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