IntroductionA 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.

BackgroundPharmaceutical 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 Pharmaceutical ApplicationsBrominated isobutylene isoprene copolymer

Experimental Conditions

Extraction Soxhlet Reflux Sonication Sealed Vessel

IPA 5g/125mL 16 hrsHPLC/UV/MSGC/MS

3.5g/125mL3 hrsHPLC/UV/MSGC/MS

N/A N/A

Hexane 5g/125mL 16 hrsHPLC/UV/MSGC/MS

3.5g/125mL3 hrsHPLC/UV/MSGC/MS

N/A N/A

1:1 IPA:H20 N/A 5g/200mL55C/3daysHPLC/UV/MSGC/FID‡†

N/A 5g/200ml55C/3daysHPLC/UV/MSGC/FID‡†

pH2.5 N/A N/A 5g/200ml 2hrsICP/MS

5g/200ml 121C/1hrHPLD/UV/MSGC/FID‡†

pH9.5 N/A N/A 5g/200ml2hrsICP/MS

5g/200ml121C/1hrHPLC/UV/MSGC/FID‡†

Sample Solvent Ratio

3.5g = 15cm2/125mL; 5g = 20 cm2/125mL; and/200mL

Analytical Techniques

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, Dethylhex-ylphthalate (DEHP), Bisphenol A, Irganox 1010, Irganox 168

Headspace Standard: Methanol, Acetic Acid, Cyclohexanone, Toluene, Trimethylsilianol, 2-ethylhexanol

GC Standard: Grob MixtureTMS Derivitized ‡

Underivitized†

N/A= Not analyzed

Headspace 1g/20mL @ 80-120C GC/FID/MS

Headspace AnalysisA 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

Table 1 Headspace First Pass Results

RT, min

Conc, μg/g

ID FormulaStructure/

Fragmentation CAS# Status

4.59 0.99 Unknown - no ms signal

NA NA NA Unknown

5.68 1.19 Cyclopentane, methyl-

C6H12 96-37-7 Confident

6.75 0.48 Cyclohexane C6H12

110-82-7 Confident

19.95 0.14 Unknown NA m/z 39, 43, 45, 58 NA Unknown

33.22 20.8 1,4-Dioxane C4H8O2 123-91-1 Internal Standard

37.21 0.77 Unknown – Butyl Oligomer

related

NA m/z 29, 41, 57, 68, 82, 97, 109, 124, 137, 165, 180

NA Unknown

40.82 0.46 Unknown – Butyl Oligomer

related

NA m/z 29, 43, 57, 69, 81, 97, 109, 123, 137, 151, 165, 180

NA Unknown

41.67 0.16 Unknown NA m/z 29, 41, 57, 67, 79, 97, 109, 123, 133, 145,

160, 179, 191

NA Unknown

Gas Chromatography (GC) AnalysisRelatively 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.

Standard chromatograms of Derivatized and Undervatized Grob Mixture (initial and final runs) in sealed vessels are shown in Figures 3 and 4, respectively.

Table 2 GC System Suitability Requirements

Criterion Acceptance Criterion Early Runs, Un-Derivatized(DB-5HT column with 0.1 µm film)

Final Runs, Derivatized(DB-5MS column with 0.25 µm film)

Signal to Noise (S/N)

All peaks, S/N GT 10 2-Ethyl-hexanoic acid has lowest S/N, observed over a range from 13 - 21

2-Ethyl-hexanoic acid has lowest S/N, observed over a range from 13 - 21

Resolution Protocol: GT 1.5 for critical pair Amended by study coordinator: 1.0 or better

Two critical pairs: 1.0 be-tween undecane and 1-non-anal 1.0 between 1-nonanal and 2,6-dimethyl phenol

Undecane and 1-non-anal is critical pair: 1.5 to 1.6

Peak Shape Tailing factor LT 2.0 All peaks, tailing LT 2.0 Dicyclohexylamine is worst case: 1.5 to 1.7

Consistency No significant differences between chromatograms at beginning and end of run

Degradation of critical pair resolution over course of run

Consistent resolution throughout run

Table 3 System Suitability Chromatographic Peaks

Compounds Observed in Grob Mix

Peak ID (1) Derivitized Compound Figure 3 Underivitized Compound Figure 4

1 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 3 Dervatized Grob Mixture Initial and Final Runs

GC/FID ObservationsTentatively identified compounds from the sealed vessel extractions are listed in Table 4, concentrations estimated relative to BisphenolM.

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).

Table 4 GC Peaks Associated with Organic Extractables from RE Material; Sealed Vessel Extraction

Information Related to the GC Peaks Associated with Organic Extractables from RE Material; Sealed Vessel Extraction (See Figure 2)

Peak #

RT (min) Tentative Identification CAS RNMaximum Conc, mg/L2

pH2.5 pH9.5 IPA/W

1 6.06 Unknown – 0.05 0.02 –

2 12.97 Unknown – – – 1.03

3 13.37 Unknown – – – 0.52

4 13.59 Unknown – – – 0.37

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 15.271,2-Benzenecarboxylic acid,

monoethylester2306-33-4 – 0.08 –

14 15.88 Unknown – – – 2.06

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

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

23 18.52 Unknown – – – 0.04

24 – Hexadecanoic acid, 1-methyl ester 142-91-6 – – 0.16

25 18.87 Unknown – – – 0.44

26 19.72 Methyl Octadecanoate 112-61-8 – 0.02 3.86

27 19.89 Heptadecanoic acid 506-12-7 – – 0.15

28 20.80 Octadecanoic acid [TMS] 57-11-4 – 0.66 9.68

29 – Octadecanoic acid, Isopropyl ester 3654-92-2 – – 0.21

30 20.93 Unknown# [TMS] – – 0.11 0.13

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 23.42Hexadecanoic acid-2,3-

dihydroxypropyl 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 25.95Nonadecanoic acid-2,3-

dihydroxypropyl ester62927-07-5

– – 0.07

Figure 5 Underivatized IPA/H2O, pH9.5 and pH2.5 Sealed Vessel Extracts

GC/MS Observations 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 confi-dent 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 7 Rubber Elastomer: GC/MS-TIC Reflux/IPA Inverted overlay with corresponding blank

Figure 8 Rubber Elastomer: GC/MS-TIC Soxhlet/IPA Inverted overlay with corresponding blank

Figure 9 Rubber Elastomer: GC/MS-TIC Reflux/n-hexane Inverted overlay with corresponding blank

Figure 10 Rubber Elastomer: GC/MS-TIC Soxhlet/n-hexane e Inverted overlay with corresponding blank

Table 6 First Pass Compounds Relux (R)and Soxhlet (S)Extractions

RT min Confirmed and Confident Compounds μg/g IPA R/S Hexane R/S

3.44 Morpholine 51/26 2/3

12.08 C13 90/130 112/185

14.07 C21 180/272 205/308

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: 16.8 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: 10.70 min 2-Fluorobiphenyl 21.09 min Irganox 415 22.9 min Bisphenol M

Figure 11 IPA Reflux Chromatogram with Retention Time

Figure 12 Mass Spectra

RRT 0.157 RRT 0.549Morpholine C13-oligomer, brominated

RRT 0.640 1-Isopropenyl-2,2,4,4,6-tetramethyl- 6-(2,2,4-trimethylpentyl-1-1 RRT 1.053 cyclohexane (C21-Oligomer) 4,4’-Dioctyl-diphenylamine

LC/UV/MS AnalysisA 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 Figure 14 Hexane Reflux

LC/MS ObservationsHPLC/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.

Figure 15 Positive Ion IPA/H2O; pH9.5 ; pH2.5 extracts

Table 7 LC/MS ResultsInformation Related to the LC Peaks Associated with Organic Extractables from RE Material

Peak #

Rt. Min (from MS traces)

Tentative ID CAS RNMW (or ion observed)

Observed in extracts

pH2.5 pH9.5 IPA/H2O

1 0.77 Morpholine 110-91-8 87 x x

2 7.9 Unknown 298 x x

3 8.5 Tetradecanoic acid 544-63-8 228 x x

4 8.5 Unknown ( 293 ) x x

5 8.8 Unknown polymers ( 424, 468, 512 .. ) x

6 9.3 Unknown polymers ( 438, 482, 526.. ) x x x

7 9.9 Hexadecanoic acid 57-10-3 256 x x

8 9.9 Unknown ( 663 ) x

9 10.5 Unknown ( 266 ) x x

10 10.7 Unknown ( 663 ) x

11 10.8 Unknown ( 326 ) x

12 11.5 Octadecanoic acid 57-11-4 284 x x

ICP/MS AnalysisSingle 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.

Alkali Na K Li

Alkali Earth Be Mg Ca Sr Ba

Metals Al Sn Pb Bi

Transition Ti Zr V Cr Mo W Fe Cd Ni Pt Cu Zn Hg

Non metals Si As Cl Br

Table 8 ICP/MS Semi-quantitative Results

Trace Elements and Metals Results, Rubber

Element

Extracted Amount, μg/g

pH2.5 Extracts pH9.5 Extracts

Sonication Sealed Vessel 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

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.

ConclusionsSources 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:

Supplier Rubber Formulation Informationbrominated isobutylene isoprene copolymer 57.3% (C13 and C21 oligomers)

calcined aluminium silicate 38.2% (Ca, Al)

titanium dioxide 1.2%

paraffinic oil 1.2% <10ug/g (Hydrocarbons)

zinc oxide 0.6% (Zn)

polyethylene 0.6% (Hydrocarbons)

SRF Carbon black mixture 0.4%

calcined magnesium oxide 0.3% (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.”

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. Oth-er 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.

6025

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.

Gaskets

Closures

Pistons

Figure 4 Underavatized Grob Mixture Initial and Final Runs

Figure 6 Dervatized IPA/H2O, pH9.5 and pH2.5 Sealed Vessel Extracts

Figure 16 Negative Ion IPA/H2O; pH9.5 ; pH2.5 extracts

Figure 2Headspace Late Eluting Compounds