1. Cobalt ion at low ppm levels (less or equal than 10 pprn) cm act as a potent catalyst for

the oxidation of UHMWPE particles at temperatures which reflect those reported near the

pol yrned metal interface during loading.

2. In a hydrolytic environment. the oxidized polyrners will be subject to the release of a host

of organic molecules with unknown chemical character and biological function.

3. The oxidized substrates will be less resistant to oxidative chemicals of the inflanmatory

cells than native UHMWPE products

in order to address the above hypotheses the following objectives will be pursued in this

shid y:

1. Optimize an in vitro oxidation mode! for measunng the effect of cobalt ions on the

oxidation processes of UHMWPE particles under themal and chemical oxidation.

2. Degrade the oxidized UHMWP E with a bio-chemicall y-relevant oxidative agent, i.e.,

hydrogen peroxide and a control phosphate buffer solution.

3. Isolate and identifi the dominant degradation products fiom the hydrolysis reactions.

2.0 BACKGROUND AND LITERATURE REVIEW

2.1 Büstory of the Implants and Wear Problem

Early total hip replacements which combined UHMWPE with cobalt/chromium alloy (or

stainless steel, Ti/6A1/4V alloy) developed by Charnley in 1962, had relatively good success

rates (97% survival afier 1 1 years) [Harris, 19911. Further applications of polyethylene have

included the Total Condylar (Knee) Prosthesis, which was developed at the Hospital for

Special Surgery (New York) and was b t implanted in 1974. It demonstrated a 91094%

success rate afier 15 years [Ranawat et al., 19931. However, despite these reported success

rates for the UHMWPE system, it has been shown that the polymer chemically degrades

before and after implantation. Wear debris resulting frorn surface damage of both UHMWPE

and the metal components is recognized as the greatest factor resûicting the Iongevity of total

joint replacements [Wright et al., 19921.

Since the advent of total hip replacements in the 1950's. relatively few polymenc matends

have been used in total joint replacements. These polyrnenc materials have included

polytetrafluoroethylene (PTFE), polyacetal (polymethylene oxide), high-density polyethylene

(HDPE), polyesters, UHMWPE and carbon-fiber-reinforced UHMWPE. During the past four

decades, investigators have observed that with the exception of UHMWPE, d l of these

materials have had significantly high rates of failure. The reason for the failure of PTFE,

polyacetal and HDPE was the hi& Wear resulting fiom the reported ection. The failure of

the carbon-fiber-reinforced UHMWPE system was attributed to poor adhesion between the

carbon fibers and the UHMWPE matrix [Li et al., 19941. Thus, UHMWPE has remained the

material of choice despite the generation of wear debns particles.

From the 19803, the focus of the joint-replacement industry was on the fixation of the

implants since there had not been any demonstration of widespread clinicai problems with

the exception of osteolysis. It was found that osteolysis was cornmody associated with

polyethylene Wear particles fiom surface damage [SchrnalPied et al., 19921. Since the

degenerative Wear process slowly erodes the original profile of the component, the result in

many cases is ultimate failure. The metal component bores through the UHMWPE until it

completely wears through to the underlying bone or metd backing of the polyethylene

component. Usually, before such catastrophic failure occurs, the body mounts a response to

the presence of Wear debns in the sofi tissues surrounding the implant, often resulting in

infection, osteolysis, and late implant loosening [ Xenos et ai., 19951.

Manley et al. and Willert et al. have desaibed a mode1 of long-term implant loosening and

late revision. They indicated that üHMWPE debris are released fiom the implant surface and

steadily accumulate in the joint capsule, gradually polluting the surrounding tissues.

Microscopie debris particles are initially phagocytosed and transported away fiom the joint

capsule, however, in the f i a l stages of "polyethylene disease", the concentration of Wear

debns around the joint stimulates macrophages to elicit a local imrnunological response and

attack nearby bone cells. While the bone underlying the implant slowly resorbs under

osteolytic assault, the patient experiences mounting discornfort as the joint becomes painfully

inflamed and the implant loosens. Ultimately, the joint replacement is surgically revised to

clear the joint capsule of debris and implant a new prosthesis [Manley et al., 1994, Willert et

al., 19901.

The surface damage to UHMWPE components has been studied for many years, and

burnishing, scratching, abrasion, embedded debns, creep, pitting and delamination have been

observed by several researchers [Blum et al., 199 1; Bostrom et al., 1994; Rimnac et al.:

19941. Along with the Wear of polyethylene, some investigators have demonstrated the

presence of metai Wear debns particles in periprosthetic tissue samples [Meldrum et al.,

19931. The presence of metallic debris particles have been associated with an elevation metal

ion concentrations in systemic fluids, synovial Buid and periprosthetic tissue because of their

corrosion [ Betts et al., 19921. Brodner et al. found senun cobalt levels h m one-year implant

patients were below 0.3 p&. The presence of these metal ions is important because at

defined concentrations. It is known that metal ion accumulation can induce ce11 death

[Granchi et al., 19981. Another metal ion source was derived fiom the UHMWPE itself

meldrum et al., 19931. When comparing the cobalt ion concentration of rnanufactured

inserts with retrieved inserts, Meldnim et al. found that the metal ions concentration levels

changed &er many years of implant life. In some cases, original levels were found to be

higher than levels detected following 12-year implants, therefore, this implying the release of

metal ion fiom the UHMWPE component over t h e . Metal alloys, corroding in aqueous

environments, do not in general, release metai ions in propomon to the elementfs abundance

in the dloy [Betts et al., 19921. For example, stainless steel implant materials were found to

release significant quantities of cobalt, even though cobalt was present in the alloy as a trace

impurity wichel et al., 199 11. Metd binding proteins cm be expected to have relative higher affbities for cobalt and chromium versus nickel [Betts et al., 19921. Hence, tracing these

contaminants is often a difficult task. Likewise, it can be conceived that isolating the organic

degradation products of UHMWPE components will also be a difficult talk. Therefore, most

studies to date have focused on defining the degree of Wear for polymeric and metallic

materials, and associating the influence of size, shape and type of Wear particle debris,

generated (i.e., polyethylene, polymethymethacrylate, etc.) on ce11 function.

While studies specifically looking at the generation of degradation products fkom the native

metai and UHMWPE components are rare, work relating the influence of released metal ions

on the generation of üHMWPE particle degradation is even more spane. Partly for this

reason, the role of metal ions, and specifically cobalt ions and their influence on the

UHMWPE particles will be a principle focus of this thesis.

2.2 Polyethylene Materials

For any polyrner system, the properties of the material are inextricably linked with its

chernical structure, molecular architecture, crystalline organization and thermal history

during processing and manufachiring of components containing the material.

The various forms of commercially available poiyethylene belong to a broad family of

polyethylenes, whose members differ by their rnolecular structure. Depending upon their

polymerization process, polyethylenes can be created as short linear, highly branched or long

linear chains. The physical and material properties of branched and linear polyethyIenes are

quite different.@Xodriguez, 1989; Young, 19831. Highly branched polyethylenes are typically

referred to as 10 w-density polyethy lenes (LDP E), since branching hinders the molecular

chains from forming high-density crystalline regions in the material. Branched polyethylenes

are softer, more ductile and more sensitive to creep than their linear equivalents. While

branched polyethylenes have found many applications outside orthopaedics (in film,

package, containers), their comparatively poor material properties preclude their use in high

load bearing devices, such as total hip and knee replacements, for which linear polyethylenes

are most suitable [Eyerer et al., 19901. Table 2.1 lists some properties for LDPE and HDPE

[Wang WB et al., 19961.

Table 2.1 Properties of LDPE and HDPE [Wang WB, 1996)

1 Properties 1 LDPE 1 HDPE I

1 Degree of transparent 1 Serni-transparent 1 Non-transparent 1 Average molecule weight 3~ lo5 1 1 .25x10'

Elongation at break

UHMWPE and high-density polyethylene (HDPE) belong to the fmily of linear

polyethylenes. Despite HDP E 's ease of processing (due to low molecular weights), when

compared with UHMWPE, it has only been used in biomedical devices which experience

minimal loads, such as catheters and coatings for pacemakers [Eyerer et al., 1990; Li et al.,

19941. This is because HDPE has a lower impact strength and abrasive Wear resistence.

Biomedical components, which may be subjected to high in vivo loads, such as total joint

replacements for hip, knee, shoulder and elbow are manufactured from UHMWPE.

I

Hardness (shore D)

Density (g/cm3)

Pol yethylenes are in genmal, reported be relatively biocornpatible. This has been amibuted to

their hydrophobie character which results in the ineversible binding of proteins in a manner

that minllnizes tissue response [Laling, 19731. Conversely, hydrophilic surfaces have been

shown to easily desorb proteins and often can induce a more active cellular response fiom the

materiais. As a result, in their native b d k form, UHMWPE devices are associated with a

relatively low level of tissue response [Laling, 19731.

90-800% 15-100%

41-46

0.9 1-0.925

1

60-70

0.94-0.965

UHMWPE is a long Iinear chah molecule, composed of repeating ethylene (-CH2-CH2 -)

units (Figure 2.1). In the polymerization, the reactive monomer, CH2=CH2, loses its

intracarbon double bond to generate a polymer with molecular weight values on the order of

6 million. This is relatively more than other types of polyethylenes, which have molecular

weights in the range of 10,000 to 500,000.

H H Polymerization

nCH2=CH2 -+

H n

Figure 2.1 Chernical Structure of UHMWPE

Despite UHMWPE's deceptively simple chernical structure, which is similar with other types

of polyethylene resins having lower molecular weights, the material's properties are quite

unique. In general, the physical properties of polyethylene change proportiondly with

increasing molecular weight, until a moiecular weight of 1 million is reached. At this point,

there is a sudden Uicrease in melt viscosity where the material's processibility changes and

hence the charactenstics of the final product are substantially different.

Virgin UHMWPE has the following qualities, which have made it quite attractive as an

implant matmial [Wang et al., 1997](A).

0' Low friction coefficient

* High abrasion resistance ma High impact resistance

High ductility and biocompatibility

Good resistance to biological degradation

0' Hydrophobie and good resistance to aggressive media

22.1 Polymerization of UHMWPE

UHMWPE in its most basic form is a plain white powder. In North Arnenca, three common

grades of UHMWPE resins are used in orthopaedic implants. These include GUR412 and

GUR415 &orn Hoechst Celanese as well as Himont 1900 [Kurtz et al., 19951.

UHMWPE is polymerized in an inert gas atmosphere [Bimkraut et al., 19901. Pure ethylene

eas is suspended in a hydrocarbon solvent (i.e. hexane) fiee of polar ùnpurities and in the - presence of a Ziegler-Natta catalyst system. Polymerization occurs on the catalyst surface at

temperatures between 66 to 80°C and pressures between 4 to 6 Pascals [Li et al., 19941. In

order to initiate the chah polyrnerization, free radical species are produced to react with the

monomenc system using initiator molecules [ Munk, 19891. While there are no specific

references to the initiators used for the commercial UHMWPE of interest to the biomedical

field, the aromatic peroxide, benzoyl peroxide, is an optimal free radical initiator in the

temperature range of 60-80 OC [Munk, 19891. Varying the temperature or adding small

amounts of hydrogen controls the polymer molecular weight. GUR415 has a higher

molecular weight (6 million) when compared to GUR412 (4 million) and 1900 (2-4 million)

[Li et al., 19941.

After polymerization, the solvent suspension goes through a series of centrifuging, stripping

and drying steps where UHMWPE is separated fiom the suspending agents and other

residues. The powder is passed through a 500pm filter to rernove large particulate and yields

a product with a mean particle size of 100pm. The regulation of the degree of purity and the

specification of properties for UHMWPE are in accordance to ASTM F648-84, and are Iisted

in Table 2.2.

2.2.2 UaMWPE Processing and Result in Properties

The propexties of a UHMWPE component depend not only on its original resin source, but

dso upon its rnethod of manufacture. Common processing methods use high pressures and

temperatures well above the melting point in order to fuse or consolidate the resin particles

together. Total joint replacement components can be machined out of compression-molded

sheets or ram extruded rod stock materid. As well, they can be compression-rnolded directiy

into their final shape. As a h a 1 manufacturing step, some companies' heat press or anneal

the d a c e of their components to provide a smooth and glossy finish. However, it has been

demonstrated that annealhg UHMWPE alters the physical properties of the polymer (i.e.,

defoms the crystailites) and may be implicated in reducing their clinical performance

[Young, 19831.

Table 2.2: Standard Properties of UHMWPE Powder [HPIl

I Properties Type of Powder

Molecular Weight

Number of particles of contaminants

Trace elements

Homopolymer of ethylene as

specified in ASTM D4020

Relative solution viscosity>2.3

(ASTM D 4020)

~ 2 5 particles per 300grn

~ax.content (ppm)

Aluminum 100

Ti tanium 300

Calcium 1 O0

Chlonde 120 -

Particle size

UHMWPE is semi-crystalline. In the amorphous region, the long chains hinder the ability of

molecules to order themselves into crystalline arrays, thus lirniting the extent of the material's

crystallinity. The strands are held together by random mechmicd entanglernents and

occasional chernicd crosslinks Wang et al., 1997](B). Surrounding the crystalline regions

and connecting the separated crystals located in the amorphous region are bridging tie

molecules which act as crosslinks between the crystalline domains and provide load bearing,

stress transfer as well as physical and chemical strength P I , Howmedica Product

Information]. The number of tie molecules is higher in UHMWPE as compared to HDPE.

--

Al1 powder shall pass through a

No. 16 (1.18 mm) sieve

Therefore, the crystallinity of UHMWPE (55-60%) is lower than that of HDPE (85-90%) [Li

et ai., 19941.

For purposes of medical implants, the final fabricated fom must adhere to the ASTM F 648-

84 standard, as listed in Table 2.3.

Table 23: Properties of UHMWPE Fabricated Form [HP1 and Lee et al., 19981

Properties

Additives of processing aids

Number of extraneous particles

Number of light patches

Hardness (shore D)

Mechanical requirements(Min.)

Tensile yield strength

Ultimate tensile strength

Elongation at break

Izod impact strength (double notch)

Deformation under Ioad

Density

ASTM F 648-84 Standards

No stabilizers or processing aids to be added

NO particle300pm; no more than 10 particles of

300pm or less

# light patch>than 300pm in a 400 cmL sample

2800 psi

4000psi

200%

1070.JlM (20 Mb)

2% afler 90 min. recovery (1000 psi for 24 hours)

Between 0.930 and 0.944gm/cm3

Incomplete consolidation of the resins greatly shortens the life of the implant [Li et al.,

19941. Recent studies on the analysis of physical properties has led some investigators to

conclude that compression-molded (CM) UHMWPE was more thoroughiy and consistently

consolidated than ram-exmided (RE) material [ Swarts et al., 19961. Also, Bankson et al

(1995) and Rentfiow et d.[1996] found that direct molded or CM material implants have

been shown to be less susceptible to particle debris production when compared to RE

material.

23 Sterilization of UHMWPE

Any matenal implanted into the body must be sterilized. Since LJHMWPE is a thermoplastic,

methods available for sterilization of these implants are: gamma irradiation, ethylene oxide

(ETO) gas and gas plasma (GP). Gamma irradiation has been a standard sterilization method

for orthopaedic implant devices for the past thirty years [Wang et al., 1997](A) and a

common source has been "cobalt, at a minimum dose of 2.5 Mrad. The irradiation period

can Vary fiom 1.5 to 18 hours depending on the radiation source [Streicher et al., 19881.

Gamma radiation sterilization has been shown to chemically damage the polymer by

generating free radicals on the molecular chahs [Del Maestro, 19801. These free radicals

remain trapped or "caged" with the polymer maaix until they react with an adjacent polymer

chah or other electron source (i.e., O?) [ Iring et al., 19901. Investigaton have suggested that

gamma irradiation can increase the Wear rate of UHMWPE since it has been documented to

change the structural and physical properties of polymers [Costa et al., 19981. in

cornparison, the Wear behavior of UHMWPE was not changed by ethylene oxide and gas

plasma stedization [Costa et al., 19981. investigators have shown that gamma -irradiation in

air has caused both oxidative chah scission and crosslinking [Streicher et al., 19881. Chain

scission has been shown to impoverish the mechanical properties of UHMWPE [Kurtz et al.,

19951. Meanwhile, Wang et al. found that the radiation sterilization of the polymers in

controlled environments could provide a potential benefit to UHMWPE since the Wear rates

were shown to be significantly lower than for both untreated and ET0 sterilized UHMWPE

cups [Wang et al., 1997](A).

2.4 Degradation of UHMWPE

The degradation of biomaterials c m be subdivided into several categories according to their

various modes of initiation. These comprise thermal, mechanical, photochemical, radiation

chernical, biological and chernical degradation [Schnabel, 1 98 11.

The tenn degradation in this study is used to denote changes in chemical properties involving

bond scission and the generation of new chemical groups in the backbone of the

macromolecule. In linear UHMWPE, these chemical reactioas lead to a reduction in

molecular weight Wurtz et al., 19951. The shortened chahs result in an efficient folding of

polymer segments, therefore, bringing about more crystalline domains [Kurtz et al., 1995;

Shinde et al., 19851. This leads to an increase in the polymer's density as degradation

proceeds.

Several investigatoa [Kum et al., 1995; Rimnac et al., 19941 have attributed the mechanicd

degradation of UHMWPE to the presence of free radicals that are generated from the

material absorbing extemal energy fiom the load or cyclic stress applied ont0 the material.

This process of degradation promotes the formation of surface roughness, surface creep

deformation and increases the arnount of Wear generated fiom the implant [Wang et al.,

1995; Deng et al., 19981.

2.4.1 Oxidative degradation of UKMWPE

The oxidation of UHMWPE has been shown to initially occur in the amorphous structure of

the material because it is more accessible to oxygen diffising into the material [Scheirs et al.,

199 11. The oxidation propagates non-unifomly throughout the polymer because the

amorphous structure is defined by a series of loosely packed tie molecules that exist within

the material and bridge the crystalline phases. After oxygen difision occurs, the oxidation

reaction c m propagate throughout the material in an autocatalytic process [Fodor et al.,

199 11.

The principle reactions involved in the oxidation of UHMWPE have been summarized in the

following steps: initiation, propagation and termination (see equations 2.1 to 2.6). [Schnable,

198 1 ; Fodor et al., 199 11

In the initiation reaction, Bee radicals are formed:

Equation 2-1

(Note: in the reactive environment, X* cm be any number of factors, including light, heaî,

ultasonics, energy, radiation, certain transition metais, oxygen, oxygen radicals or other fiee

radicals.) Here R-CHrCH2-R' represents polymer.

In the chah propagation step, oxygen is usually involved. The radicals are oxidized

(Equation 2-2) and chain-c-ng peroxyl radicals are formed [Fodor et al, 199 LI:

P'

The reaction of peroxyl radicals with the substrate results in polymer hydroperoxide

formation (equation 2-3). A fraction of the peroxyl radicals cm convert directly into

carboxylic groups with simuItaneous chain scission [Fodor et al., 199 11. The peroxyl radicals

propagate the reaction or fom alkyl radicals:

R- 6~-0-0 + R" - R- CH-O-OH + -C + Equation 2-3 Equation 2-4

OOH OH OH

Chain termination occurs in the bimoIecular cross or homo reaction of the radicals:

2 R-CH2* -b R-CH2-CHrR Equation 2-5

R-CHrO-0. + R'-CH2* R-CH2-CH2-R' + O2 Equation 2-6

Many clinical retrieval studies of failed orthopaedic components have shown clear evidence

of oxidation as measured by FTIR [Sun et al., 1995; Eyerer et al., 19841. Therefore, the

oxidative degradation of UHMWPE is an important aspect of investigations on the

degradation of UHMWPE.

2.4.1.1 Gamma Irradiation and Its Oxidative Degradation

Sterilization of UHMWPE components in air by gamma radiation has received considerable

attention in recent years due to evidence linking this process with inaeased oxidative

degradation. When UHMWPE undergoes 60~o-irradiation in a nitrogen environment,

oxidation of the polyethylene is reduced and thus Wear is also decreased [Streicher et al.,

19881.

The severity of the oxidation is dependent on the irradiation source, the absorbed dose and

the amount of oxygen accessible to the generated radicals [Streicher et al., 19881. Some

authors [Streicher, 1988; Kurtz, 19951 have reported extensively on the relationship between

decreased mechanical properties and the oxidation of UHMWPE induced by irradiation.

On a molecular level, the effects of irradiation on mechanical prope~ties are a consequence of

chah scission due to oxidation and crossliking. This brings about changes in crystallinity and

decreases the entanglernent density and the nurnber of tie molecules. Tende behavior, visco-

elastic properties, hcture strength, fatigue and finally Wear are af5ected [ Premnath et al.,

19961.

2.4.1.2 In Vivo Chernical Oxidation

Following implantation, UHMWPE is believed to be subjected to oxidation process as

involving oxidants such as hypochlorous acid, hydrogen peroxide, nitric acid produced by

phagocytic cells and saline present in the synovial fluid [Nathan, 19861. This oxidation can

be fllrther accelerated by thermal [Jelinski et al., 19841 and Wear [Bankston et al., 1995,

Rimnac et al., 19941 mechanisms associated with fiction between the polymer and metalIic

surfaces.

Meldnim et al. (1993) reported that polyethylene particles were the major constituent of the

material debris formed as a result of orthopaedic implant Wear. These particles vary in size

fiom submicron values up to 100 pm and in trace metal content (Co, Ti, Cr, Va, Mo, Ni,

etc.). The Wear particles can be phagocytosed by macrophages, which can be activated to

release cytokines and other agents at the same time poronov et al., 19981.

Stokes et al. [1990] has s h o w that transition metai ion salts and sodium chloride cm play a

role in catalyzing the oxidation of polyrners. Specifically, Stokes found that polyether-

urethane elastomers exposed to metal conductor wire devices (in other work, related to

cardiac and neurologie pacing leads) undenvent auto-oxidation in vivo because of the metal

ions derhed from corrosion processes. Henry and colleagues found that when polyethylene

was incubated in aqueous salt solutions such as sodium chloride, the materials were s h o w to

undergo heavy oxidation when compared with water. Furthemore, the level of oxidation

increased as a fiinction of sodium chloide concentration and the materid underwent fiuther

changes in density [Henry et al., 19901. Hence, the susceptibility of UHMWPE to oxidation

and the presence of trace metal content raises the concern in regards to the possibility of

accelerated oxidative degradation in UHMWPE. This point will be fuaher discussed in

section 2.4.2.

2.4.1.3 Shelf Storage Environment

Many authors [ Streicher et al., 1988; Rirnnac et al., 1994; Kurtz et al., 19951 have

investigated the relation between storage t h e and the oxidative degradation process, afier

UHMWPE was irradiated. Al1 of them found that the density of polyethylene increased with

storage time. As well, most of the other material properties undenvent changes. Therefore,

the oxidative degradation of UHMWPE progressed during storage tirne.

Streicher [1988] performed a storage experiment with irradiated samples in both air and

nitrogen at 2 1 OC, and in water at 37°C. Their results demonstrated that oxidation was greatest

for the samples stored in water while the least oxidation was found for samples stored in the

nitogen atmosphere.

2.4.1.4 Thermal Oxidation

Thermal oxidative degradation of polyethylene is a well-documented phenomenon, and is

related to the elevated temperatures (over 200-250°C) used for processing the UHMWPE

implant device [Hawkins et ai., 19711. Meanwhile, thermal oxidation is also a concem in

vivo. It has been proposed that the free radical reaction is accelerated due to frictional heating

and stress in the loading zones. This ûictional heating is consistent with observations of

temperature rise in acetabular cups during in vitro fictional Wear stress tests and in vivo

telemetry observations [Janan et al., 199 11. Studies showed a rise in temperature of 4 to 7 O C

in the overall material depending on the type of articulating surfaces and an increase of up to

50°C right at the articulating surface [Lanzer et al., 19921.

In vitro studies by Lee reported that samples of UHMWPE particles which underwent

heating at 105 O C for 170 hours exhibited a significant elevation of oxidation index values

[Lee et al., 19981. However, samples treated at 37, 60 and 80°C showed no significant

increase in the oxidation index values [Lee et ai., 19981. Another observation in the latter

study was that it took appropriately 100 hours, at lOS0C, in order to induce a substantial

increase in the oxidation index. Beyond 100 hours at 105OC, the change in the oxidation

index value became exponential in nature. This suggested that a threshold condition had been

achieved which allowed for an accelerated reaction to occur. This condition could be

attributed to the sample attaining a critical concentration of free radicais, which then initiated

the oxidation reaction throughout the material [Lee et al., 19881.

2.4.2 Metal Ions and their Role as a Catalyst in Oxidation

Cobalt-chromiurn metal alioy is a cornmonly used material for the fabrication of the stem

portion in joint replacements. As such, traces of cobalt and chromium ions have been found

in retrieved implants weldrum, et al., 19931. Interestingly, it has been found that in retrieved

pol yethylene cups of the Co-Cr-UHMUrPE s ystem ( following 1 2 years afier implantation)

there was a drop in cobalt content associated with the polyethylene components of retneved

polyethylene cups weldnim et al., 19931. It was considered that the original metal

contamination was acquired during the machhing of the polyethylene components and that

subsequent Wear released the metal contaminant with the polyethylene.

A study by Michel et al. (1991) has show that the concentration of cobalt ion in s e m was

different for two groups of patients which diflered in that one group did not have implants

and the patients in the second group al1 had cobalt-chromiurn implants. Patients without

implants had a base Ievel cobalt concentration of 0.33*0.16 ( 1 0 ' ~ m l ) while patients with

implants had levels of 1.13 ( lu9 g/ml). It has been reported that cobalt ions in elevated

concentrations (Le. 10 to 40 ppm) are toxic to human gingival fibroblast cells [Lacy et al.,

1 9961.

Zhao et al. (1 995) found that cobalt could accelerate the oxidation of polyurethanes by acting

as a catalyst in oxidation reactions. Stokes et ai. also reported that the cobalt ion, present

because of corrosion processes, in a polyether-urethane biornaterial system could accelerate

the oxidative degradation of the polymer. While serious surface damage to the polyurethanes

has been observed in cobalt bearing alloy systems, there has been linle evidence of

significant auto-oxidation found in the presence of silver, nickel, chromium, molybdenum,

iron, titanium and platinum [Stokes et al., 19901.

Stokes et al. (1990) concluded that the cracking of the polyurethane biomaterials occuned as

a result of corrosion processes, which first liberated cobalt ions that could react with water to

fom the oxygen needed in the auto-oxidation reaction. Subsequently, free radicals would be

fonned and this would initiate and propzgate polymer oxidation. Here, metal ion was acting

as a catalyst in the oxidation of the polymer [Stokes et al., 19901. The proposed chernical

reactions involved are expressed in the following equations 2-7 to 2-20.

Formation of metd ion (m fiom metd (M) in various environments [Coury et al., 19861:

--+ M * + ne- (electrolysis) Equation 2-7

2l@ + 2 ~ ' - ~ M ? + H ~ Equation 2-8

~ M O + O ~ + ~ H ~ O - 4 ~ + + 4 0 K Equation 2-9

M! + HOOH -+ M + HO. + OH- Equation 2- 10

I@ + HOCI + M * + HO. +cl- Equation 2-1 1

While several metai ions have been associated with polyethylene Wear [Meldrum et al.,

19931. Cobalt is of particular interest because it can readily establish an equilibrium state

redox reaction systern in an aqueous medium (Equation 2- 12 and 2- 13) [ Stokes et al., 19901.

C O " co3' + e(~O=-1.8v) Equation 2- 1 2

2Hz0 O2 +4FIi +2e (E' =- 1.2~) Equation 2- 1 3

Metallic ions such as cobalt ion can react with endogenous oxygen and oxygen free radicals

to initiate and propagate the oxidation of polymers in several ways including the following

equations which show the cycle between Co ?' and Co '' [Stokes et al., 1 9901:

co2+ +02 -c03+ +oz-. Equation 2- 14

3+ Co + R-CH2-Rt R-CH*-R' + Co " + Hm Equation 2- 16

These metal ion catalyzed processes can also propagate oxidation reactions related to

intlarnmation [Stokes et ai., 19901. The inflammatory process involves a phagocytic

mechanisrn by macrophages, which is laiown as the 'foreign body responset. During this

process, cells will release hydrolytic enzymes, hypochloric acid, oxygen radicals and

hydrogen peroxide, directly on the device d a c e mathan et al., 19861. In this situation,

some chernical reactions will be propagated by the presence of metai ions such as cobalt

[Stokes et al., 19901. Their reactions with hydrogen peroxide and other compounds could

include any of the following:

Equation 2-17

Equation 2-18

Equation 2-19

Equation 2-20

Equation 2-21

Equation 2-22

The above f?ee radicals can initiate and propagate oxidation of polymeric biomaterials in in

vivo systems [Stokes et al., 19901.

3.0 MATERIALS AND METEIODS

3.1 Material Selection

Ultra-hi& molecular weight polyethylene (UHMWPE) particles were obtained from the

Hospital for Specid Surgery (HSS), New York. The properties of the UHMWPE are listed in

Table 3.1.

Table 3.1 Material Properties for the Selected UHMWPE [Hoechest Celanese Product

S pecXcationl

Property

I~ver. Particle size I

Resin

Resin Manufacturer

4 150HP

Hoechst Celanese

I~ensile modulus MPA 19 15.8 I

Melting point

Specific gravity*

135.6*0.4"C

0.93cm3/g

*nie specific gravity was measured relative to that for water which has a specific gravity of 1 .O0 at

Tensile yield MPA

Elongation to break %

UHMWPE particles were selected for this study since this type of polyethylene is commonly

used in the fabrication of articular components for total joint replacements. And it is the most

common Wear particle debris found at the bearing d a c e s of implants [Blum et al., 1991;

Bostrom et al., 1994; Tanner et al., 19951.

23.8

390

3.2 Test Sample Preparation

3.2.1 Steriiization of Samples

Sterilization by irradiation is the most cornmon method used to sterilize UHMWPE

components. Other methods such as ethylene oxide treaûnent are also in use, but there has

been a cal1 by the FDA to phase out its use due to the toxic residues which remain following

sterilization [Hui et al., 19971. In this shidy, sampies were gamma-irradiated with a 2.5 Mrad

cobalt-60 source in the presence of air at the department of Chernical Engineering and

Applied Chemistry, University of Toronto. It was not possible to perform the sterilization

under nitrogen atmosphere at these facilities since they were not equipped for such a task.

The selected dosage of radiation was 2.5 Mrad 60~obalt, which is standard for the

sterilization of d l medical implant systems [Streicher, 19881. There have been studies which

suggest that storage time (days to months) after irradiation increases the susceptibility of

UHMWPE oxidation [Kum et al., 19951, hence for the current study of the particles were

used within 5 hours of irradiation.

3.2.2 Coating of UHMWPE Particles with Cobalt Chloride

Experirnents were carried out to detemiine the effect of metal ions on the oxidation of

UHMWPE. Cobalt ion was of particular interest because of its oxidation potential [Zhao et

al., 19951 and since analytical studies of UHMWPE have indicated its presence at the

interface between the UHMWPE cup and the cobalt-chromium bal1 in total hip replacements

@ielcirurn et al., 19931. Cobalt chlonde hexahydrate in 99.99% punty (ASC reagent grade,

BDH Inc., Toronto, Ont.) was selected as a source of cobalt ion for the work in this thesis.

The mass ratio of metallic salt to UHMWPE particles selected for most of this work was

1 : 100. The metallic sd t and polymer particles were added to a 50 mL beaker. Approximately

30 mL of HPLC grade ethanol (Sigma-Aldrich, St.Louis, MO) were used to dissolve the salt.

The solution mixture was stirred u n i f o d y using a Vortex-Genie vortex [Scientific

Industries Inc., Bohemia, NY] for 10 minutes in order to completely dissolve the salt. This

mixture was then left under the fume hood for several days until the ethanol was totally

evaporated. Kimwipes@ were placed on the beaker to prevent any contamination fiom

airborne particles d h g the evaporation process.

The powder mixture was then washed 10 times using 30 mL of HPLC-grade water and the

water was rernoved from the particles in a filtration system with a 0.5 pm Teflon filter

(FHUP04700, Millipore Corporation). The filter was used to collect the coated particles,

which then were placed in a covered petri dish and lefi to dry overnight in an oven at 37°C.

0 t h metal ions, such as titanium oxide (rv) (Aldrich, Milwaukee WI, Cat#25, purity 43 1-

2). chromium chloride (III) (Aldrich, Milwaukee WI, CatR2, purity 957-l), nickel chloride

(II) (Aldrich, Milwaukee WI, Cat#22, purity 338-7) were also coated with WMWPE

particles by using a similar procedure. Work with the latter metal ions was canied out by

Zaheer Ahmed [Thesis, 19991 and the work was supervised by the M.Sc. Candidate. These

metals were selected because they have been reported to be present in high concentrations

isolated in retrieved implants [Meldrum et al., 19931.

3.23 Metal Ion Concentration (Atomic Absorption Spectroscopy)

Cobalt-chromium metal is comrnody used for the stem portion of total joint replacements

[Meldrum et al., 19931. As such, mces of cobalt ions have been found in retrieved implants

bond with the particulate Wear debris [ M e l d m et al., 19931 and bey were also found at the

site of inflamrnatory tissues around loosened implants [Meldrum et ai.,1993]. The

concentration of cobalt from retrieved polyethylene cups of Co-Cr-LJHMWPE systerns was

approximately 64 * 2 1 ppb after 12 years [Meldrum et al., 19931, while the concentration of cobalt in smounding tissues ranged from 0.06 to 171 pg/g (ppm) dried tissue [Betts et al.,

19901.

In this work, the cobalt ion concentration was set at a level of about 10 ppm because this

level was the lowest concentration that could be achieved within the prelirninary studies (see

resuIts section). Co bah levels were measured using an atomic absorption spectrometer ( AA)

located in the Department of Chernical Engineering and Applied Chernistry, University of

Toronto. AA has a high sensitivity t e h g the rnetd ion concentrations between ppb to ppm

levels.

In the atomic absorption (AA) method, a weighed amount of the sample for analysis was

atomized in a Barne, and the absorbance of the light by this vapor was measured at a specific

wavelength which was characteristic of the element to be determined. The unlaiown

concentration was given by cornparison with absorbance measurements for standards of

known composition [Ebdon et al., 19981.

3.3 Oxidation of UHMWPE Particles

Oxidative degradation has been identified as a prevalent mechanism of UHMWPE

degradation [Li et al., 19941. There are several sources of oxidation that can contribute to the

degradation of UHMWPE. While UHMWPE has suitable chemical, mechanical and

physical properties for orthopaedic devices in its virgin state, it is found that gamma

irradiation during sterilization; thermal oxidation during the process of the implant as well as

chemical oxidation degradation due to the presence of salts and oxidants such as hydrogen

peroxide in the physiological environment alters its pnstine condition. The purpose of this

study was to oxidize UHMWPE particles containing cobalt ion and to isolate and identify the

nature of particdate degradation products produced by both oxidative and hydrolytic

processes.

3.3.1 Oxidation b y Thermal Treatment

Oxidation by heat-treatment was performed to determine a relationship between the

oxidation index and incubation time for different ternperatures. Samples of UHMWPE were

placed in an airflow oven for various t h e periods and temperatures (i.e., 37OC, 60°C, 7O0C,

75OC, 80°C, 90°C, M°C and 105OC). A list of the heat-treated samples and their incubation

times are given in Table 3.2. The 37OC temperature was chosen because it represents a

physiological temperature for the human body. The remaining temperatures were chosen

based on previous accelerated aging studies for UHMWPE [Poggie et ai., 1997; McKellop et

al., 1997; Dwyer et al., 19961.

Table 3.2: Experimental Heat-treatment Periods for UEMWPE Particles with Cobalt

Ion Samples

1 Temperatures 1 Times tested 1

#Experiments were performed once for exploration purposes

*Experiments were performed in triplicate.

37"C# 60°C# 70°C# 75T# 80°C*

9o0c* 95OC* ' 1OS0C*

33.2 Chernical Oxidation of UEEMWPE particles

The samples were subjected to hydrogen peroxide in phosphate buffer (pH=7) for 20 days.

Studies have s h o w that hydrogen peroxide is a chernical agent which is present in

significant amounts during the inflammatory response at sites of inflammation near

orthopaedic implants Weiss et al., 1982; Iwasaka et al., 19981. While the H20r is produced

continuously, it is also consurned. Therefore, while commutative amounts of hydrogen

peroxide generated can be quite hi&, the actual hydrogen peroxide levels at any point in time

is much lower than 10 w/w%.

6 months 1 month; 6 months 13 days; 2 1 days; 30 days 1 3 days; 2 1 days; 30 days 18 hours;24 hours; 30hours; 38 hours; Zdays; 3 day; 4days; Sdays 8 hours; 13 hours; 18 hours 8 hours; 13 hotus; 18 hours 8 hows; 13 hours; 18 hours

In the current study, the concentration of hydrogen peroxide was chosen as 10 w/w% [Lee et

al, 19981. While this concentration is very high in cornparison to what may be expected

physiologically, calculations in Appendix B show that the experimental parameters in this

study are such that the achial particle exposure to hydrogen peroxide is similar to that which

may be expected for phagocytosed particles. It is reported that macrophages cm release

hydrogen peroxide continuously during four contrasting states of activation [Nathan et ai.,

19861. The levels of hydrogen peroxide generated by macrophages are shown in Table 3.3

based on an ideal choice of an oxidant for this study. As well, similar concentrations of Hz02

have been used by others to study the oxidation of UHMWPE implant components [ Rimnac

et al., 19941.

Table 33: Hydrogen peroxide released in macrophages i t four different contrasting

States of activation (Data obtained from Nathan, 1986)

kel l State 1 ~ ~ 0 ~ released per hour, nmoUmg protein

l Non-activated 50- 1 O0

1 Activated 13 50-650 1

33.2.1 Determination of Bydrogen Peroxide Activity

Hydrogen peroxide activity was determined using a method based on the spectrophotometric

determination of I*, formed when hydrogen peroxide is added to a solution of iodide (1-)

rneasured at a wavelength of 35 1 nm [Klassen et al., 19941. The reaction proceeds as follows:

Deactivated Inactivated

H202 + 2r + 2 H I2 + 2Hz0 Equation 3- 1

1-100 0-20

and the iodiddiodine species are in equilibrium:

I~ + r - I ~ - Equation 3-2 The chernicals for this assay are listed in Table 3.4. Solution A was made up in a 500 mL

volumetric flask and contained: 33g of potassium iodide (99.99+%), Ig of sodium hydroxide

(99.99%), and O.lg of ammonium molybdate tetrahydrate (reagent grade) dissolved in

filtered water. This solution was stirred for 10 minutes to dissolve the molybdate and poured

into a foil-covered bottie to prevent photochernical oxidation of T. Solution B contained 1 0g

of potassium hydrogen phthdate dissohed in 500 rnL of water, in a volumetric flask. A

calibration curve was created using equal amounts (by weight) of solutions A and B and

varying the concentration of hydrogen peroxide. The absorbance of the resultant solution was

then rneasured at 35 1 nm. The cdibration curve can be found in Appendix C.

Table 3.4 Chemicals for Hydrogen Peroxide Assay

C bedcals

i Sodium hydroxicie, 99.99%

Supplier

Po tassiurn iodide, 99.99+%

' ~ idr ich Chernical Company, Milivaukce, I IWI I

Aldrich Chernical Company, Milwaukee, WI

1 Ammonium mol ybdate tetrahydrate, ACS reagent [Aldrich Chemical Company, Milwaukee, 1 I grade Potassium hydrogen phthalate, 99.95%

33.2.2 Half-üfe Study for Hydrogen Peroxide

A half-life study was performed to determine the activity of the oxidant over the incubation

paiod when in the presence of the üHMWPE particles (with trace levels of CoClr). The

half-life value was chosen to be the time at which the hydrogen peroxide would be

replenished to yield the original activity of the experiment.

WT Aldrich Chernical Company, Milwaukee, WI

Hydrogen peroxide, 30 w/w%

Using a Mettler-Toledo anaiytical balance (Greifensee, Switzerland), approximately 0.240g

sample of UHMWPE particles was weighed, placed in a glass French bottle (VWR

Scientific, #363 19-760, Mississauga, ON), and layed on its side. The lid of the bottle was

TeflonB-lined. A 15 ml aliquot of 10 w/w% hydrogen peroxide solution in phosphate buffer

@H=7) was added to the polymer. The concentration of the hydrogen peroxide was read in

triplicate at periodic intervals (e.g., tirne = 0, 2, 4, 6, 12, 24, 48 hours). The half-life was

determined to be 3 days. The half-life data are plotted in Appendix C.

Aldrich Chemical Company, Milwaukee, WI

3.33 Incubation Experiments

The sample groups used in this study and their treatrnents are given in Table 3.5.

Table 3.5: Chemical Treatments for UHMWPE Particles

1 Pre-Treatment of Samples l Incubation conditions (8 ho~rs(l05~C) heat with cobalt* 1H2O2 10 W/W% in buffer*; buffer* 1

* ~am&s were perfokned% triplicate

I~on-heat with cobalt* 1 ~ ~ 0 2 10 w/w% in buffee; buffer*

The method of UHMWPE particle incubation was previously developed by Angela Lee [Lee,

20001. However, some changes were made to the incubation times. A wide-mouth French

square glas bottle with the dimensions of 5.5 an x 5.5 cm x 11.4 cm (VWR Scientific,

Mississauga, ON) was used with a Teflon@-lined lid. The low density of UHMWPE made it

diEcult to completely expose the particles to the solutions. Hence, this bottle was selected to

maximize the particle exposure by providing a high surface area per volume ratio. The

surface area available at the particle-solution interface was 52.80cm" n e mass of particles

used to form a monolayer in this surface area was calculated as being 0.230g (See Appendix

C). This m a s was calculated based on an average particle size 250 Pm in diameter [ SEM

measurements, Lee, 1 9981.

8 houn( 105°C) heat, non-cobalt Non-heat, non-cobalt

An aliquot of 0.23g of LTHMWPE particle was weighed and placed into three bottles for each

sample. Then an aliquot of 15 mL of 10 w/w% hydrogen peroxide in buffer or buffer control

solution was carefully added to the bottie to ensure that a monolayer of polymer was floating

on the surface of the solution. The botîles were capped tightly and covered with aluminurn

foi1 because the hydrogen peroxide was light sensitive. Al1 samples were incubated at 37OC

for 20 days. The samples incubated with hydrogen peroxide were replenished every three

days, based on half-life data [Appendix Cl with 2.5 ml of 30 w/w% hydrogen peroxide.

H2O2 10 w/w% in buffer; buffer H202 10 W/W% in buffer, buffer

Following the end of the incubation period, the incubation solutions were separated f?om the

particles. This was done using a filtration system with a 0.5 pm Tefion@ membrane filter.

The particles were collected on the filter, placed in a covered petri dish and left to dry in a

37OC oven for one day or until the residual water evaporated. The incubation solutions were

prepared for hi& performance liquid chromatography (HPLC) analysis (see section 3.5) and

the particles were analyzed ushg FTIR ( see Section 3.4).

Oxidized UHMWPE particle sarnples were incubated with phosphate buffer @H=7) or

h y d r o p peroxide !10w/w%) in buffer for 20 days at 37OC. These conditions provided both

hydrolytic and oxidative conditions. The sarnples were previously listed in Table 3.5. The

incubation conditions were selected in order to mode1 aspects of the physiological

environment. For instance, it has been reported that monocyte-derived macrophages will

release hydrogen peroxide after particles are phagocytosed by the cells pathan et al., 19861.

At the end of the incubation periods, particles were isolated fiom the solutions using a filter

system with a 0.5pm Tefion0 membrane (0.5 pm, FH type, Millipore Corp.). The details of

this step were desaibed in section 3.3.3. The polymer was dried in a oven at 37°C overnight

and stored at room temperature in dark until FTIR analysis was perfomed within 7 days. The

incubation solutions were kept for analysis of the degradation products using HPLC.

3.4 Characterization of UELMWPE Oxidation by Fourier Transform Infrared

Spectroscopy (FTIR)

Fourier transfomi i&ared spectroscopy is an analytical technique b a t is used to identify the

various chemical groups present in a substance. The theoretical principle is based on the

ability of chemical groups to absorb energy that will be used to increase the vibrational

motion of the bonds pavia et al., 19791. However, only chemical groups that contain a

dipole moment can absorb infrared radiation. These diflerent groups can absorb energy to

increase the vibrational motion at a characteristic wavelength or wavenumber. IR

spectroscopy can be used to fhgerprint a specific compound and the types of vibrational

motion can be used to elucidate the chemical structure of the material.

In this study, there were two methods of FTIR applied to quanti@ the amount of oxidation

found in UHMWPE particles: transmission FTIR and diffise reflectance FTIR @RIFT)

spectroscopy. In transmission FTIR, a s p e c t m is generated h m the vibrational energy that

is detected when the infrared radiation passes through a sample. However, DRIFT generates

an FTIR spectnim by using the reflectance of the radiation fiom the surface of the sample.

Reflectance consists of regdar reflection and diffuse reflectance. Diffise reflectance results

when light enters a substance where it is partially absorbed and the energy emerges f?om the

substance afler it has been scattered. The reading obtained using the diffise retlectance mode

is given in Kubelka-Munk nits ( K-Munk ). These units are linearly proportional to the

sample concentration [ GRAMSI 386User's Guide] and are analogous to transmission in

normal transmission FTIR. K-Munk can be converted fkom transmission data using the

following relation:

K- Mun k =[I - ~ r a n s m i~sion]~/[2~r~ramrnissionj Equation 3-3

Following some preliminary measurements, it was decided to analyze the particles using the

reflectance mode because the sample preparation was quicker and easier, and the data tended

to be more reproducible than results acquired by transmission FTIR.

For the sample preparation, the particles were diluted with FTIR grade potassium bromide

(Aldrich Chernical Company, Milwaukee, WI) in a ratio of 1 :5 (mass of particles to mass of

potassium bromide), a typical mixture would consist of 6ûmg of üHMWPE. The samples

were ground together using a m a d e mortar and pestle and placed in a sample holder (5 mm

in diameter, 2.5 mm in depth). The surface of the sample was pressed and flattened using a

spatula and force. The sarnples were s c a ~ e d using a nitrogen purge to remove most of the

moishire and carbon dioxide fiom the sample chamber. In reflectance mode the IR scan

provide an analysis of the upper 10 pm of the surface [Lee, 19981, depending upon the ability

of the materials to absorb IR radiation.

The experiments were c&ed out using a Bomen Hartmann & Braurn, MS-Series FT-Raman

instrument and GRAMSI386 software at Photonics Research of Ontario, University of

Toronto. All samples were scanned in the mid-IR region (500 to 4000 cm"). The number of

scans taken up for each sampIe was 10-25.

3.4.1 Definition of Oxidation index for UHMWPE Particles

In order to quantifi the degree of oxidation in the experiments, a convenient measure of this

value was needed. Various dennitions have been provided for the oxidation index values of

UHMWPE over the past decade, and most of those have involved the height and area

measurements of various peaks. Usually, the definition includes peaks within the 1650- 1850

cm-' range which are associated with the carbonyls. As well, these latter values are

normalized usine selected peaks. which are associated with methylene or methyl groups.

Personal communication with the chairman of the ASTM cornmittee on UHMWPE, Dr.

Steve Kurtz has indicated that eight of the most comrnon methylene or methyl peak

selections used to norrnalize the carbonyl region are givan in Table 3.6 [Kurtz, 19951.

Table 3.6: Summary of Oxidation Index Defmition for UHMWPE*

Method 1

Normalization for methyl vibration Area of 2020m"

2 3 4 5

1 8 IHeight of 425 1 cm" 1 * This surnmary was obtained fiom persona1 communication with Dr. Steve Kurtz (chaimai of

Height of 2020m" Area of 1468cm-l Height of 1468cm-' Area of 1370cm-'

6 7

ASTM for UHMWE)

Height of 1370cm" Area of 425 1 cm"

The oxidation index (0.1.) selected for the curent work was defined as the ratio of area under

the peak for the carbonyl region, between 1650 -1850 m", to the methyl peak vibration

between 1330-1390cm" (Figure 3.1). This was based on the Arnerican Society of Testing

and Material (ASTM F04.15.12) standard. A higher 0.1. value defines a material with more

extensive oxidative damage to the particles. The 0.1. equation can be expressed as equation

3-4:

3.4.2 Typical FïIR Spectra Diagrams Associated with the particles

A typical FTIR spectrum for virgin and oxidized UHMWPE particles is s h o w in Figure 3.1.

Its contains a sharp asymmetric stretching at 2915 cm-', rnethylene in-plane defornation at

1466 cm", a light methyl urnbrella defornation at 1370 cm-' and a strong in phase rock at

722 cm-' [James et al., 19931.

Virg in

Figure 3.1 Typical FI'IR Spectra of Non-Oxidized and Oxidized UBMWPE

The oxidized sample in Figure 3.1 contains an additional peak at 17 10 cm'' (carbonyl peak),

which is characteristic of the oxidation process. The samples that were anaiyzed using FTIR

are listed in Table 3.2.

3.43 ATR-FTLR Analysis of IsoIated Degradation Products

FTIR was also used to analyze solution samples. The sample holder was changed to a

solution chamber (50 mm in length, 5 mm in width, 2.5 mm in depth), that had a high

rehctive index crystal between the sample and the light source. In this method, the

background was changed to a solvent solution in place of the potassium bromide used for

particles. The data are presented as a measue of transmission. This method is also called

Attenuated Total Reflectance FTIR (ATR-FTIR).

in order to identify the nature of chernical fiinctional groups within the isolated degradation

products, specified HPLC fractions were collected h m the incubation solutions (Figure

4.10) and concentrated in order to analyze the products by FTIR. The specified samples are

listed in Table 3.7.

Table 3.7: Samples Using ATR-FTIR Chromatogram A in Figure 4.10

Sample description Retention times fiom 53-55 minutes of #A0 Retention times fiom 60-67 minutes of #.4O Retention times fiom 69-70 minutes of #.4O

Note: Al1 products were derived h m the incubations of particles that were treated with CoCI2, heated for 8 hours in air at i OSaC and then incubated in (IOw/w%) hydrogen peroxide solution for 20 days.

3.5 Extraction of Degradation Products

In order to prepare the incubation solution sarnples for high performance liquid

chromatography (HPLC), contaminants such as salts in the incubation solutions were

rernoved. Following this, the solutions were concentrated and then dissolved into a suitable

mobile phase. Al1 solutions were processed in the same manner. Triplicate solutions f?om the

same reaction conditions were pooled together in order to increase the intensity of the HPLC

and mass spectroscopy (MS) signals. The preparation scheme for the HPLC samples is

shown in Figure 3.2. Two extracts of the incubation samples were analyzed for degradation

products. The first extract consisted of samples taken fbm the organic phase of

chloroform/incubation solution extraction and the second was a rnethanol extraction of the

fieeze-dned aqueous phase firom the chlorofodincubation solution extraction.

The solution extracts were prepared in the following manner. A 10 ml aliquot of HPLC grade

chloroform (Caledon Labonitories Ltd., Georgetown, ON) was added to the incubation

solutions. The chloroform/aqueous mixture was vortexted for 5 minutes. Upon resting this

sample immediately separated into two phases. The chloroform phase was removed with a

Pasteur pipetîe and was evaporated with a stream of high pwity, dry nitrogen (Grade 4.8,

BOC Gases, Mississauga, ON) until a residue remained. The incubation solutions were

extracted with chloroform three tunes and the residues were pooled together. The solid

residue was dissolved with the starting HPLC mobile phase (100~1 of methanol and 900 p1 of

ammonium acetate buffer. pH=7.0). These samples were labeled as the symbol "organic

phase" denoted following the sample number (e.g., #40 organic phase).

The aqueous phase of the incubation solution was frozen in liquid nitrogen and fieeze-dried

(WDS-54A, FTS Systerns Inc., Stone Ridge, NY). The recovered solids were extracted with

methanol solution. The methanol solution was separated from the solid and subsequently

evaporated, using a stream of Grade 4.8 nitrogen, to recover the products. Finaily, the

residues f?om thermal evaporation were dissolved with the starting HPLC mobile phase

(100~1 of methanol and 90 pl of ammonium acetate buffer, pH 7.0). These samples were

labeled as " polar phase" before its number ( e g , #40 polar phase). AI1 HPLC samples were

stored in a fkidge at 4OC until ready for HPLC analysis. Since the organic phase samples

contained most of the oxidation products and the polar phase samples contained almost

nothing observed in prelirninary HPLC work, subsequent experiments were only camed out

fiom the organic phase, and the "organic phase" samples label were not separated used.

Incubation solution filtered with 0.5 pm Teflon

membrane

Particles P Solution extraction

I

Organic Phase C hl0 ro form

evaporation

methanol, 90% buffer.

Aqueous Phase Freeze-dried

Solid extraction( 100% methanol)

Solvent

Residue dissolved in ATR-FTR Soluble products mobile phase ( 10% analysis HPLC analysis methanol, 90% buffer)

A

Figure 3.2: Preparation of EIPLC Samples

3.6 EUgh Performance Liquid Chromatography (HPLC)

High performance liquid chromatography is a common technique used in analytical

chemistry to separate various compounds fiom a mixture of compounds with molecular

weights less than 5000. The separation is based upon the affuiity of the compounds for either

the stationary phase (colum) or the mobile phase (solvent system). A block diagram of the

HPLC system used in this study is described in Figure 3.3.

A Waters TM HPLC systern was used Ki the separation of biodegradation products. It was

composed of a 600E multi-solvent delivery system and used methanol, ammonium acetate

b d e r @H=7.0) and water as the solvents making up the mobile phases. The solvents were

sparged with helium to remove air bubbles that could interfere with the hc t i on of the

co1umn. Pnor to use, al1 the solvents were filtered to remove particulate contamination and

degassed.

A UK6 injector (Waters TM) delivered the sarnple through an in-line column filter (SS,

0.22pm), past a guardpack column filter (pBondapak, Ci*, Waters TM) and through a Waters

pBondapak TM Cig steel column (4.6 by 25ûmm) packed with dimethyloctodecylsiy1 bonded

amorphous silica. The in-line column filter and guardpack filter protects the Ci column fkom

particulate and hi& column loading. The products are eluted off the column at different

retention rimes and are detected using a photodiode array detector (PDA)(Waters PDA 996).

The pump system controller (Waters 600 controller) delivered and mixed various solvents at

specified compositions and flow rates. MiIlenniun 320 software was used to control the

system and acquire as well as process the original data.

The retention time and the degree of separation can be varieci depending upon the solvent

system used (polar or non-polar), the type of system run (isoratic or gradient) and the type of

columns used (polar or non-polar). In this thesis, the stationary phase (column) had a lower

polar@ than the mobile phase. The mobile phase was a mixture of methanol and 2mM

ammonium acetate buffer @H=7.0).

Al1 sarnples were run using a mobile phase gradient method because they each contained

many degradation products. The gradient program method is given in Table 3.8. The

program has a 10% methanol /90% acetate buffer isocratic system in the first 10 minutes,

which allowed many of the relatively polar products to be separated. The gradient over 50

minutes allowed for the separation of the non-polar products found in the rest of the sarnple.

The 100% methanol isocratic gradient at the end of the run allowed for the elution of any

non-polar products that were strongly bonded to the column.

Mobile phase -4 Solvent ~ Reservoirs Pump, gradient

controller s ystem (Waters 600)

Manual hj ector (or Auto injecter)

1 in-line co~umn 1

(Stationary phase)

Waste Detector (PDA 996) Printer or recorder

Fluid connection 4 Elecnical signal

Figure 33: HPLC block diagram in this thesis work.

Table 3.8: Gradient Program Run for HPLC

1 Time 1 Flow ( Methanol (%) 1 Acetate buffer (%) (

Al1 HPLC chromatograms were displayed at 2 10 nm, as acquired from the PDA detector. At

this wavelenth, some of the degradation products of interest were clearly differentiated. The

peaks of interest were isolated and prepared for liquid chromatography mass spectrum

(LC/MS) analysis.

3.7 Identify Biodegradation Products using LCIMS, (Liquid Chromatography-Mass

Spectrometry)

The coupling of mass spectrometry (MS) to LC is a universal detector and has the advantage

of being used to detect products that are not absorbing in the WNIS range or do not

undergo fluorescence. n i e mass spectrometer can be a very selective detector since it

provides specific information on the molecular structure of an unknown component.

Dr. Men Linjie in the Carbohydrate Research Laboratory performed the LCIMS and mass

spectrometry experiments at the University of Toronto, faculty of Medical Science.

3.8 Identification of Biodegrsdation Products using MSlMS

M a s spectrometry is a technique used to determine the molecular weight of a pure sample.

Ion spray mass spectrometry bombards a sample with a hi& energy bearn of electrons to

produce in fragments of positive ions which are generated fiom molecules by the removal of

an electron Favia et al., 19791. The ions are accelerated in an electnc field and separated

according to their mass to charge ratios. Finally, ions with a particular mas-to-charge ratio

are detected. The mass spectnrm is recorded as a measurement of relative intensity vernis

rnass-to charge ratio (rd).

The relative intensity is cornpared to the number of counts of the most intense peak. The

h p e n t a t i o n pattern is charactenstic for each compound. Multiple peaks can be associated

with the same molecular ion when combined with different salts, which are present in the

mobile phase or the sample (e.g., MN^', MK+, bNl+~). By piecing together the fragments, a

general structure of the compound can be elucidated. Further fragmentation of selected mass

fiagrnents can occur using tandem mass spectroscopy (MS-MS). This provides a mass

spechum with minimal contamination.

Mass spectrometxy was camed out on an API-III triple quadrupole mass spectrometer (MS-

MS) (Perkin-ElmedSciex, Concord, ON). The first quadrupole detects the initial molecular

ions [Pavia, 19791. Ln MSIMS, the second quadrupole is the reaction region for the ionic

collisions of a selected molecular or parent ion kom the first quadnipole. Ions f?agmented

nom the parent ion are narned daughter ions. The third quadrupole is used as a molecular

weight analyzer of fragments fiom both the MS and MS-MS sam

of polyeteylene (uhmwpe) of ion · hawig an o.i.= 1.1 5 for di fferent temperature figure 4.8:...

Documents