Controlled Extraction on Rubber Elastomer (RE)
Diane Paskiet, Associate Director of Scientific Affairs; Kimberley Miller, Senior Thermoset Formulation Development Chemist; and Samuel Conway, Principal Chemist; all of West Pharmaceutical Services, Inc.
Introduction
RT,
min
ID
Formula
Structure/
Fragmentation
Unknown no ms signal
NA
NA
C6H12
Conc,
μg/g
4.59
0.99
5.68
1.19
Cyclopentane,
methyl-
6.75
0.48
Cyclohexane
Background
Pharmaceutical elastomers are compounded to impart certain physical properties such as
resilience, resistance to abrasion, compression set, and low permeability to vapor and gases.
This formulation was made on a pilot scale for PQRI with the following ingredients added to a
brominated isobutylene isoprene copolymer, cured with 4,4’-dithiodi-morpholine/polyisobutylene:
aluminum oxide, titanium dioxide zinc oxide, magnesium oxide paraffinic oil and carbon black.
Elastomer Material
Brominated isobutylene isoprene copolymer
19.95 0.14
Unknown
33.22 20.8
1,4-Dioxane
37.21 0.77
Unknown –
Butyl Oligomer
related
NA
Unknown –
Butyl Oligomer
related
NA
NA
Pharmaceutical Applications
40.82 0.46
Gaskets
41.67 0.16
m/z 39, 43, 45, 58
C4H8O2
Closures
Pistons
Experimental Conditions
Extraction
IPA
Hexane
1:1 IPA:H20
pH2.5
pH9.5
Soxhlet
Reflux
Sonication
5g/125mL
16 hrs
HPLC/UV/MS
GC/MS
3.5g/125mL
3 hrs
HPLC/UV/MS
GC/MS
N/A
5g/125mL
16 hrs
HPLC/UV/MS
GC/MS
3.5g/125mL
3 hrs
HPLC/UV/MS
GC/MS
N/A
N/A
5g/200mL
55C/3days
HPLC/UV/MS
GC/FID‡†
N/A
N/A
5g/200ml
2hrs
ICP/MS
N/A
N/A
N/A
Sealed Vessel
N/A
N/A
5g/200ml
2hrs
ICP/MS
5g/200ml
121C/1hr
HPLD/UV/MS
GC/FID‡†
5g/200ml
121C/1hr
HPLC/UV/MS
GC/FID‡†
Sample
3.5g = 15cm2/125mL; 5g = 20 cm2/125mL; and/200mL
Solvent Ratio
GC/FID/MS; LC/UV/MS; ICP/MS
System
Suitability
Internal Standards: Irganox 415 and Bisphenol M and 2-Fluorobiphenyl,
1,4 Dioxane
LCStandard: Caprolactam, Butylated Hydroxy Toluene (BHT), Diphenylamine,
Monoethylhexylphthalate (MEHP), Stearic Acid, Dethylhexylphthalate (DEHP), Bisphenol A, Irganox 1010, Irganox 168
Headspace Standard: Methanol, Acetic Acid, Cyclohexanone, Toluene,
Trimethylsilianol, 2-ethylhexanol
GC Standard: Grob Mixture
TMS Derivitized ‡
Underivitized†
1g/20mL @ 80-120C GC/FID/MS
Headspace Analysis
A System Suitability mixture was prepared in PEG 200. A standard mixture of methanol,
acetic acid, cyclohexanone, toluene, trimethylsilanol and 2-ethylhexanol was prepared at a
concentration 10-20µg/mL; the sensitivity concentration was 0.5-1µg/vial. 1,4 Dioxane was
used as an internal standard. Trace amounts of C6H12 (1µg/g) was the main constituent found
in the headspace; this would be typical of a solvent used in the polymerization process. Other
minor peaks <1µg/g were seen mostly related to butyl oligomers. The results are tabulated in
Table 1 Headspace First Pass Results; the chromatograms are shown in Figure 1 Early Eluting
Compounds and Figure 2 Late Eluting Compounds.
Figure 1 Headspace Early Eluting Compounds
Li
Mg
Ca
Sr
Ba
Metals
Al
Sn
Pb
Bi
Peak
#
Maximum Conc, mg/L2
Transition
Ti
Zr
V
Cr
Mo
pH2.5
pH9.5
Non metals Si
As
Cl
Br
Tentative Identification
CAS RN
1
6.06
Unknown
–
0.05
0.02
–
2
12.97
Unknown
–
–
–
1.03
3
13.37
Unknown
–
–
–
0.52
Unknown
4
13.59
Unknown
–
–
–
0.37
123-91-1 Internal
Standard
5
13.64
Unknown
–
–
–
0.32
6
13.67
Unknown
–
0.04
0.04
–
7
13.82
Unknown
–
–
–
0.55
8
–
2,6-di-tert-butyl-4-methyl phenol
128-37-0
–
–
0.22
9
–
Methyl dodecanoate
111-82-0
–
–
0.11
10
15.32
Dodecanoic acid
143-07-7
–
0.29
0.35
11
14.70
Diethyl phthalate
84-66-2
0.62
0.02
0.24
12
14.92
[TMS] Compound
–
–
–
0.05
13
15.27
1,2-Benzenecarboxylic acid,
monoethylester
2306-33-4
–
0.08
–
14
15.88
Unknown
–
–
–
2.06
96-37-7
Confident
110-82-7
Confident
NA
m/z 29, 41, 57, 68, 82,
97, 109, 124, 137, 165,
180
NA
m/z 29, 43, 57, 69, 81,
97, 109, 123, 137, 151,
165, 180
NA
m/z 29, 41, 57, 67, 79,
97, 109, 123, 133, 145,
160, 179, 191
NA
Unknown
Unknown
Unknown
15
–
Methyl Tetradecanoate
124-10-7
–
–
0.12
16
17.26
Tetradecanoic acid [TMS]
544-63-8
–
0.28
0.24
17
–
Tri-tert. Butyl-di-hydroxy benzene
24851-96-5
–
–
0.38
18
–
Unknown
–
0.10
–
0.11
19
16.64
Unknown†
–
0.03
–
0.04
Standard chromatograms of Derivatized and Undervatized Grob Mixture (initial and final runs)
in sealed vessels are shown in Figures 3 and 4, respectively.
20
17.95
Methyl Hexadecanoate
112-39-0
–
0.05
3.19
21
19.13
Hexadecanoic acid
57-10-3
–
1.84
8.67
22
18.46
Unknown
–
–
–
0.07
Table 2 GC System Suitability Requirements
23
18.52
Unknown
–
–
–
0.04
Signal to
Noise (S/N)
Resolution
Acceptance Criterion
All peaks, S/N GT 10
Protocol: GT 1.5 for
critical pair Amended by
study coordinator: 1.0
or better
Peak Shape Tailing factor LT 2.0
W
Fe
Figure 9
Rubber Elastomer: GC/MS-TIC
Reflux/n-hexane
Inverted overlay with corresponding blank
LC/UV/MS Analysis
A system suitability test mixture was prepared with caprolactam, BHT, diphenlyamine, MEHP,
Steric acid, DEHP and bisphenol A at a concentration of 1-5 ug/mL. A 100mm x 3mm C18
column @50 ºC was used with a gradient starting at 5% acetonitrile ramped up to 100% for
10 minutes. A time of flight mass spectrometer operating with atmospheric pressure chemical
ionization (APCI) both positive ion and negative ion modes was employed, the mass range of
80-1250 amu. UV data were acquired at 205 nm.
The HPLC UV chromatograms of IPA and hexane reflux extracts are shown in Figures 13
and 14.
Figure 13 IPA Reflux
pH2.5 Extracts
Sonication
Sealed Vessel
Br
17.5
0.29
9.40
20.5
K
ME1
ME1
6.84
NP3
Ca
2.60
4.07
2.072
NP3
Mg
3.50
0.06
2.83
2.90
Al
0.66
0.03
2.19
3.56
Zn
2.89
0.04
0.49
NP3
Figure 14 Hexane Reflux
Conclusions
Figure 10
Rubber Elastomer: GC/MS-TIC
Soxhlet/n-hexane e
Sources of extractables are intrinsic to the rubber formulation ingredients as well as extrinsic
sources. Supplier information should be revisited and may show information from the N-1
supplier may or may not be inclusive of all potential extractables. Compounds detected can
be directly or indirectly related to the formulation ingredients. None of the compounds listed
on the rubber formulation list were directly identified. Compounds that can be inferred from
the data are noted (
) in the Rubber Formulation Information list origins of the known
extractables are as follows:
Inverted overlay with corresponding blank
Supplier Rubber Formulation Information
0.16
LC/MS Observations
25
18.87
Unknown
–
–
–
0.44
brominated isobutylene isoprene copolymer 57.3%
26
19.72
Methyl Octadecanoate
112-61-8
–
0.02
3.86
2-Ethyl-hexanoic acid
2-Ethyl-hexanoic acid has
lowest S/N, observed over a has lowest S/N, observed
over a range from 13 - 21
range from 13 - 21
27
19.89
Heptadecanoic acid
506-12-7
–
–
0.15
28
20.80
Octadecanoic acid [TMS]
57-11-4
–
0.66
9.68
HPLC/MS TIC Chromatograms (+ ion) for the rubber extracts are shown in Figure 15. The
green trace is IPA/H2O extract, red trace is pH9.5 extract and blue trace is pH2.5 extract. The
HPLC/MS TIC Chromatograms (- ion) for the multiple rubber extracts I are shown in Figure 16.
The LC/UV/MS results are tabulated in Table 7.
29
–
Octadecanoic acid, Isopropyl ester
3654-92-2
–
–
0.21
zinc oxide 0.6%
30
20.93
Unknown# [TMS]
–
–
0.11
0.13
Figure 15
Positive Ion IPA/H2O; pH9.5 ; pH2.5 extracts
31
20.97
Unknown# [TMS]
–
–
0.12
0.14
32
–
9-Oxo Octadecanoic acid, methyl ester
1842-70-2
–
–
0.40
33
22.08
Nonadecanoic acid [TMS]
646-30-0
–
0.49
0.29
34
22.24
Eicosanoic acid [TMS]
506-30-9
–
–
0.08
35
23.42
Hexadecanoic acid-2,3dihydroxypropyl ester [2TMS]
542-44-9
–
0.09
0.20
36
23.43
n-Nonanoyl morpholine
5299-64-9
–
–
0.20
37
23.73
Consistent with C18 fatty acid [TMS]
–
–
0.07
–
38
24.87
n-Decanoyl Morpholine
5299-65-0
–
–
0.36
39
25.95
Nonadecanoic acid-2,3dihydroxypropyl ester
6292707-5
–
–
0.07
2,3-Butanediol [2TMS]
Decane
2
Undecane
1-Octanol
3
2-Ethyl hexanoic acid [TMS]
Undecane
4
2,6-Dimethyl aniline
1- Nonanal
5
1-Octanol [TMS]
2,6-Dimethyl phenol
6
2,6-Dimethylphenol [TMS]
2-Ethylhexanoic acid
7
Methyl Decanoate
2,6-Dimethyl aniline
8
Nonanoic acid [TMS]
Methyl decanoate
9
Methyl Undecanoate
Methyl undecanoate
10
Methyl Dodecanoate
Dicyclohexylamine
11
--------
Methyl dodecanoate
Figure 4 Underavatized Grob Mixture
Initial and Final Runs
Figure 5 U
nderivatized IPA/H2O, pH9.5 and pH2.5 Sealed Vessel Extracts
GC/FID Observations
Tentatively identified compounds from the sealed vessel extractions are listed in Table 4,
concentrations estimated relative to BisphenolM.
Table 6 First Pass Compounds Relux (R)and Soxhlet (S)Extractions
IPA R/S
Figure 16
Negative Ion IPA/H2O; pH9.5 ; pH2.5 extracts
paraffinic oil 1.2% <10ug/g
(Hydrocarbons)
(Zn)
3.44
Morpholine
51/26
2/3
12.08
C13
90/130
112/185
SRF Carbon black mixture 0.4%
14.07
C21
180/272
205/308
calcined magnesium oxide 0.3%
16.01
Methyl n-hexadecanoate
12/8
2/ND
16.31
Palmitic Acid
91/149
15/1927
17.89
Stearic acid
90/169
22/3027
18.02
Hexadecanamide
15/5
9/ND
19.36
Oleamide
87/109
45/89
19.62
Tetracosane
24/42
10/8
22.36
Octacosane
36/53
17/7
22.18
Pentanoic acid, morpholide
26/50
24/31
23.16
4,4’-Dioctyldiphenylamine (ODPA)
30/25
29/25
Unknown Peaks: 1
6.8 min 70-80 ug/g in hexane extract
16.45, 18.90, 20.9, and 23.90 mins at 30-60 ug/g IPA in extract
Internal Standard Peaks: 1
0.70 min 2-Fluorobiphenyl
21.09 min Irganox 415
22.9 min Bisphenol M
The chromatograms shown in Figures 7 – 10 are of the IPA and Hexane extracts under
Soxhlet and Reflux extraction techniques. Table 6 summarizes first pass confirm and confident compounds. Figure 11 is the profile of the IPA reflux extracts with retention time labeled
corresponding with the indentified compounds. The mass spectra of selected identified
compounds are shown in Figure 12.
Figure 12 Mass Spectra
RRT 0.157 Morpholine
RRT 0.549
C13-oligomer, brominated
(C13 and C21 oligomers)
(Ca, Al)
titanium dioxide 1.2%
Confirmed and Confident Compounds µg/g
GC/MS Observations
Inverted overlay with corresponding blank
Hexane R/S
calcined aluminium silicate 38.2%
RT min
Figure 11 IPA Reflux Chromatogram with Retention Time
Figure 7
Rubber Elastomer: GC/MS-TIC Reflux/IPA
Figure 2
Headspace Late Eluting Compounds
Figure 6 D
ervatized IPA/H2O, pH9.5
and pH2.5 Sealed Vessel Extracts
Hg
Elements were tabulated if found in at least one extract. > 0.3ug/g. Trace Fe, Ni, Ti, V, Cr, Sr,
Mn were detected but could be a result of equipment or acids used in the analysis.
–
1
Zn
pH9.5 Extracts
Sealed Vessel
–
Underivitized Compound Figure 4
Cu
Sonication
142-91-6
Dicyclohexylamine is
worst case: 1.5 to 1.7
Pt
Extracted Amount, μg/g
Element
Hexadecanoic acid, 1-methyl ester
All peaks, tailing LT 2.0
Ni
Trace Elements and Metals Results, Rubber
–
Undecane and 1-nonTwo critical pairs: 1.0 between undecane and 1-non- anal is critical pair: 1.5
anal 1.0 between 1-nonanal to 1.6
and 2,6-dimethyl phenol
Cd
Table 8 ICP/MS Semi-quantitative Results
24
Derivitized Compound Figure 3
Figure 3 D
ervatized Grob Mixture
Initial and Final Runs
Na
Early Runs, Un-Derivatized Final Runs, Derivatized
(DB-5MS column with
(DB-5HT column with
0.25 µm film)
0.1 µm film)
Consistent resolution
Consistency No significant differences Degradation of critical pair
between chromatograms resolution over course of run throughout run
at beginning and end of run
Peak ID (1)
Alkali
RRT 1.053
4,4’-Dioctyl-diphenylamine
IPA/W
Relatively volatile and semi-volatile compounds were analyzed by GC using a non-polar
capillary column with a temperature range of 40-300 ºC. Detection strategies employed Flame
Ionization (FID) and Mass Spectrometry (MS). Extracts were analyzed directly and derivatized
using BSTFA with 1% TMCS. System Suitability requirements are listed in Table 2. The System
Suitability chromatographic peaks are identified in Table 3.
Compounds Observed in Grob Mix
N/A= Not analyzed
K
RT (min)
Table 3 System Suitability Chromatographic Peaks
Analytical
Techniques
Headspace
Unknown
RRT 0.640
1-Isopropenyl-2,2,4,4,6-tetramethyl6-(2,2,4-trimethylpentyl-1-1
cyclohexane (C21-Oligomer)
Alkali Earth Be
Inverted overlay with corresponding blank
Information Related to the GC Peaks Associated with Organic Extractables
from RE Material; Sealed Vessel Extraction (See Figure 2)
Gas Chromatography (GC) Analysis
Criterion
5g/200ml
55C/3days
HPLC/UV/MS
GC/FID‡†
NA
Status
NA
Unknown
CAS#
C6H12
Figure 8
Rubber Elastomer: GC/MS-TIC Soxhlet/IPA
Table 4 GC Peaks Associated with Organic Extractables from RE Material;
Sealed Vessel Extraction
Table 1 Headspace First Pass Results
A bromobutyl rubber formulation was moulded into 11cm x 11cm x 0.1cm test plates. The
plates were sandwiched between two sheets of coated paper. The plates were stacked and
placed in a plastic bag and submitted to the Product Quality Research Institute (PQRI) to
perform controlled extraction studies. The test plates were characterized by extraction using
multiple solvents and included silylation of the aqueous extracts. The extracts were analyzed
for volatiles, semivolatiles, nonvolatiles and inorganic constituents. In addition, the headspace
volatiles were evaluated. The extractables were identified as confirmed, confident or tentative.
Amounts of extractables were estimated in µg/g relative to an internal standard.
polyethylene 0.6%
(Hydrocarbons)
(Mg)
4,4’-dithiodi-morpholine/polyisobutylene 0.3%
(Morpholine)
The evolution of free morpholine is a result of sulfur as a crosslinking agent between polymer
chains. Below is a simplified diagram illustrating the role of 4,4-dithiodimorpholine as
“sulfur donor.”
Table 7 LC/MS Results
Information Related to the LC Peaks Associated with Organic Extractables from RE Material
Rt. Min
Peak
(from MS
#
traces)
Tentative ID
CAS RN
MW (or ion
observed)
110-91-8
87
Observed in extracts
pH2.5 pH9.5
1
0.77
Morpholine
2
7.9
Unknown
3
8.5
4
8.5
5
8.8
x
IPA/H2O
x
298
x
x
228
x
x
Unknown
( 293 )
x
Unknown polymers
( 424, 468, 512 .. )
Tetradecanoic acid 544-63-8
6
9.3
Unknown polymers
7
9.9
Hexadecanoic acid
8
9.9
Unknown
( 438, 482, 526.. )
57-10-3
x
256
x
x
x
x
( 663 )
9
10.5
Unknown
( 266 )
10
10.7
Unknown
( 663 )
11
10.8
Unknown
( 326 )
12
11.5
Octadecanoic acid
57-11-4
284
x
x
x
x
x
x
x
x
x
ICP/MS Analysis
Single elements in the aqueous extracts were analyzed by Inductively Coupled Plasma using
mass spectroscopy (ICP-MS); the analysis was performed consistent with USP <730>. The
resulting extracts had high amounts of Na and P and these elements could not be determined
in this study. The below listed elements were targeted; semi-quantitative results were obtained
using a standard of Cs, Co, Li, Tl and Y at 10ug/L. Table 8 is a summary of semi-quantitative
ICP/MS results.
Several fatty acids were detected, significant palmitic and stearic were found in the organic
extracts and low levels in the aqueous extracts. The source of the fatty acids was traced to
Ca stearate used by the N-2 supplier during the polymerization process. ODPA was detected
and suspected to also be from the N-2 supplier processing. In addition, cyclohexane and
methyl cyclopentane found in the headspace analysis was traced to solvents used by the
N-2 supplier.
DEHP (40ug/g) was seen in the aqueous extracts, yet not seen in the organic extracts. The
solubility of DEHP in water is 41 μg/L at 25C while it is miscible in hexane. The water extract
was likely contamination since there was no DEHP accumulation in the organic extracts. Other minor methyl esters of unknown sources were detected in the aqueous extracts. Oleamide
and hexadecanamide (slip agents) were found at about 100 µg/g, the source is unknown but
could be a result of test plate contact with packaging or mold release. Polynuclear aromatic
hydrocarbons and nitrosoamines were not detected nor specifically sought.
Properly conducted extractable studies should employ multiple solvents and analytical techniques
and be robust enough to comprehensively characterize the material, detecting both the
expected and unexpected. Formulation ingredients do not always indicate potential extractables
resulting from raw material side reactions and oxidation by products. These must also be
understood through extractable testing. First-pass extractables data can indicate tentative
leachables. This data will support the appropriate selection of materials to be in contact with a
particular drug product and compounds to target for leachable studies.
Chromatograms of the rubber extracts (pH2.5, pH9.5 and IPA/H2O) from the sealed vessels
extractions are shown in Figure 5 (underivatized) and Figure 6 (derivatized). Internal standard
peaks are (1 = Irganox 415, ≈ 25mg/L and 2 = Bisphenol M, ≈ 1 mg/L).
6025