Experimental Protocol for Qualitative Controlled Extraction Studies on Material Test Articles Repersentative of Prefilled Syringe (PFS) and Small Volume Parenteral (SVP) Container Closure Systems: Extraction Methods and Analytical Testing Procedures Dennis Jenke, Baxter Healthcare Corporation Overview The PQRI Parenteral and Ophthalmic Drug Products (PODP) Leachables and Extractables Working Group developed an experimental protocol as a means of establishing Best Demonstrated Practices for the performance of Controlled Extraction Studies, specifically relevant for PODP container closure systems and dosage forms. This protocol considered the processes by which a Controlled Extract is generated, the processes by which a Controlled Extract is analyzed and processes by which the test results are evaluated and interpreted, specifically within the context of the Working Group’s approved Work Plan and experimental hypothesis. Introduction: PODP Working Hypothesis Threshold concepts that have been developed for safety qualification of leachables in OINDP can be extrapolated to the evaluation and safety qualification of leachables in PODP, with consideration of factors and parameters such as dose, duration, patient population and additional product dependent characteristics unique to various PODP types. The “good science” best demonstrated practices that were established for the OINDP pharmaceutical development process can be extrapolated to container closure systems for PODP. Threshold and best practices concepts can be integrated into a comprehensive process for characterizing container closure systems with respect to leachable substances and their associated impact on PODP safety. A PODP Work Plan was designed to test this hypothesis by developing these scientifically justifiable thresholds and establishing best demonstrated practices. Test Articles Representing PODP Materials Test Articles (Material Type) Format Composition (Supplier Information) Application Category Polycarbonate (PC) Injection moulded plaques 0.05 PHR Irganox 1076 0.1 PHR Irgafos 168 Ports, Tubes LVP Rubber Elastomer (Bromobutyl) Sheet Brominated isobutylene isoprene copolymer (57.3%) calcined aluminum silicate, 38.2% titanium dioxide, 1.2%; paraffinic oil, 1.2%; zinc oxide, 0.6% polyethylene0.6% SRF Carbon block mixture, 0.4% calcined magnesium oxide, 0.3% 4,4’-dithiodi- morpholine/polyisobutylene, 0.3% Closures, Plungers, Gaskets SVP Cyclic Olefin Copolymer (COC) Plaques Irganox 1010 Ultramarine Blue Syringes, Vials PFS, SVP Polyvinylchloride (PVC) Pellets PVC resin DEHP 30% Epoxidized oil 7% Zn stearate 0.5% Ca stearate 0.5% Stearamide 1% Bags, Tubing LVP Low density polyethylene (LDPE) Blown Film Irganox B 215 (2:1 blend of Irgafos 168 and Irganox 1010) 1000 ppm BHT 200 ppm Calcium Stearate 500 ppm Erucamide 500 ppm Chimassorb 944 2000 ppm Overpouch, BFS, Containers BFS, SVP, LVP Aqueous Extract pH 2.5 200 ml Aqueous Extract pH 9.5 200 ml IPA/Water extract 200 ml Transfer aliquot of 50 ml to 125 mL separating funnel Add 1.0 ml surrogate internal standard solution 50 μg/mL) + 25 ml DCM Shake vigorously for 1 min Collect lower DCM layer Repeat 1 x Repeat 1 x Concentration Step Transfer extract with DCM rinses to Turbovap tube Concentrate to < 0.5 ml Add 0.5 ml aliquot of of the injection standard solution (50 μg/mL) Adjust to a final volume of 1.0 ml Withdraw aliquot of extract Add 1.0 ml surrogate internal standard solution 50 μg/mL Bisphenol M in MeOH perform suitable sample work-up Concentration Step Add 0.5 mL of injection internal standard solution: 50 μg/mL Irganox 415 in MeOH if appicable, perform concenctration step Adjust to final volume of 1.0 ml Extraction Techniques: Soxhlet (min. 10 cycles, 24 hrs, 5 g- 200 ml) Reflux (2 hrs, 5 g - 200 ml) Sonication (2 hrs, T=0°C, 5 g - 200 ml) Sealed Vessel (55 °C / 3 d, 5 g- 200 ml) Sealed Vessel Autoclaved (121 °C / 1 hr 5 g - 200 ml, 2 replicates) IPA extract 200 ml N-Hexane extract 200 ml Transfer aliquot of 50 ml to 125 mL separating funnel Add 1.0 ml surrogate internal standard solution (50μg/mL) pH 2 (5 N HCl) + 25 ml DCM Shake vigorously for 1 min Collect lower DCM layer Repeat 1 x Repeat 1 x GC/MSD GC/FID LC/MS n LC/TOF-MS LC/UV Concentration Step Transfer extract with DCM rinses to Turbovap tube Concentrate to < 0.5 ml Add 0.5 ml aliquot of of the injection standard solution (50 μg/mL) Adjust to a final volume of 1.0 ml Aqueous Extract pH 2.5 Injection Solution Aqueous Extract pH 9.5 Injection Solution IPA/Water extract Injection Solution IPA extract Injection Solution N-Hexane extract Injection Solution Sylilation Procedure Aqueous Extract pH 2.5 Injection Solution TMS-Derivatives Aqueous Extract pH 9.5 Injection Solution TMS-Derivatives IPA/Water extract Injection Solution TMS-Derivatives Sylilation Procedure Sylilation Procedure Combine all DCM layers Add anhydrous sodium sulfate to flask Test Articles: Sample Weight [5g] Sample Pre-treatment System Suitability (Test Mixtures): SST Test Mixture for HPLC SST Test Mixture for GC SST Test Mixture for HS/GC Materials: LDPE PC PVC COC Rubber Combine all DCM layers Add anhydrous sodium sulfate to flask Aq Extract pH 2.5 pH 10 (5N NaOH) Aq Extract pH 9.5 pH 2 (5N HCl) pH 10 (5N NaOH) HS-GC/MS(FID) ICP/MS(AES) Experimental Workflow Overall Study Design Matrix Extraction Procedure Sample weight 5 g / 200 ml extraction solvent Soxhlet Reflux Sonication Sealed Vessel Head- space Solvent IPA Hex IPA Hex IPA/W pH2.5 pH9.5 IPA pH2.5 pH9.5 IPA/W - LDPE - X X - X X - X X X X - - X - X - X X PC X X X X X X X X X X X - X X X X X X X PVC X X X X X X X X X X X - X X X X X X X Rubber X X X X X X - X X X X - X X X X X X X COC X X X X X X - X X X X - X(2) X(2) X(2) X(2) X(3) X(3) X GC X X X X X X X X - X - - X - X - X X --- LC X X X X X X X X - X - - X - X - X X - ICP/ - - - - - - - X - X - - X - X - - - HS/MS - - - - - - - - - - - - - - - - - X Sample Prep. (4) SP1 SP1 SP1 SP1 SP3 SP2 SP2 SP1 SP2 SP2 SP3 - Surrogate standard (total process) (5) 4,4'-(m-Phenylenediisopropylidene)diphenol (Bisphenol M, 13595-25-0) 2000 μg/ml in MeOH, add aliquot of 500 μl to extract (200 μg/g sample) 1,4- Dioxane (123- 91-1) Instrumental -injection standard (6) 4,4-Thiobis(3-methyl-6-t-butylphenol) (Irganox 415, 96-69-5) 5000 μg/ml in MeOH, add aliquot of 100 μl to final extract (100 μg/g sample) Lab #1 Lab #2 Lab #3 Lab #4 Lab #5 Lab #6 Notes : (1) An X denotes a material/solvent couple that was performed, an --- denotes a couple that was not performed. (2) Under autoclave conditions (3) Storage at 55°C for 3 days (4) 3 different sample preparation schemes (extract work-up) - (SP1, SP2, SP3) will be applied. For detailed procedure refer to section xy. SP1: Organic SP2: pH2.5, pH 9.5 SP3: IPA/Water (5) Specification of a surrogate - standard (internal standard for total analytical procedure) added to the extract after completion of the extraction step. (6) Instrumental standard or injection standard added to the final extract prior to instrumental analysis. Sample Work-up for Sealed Vessel and Aqueous Sonication Extracts, GC. Sample Preparation, Liquid-liquid Extraction; pH 2.5 and pH 9.5 Solutions. 1. A 50-mL portion of each of the solutions is transferred to a 125 mL separatory funnel. 2. A 1.0-mL aliquot of the surrogate internal standard solution is added to each sample. 3. 25 mL of Dichloromethane (DCM) is added to each funnel. 4. Each funnel is shaken for 1 minute. 5. The layers are allowed to separate and the lower (DCM) layer is collected. 6. Steps 3 through 5 are repeated. The collected DCM layers are combined. 7. The pH of each pH 2.5 sample is adjusted to 10 with 5 N NaOH. The pH of the pH 9.5 sample is adjusted to 2 with 5 N HCl. 8. Steps 3 through 5 are repeated twice for the pH adjusted samples. The collected DCM layers from all extractions are combined. 9. The DCM extracts are dried by adding anhydrous sodium sulfate to each collection flask. 10. Each DCM extract is transferred from the collection flask to a different Turbovap concentration tube with DCM rinses, and concentrated to less than 0.5 mL. A 0.5 mL aliquot of the injection internal standard is then added to the Turbovap tube. The final volume is adjusted to approximately 1 mL with DCM. 11. 0.5 mL of each concentrated extract is transferred from the Turbovap tube to an autosampler vial. 12. The remaining 0.5 mL aliquot of each of dichloromethane extract described above is transferred to separate amber autosampler vials.for TMS derivatization (see below) Sample Preparation, Liquid-liquid Extraction; IPA/Water Solutions The same basic process as noted above will be followed for the IPA/water samples. In the first extraction step, these samples will be pH adjusted to pH 2 and extracted twice. In the second extraction step, the samples will be adjusted to pH 10 and extracted twice. The resultant DCM extracts will be combined, dried and concentrated per steps 9 through 11 above. TMS Derivatization of Residues 1. Approximately 100 L dimethyl formamide is added to each amber autosampler vial prepared under step 12 above. 2. The contents of each vial are evaporated nearly to dryness using nitrogen. 3. To each of the sample extracts, and the standard solutions is added 100 μL of BSTFA w/ 1% TMCS (Pierce) 4. Each vial is capped and allowed to stand for one hour at approximately 70°C. 5. DCM is added to each auto-sampler vial to make a final volume of approximately 0.5 mL, and is mixed. Example Operating Parameters, GC Analysis of the Sealed Vessel Extracts. Operating Parameter Operating Value Column J&W DB-5HT, 30m x 0.25mm, 0.1 μm film thickness Oven Program Start at 40C, hold for 1 min; ramp at 10C/min to 280 o C, hold for 2 min; ramp at 15C/min to 310C, hold for 3 min. Carrier Gas He at 1 mL/min Injection Splitless; 2 L. Injector Temperature 300C FID Detector Temperature: 350C MS Transfer Line Temperature 310C MS Detection Details 70 eV (+), mass range of 33 – 650 amu (5.0 min or 7.5 min solvent delay used for un-derivatized or derivatized samples) Analytical Test Procedures, Organic Extractables Gas Chromatography (GC); Semi-volatiles Liquid Chromatography (LC); Semi-volatiles, non-volatiles Operating Parameters, LC/UV/MS Analysis of the Extracts. Operating Parameter Operating value Column Agilent Zorbax Eclipse Plus C 18 , 100 x 3.0 mm, 3.5m particles Column Temperature 40C Mobile Stage Components A = 10 mM ammonium acetate, B = acetonitrile Mobile Stage Gradient Time % B 0.0 5.0 8.0 95.0 11.0 95.0 14.0 5.0 17.0 5.0 Mobile Stage Flow Rate 0.8 mL/min Sample Size 60 L Detection, UV 205 –300 nm; spectra recorded at = 210, 230, 250 and 270 nm Detection, MS API-ES, positive ion and negative ion (mass range 80 – 900) Sample Preparation None, direct injection Gas Chromatography with Headspace sampling (HS-GC); Volatiles 8 9 10 11 12 13 14 15 16 100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 750000 800000 850000 900000 Time--> Abundance Signal: RS037.D\FID1A.CH Derivatized Grob 1/100 Comparison 10 10 9 9 8 8 7 7 6 6 5 4 3 5 4 3 2 2 1 1 GROB 1/100 (Bracketed) GROB 1/100 (System Suitability) Signal: RS068.D\FID1A.CH (*) 6 7 8 9 10 11 12 13 14 15 16 17 120000 140000 160000 180000 200000 220000 240000 260000 280000 300000 320000 340000 360000 380000 400000 420000 Response_ Signal: RS003.D\FID1A.CH GROB 1/100 (SYSTEM SUITABILITY) GROB 1/100 (Bracketed) 11 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 Signal: RS034.D\FID1A.CH (*) Grob 1/100 Comparison After Samples Before Samples Compounds Observed in Underivatized Grob Mix Peak ID (1) Compound 1 Decane 2 1-Octanol 3 Undecane 4 1- Nonanal 5 2,6-Dimethyl phenol 6 2-Ethylhexanoic acid 7 2,6-Dimethyl aniline 8 Methyl decanoate 9 Methyl undecanoate 10 Dicyclohexylamine 11 Methyl dodecanoate Compounds Observed in Grob Mix after TMS Derivatization Peak ID Compound 1 2,3-Butanediol [2TMS] 2 Undecane 3 2-Ethyl hexanoic acid [TMS] 4 2,6-Dimethyl aniline 5 1-Octanol [TMS] 6 2,6-Dimethylphenol [TMS] 7 Methyl Decanoate 8 Nonanoic acid [TMS] 9 Methyl Undecanoate 10 Methyl Dodecanoate min 0 2 4 6 8 10 12 14 16 mAU -600 -400 -200 0 DAD1 A, Sig=210,20 Ref =360,100 (MCI1310\MC000004.D) CAP MEHP BPA BHT DEHP DPA min 0 2 4 6 8 10 12 14 16 0 1000000 2000000 3000000 MSD2 TIC, MS File (MCI1310\MC000004.D) API-ES, Pos, Scan, Frag: 100, "Positive" CAP MEHP DPA DEHP min 0 2 4 6 8 10 12 14 16 0 200000 400000 MSD2 114, EIC=113.7:114.7 (MCI1310\MC000004.D) API-ES, Pos, Scan, Frag: 100, "Positive" CAP min 0 2 4 6 8 10 12 14 16 0 10000 20000 MSD2 170, EIC=169.7:170.7 (MCI1310\MC000004.D) API-ES, Pos, Scan, Frag: 100, "Positive" DPA min 0 2 4 6 8 10 12 14 16 0 200000 400000 MSD2 391, EIC=390.7:391.7 (MCI1310\MC000004.D) API-ES, Pos, Scan, Frag: 100, "Positive" DEHP 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 T im e --> Abundance Signal: 26JAN2010004std1.D\FID1A.CH 16.662 31.334 32.051 33.229 37.971 39.070 39.194 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00 40.00 41.00 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 Time--> Abundance Signal: 26JAN2010004std1.D\FID1A.CH 31.334 32.051 33.229 37.971 39.070 39.194 Analytical Test Procedures, Trace Element and Metallic Extractables The following primary target elements were included in the ICP analysis: Al, As, Be, B, Cd, Cr, Co, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pd, Pt,Sb, Se, Si, Sn, Sr, Ti, V, W, Zn, and Zr. These are all the elements that were included in the study protocol, except for S. Additional secondary interest elements that were captured in the ICP-MS scan included: Ba, Bi, Br, Ca, Hg, K, Na, and Pb. The ICP-MS scan included several elements which were not relevant to plastics characterization and whose results will not be reported. The analysis conditions were such that these elements can be measured at the appropriately low levels, typically 0.25 g/mL or less in the material extracts. The extracts were analyzed by ICP-MS in semi-quantitative mode as ‘calibrated’ using a 10 µg/L cerium, cobalt, lithium, thallium and yttrium standard. Purified water (DI water) was analyzed as a sample. This elemental result was subtracted in the software for all sample results as ‘background’. The method blanks were analyzed with the samples and the average result of the method blanks was subtracted from all sample results. A 500 CPS peak threshold was employed during data processing. After the method blank result was subtracted from the sample result, only those elements that had values ≥ 0.1 µg/L remaining were reported, unless they were eliminated due to confirmation of poly-atomic interference as verified by isotopic abundance ratio template analysis. Operating Conditions, ICP/MS Analysis Parameter Parameter Instruments Agilent model 7500C and 7500A ICP-MS Forward Power 1300 watts (7500a) 1500 watts (7500c) Acquire Integration Time 0.10 seconds per point Integration Mode Auto Replicates 1 Points per Peak 6 Rinse Time 180 seconds Rinse Rate 0.5 rps Uptake Time 35 seconds Stabilization Time 0.5 rps Analysis Pump Rate 20 seconds Sample Introduction 0.1 rps Nebulizer Polypropylene Spray Chamber/Platinum Injector Nebulizer Flow Rate Cross Flow All Other Settings 1.1 L/min Typical Headspace GC/MS Chromatograms of System Suitability Test Mixture Typical LC Chromatograms of System Suitability Test Mixture Typical GC Chromatograms of System Suitability Test Mixture. The test mixture is a commercial mixture (Grob Mix) of 12 compounds, prepared at a level from 14 -27 ppm. Key: At 1 ppm, CAP = caprolactam, MEHP = mono-(2-ethylhexyl) phthalate, BPA = Bisphenol A, DEHP = di-(2-ethylhexyl) phthalate; at 5 ppm, BHT = Butylated- hydroxytoluene, DPA = Diphenylamine, SA = Stearic acid. Expanded Chromatogram Full Chromatogram Key: At 10 ppm, toluene at 32.1 min, cyclohexanone at 38.0 min; at 20 ppm, methanol at 16.7 min, trimethylsilanol at 31.3 min, acetic acid at 39.1 min, 2-ethyl-1-hexanol at 39.2 min. The peak at 33.2 min is the internal standard, 1,4-Dioxane. Additional Aspects of the Study Design and Implementation 1. Multiple layers of quality control. a. Standardized methods (modified appropriately as necessary). b. Use of instrumentation qualified per participating lab’s procedures. c. System suitability testing. 2. Concentration estimation via internal standards (chromatographic assays). 3. Reporting limit of 10 g/g, although lower levels were reported consistent with method capabilities. 4. Identifications made based on OINDP Best Demonstrated Practices. Operating Parameters, Headspace GC-FID-MS Analysis for Volatiles. Operating Parameter Operating Value Headspace Autosampler Oven Temperature 80C Temperature 120C Transfer Line Temperature 155C Carrier gas He at 2.4 mL/min, constant flow Equilibrium Time 120 min Inject time 0.5 min Loop equilibration time 0.30 min Loop fill time 0.30 min Vial pressurization time 0.30 min GC/MS Analyzer Column J&W DB-WAXETR, 60 m x 0.32 mm I.D., 1 m film Oven Program Start at 35C, hold for 7 minutes. Ramp at 1C/min to 40C, hold for 15 minutes. Ramp at 10C/min to 100C. Ramp at 25C/min to 240 C, hold for 5 min. MS Ionization Mode EI+, 70 eV MS Transfer Line Temp. 240C MS Detection Mass Range 25 – 200 amu Solvent Delay 0 min FID Temperature 260C FID Hydrogen Flow 40.0 mL/min FID Air Flow 400.0 mL/min FID Mode: Constant Makeup Flow FID Makeup flow: 30.0 mL/min FID Makeup gas: Helium Splitter make up gas Helium at 4.0 psi