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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 xRepeat 1 x
GC/MSD
GC/FID
LC/MSn
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 (TestMixtures):
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 ProcedureSample 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 XPC X X X X X X X X X X X - X X X X X X XPVC X X X X X X X X X X X - X X X X X X XRubber X X X X X X - X X X X - X X X X X X XCOC X X X X X X - X X X X - X(2) X(2) X(2) X(2) X(3) X(3) XGC 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
SamplePrep. (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: OrganicSP2: pH2.5, pH 9.5SP3: 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 280oC, 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 C18, 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
Time
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
min0 2 4 6 8 10 12 14 16
mAU
-600
-400
-200
0
DAD1 A, Sig=210,20 Ref =360,100 (MCI1310\MC000004.D)
CAPMEHPBPA BHT
DEHP
DPA
min0 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, "Positiv e"
CAP MEHP
DPA
DEHP
min0 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, "Positiv e"
CAP
min0 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, "Positiv e"
DPA
min0 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, "Positiv e"
DEHP
5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
4 5 0 0 0 0 0
T im e -->
A b u n d a n c e
S ig n a l: 2 6 JA N 2 0 1 0 0 0 4 s td 1 .D \ F ID 1 A .C H
1 6 .6 6 2 3 1 .3 3 4
3 2 .0 5 1
3 3 .2 2 9
3 7 .9 7 1
3 9 .0 7 0
3 9 .1 9 4
3 1 .0 0 3 2 .0 0 3 3 .0 0 3 4 .0 0 3 5 .0 0 3 6 .0 0 3 7 .0 0 3 8 .0 0 3 9 .0 0 4 0 .0 0 4 1 .0 0
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
4 5 0 0 0 0 0
T ime -->
A b u n d a n c e
S ig n a l: 2 6 JA N 2 0 1 0 0 0 4 std 1 .D \ FID 1 A .CH
3 1 .3 3 4
3 2 .0 5 1
3 3 .2 2 9
3 7 .9 7 1
3 9 .0 7 0
3 9 .1 9 4
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