A11101 llOMflq

NBS

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NAT'L INST. OF STAND & TECf

AlllOb 407373

NBSIR 81-2436

Low Molecular Weight Leachables

from Medical Grade Polymers

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

National Measurement Laboratory

Center for Analytical Chemistry

Washington, DC 20234

Aprii 1931

Final Report

Issued Aprii, 1 982

Prepared for Food and Drug Administration

Center of Medical Device Analysis

Bureau of Medical Devices

Rockville, MD 20582

NBSIR 81-2436

LOW MOLECULAR WEIGHT LEACHABLES

FROM MEDICAL GRADE POLYMERS

NATIONAL BURRA l Of STANDARDS

LIBRARY MAY 14 1982

floe Cuu

S* \a

B. F. Howell, S. Chesler, L. Hilpert, and D. J. Reeder

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

National Measurement Laboratory

Center for Analytical Chemistry

Washington, DC 20234

April 1981

Final Report

Issued April, 1 982

Prepared for Food and Drug Administration

Center of Medical Device Analysis

Bureau of Medical Devices

Rockville, MD 20852

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler. Director

CONTENTS

Page

1. Introduction . . 1

1.1 Silicone ........ ....... . 1 1.2 Polyethylene ................. 2 1.3 Polyurethane .................... 3 1.4 Polycarbonates . . 3 1.5 Prolyl Hydroxylase ... ................. 3 1.5.1 Objective ... .................. 4

2. Experimental Section . ......... 4

2.1 Materials and Reagents ................. 4

2.2 Leaching Experiments .............. 5 2.2.1 Polyethylene .................... 5 2.2.2 Polyurethane and Polycarbonate ........ 5 2.2.3 Silicone Gel ................ 14

2.3 Prolyl Hydroxylase Studies ......... 22

2.3.1 Enzyme Preparation ........ . 22

2.3.2 Protocollagen Substrate Preparation ... 22

2.3.3 Enzyme Assay ......... . 26

2. 3. 3.1 Conway Cells ..... 26

2. 3. 3. 2 Microstills 26

2. 3. 3. 3 C02 Assay . 27

2. 3. 3. 4 Column Separation 27

2.3.4 Enzyme Purification ............ ... 28

2. 3. 4.1 Sepharose 4-B ............. 28

2.3.4. 2 Dye Ligand Chromatography . . 28

2. 3. 4. 3 Affinity Chromatography . ...... 28

3. Discussion and Conclusions .................. 29

4. References .......... 29

I

1i
LIST OF FIGURES

Figure No. Page

1 Total ion current profile vs. probe temperature for the methanol

soluble portion of the aqueous leachate of polyethylene blood

bag . . 6

2 Total ion current and m/z 149, 167, and 279 (indicative of

phthalate ester) vs. probe temperature for the methanol soluble

portion of the aqueous leachate of polyethylene blood bag 7

3 Total ion current and m/z 105, 163, and 77 vs. probe temperature

for the methanol soluble portion of the aqueous leachate of

polyethylene blood bag 8

4 Mass spectrum at probe temperature of 40 °C corrected for

background (polyethylene) leachate ...... 9

5 Mass spectrum at probe temperature of 26 °C (polyethylene

leachate). Unidentified materials 10

6 Mass spectrum at probe temperature of 50 °C (polyethylene

leachate). Unidentified materials 11

7 Gas chromatogram of polycarbonate leachate, methylene chloride

extract . 12

8 Gas chromatogram of polyurethane leachate, methylene chloride

extract 13

9 GC/MS total ion current profile, polyurethane leachate,

methylene chloride extract 15

10 Gas chromatogram of silicone leachate blank, methylene chloride

extract 16

11 Gas chromatogram of silicone leachate, methylene chloride

extract 17

12 Total ion current, m/z 73, 147, 207, 221, 281 and 295 vs. probe

temperature for silicone gel 19

13 Mass spectrum at a probe temperature of 90 °C silicone gel

leachate 20

14 (upper) Total ion current and m/z 149 vs. probe temperature for

silicone gel leachate, (lower) Mass spectrum at probe temperature

of 45 °C showing the presence of ions characteristic of phthalate

esters 21

15 Total ion current vs. probe temperature for silicone gel 23

16 Total ion current and selected mass chromatograms for silicone gel . 24

17 Mass spectrum at probe temperature of 50 °C (silicone gel) 25

11 i
Low Molecular Weight Leachables from Medical Grade Polymers

B. F. Howell, S. Chesler, L. Hilpert, and D. J. Reeder

Center for Analytical Chemistry

National Bureau of Standards

Washington, DC 20234

Four medically important polymeric materials (silicone polymer from

mammary prostheses, polyurethane, polyethylene, and polycarbonate) v/ere

placed in contact with water for time periods ranging from four hours to seven days. Substances leaching into water were separated by extraction

with methylene chloride, or by evaporation of water, and identification of

species in the leachate was attempted by a number of analytical techniques, with extensive use of combined gas chromatography/mass spectrometry

(GC/MS). Low molecular weight fragments of dimethyl si 1 oxane were identified from mammary prosthesis gel. Spectra are also included for leachate

from the other three polymers; identification of these compounds is still

in process.

Prolyl hydroxylase and tritium-labeled protocol! agen substrate were

prepared from chick embryos, and an enzyme activity assay system was developed with use of Bio-Rad AG 50W-X8 cation exchange resin. No effect

on enzyme activity was seen when silicone gel leachate was added to the

assay mixture.

Key words: enzymatic assay, gas chromatography/mass spectrometry,

leachables, mammary prosthesis, polymeric implants, prolyl hydroxylase.

1. Introduction

Because of their low cost and versatility, plastics of various kinds have found

increasing application for medical purposes; e.g., polyethylene blood bags, polycarbonate centrifuge bowls, extracorporeal shunt tubing and ul traf il tration membranes

In addition, implantation of devices for cosmetic purposes or to sustain life is done

2-3 million times annually with a variety of substances being employed as implant

materials. Implanted materials include stainless steel and high density polyethylene

for femur prostheses, siloxane for heart valve poppets, silicone polymers for breast

prostheses, and a host of other materials for a wide variety of purposes [l] 1

.

1.1 Silicone

Of the many plastic materials which have been employed for implant purposes,

sil icone polymers have proven to be among the most biologically inert [2]. However,

problems are still being encountered with mammary prostheses which consist of

sil icone gel enclosed in a silicone film. For these, capsular contraction, caused by

collagen buildup around the implant, has sometimes been a cause for complaint. Work

figures in brackets indicate the literature references at the end of this paper.

1
has been done to elucidate the causes of encapsulation but without shedding much

light on the nature of the problem. For example, recent work at Battelle, Columbus

C3], investigated low molecular weight leachables from silicone gel mammary augmentation devices. Using ethanol and tetrahydrofuran as solvents, the Battelle researchers

identified methyl and phenyl -si 1 icones but no non-silicone materials except for

phthalate esters, which may have been an artifact. Their work included examination

of the rheological properties of the gels, with complex modulus and viscosity

measurements.

Medical grade silicone gels originating from linear or branched polysi 1 oxanes with methyl groups alone or with phenyl and/or hydroxyl groups on the silicon atom can be obtained in quite pure form. Since condensation polymerization accelerators

are usually simple acids or bases, and cross-linking agents are peroxides such as benzoyl peroxide, dichlorobenzoyl peroxide, dicumyl peroxide or tert-butyl cumyl

peroxide [4], complex organic leachables are not expected to be present. Likewise,

fill ers such as titanium dioxide are not present.

1.2 Polyethylene

Polyethylene may be prepared by a variety of methods depending on the properties

desired for specific applications. Medical grade polyethylene, such as that used in

blood bags, is expected to have low amounts of organic extractables. However,

production of "clean" polyethylene is somewhat more difficult than for silicone

polymers. A variety of additives may be used to retard degradation. Peroxides,

carbonyl compounds, and stabilizers in a variety of forms can be present in polyethylene. Recent work by Schabron and Fenska [5] demonstrated the capability of

extracting the three most commonly used hindered-phenyl -type antioxidants added in

the manufacturing of polyethylene: 2 ,6-di -tert-butyl -4-methyl phenol (BHT), octadecyl

3, 5-d i- tert- butyl -4-hydroxy-hydrocinnamate (irganox 1 076

)

2 and tetrakis

[methyl ene( 3 ,5-ditert- butyl -4-hydroxyhydroci nnamate) ] methane (Irganox 1010) 2. In this analytical procedure, a 2 g sample of polyethylene was dissolved in decal in

(decahydronaphthalene) at 110 °C and then stirred as the solution cooled and the

polyethylene precipitated. The filtrate was analyzed by normal -phase high perfor- mance liquid chromatography (HPLC) on a p-Porasil column with the mobile phase

consisting of a heptane to methylene chloride gradient.

Other methods of chemical analyses recently have been reported for additives in

polyethylene. Lehotay, et al. [6] used thin layer chromatography (TLC), HPLC, gas

chromatography (GC) and mass spectrometry (MS) to separate and detect polyethylene

additives. Their extraction scheme used Soxhlet extraction of 100 g of powdered

polyethylene with 500 mL of n-hexane (or chloroform). The fractions were filtered

and evaporated and soluble fractions of low molecular weight polymers were removed by

methanol extraction. Unfortunately, detection limits for each method of analysis were not provided.

Qualitative studies of water leaching of polyethylene were reported as early as 1976 by Kandpal, et al. [7]. They reported that water contact with polyethylene

containers for even one hour at room temperature was sufficient to leach watersoluble low molecular weight compounds having hydroxyl, carboxyl or carbonyl groups

which caused measurable decreases in surface tension of the leachate. Further

storage of the water (6 hours or longer) in the containers resulted in strong absorbance in the ultraviolet region (200-300 nm). However, further identifications of the

leachates were not reported in this publication.

2 In this report, in order to describe procedures adequately, it has occasionally

been necessary to identify commercial products and equipment. In no case does such

identification imply MBS recommendation or endorsement, nor does it imply that the

item identified is necessarily the best available for the purpose.

2
1.3 Polyurethane

A wide range of medical grade polymers may be obtained by reacting polyesters,

polyethers or polyols with di i socyanates . Resulting polyurethanes may have properties ranging from rubber-like resins to foams, to clear, flexible sheeting [4]. As described in a recent publication, medical -grade polyurethane blood bags were

fabricated from polyurethane prepared from a polyether diol and 4

,4 ' -di phenylmethane

diisocyanate (MDI). When the blood bags were autoclaved (121 °C) in an aqueous

solution for 2 or 5 hr, an aromatic amine analog of MDI was obtained [8]. This

analog was chemically identified by GC, MS, and nuclear magnetic resonance spectroscopy (NMR) as 4,4'-diaminodiphenylmethane, also known as 4 ,4 1 -methylenediani 1 i ne (MDA)

.

In another study [9], extraction of an unspecified grade of polyurethane with

toluene has been employed for characterization of low molecular weight oligomers.

The extraction solvent was evaporated under nitrogen. The residue, dissolved in tetrahydrofuran , was subjected to field desorption mass spectrometry (FD-MS). FD-MS

revealed cyclic esters (unit mw=200) derived from adipic acid and 1 ,4-butanedi ol , and

related cyclic esters. The polyurethane was shown to be derived from the incorporation of one and two 4 ,4

1 -di phenylmethane diisocyanate units into a basic cyclic

polyurethane with a molecular weight of 200. Solvent extraction allowed isolation of

residual monomers and solvents, chemical additives, and low molecular weight

oligomeric material.

Based on the above reports, it is expected that polyurethane may be the richest source of extractable materials.

1.4 Polycarbonates

Polycarbonates are essentially linear thermoplastic polyesters of carbonic acid

with aliphatic or aromatic dihydroxy compounds and have numerous applications in the

medical and dental fields. They may be synthesized by a variety of procedures, but

most synthetic methods employ aromatic dihydroxy compounds such as 2,2' -bis(hydroxyphenyl-)propane, commonly known as bisphenol A. Condensation may be achieved by a variety of procedures, involving organic solvents such as pyridine or methylene

chloride. The mechanical properties of medical grade polycarbonate make it suitable

for durable appliances such as centrifuge bowls, blood separators, syringes, and

various receptacles. Such articles may be sterilized by autoclaving at 121 °C.

Polycarbonate materials have been more widely used in dental prostheses than in

implantable prostheses.

A search of the recent literature failed to reveal any studies that sought to identify leachable products from polycarbonate materials.

1.5 Prolyl Hydroxylase

The enzyme, prolyl hydroxylase (EC 1.14.11.2; proline 2-oxoglutarate dioxygenase)

,

which hydroxylates prolyl residues in newly synthesized collagen, is essential in the

biosynthesis of collagen. In fact, hydroxyprol ine is found in vertebrate tissues

almost exclusively in collagen. Changes in prolyl hydroxylase activity have been

studied in a number of experimental and pathological states. Of particular interest

is the observation that enhanced collagen synthesis has been detected in human tissue

in contact with a variety of polymeric breast implants. In some cases, hardening of

the collagenous scar tissue was sufficient to require surgical removal of the

prosthesis.

3
It is postulated that prolyl hydroxylase is stimulated by chemical agents

released from polymeric implants, thus promoting excess collagen formation. Such

Stimulating agents may be from unreacted monomers, from various additives, or from

substances introduced into the device after it is prepared for implantation.

1.5.1 Objective

Work at NBS was focused on providing the Center for Medical Device Analysis,

Bureau of Medical Devices with data on the identification of low molecular weight

leachables from polymeric prostheses and medical devices. Because physiol ogical

systems are mostly aqueous in composition, we chose to employ water leaching to study

the four polymeric materials described above. Extension to saline leaching, followed

by more complex leaching solutions, (e.g., complex salts, pure proteins or serum) is

anticipated for future studies.

In a parallel investigation we focused our efforts on establishing a reliable

assay for prolyl hydroxylase as well as investigating methods for purification of

this enzyme. Preliminary work has been performed to test the influence of low

molecular weight leachables on the enzymic activity.

2.

Experimental Section

2.1 Materials and Reagents 2

The four types of polymeric materials obtained for study were polyurethane,

polyethylene, polycarbonate, and silicone elastomer and gel. A list of the

materials, descriptions, and the suppliers follows:

1. Polyethylene - sterile blood freezing bags. Style 8000-2, Lot 4016, Delmed Corp.,

Holbrook, MA 02343

SRM 1482, MW 13,600, National Bureau of Standards

SRM 1484, MW 119,600, National Bureau of Standards

2. Polyurethane - 0.5 cm pellets, 2363-80A, Lot 3091 R , Upjohn Co., North Haven,

Conn. 06473

3.

Polycarbonate- 0.5 cm pellets, Hemonetics, Braintree, MA 02184

4

.

Silicone - Q7-21 59A-cured gel, lot No. H020059 #22, prosthesis envelopes

Elastomer & (Prod. #MDF0077) Lot #H11-8313; silastic R gel filled mammary

Gel prosthesis (200 cc size) Lot No. H099686; silastic R block,

7 x 25 cm, Lot No. HI 0901 3 , #802 PN96800-02; all from Dow-Corning

Corp, Midland, Mich. 48640

Chick embryos were the source of prolyl hydroxylase and substrate used to perform enzymatic assays. The embryo-containing eggs were obt ained from T ruslow

Farms, Chestertown, MD. Tritium labeled proline, L-[3,4- 3 H]-HN-CH 2 CH 2 CH 2 CHC00H , was obtained from New England Nuclear Co., Boston, MA 02118, as was carbon-14 labeled

a-ketogl utaric acid. All other reagents were of the highest quality commercially

obtainable.

4
2.2 Leaching Experiments

2.2.1 Polyethylene

One hundred mL of water processed for use in high performance liquid chromatography (HPLC) was added to each of five polyethylene blood bags and these were

placed horizontally on a slowly rotating mixing table. Before filling, tubes

connected to the bags were snipped off and bag openings were closed with clamps.

Nevertheless, water leaked from some of the bags and left a white powder residue

where drops evaporated.

In a follow-up experiment, ten polyethylene blood bags were leached with 20 mL portions of HPLC-grade water for one week. The 20 mL portions were pooled and water

was evaporated at 30° on a rotary evaporator under vacuum. A few milligrams of a light tan powder remained. The manufacturer was consulted about the possible nature

of this material. It could not be an agent to prevent adherence of plastic layers or

a mold release agent since such substances were not used in the manufacturing process.

Suggestions were made by the manufacturer to check for polymers of ethylene oxide,

ethylene glycol or ethylene chlorohydrin. Ten to 25 ppm of these three substances is

a permissible load, according to the manufacturer.

Analysis by mass spectrometry of a methanol soluble portion of the tan powder

did not yield any good matches to compounds present in the NBS/NIH/EPA mass spectrometry data base. The spectra are consistent with the presence of an octyl

phthalate, a compound with a benzoyl group and other unknown constituents. Figures

1-6 present the mass spectral data.

In considering that the contaminating powder was talcum powder (because of its use in dusting polyethylene surgical gloves) samples of the powder were subjected to atomic absorption spectrometry (AAS) and infrared analysis (IR). Specifically, AAS was used to analyze for the presence of zinc (from zinc stearate in talcum powder).

The results indicated the presence of ^0.03 ng Zn/20 pL sample, or 1.6 ug in a single

blood bag, implying that zinc stearate was not present to any great extent. Likewise, IR spectra showed no resemblence to a stearate-containing molecule.

2.2.2 Polyurethane and Polycarbonate

For the first series of studies, polyurethane and polycarbonate were leached at room temperature by weighing 20 g quantities into separate Erlenmeyer flasks, adding

20 mL of HPLC-grade water to each flask, covering with aluminum foil to prevent loss

of water, and shaking at room temperature for one week. At the end of the week,

water from each beaker was filtered through #1 Whatman filter paper and 0.3 pm

Millipore filters and then extracted with 20 mL of methylene chloride which was evaporated to ^1 mL under a nitrogen stream. Samples thus concentrated were examined

by gas chromatography. In addition, mass spectrometry was used for identification of

constituents of the polyurethane extracts.

Gas chromatography employed a 3 percent Dexsil 300 packed column (1.8 m x 2 mm).

The injector and detector temperatures were 250 °C with an initial column temperature

of 70 °C for 4 min, followed by an increase of 8 °C/min until the final temperature

was achieved, then held for 16 min. Several small peaks were observed in the polycarbonate leachate (Fig. 7).

An additional leaching technique was employed for polyurethane. Ten gram

samples (to which 10 mL water were added) were leached at 56° for 75 hours. Twentyfive mL of methylene chloride were used to extract each 10 mL of water.

5
Polyethylene

Bag

Extract 6

leachate

polyethylene

blood

bag.
Polyethylene

Bog

Extract

DIP

50,1.-30,350 7

Figure

2.

Total

ion

current

and

m/z

149,

167,

and

279

(indicative

of

phthalate

ester)

vs.

probe

temperature

for

the

methanol

soluble

portion

of

the

aqueous

leachate

of

polyethylene

blood

bag.
Polyethylene

Bag

Extract

DIP

50,

1..

30,

350 8

the

aqueous

leachate

of

polyethylene

blood

bag.
m/z

51, 7

7

ore

indicative

ofa

phenyl

group

m/z

105

is

indicative

ofa

benzoyl

group

A1ISN31NI 3 AllV13iJ

9

Figure

4.

Mass

spectrum

at

probe

temperature

of

40

°C

corrected

for

background

(polyethylene)

leachate.
10

Figure

5.

Mass

spectrum

at

probe

temperature

of

26

°C

(polyethylene

leachate).

Unidentified

materials.
11

Figure

6.

Mass

spectrum

at

probe

temperature

of

50

°C

(polyethylene

leachate).

Unidentified

materials.
12

Figure

7.

Gas

chromatogram

of

polycarbonate

leachate,

methylene

chloride

extract.
Methylene chloride extracts were evaporated to ^50 uL under a stream of dry

nitrogen and one to two uL aliquots were analyzed by GC. Chromatographic separations were carried out on a 30m SE-30 fused silica capillary column. The GC column was temperature programmed from 80 °C to 250 °C at a rate of 8°/min. In this procedure

(and in comparison with the previous leaching technique) four major peaks were observed (Fig. 8).

13
Based on this separation, a similar SE-30 column was used in the GC inlet to the mass spectrometer. Mass spectra were scanned repetitively every 2.5 s over the course of the GC run. Peak identifications were made based on a comparison of the mass spectra with those in the NBS/NIH/EPA Mass Spectral Data Base and confirmed by

manual interpolation. Peaks where no identification was made are listed as UNK.

These data appear in Fig. 9 and Table 1.

Table 1. Peak Identifications from GC/MS Analysis of Polyurethane Extract U-l (FRN

6232)

Peak RT Five Most Intense Ions Identification

3.3 min. 36, 43, 56, 41, 85 ethylene urea

7.5 88, 73, 87, 57, 101 2-ethyl hexanoic acid

14.4 71, 73, 100, 85, 55 unk

15.0 105, 135, 75, 45, 107 unk

18.3 149, 223, 150 phthalate ester (also seen in blank)

21.2 71, 73, 55, 85, 100 unk

21.6 71, 73, 55, 85, 100 unk

27.5 71, 73, 55, 85, 100 unk

Unknowns at 14.4, 21.2, 21.6, and 27.5 min are clearly related.

2.2.3 Silicone Gel

For silicone samples, 10 g of the silicone gel were extracted with 10 mL of HPLC

grade water at 56 °C for 48 hours and room temperature for an additional 96 hours.

The aqueous leachate was decanted into a 250 mL separatory funnel and 20 mL of

CH2 C1 2

. The methylene chloride extract was reduced in volume under a stream of dry

nitrogen to ^1.5 mL. Both silicone gel (S-l) extract and blanks were treated

similarly.

For gas chromatographic analysis, run conditions were similar to those used for

polycarbonate and polyurethane, except that a SE-52 capillary column was used.

Results are shown in Figures 10 and 11. The chromatographic traces for the silicone

gel are considerably more complex than those for the polycarbonate and polyurethane.

14
Time (minj

Figure 9. GC/MS total ion current profile, polyurethane leachate, methylene chloride

extract.

15
16

Figure

10.

Gas

chromatogram

of

silicone

leachate

blank,

methylene

chloride

extract.
4-> U

(V

s4-> X

<uCD “O

•r~s-oo

CDc

<D

CDE

CD

4-> ro SZu

rOCD

CDCoo

</) 4-OE

fOCDo

4-> roEO

i- JC

CJ

l/) fO o

<D

s~13CD l

17
For mass spectrometric analysis, 10 uL of the methylene chloride solution was placed in a clean capillary tube, and the solvent removed under house vacuum. The

capillary was placed in the direct insertion probe of a mass spectrometer and heated

from 50 ° to 300 °C at 30°/min. Mass spectra were scanned repetitively at 200 mass units per second over the course of the run. Figure 12 shows the total ion current

profile and m/z 73, 147, 207, 221, 281 and 295 mass chromatograms versus the probe

temperature. The 147-295 mass chromatograms were fragments from a dimethyl si 1 icone.

,The presence of m/z 73 suggests that the ends of the dimethyl si loxane chains are capped with trimethyl si lyl groups.

Figure 13 is a representative spectrum of the polymer. The same major ions are observed at all temperatures examined.

As with many polymeric materials, the presence of phthalate esters was observed.

Figure 14 (upper) shows the total ion current profile and m/z 149 mass chromatogram

versus probe temperature. At 45 °C, the mass spectrum (Fig. 14, lower) shows the

presence of ions characteristic of phthalate esters.

For a second leaching study, 10 g of the gel and 10 mL of water were stirred

slowly at room temperature for four hours. The leachate was decanted into a flask

and the water was evaporated under vacuum. The residue was subjected to gel permeation chromatography (GPC), spectroscopic, GC/MS, and NMR analysis.

A tetrahydrofuran solution of this residue was used for GPC analysis (refractive

index detection) which showed two very low, broad peaks, indicating substances with

molecular weights of approximately 200 or less. Chemical spectroscopic analysis of

the remaining portion of this sample showed the presence of sodium and silicon.

GC/MS analysis confirmed our previous results with no new information being obtained

from this leaching experiment.

The leachate studies on silicone gel prompted a series of experiments to examine

the silicone gel itself, without leaching. For this purpose, a small amount of the

silicone gum was placed on the tip of a clean capillary tube. The capillary tube was heated from 50 to 300 °C and mass spectra were scanned repetitively every 2 s during

the elution- of material. The mass spectra showed the material to be a dimethyl

silicone polymer of the type:

ch

3 | ch3

| ch

3

I CH, i ch

3

CH

3

CH

3

CH

3 CHj—Si-j-0—Si—fO —Si—PO—Si-40—Si

CH

3—Si—0--Si—0--Si—CH

3

ch

3 | ch3 , ch

3

| ch

3

| ch

3

CH

3

CH

3

CH

3

147 —

n

18
19
20
I N

T

E

N

S

T

Y

MiukfctAltlii rtfLkUfcMM

40 60 80 100 * 120 MO 160

Figure 14. (upper) Total ion current and m/z 149 vs. probe temperature for silicone

gel leachate, (lower) Mass spectrum at probe temperature of 45 °C showing the presence of ions characteristic of phthalate esters.

21
Mass chromatograms for characteristic ions for the methyl silicone polymer (e.g., m/z

73, 147, 221 and 295) show the same elution profile as the total ion current trace.

Spectra obtained near the beginning and end of the run are similar, although there

are some differences in relative abundances of some of the ions. Results are shown

in figures 15-17.

2.3 Prolyl Hydroxylase Studies

2.3.1 Enzyme Preparation

Prolyl hydroxylase was prepared by the method of Bentley and Weiser [11] from

10.000 x g supernatant fractions of the homogenates of 10 day old eviscerated chick

embryos. After evisceration, chicks were weighed, cut up with scissors and homogenized with extraction buffer in a homogenizer obtained from Bellco Glass, Inc. (Vineland, N.J. 08360). Half a gram of chick was extracted with 2 mL of extraction

buffer which contained 0.23 mol/L sucrose, 9 pmol/L dithiothreitol , 23 pmol/L EDTA,

0.28 mmol/L a-toluene sulfonyl fluoride in tris(hydroxymethyl )aminomethane hydrochloride (TRIS-HC1) (pH 7.5, 0.05 mol/L). The homogenate was centrifuged at 10,000 x g for 25 min at 4 °C. The supernatant liquid was used as the enzyme solution and

stored in a deep freeze at 20 °C. Five hundred mL of this crude enzyme preparation

were prepared and some of it has been used for tests and for purification attempts.

2.3.2 Protocollagen Substrate Preparation

a. Initial enzyme substrate was prepared with minor modifications by the method

of Hutton, Tappel , and Udenfriend [12]. To 5-6 g of 10-day-old decapitated chick

embryos was added 8.5 mL of buffer which contained 0.022 mol/L NaCl , 0.003 mol/L KC1 , 0.0012 mol/L MgS04 , 0.0013 mol/L CaCl 2 , 0.0004 mol/L KH 2 P0 4 , 0.025 mol/L NaHC0 3 , 0.01 mol/L D-glucose, 1 mmol/L a ,a ' d i pyridyl and 500 pCi of L-[3 ,4- 3 H]prol i ne . Ten

bottles each containing 8.5 mL of chick homogenate were incubated aerobically in

separate beakers for 90 min at 37 °C. The contents of the 10 beakers were pooled and

centrifuged, and the supernatant liquid was discarded. The pellets were extracted

with 0.5 mol/L acetic acid (50 g tissue per 100 mL acid) for 90 min at 0 °C. The

supernatant liquid from the extraction was dialyzed for 18 hours against three changes

of water, and the pellet reextracted with acetic acid and dialyzed also. Typically

we obtained 146,000 dpm/mg of protein for the first extraction and 265 dpm/mg of

protein for the second extraction, with a counting efficiency of 46.3 percent.

b. The substrate preparation procedure of Peterkovsky and DiBlasio was also

employed [13]. For this 2.13 g of frontal bones (calvaria) were obtained from 80 15-

day-chick embryos. These were incubated with shaking at 37 °C in 25 mL of Eagle's

minimal essential medium containing 0.5 mmol/L a ,a

1 -di pyridyl and 1 mCi of

L-[3,4- 3 H]prol ine. The solution was chilled to 0 °C, the bones were separated and

rinsed with cold 0.11 mol/L NaCl-0.05 mol/L TRIS-HC1 , pH 7.4 containing 1 mmol/L

proline. The bones were homogenized in 24 mL of 0.5 mol/L acetic acid in a Polytron

tissue homogenizer for two 15s intervals at 0 °C, and the suspension was stored at

4 °C overnight. It was centrifuged at 20,000 x g for 30 min, and the supernatant

solution was dialyzed against two 3.5-L changes of 0.4 mol/L NaCl-0.1 mol/L TRIS-HC1

buffer (pH 7.6). The dialyzate was precipitated with ammonium sulfate, the precipitate was dissolved in 11.25 mL of 0.2 mol/L NaCl-0.05 mol/L TRIS-HC1 (pH 7.6) and

dialyzed against this buffer. The dialyzed solution was heated at 37 °C for 1.5 hours

to induce precipitation of fibrils. These were removed by centrifugation at 20.000 x g for 10 min. The radioactive (16,300 dpm/mg), unhydroxylated , denatured

collagen remained soluble and the resultant 10 mL of solution was stored at -20 °C

for future use. Use, however, was limited due to the lower specific activity of this

preparation.

22
*— 21 h** lu ~ZL to ~

23

Figure

15.

Total

ion

current

vs.

probe

temperature

for

silicone

gel.
ooZ

m

-h Z

—

-<— i

—

coZ^-hZ

Figure 16. Total ion current and selected mass chromatograms for silicone gel.

24
Figure 17. Mass spectrum at probe temperature of 50 °C (silicone gel).

25
2.3.3 Enzyme Assay

Enzyme activities were initially measured using the following assay mixture

[ 11 ]: 0.05 mL of tritiated proto'collagen substrate, 0.1 mL of enzyme preparation,

and 0.05 mL of cofactor solution. The cofactor solution contained: 2 mmol/L sodium

a-ketogl utarate, 4 mmol/L Fe(NH4 ) 2 (S04 ) 2 , 20 mmol/L ascorbic acid, 15 mg bovine serum albumin/5 mL, 4 mg/5 mL of catalase, and 0.4 percent v/v Triton X-100. The components were dissolved in 5 mL of 0.4 mol/L TRIS-HC1, pH 7.5 immediately before the

assay was to be performed. Typically the reactants were added to ten plastic test

tubes (10 x 75 mm) and these were capped and shaken in a 37 °C bath for one hour.

The reaction was stopped by addition of 0.02 mL of 50 percent trichloroacetic acid.

Tritiated water from each sample was separated from the other reactants by one of

several methods detailed in this section, and placed in a scintillation vial, to which 15 mL of Aquasol II (New England Nuclear, Boston, MA 02118) were added.

Samples were counted in a scintillation counter.

2.3. 3.1 Conway Cells

Initially we attempted to use the assay method of Bentley and Weiser [11]. For

this the tritiated water was separated from the other reactants by placing the

reactants into the outer well of a Conway cell. Concentrated sulfuric acid (18.4

mol/L) was placed in the center well to trap the tritiated water. The cells were covered with a lightly greased, ground glass cover and placed in a 78-85 °C oven for one hour. The cells were allowed to cool and the center well contents placed in a scintillation vial. The vial contents were frozen by placing the vial in a dry ice

acetone bath, 0.70 mL of 13.4 mol/L potassium hydroxide was added, the vial cap

quickly replaced, and the neutral ization allowed to occur at room temperature. The

vials were placed in a refrigerator for 5-10 min; then 15 mL of scintillation cocktail were added. After the addition of K0H, we tested the pH of samples processed

according to this procedure and found that it varied from 2 to 12. Since acidic

scintillation samples produce spurious counts, we attempted to refine the technique

by making minor procedural modifications such as doing test titrations, changing the

reactant volume, etc. However, we were never able to obtain good precision with this

technique; at best the relative standard deviation was 12 percent (700 cpm) , but

-there were often samples with count rates three times the value of the mean when the

high sample was excluded from calculation of the mean.

2. 3. 3. 2 Microstills ,

In an attempt to achieve better separation; we built a manifold vacuum still to separate tritiated water from the other reactants. The plastic tubes containing

incubated assay samples were connected to 5 mL capacity traps. The system was designed to allow heating of 14 sample tubes from which distilled water was trapped

by dry ice-acetone cooling. Trapped tritiated water was transferred to scintillation

vials and counted as before. With this experimental system, blank samples (those

containing only cofactor and substrate) typically gave 50 cpm, those with enzyme gave

200 cpm (relative standard deviation of 10 percent). Abnormally high sample

activities were infrequently seen and were traceable to instances of faulty sample

handling or inadequate cleansing of reused glassware. The principal drawback was that performing the assay was laborious; the distillation step required approximately

two hours, and it was found necessary to subject the reused glass parts of the

apparatus to acid washing. The low level of tritium recovered was also a disadvantage because long counting periods were required for good statistics. In total,

however, the method was reliable and was the method we used for monitoring enzyme

activity resulting from various purification procedures.

26
2. 3. 3. 3 C02 Assay

Because the distillation assay was time consuming, we attempted to assay enzyme

activity by trapping C0 2 [14] in a coupled enzyme assay. For this assay we used

0.25 mL of crude enzyme preparation, 0.25 mL of solution containing 0.1 mg of

poly(L-prolyl -glycyl -L-prol ine) , (Miles-Yeda Ltd., Elkart, Ind. 46514) and 0.5 mL of

cofactor solution such that the final concentrations of components were: 0.05 mmol/L

FeS04 , 0.1 mmol/L 2-oxo[l

14 C]-glutaric acid (adjusted to a specific activity of

60,000 dpm/0.1 pmol), 2 mmol/L ascorbic acid, 0.1 mg/mL of catalase, 0.1 mmol/L

di thiothrei to! , and 2 mg/mL bovine serum albumin in 0.05 mol/L TRIS-HC1 (pH 7.8 at

5 °C). The peptide substrate was thermally denatured by heating at 100 °C for

5 min and quenching to 0 °C immediately prior to addition to the reaction system.

Mixed reactants were heated at 37 °C for 30 min in cork-capped test tubes. To detect

evolved C02 , fluted filter paper (2.5 x 10 cm) was impregnated with 0.2 mL of

1 mol/L hyamine hydroxide® (Calbiochem, La Jolla, CA). These were inserted into the

test tube, 0.2 mL of 2.5 mol/L H 2 S04 was added from a syringe equipped with a long

needle and the cork was quickly replaced. The tubes were shaken several times during

the half hour C02 evolution period. Filter papers from the test tubes were added to separate scintillation vials, to which 15 mL of scintillation fluid was also added.

These were counted in the scintillation counter. The entire experiment was repeated

a number of times. However, no enzyme activity could be measured reliably by this

method because the enzyme preparation was too low in activity.

2. 3. 3. 4 Column Separation

Realizing that the distillation assay was time-consuming and gave a low tritium

recovery, we attempted to develop a faster procedure in which the tritiated water

would be separated by means of a short chromatography column.

(1 ) Sephadex G-25

We made a number of trials with prepacked columns containing G-25 Sephadex. For

these the assay mixture described in part (3) (below) was used. For experimental

mixtures of substrate and tritiated water, tritium counts in the third mL eluted

analyzed 1711 ± 77 cpm or a relative standard deviation of 4.5 percent. In different

assays, however, the tritium activity of the third mL of eluent varied from 1571 to 5284 cpm. If total activity of all three mL of eluent was considered, the mean value was 17,050 ± 1721 with a blank value (no enzyme) of 8098 cpm. This was not considered

adequate as a separation procedure.

(2) Sephadex C-25

We attempted to make a separation on C-25 Sephadex, a cation exchange gel, after

treating the gel with acid to replace bound sodium. The tritiated water was eluted

after the tritium-labeled substrate, as it had been from G-25 Sephadex, making

consistent collection of tritiated water in a specified fraction impossible.

(3) Bio-Rad AG 50W-X8

i Due to our unsuccessful attempts to separate tritiated water satisfactorily with

Sephadex, we decided to try the assay method of Peterkovsky [13] which makes use of

Bio-Rad AG 50W-X8 as column packing material. For this assay the final volume of the

reacting mixture is 100 pL of which 15 pL is L-[3,4- 3 H] proline-labeled substrate

(denatured for 15 min at 37 °C prior to addition), 60 pL of enzyme and 25 pL of

cofactor solution containing: 1 mmol/L sodium ascorbate, 1 mmol/L sodium a-ketoglutarate, 0.2 mmol/L ferrous ammonium sulfate, 0.4 mg/mL catalase, 2 mg/mL bovine serum

albumin, and 0.5 mmol/L di thiothrei tol dissolved in TRIS-HC1 (0.04 mmol/L, pH 7.6).

27
The reactants were incubated for 15 min at 37 °C, then the reaction was stopped by

addition of 0.4 mL of 6.25 percent trichloroacetic acid. Fifteen uL of 25 mg/mL

bovine serum albumin was added to. each tube, reactants were mixed, allowed to stand

for 5 min' at 0 °C and centrifuged at 1.5 g for 5 min. The supernatant liquid was removed, an additional 0.5 mL of 5 percent trichloroacetic acid was added to the

resuspended pellets, and the mixture centrifuged as before. The supernatant portions were combined and added to small chromatography columns (0.5 x 2 cm). Scintillation

.vials were used to collect the effluent and 0.5 mL of water wash. Scintillation

fl uid (15 mL) was added and the solutions counted. Blank solution (containing no enzyme) gave a count rate of 28.5 ± 2.5 cpm, whereas assay mixtures gave a rate of

'85.3 ± 9.6. Longer counting times than 10 min for these samples would have yielded

smaller standard deviations. Since we desired larger count rates, we tested the

separation with Bio-Rad AG 50W by adding mixtures containing 50 yL of substrate and

50 uL of tritiated water. These were placed on the column, rinsed through with

1 mL of water and collected. An additional 1.5 mL of water was added, collected and

counted as a second fraction. The second fraction contained about 95 percent of the

activity; however, the count rate for the second sample varied from 1367 to 1648 cpm.

When the percent of the activity of both eluted fractions was calculated it was found

that 94.5 percent ± 0.9 percent was in the second sample. We believe this separation

method provides a basis for a suitable assay technique.

2.3.4 Enzyme Purification

2.3.4. 1 Sepharose 4-B

Chromatography of a crude prolyl hydroxylase ammonium sulfate fraction

(30-65 percent) on a Sepharose 4B column with buffer consisting of 0.1 mol/L NaCl,

0.1 mol/L glycine, 10 umol/L di thiothrei tol , 0.01 mol/L TRIS-HC1 (pH 7.8, 4 °C)

yielded enzyme with specific activity increased 13,000 times. However, all activity

was lost after storage at -12 °C for one week.

2. 3. 4. 2 Dye Ligand Chromatography

Promising results were obtained from an initial experiment in which we attempted

'to enhance -enzyme specific activity by use of dye-ligand chromatography (Amicon

Corp., Lexington, MA). Enzyme activity was doubled when it was eluted from a gel

matrix to which Blue A was bound. However, we have not been able to duplicate these

results in a larger scale system.

2. 3. 4. 3 Affinity Chromatography

Affinity chromatography was also employed for enzyme purification [14]. To assess the efficiency of the coupling method used in preparing the affinity columns,

we performed amino acid analysis on a sample of the proline-labeled gel. Analysis

indicated 13 percent w/w proline, suggesting that attachment of the poly-L-prol ine

(Type IV, MW >30,000) to the gel was successful.

Prior to addition to the affinity column, crude enzyme solutions were purified

by ammonium sulfate fractionation of 100 mL of the crude preparation. The precipitate from 30-85 percent saturation was redissolved in 25 mL of buffer, and then

dialyzed against 0.1 mol/L glycine, 0.1 mol/L NaCl in buffer (0.05 mol/L TRIS-HC1,

pH 7.8, 4 °C). The 35 mL of dialyzate, which was found to have a 230 nm absorbance

of 12.8, indicating a protein concentration of ^10 mg/mL, was added to the affinity

column. For the first trial with the affinity column, 5 mL of dialyzate was added to the column. In later trials larger amounts were added, e.g., 35 mL. The enzyme was extracted from the column with poly-L-prol ine, type II. Poly-L-prol ine was separated

from the enzyme by rinsing the enzyme containing portions of the eluent through a

28
Bio-gel column (A 1.5, 200-400 mesh). Enzyme containing eluent from the Bio-gel

column was concentrated by use of an Amicon filter. Initially the enzyme had a net

activity of 155 cpm (assay by distillation); after the affinity procedure and

concentration, the most active fraction produced an activity of 70 cpm, and a protein

concentration of 0.02 ng/mL. Three repetitions of this experiment gave the same

result; most of the enzyme activity was lost when the extraneous protein was removed.

Other investigators have also found prolyl hydroxylase to be unstable when purified.

Therefore added protein may be necessary for stabilization.

3. Discussion and Conclusions

Identification of the leachate content from the four polymeric substances has

not been completed, although a number of substances have been identified. However,

it appears certain that the silicone gel leachate contains a low molecular weight

dimethylsiloxane polymer and a phthalate ester. More definitive measurements need to be made to determine the influence of leachate on the enzymatic activity.

Vie have identified some minor components which leach from polyurethane into

water. These are ethyleneurea , 2-ethylhexanoic acid, and a phthalate ester. No methylenedianal i ne (MDA) was detected, although this doesn't preclude that MDA was present in the polymers. The long-term extraction procedures that we used were not

suitable for preservation of MDA in the leachate if it were present. Experiments are

planned in which extraction is performed with ethyl ether and where the methylene

chloride extraction is eliminated.

We found that a tan-white substance leaches from a particular lot of polyethylene blood bags. We have not yet been able to identify this material. Collection of more material and further identification by infrared and other methods is planned for

the next series of experiments.

Identification of components leaching from polycarbonate was assigned the lowest

priority. Preliminary work with polycarbonate indicates low molecular weight

leachables are present.

Leaching experiments of the polymers with physiological saline and Eagles

minimal essential media are indicated as an extension of studies already performed.

Results of these experiments provide an additional basis for evaluating the

suitability of the materials tested for their intended medical applications, adding

to the body of information which can be used as a basis for making better decisions

regarding polymer use.

4 . References

[1] Hench, L. L. Biomaterials. Science 208: 826-831; 1980.

[2] Homsy, C. A.; Ansevin, K. D.; 0'Bannon, W.; Thompson, S. A.; Hodge, R.; Estrella, M. E. Rapid in vitro screening of polymers for biocompatibility.

Biomedical Polymers, A. Rembaron and M. Shen, eds. New York, NY: Marcel

Dekker; 1971. 121 ff

.

[3] Thompson, R. M.; Howard, C. C.; Brewer, E. J.; DeRoos, F. L.; Mattery, G. R.; Leininger, R. I. unpublished report, Battelle, Columbus Laboratories, Columbus,

Ohio; 1979.

29
[4] Lefaus, R. Practical Toxicology of Plastics. London: ILIFFE Books, Ltd.;

1968.

[5] Schabron, J. F.; Fenska, L. E. Determination of BHT, Irganox 1076, and Irganox

1010 antioxidant additives in polyethylene by high performance liquid

chromatography. Anal. Chem. 52: 1411-1415; 1980.

[6] Lehotay, J.; Danecek, J.; Liska, 0.; Lesko, J.; Brandsteterova , E. Analytical

study of the additives system in polyethylene. J. Appl . Polym. Sci . 25(9):

1943-1950; 1980.

[7] Kandpal, L. D.; Tewary, A. K.; Balakrishna, K. J. Leachility of moulded polyethylene. Indian J. Technol. 14(7): 359; 1976.

[8] Darby, T. D. ; Johnson, H. J.; Northup, S. J. J. Toxicol. & Appl. Pharmac.

46: 449-453; 1978.

[9] Lattimer, R. P.; Welch, K. R. Direct analysis of polymer chemical mixtures by

field desorption mass spectroscopy. Rubber Chemistry and Technology 53(1):

151-159; 1980.

[10] Autian, J. Toxicology of Degradation Products of Plastics, in Corrosion and

Degradation of Implant Materials. ASTM STP 684, B. C. Syrett and A. Acharyo,

eds., American Society for Testing and Materials; 1979. 5-19.

[11] Bentley, J. P.; Weiser, K. A. A simple microassay of Prolyl hydroxyl ase applicable to skin punch biopsy specimens. Conn. Tissue Res. 4: 255-259; 1976.

[12] Hutton, J. J.; Tappel, A. L., Jr.; Undenfriend, S. A rapid assay for collagen

proline hydroxylase. Anal. Biochem. 16: 384-394; 1966.

[13] Peterkovsky, B.; DiBlasio, R. Modification of the tritium-release assays for

prolyl and lysyl hydroxylases using Dowex-50 columns. Anal. Biochem. 66: 274

286; 1975.

[14] Tuderman, L.; Kuuti, E.-R.; Kivirikko, K. I. An affinity^column procedure using

poly(L-prol ine) for the purification of prolyl hydroxylase. Eur. J. Biochem.

52: 9-16; 1975.

30
NBS-114A (REV. 2-BC) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

1. PUBLICATION OR

REPORT NO.

2. Performing Organ. Report No. 3. Publication Date

SHEET (See instructions) NB5IR 81-2436 April 1982

4. TITLE AND SUBTITLE

Low Molecular Weight Leachables from Medical Grade Polymers

5. AUTHOR(S)

B. F. Howell, S. Chesler, L. Hilpert, and D. J. Reeder

6. PERFORMING ORGANIZATION (If joint or other than NBS, see instructions)

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

7. Contract/Grant No.

8. Type of Report & Period Covered

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City. State, ZIP)

10. SUPPLEMENTARY NOTES

_~| Document describes a computer program; SF-185, FIPS Software Summary, is attached.

11. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here)

Four medically important polymeric materials (silicone polymer from mammary

prostheses, polyurethane, polyethylene, and polycarbonate) were placed in contact

with water for time periods ranging from four hours to seven days. Substances

leaching into water were separated by extraction with methylene chloride, or by

evaporation of water, and identification of species in the leachate was attempted

by a number of analytical techniques, with extensive use of combined gas

chromatography/mass spectrometry (GC/MS). Low molecular weight fragments of

dimethyl si 1 oxane were identified from mammary prosthesis gel. Spectra are also

included for leachate from the other three polymers; identification of these

compounds is still in process.

Prolyl hydroxylase and tritium-labeled protocollagen substrate were prepared

from chick embryos, and an enzyme activity assay system was developed with use

of Bio-Rad AG 50W-X8 cation exchange resin. No effect on enzyme activity was

seen when silicone gel leachate was added to the assay mixture.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

enzymatic assay; gas chromatography/mass spectrometry ; leachables; mammary

prosthesis; polymeric implants; prolyl hydroxylase

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