PODP Approach to Acquire Extractable Profile Data - PQRI

PODP E&L 

Working Group 

PODP Approach to Acquire 

Extractable Profile Data 

Thomas Egert 

Alan Hendricker 

Chris Houston 

T. Egert, A. Hendricker, 

C. Houston 

Bethesda, February 2011 

Thresholds and Best Practices for 

Parenteral and Ophthalmic Drug 

Products (PODP) 

February 22-23, 2011


Working Group 



Part 1: The Experimental Protocol 

Thomas Egert 

Research Scientist 

Boehringer Ingelheim Pharma GmbH & Co.KG 



Bethesda, February 2011


Working Group 

PODP 

Experimental 

Protocol 

Summary of Results 

Recommended Best 

Practices for 

Extractables Testing 




PODP Working Plan 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. 

3


Working Group 


Experimental 



Work Plan Outline 

• “. . . In order to test the hypothesis that 

best demonstrated practices for the 

characterization and analytical evaluation 

(of PODP) exist, the Working Group must 

establish such practices …“ 


Practices for 


Motivation for a Experimental 

Program (Phase I): 




• Controlled Extraction 

Studies ! 

4


Working Group 

Coming from OINDP Best Practices: 

Controlled Extraction Studies are the first 

experimental milestone . . . 


Experimental 


Select components and/or raw 

materials 

Conduct rusk assessment on 

information from supplier 

Individual 

ingredient poses 

unacceptable 

risk 

YES 

• Safety Thresholds and 

Best Practices for 

Extractables and 

Leachables in Orally 

Inhaled and Nasal Drug 

Products 2006 


NO 

Conduct controlled extraction 

studies on components 

Individual 

extractable greater 

than or equal to the 

AET/SCT 


Practices for 


Develop and validate 

extraction studies on 

components 

NO 

No further safety assessment 

NO 

YES 

Conduct leachables studies on 

drug product and placebo 

Individual 

extractable greater 

than or equal to the 

AET/SCT 

Establish correlation between 

leachables and extractables 

profiles 

YES 

Report leachable to 

toxicologist for risk 

assessment 

Report extractable to 

toxicologist for risk 

assessment 




Establish acceptance criteria 

for leachables and extractables 

Go to safety qualification 

process 

5


Working Group 


Experimental 




Practices for 


Recapitulation: 

What is the Purpose of a Controlled 

Extraction Study 

• Verify and complement supplier information about 

material 

• Establish a basis for the development and 

validation of routine quality control methods and 

acceptance criteria for critical components 

extractables profiles (consistency in composition) 

• Establish a basis for the development and 

validation of leachables methods 

• Allow for correlation of extractables and leachables 




6


Working Group 

Investigation of Workplan Hypothesis 

Implies to Investigate: 


Experimental 



• Nature of typical PODP materials and their 

(unique?) interactions with typical PODP drug 

product formulations (implications for 

extraction solvents and – techniques) 

• Universe of substances encountered 


Practices for 


• Applicability of AET – related concept (OINDPparadigm) 

to extraction studies on PODP 

materials 




7


Working Group 

Investigation of Workplan Hypothesis 

Implies to Investigate (cont‘d): 

Analytical methods: 


Experimental 




Practices for 


• Appropriate range of instrumental techniques 

(Likelihood of comprehensive evaluation of 

extractables) 

• Identification performance, suitable to generate 

data for safety assessment based on: 

(i) SAR endpoints 

(ii) confirmed identification 




• Sensitivity (in terms of lowest level for 

identification) 

• Specificity (matrix interferences) 

• Limitations (critical substances/mixtures)? 

8


Working Group 

PQRI PODP Experimental Protocol 

The General Concept 


Experimental 




Practices for 





• Test Articles representing PODP materials 

• Appropriate extraction techniques 

• Appropriate solvents 

• Various analytical techniques 

• Various participating laboratories 

(experienced in the field of E&L) 

• Comprehensive and detailed experimental 

protocol 

• Semiquantitative approach – 

Reporting Limit 10 µg/g 

• Quality requirements (system suitability) 

9

PODP E&L Test Articles 

Working Group 

(Material Type) 

Test Articles Representing PODP Materials 

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’-dithiodimorpholine/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 

10


Working Group 


Experimental 


Solvents should mimic drug product 

formulation 

• The majority of PODP are represented by aqueous based 

formulations ! 

• Cosolvents can be subdivided into two groups: 

A: Polarity Neutral 

B: Polarity Impacting 


Primary function of excipient is not drug 

solubilization 

Generally compounds with high 

aqueous solubility: 

Components primary function is to 

increase the solubility of the drug 


Practices for 


• Diluents (dextrose, saline) 

• Buffers (acetate, lactate, bicarbonate, 

phosphate) 

• Amino acids 

• Vitamins 

• Tween 80 

• Cyclodextrins 

• SDS 

• Lipids up to 20% wt/wt 

• Surfactants, Emollients 




Aqueous pH 2.5 / 9.5 

Isopropanol / Water 

11


Working Group 

Extraction/Solvent Map 

Solvent Polarity / 

Drug Product Similarity 

Temp. 

Thermal 

n‐ Isopropanool/Water 

pH 2.5 pH 9.5 

Isopropan‐ 

Aqueous Aqueous 

Hexane 

Headspace X ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 

Reflux ‐‐‐ X X PC/PVC only ‐‐‐ ‐‐‐ 

Soxhlet ‐‐‐ X X ‐‐‐ ‐‐‐ ‐‐ 

Sealed Vessel ‐‐‐ ‐‐‐ ‐‐‐ 55°C/3d 

(121°C/ 

1hr) 1 

Sonication ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ x x 

1 : autoclave conditions: (121°C/1hr) 

(121°C/ 

1hr) 1 




12


Working Group 

Extraction “Paradigms*” in Laboratory Practice 

• Vigorous/exaggerated/- 

exhaustive conditions 

• no material deformulation 

‣„Hot“ extraction techniques 

but no sample dissolving 

solvents 

• Solvents should be 

attributed to the 

expected universe of 

substances (cover 

wide range of polarity) 

• Solvents 

should mimic 

drug product 

formulation 

Thermal 


pH 2.5 pH 9.5 

Isopropan‐ 


Hexane 

Headspace X ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 


Soxhlet ‐‐‐ X X ‐‐‐ ‐‐‐ ‐‐ 


(121°C/ 

1hr) 1 

Sonication ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ x x 


(121°C/ 

1hr) 1 




13


Working Group 

Analytical Methods – General Aspects 

Primary Focus: 

• Specific analytical procedures 


Experimental 


(non specific could have supplemental character (e.g. 

gravimetry, photometry, total organic carbon, alkalinity, acidity, 

reducing subtances, infrared, thermal gravimetry etc.) 

pharmacopoeial testing 



Practices for 





•Non-Target Analysis in addition to targets known from 

composition 

•“Small“ molecules (< 1000 Da) 

•Trace (Organic) Analysis 

•Standard – chromatographic conditions suitable to 

efficiently separate the majority of the log Po/w range to 

be expected. 

•What you might been missing . . .? 

14


Working Group 

Analytical Methods 

…choosing the most adequate tools … 


Experimental 




Practices for 


Inorganic 

Trace elements and metals 

ICP/MS 

Organic 

Volatiles substances: 

Static Headspace-GC/MS 

Semivolatiles (GC-amenable) 

GC/MS 

Semivolatiles (not GC-amenable) 

LC/UV 

LC/MS n , HRMS 




Philosophy: 

Identification to an extent practicable . . . 

(OINDP Best Practices) 

15

Conditions 

for Analytical 


Working Group 

Methods 

(Chromatography) 

Headspace-GC/MS 

Column: 

DB WAXETR 

60 m x iD 0.32 mm, FT 1.0 µm 

He 5 psi 

GC/FID (MS) 

Column: 

DB 5HT 

30m x 0.25 mm, FT 0.25µm 

LC/UV (MS) 

Column: 

Agilent Zorbax Exclipse Plus 

C18, 100 x 3.0 mm, 

3.5 µm particles 

Oven-Program 

35°C-7 min-1 K/min - 40°C – 15 min - 

10 K/min – 100 - 25 K/min – 240 - 5 min 

MS: EI 70 eV, 25 – 200 amu 

Headspace-Cond.: 80°C - 120 min 

Oven-Program 

50°C – 5 min – 10 K/min – 330°C – 5min 

Inj.-Vol. 1 µl, splitless 

Injector: 310 °C 

FID: 150°C 

MS: EI 70 eV, 33-650 amu 

Mobile-Phase 

A : 10 mM ammonium acetate 

B : acetonitrile 

Flow rate: 0.8 mL/min 




Column Oven : 40 °C 

Gradient: 

Sample Size: 10 µl Time: %B 

0.0 5.0 

Detection 8.4 100.0 

UV 205 - 300 nm 35.0 100.0 

MS API-ES positive and 36.0 5.0 

negative ion (mass 39.0 5.0 

range 80 - 1200) 

16


Working Group 

Identification to “the Extent Practicable”… 

Identification attributes according to OINDP Best Practices 

have been applied 

Category 

Supporting Identification Data 


Experimental 


A 

B 

Mass spectrometric fragmentation behaviour 

Confirmation of molecular weight 

C 

Confirmation of elemental composition 


D 

Mass spectrum matches automated library or 

literature spectrum 


Practices for 





E 

Confimed 

Confident 

Tentative 

Mass spectrum and chromatographic retention 

index match authentic specimen 

Categories A, B(or)C and D(or)E fulfilled 

Sufficient Data to preclude all but the most 

closely related structures have been obtained 

Data have obtained that are consistent with a 

class of molecule only 

Confir 

med ID 

SAR 

Endpoints 

17


Working Group 

System Suitability Requirements were 

applied to monitor result integrity 

Level 1 - Qualified Instrumentation 


Experimental 




Practices for 





•Proper instrument condition and instrument suitabilty will be demonstrated 

by each participating laboratory by following its proprietay (inhouse) 

procedures 

Level 2 – System suitability mixtures 

•Specific test mixtures to be analyzed by HS-GC, GC, LC and ICP 

•Test mixtures are suitable to demonstrate adequate and effective analytical 

performance (separation efficiency, selectivity, sensitivity) 

•Results be evaluated against defined acceptance criteria 

Level 3 - Internal standardization 

•Surrogate Internal Standard – added to the extract 

control of effectiveness of the sample preparation process 

•Injection Internal Standard – added to the injection solution 

control of the sample introduction and chromatographic process for each 

sample run. 

18


Working Group 

Suitability Mixtures HS-GC and GC 

HS- 

GC Custom-Mix: (µg/vial) 


Experimental 


methanol 2 

acetic acid 2 

cyclohexanone 1 

toluene 1 

trimethylsilanol 2 

2-ethyl hexanol 2 

dance 

2e+07 

1.9e+07 

1.8e+07 

1.7e+07 

1.6e+07 

1.5e+07 

1.4e+07 

1.3e+07 

1.2e+07 

1.1e+07 

1e+07 

9000000 

8000000 

7000000 

6000000 

5000000 

4000000 

3000000 

2000000 

1000000 

16.608 

TIC: 9NOV2009003.D\ data.ms 

33.201 

32.014 

31.309 

39.182 

37.957 

39.064 


GC “Grob”-Mix: (µg/ml) 

> 

10.00 15.00 20.00 25.00 30.00 35.00 40.00 


Practices for 


L(+)-2,3-butanediol 27 

n-decane 14 

2,6-dimethylaniline 16 

2,6-dimethylphenol 16 

methyl decanoate (C10:0) 21 

methyl docecanoate (C12:0) 21 

methyl undecanoate (C11:0) 21 

nonanal 20 

1-octanal 18 

n-undecane (C11) 14 

1300000 

1200000 

1100000 

1000000 

900000 

800000 

700000 

600000 

500000 

400000 

300000 

1 

200000 

100000 

Time--> 

2 

Signal: 1201014.D\FID1A.CH 

6 

7 

5 

9 

11 

3 

8 

12 

10 

4 

Grob Mix (1/100) 

8 9 10 11 12 13 14 15 




19


Working Group 

Suitability Mixtures LC and ICP 

LC 

(UV/MS) Custom-Mix: (µg/ml) 


Experimental 



caprolactam 1 

butylatedhydroxytoluene 5 

diphenylamine 5 

mono-(2-ethylhexyl) 

1 

phthalate 

stearic acid 5 

di-(2-ethylhexyl phthalate) 1 

bisphenol A 1 

DAD1 A, Sig=210,20 Ref =360,100 (JIJ2009\JI000003.D) 

mAU 

-500 

0 2 4 6 8 10 12 14 16 

MSD1 TIC, MS File (JIJ2009\JI000003.D) API-ES, Neg, Scan, Frag: 100, "Negativ e" 

50000 

0 2 4 6 8 10 12 14 16 

MSD2 TIC, MS File (JIJ2009\JI000003.D) API-ES, Pos, Scan, Frag: 100, "Positiv e" 

200000 

0 

0 2 4 6 8 10 12 14 16 

MSD2 114, EIC=113.7:114.7 (JIJ2009\JI000003.D) API-ES, Pos, Scan, Frag: 100, "Positiv e" 

min 

min 

min 

0 

0 2 4 6 8 10 12 14 16 

MSD1 227, EIC=226.7:227.7 (JIJ2009\JI000003.D) API-ES, Neg, Scan, Frag: 100, "Negativ e" 

min 


Practices for 


0 

0 2 4 6 8 10 12 14 16 


0 

0 2 4 6 8 10 12 14 16 


min 

min 

20000 

0 

0 2 4 6 8 10 12 14 16 


min 

20000 

0 

0 2 4 6 8 10 12 14 16 

MSD2 391, EIC=390.7:391.7 (JIJ2009\JI000003.D) API-ES, Pos, Scan, Frag: 100, "Positiv e" 

min 




ICP all target elements 0.25 

100000 

0 

0 2 4 6 8 10 12 14 16 

20 

min


Working Group 

Surrogate Internal Standard 

Internal Standards 

Objective Requirements Substance used 


Experimental 


Monitoring of 

sample 

preparation 

process and 

instrumental 

performance 

• sufficiently stable 

• sufficiently soluble in all extraction 

solvents 

• amenable to back-extraction from 

aqueous extracts by organic solvents 

• amenable to TMS-derivatization 

• semi-volatile 

• amenable to all detection principles 

• selectively detectable 

Bisphenol M 

CAS 13595-25-0 

MW: 346.46 


Injection Internal Standard 

Objective Requirements Substance used 


Practices for 


Monitoring of 

instrumental 

performance 

• sufficiently stable 

• sufficiently soluble in final extract 

• semi-volatile 

• amenable to all detection principles 

• selectively detectable 

Irganox 415 

CAS: 96-69-5 

MW: 358.538 




Headspace Internal Standard 

1,4-Dioxane 

CAS: 123-91-1 

MW: 88.11 

21

Experimental 

Workflow 


Working Group 




22

Experimental 

PODP Workflow E&L 

Working Group 

Phase 1/3: Extract Preparation 

Thermal 


pH 2.5 pH 9.5 

Isopropan‐ 


Hexane 

Headspace X ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 


Soxhlet ‐‐‐ X X ‐‐‐ ‐‐‐ ‐‐ 


(121°C/ 

1hr) 2 

(121°C/ 

1hr) 2 

Sonication ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ x x 

1 : All test articles (materials) were extracted following this scheme if not indicated otherwise 


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) 

Test Articles: 

Sample Weight [5g] 

Sample Pre-treatment 

Materials: 

LDPE 

PC 

PVC 

COC 

Rubber 

HS-GC/MS(FID) 

ICP/MS(AES) 

Aqueous Extract 

pH 2.5 

200 ml 

Aqueous Extract 

pH 9.5 

200 ml 

IPA/Water extract 

200 ml 

IPA extract 

200 ml 

N-Hexane extract 

200 ml 




23

Experimental 


Working Group 

Phase 2/3: Sample Preparation 




24

Experimental 


Working Group 

Phase 3/3: Instrumental Analysis 




25


Working Group 

Challenges Ahead: 

What could have been missed? 

Example: Epoxidized soybean oil (specif. PVC stabilizer): 


Experimental 


Mixture of glycerol esters – 

75 possible structures 

consisting of: (~ %) 

Epoxidized trilinolein 

C 57 H 98 O 12, MW 975.45 g/mol 

O 

Palmitic acid C16:0 11 

O 


Stearic acid C18:0 5 


Practices for 


Epoxidized oleic acid C18:1 8 

Epoxidized linoleic acid C18:2 23 

Epoxidized linolenic acid C18:3 54 

O 

O 

O 

O 

O 

O 

O 

O 

O 

O 




• Identified and quantified ? 

• Almost missed ? 

• Completely missed ? 

26


Working Group 



Part 2: Summary of Results 

Alan Hendricker 

Catalent Pharma Solutions 




February 22-23, 2011 



Bethesda, February 2011


Working Group 


Experimental Protocol 



Practices for 





Test Articles for Extractables Studies 

Material Type 

Low density polyethylene 

(LDPE) 

Material 

Application 

Test Articles. 

Material 

Format 

Composition 

Overpouch Blown Film Dow 640-I LDPE resin; 

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 

Cyclic Olefin (COC) Syringe barrels, vials Plaques Irganox 1010, Ultramarine 

Blue 

Polycarbonate (PC) Port Tubes Injection 

molded plaques 

Poly (vinyl chloride) 

(PVC) 

Rubber (Elastomer) (RE) 

Solution Bags, 

tubing 

Gaskets, stoppers, 

closures 

0.05 Parts per Hundred (PHR) 

Irganox 1076, 0.1 PHR 

Irgafos 168 

Pellets PVC resin; DEHP 30%; 

Epoxidized oil 7%, Zn 

stearate 0.5%; Ca stearate 

0.5%; Stearamide 1% 

Sheets 

Brominated isobutylene isoprene 

copolymer (57.3%); calcined 

aluminum silicate, 38.2%, titanium 

dioxide, 1.2%; paraffinic oil, 1.2%; 

zinc oxide, 0.6%; polyethylene, 

0.6%; SRF Carbon block mixture, 

0.4%; calcined magnesium oxide, 

0.3%; 4,4’-dithiodimorpholine/polyisobutylene, 

0.3%


Working Group 

Poly(vinyl chloride) PVC Test Material 

Metals Results all less than 5 µg/g. Calcium and Zinc 

(known additives) detected in most extracts. Maximum 

Calcium 1.72 µg/g, Zinc 1.33 µg/g 





Practices for 


Volatiles testing (HS-GC-MS) showed primarily aliphatic 

hydrocarbons of short chain length, branched and 

unbranched. One volatile identified was attributed to 2-ethyl- 

1-hexanol, a known extractable from PVC. 

Semivolatiles testing (GC-MS) showed a large number of 

peaks (up to 50) for each extraction condition. Peaks were 

attributed to fatty acids, fatty amides, fatty alcohols, fatty 

acid esters, fatty aldehydes, aliphatic hydrocarbons, BHT, 

phthalates, phthalate esters and phthalate degradation 

products. Fatty acids and related peaks were observed at 

the highest concentrations (up to around 500 ppm). 

Non-volatiles testing (LC-MS) confirmed GC-MS results, 

peaks detected were attributed to fatty acids, fatty amides 

(erucamide, (z)-9-Octadecenamide), and phthalates. 

Example of a PVC 

IV Bag 




29


Working Group 

Polycarbonate (PC) Test Material 

Metals results showed only trace level metals except in one 

extraction condition (Autoclave/pH 2.5) which showed 

approximately 50 µg/g of Zn and 25 µg/g of Ca. 



Volatiles testing using HS-GC-MS showed only low levels of a few 

volatiles with a maximum concentration of 0.12 µg/g for nonanal. 

Acetone and acetonitrile were also observed at trace levels. 



Practices for 





Semivolatiles testing of extracts using GC-MS showed a 

significant number of peaks, including several phenolic peaks 

which are likely degradation products of the polymer itself. Most 

notable of these was Bisphenol-A, a compound of significant 

potential toxicity. It is not known the extent to which this species 

is generated via the extraction versus being present at 

endogenous levels. Other species detected included Irgafos 168 

and its oxidation product, Irganox 1076, 2,4-di-t-butylphenol and 

dimethyphthalate. 

Non-volatiles testing (LC-MS) showed a significant number of 

peaks, many of which overlapped and for which first pass 

identification was not possible. They may be various oligomeric 

fragments related to the base polymer. 

Example of a 

Polycarbonate 

Baby Bottle (no 

longer marketed) 

30


Working Group 



Cyclic Olefin Copolymer (COC) Test 

Material 

Metals results showed only trace levels of metals in general. 

One extraction condition (Autoclave/pH 2.5) showed 

approximately 25 µg/g of Mg 44 µg/g of Zn and 68 µg/g of 

Bromine. 

Volatiles testing using HS-GC-MS showed very clean 

profiles, only one peak attributed to Cis-decahydronaphthalene 

was observed at 0.03 µg/g. 



Practices for 


Semivolatiles testing of extracts using GC-MS showed a 

large number of peaks, especially via sealed vessel 

extraction. Peaks detected were attributed to fatty acids, 

fatty acid esters, siloxanes, phthalate related peaks and a 

significant number of peaks which could not be given first 

pass identifications. The intensity of peaks observed in all 

extracts was very small, typically less than 0.1 µg/g. With 

several extraction technique, no discernable extractables 

could be detected using this method. 

COC Syringe 




Non-volatiles testing (LC-MS) showed no peaks in sealed 

vessel or sonication extractions. Reflux extraction in IPA 

produced several low level peaks which could not be 

identified (less than approximately 0.1 µg/g). 

31


Working Group 




Low density polyethylene (LDPE) 

Test Material 

Metals testing results showed no significant metal 

extractables (all less than 1 µg/g). Calcium (known 

additive) was not detected under any extraction 

condition. 

Volatiles testing using HS-GC-MS showed only one peak 

above the AET, which was late eluting and could not be 

identified. It was present at 0.26 µg/g. 


Practices for 


Semivolatiles testing of extracts using GC-MS showed 

BHT (antioxidant), Z-9-octadecenamide (antistatic) 

erucamide (antistatic) and Dilauryl-3,3'-thiodipropionate 

or Irganox PS 800 (heat stabilizer and antioxidant). A 

number of aliphatic hydrocarbons branched and 

unbranched can be observed, oligomeric fragments of 

the PE itself. 

Polyethylene 

resin beads 




Non-volatiles testing (LC-MS) showed no additional 

identifiable extractables for the tests performed, though 

several peaks were observed at 220nm. 

32


Working Group 



Rubber Elastomer (RE) Test Material 

Metals results showed significant amounts of Bromine, up to 20 

µg/g, which was not surprising, since it was a bromobutyl 

elastomer. Smaller amounts (all less than 10 µg/g) were detected 

of the other known metal additives, including Mg, Al, Ca, Ti and 

Zn. The pH 2.5 sonication extraction showed the highest levels of 

these metal species. 



Practices for 


Volatiles testing using HS-GC-MS showed several peaks 

attributed to butyl oligomers. Peaks attributed to methyl 

cyclopentane and cyclohexane were also observed. All peaks 

except cyclohexane were less than 1 µg/g, except 

methylcyclopentane at 1.19 µg/g. 

Semivolatiles testing of extracts using GC-MS showed an 

extremely complex extractables profile, with large numbers of 

unknowns. Peaks identified were attributed to fatty acids, fatty 

acid esters, fatty amides, aliphatic hydrocarbon species, BHT, and 

brominated oligomers. 

Example of 

Elastomeric 

Stoppers 




Non-volatiles testing (LC-MS) showed a complex unresolved 

envelope of dozens of peaks, especially for organic extracts. The 

method utilized was insufficient for peak identifications in most 

cases. 

33


Working Group 

Observations 



• Profiles can be complex! With 

multiple extraction solvents and 

techniques hundreds of peaks can be 

extracted using the chromatographic 

methods 


• AET should be employed to help 

simplify data analysis 


Practices for 





• In general, residual solvents and 

metals testing showed low levels of 

extractables and were generally 

innocuous species, but provided 

valuable information 

See the forest 

through the 

trees? 

34


Working Group 

Observations 





Practices for 





• Understand that the peaks you are 

detecting are not necessarily what was 

added to the polymer originally (e.g. 2,4- 

di-t-butylphenol and palmitic acid can 

originate from Irgafos 168 and Calcium 

Stearate, among others) 

• Use “realistic” extraction solvents to 

understand risk. Use aggressive solvents 

to facilitate identifications 

• Oligomeric fragments represent 

challenging species for complete 

structural identification. However, 

compound class may be enough to 

assess toxicological risk 

Abundance 

Time 

55000 

50000 

45000 

40000 

35000 

30000 

25000 

20000 

15000 

Time--> 

Response_ 

1300000 

1200000 

1100000 

1000000 

900000 

800000 

700000 x 

600000 

500000 

400000 

300000 

x 

1 

1 

3 

Signal: RS015.D\FID1A.CH 

Signal: RS020.D\FID1A.CH (*) 


Underivatized PVC pH9.5 Samples Overlay 

A - Internal Standard 1 (Irganox 415) 

27 

B - Internal Standard 2 (Bisphenol M) 

9 

13 

14 

22 

18 21 

19 23 

25 

6 8 10 12 14 16 18 20 22 24 26 

UNDERIVATIZED RUBBER ELASTOMER SAMPLES OVERLAY 

Signal: RS006.D\FID1A.CH 



x - Peaks with this symbol are similar in size, Extract vs Extraction blank 

x 

x 

x 

2 

3 

4 

5 

8 

9 

11 

10 

10 

11 

11 

14 

16 

15 

18 

17 

200000 1 

x 

18 

RE-pH2.5-1 

100000 

6 8 10 12 14 16 18 20 22 24 26 

16 

Do you see the 

trees now? 

20 

20 

21 

21 

30 

24 

25 

32 

26 

28 

26 

28 

29 

32 

36 

30 

ISTD1 

38 

A 

ISTD2 

B 

RE-IW-2 

RE-pH9.5-2 

35 

PVC-pH9.5-2 

PVC-pH9.5-1 

pH9.5-Blank-2


Working Group 

Observations 

• Solvents pH may change extractables 

(specific example shown subsequently) 

DAD1 A, Sig=220,4 Ref=550,50 (F:\3\500_B...SETS\50_RESULTS_MATERIALS\03_RE\LC-UV\013-1101_REF3_NHEX_RE.D) 

mAU 

400 

300 

200 

7 . 5 5 3 7 . 7 5 2 

8 . 6 6 0 

1 0 . 1 4 9 




• Use HPLC-MS to complement GC-MS. As 

a standalone technique data interpretation in 

LC-MS can be difficult 

100 

0 

-100 

DAD1 A, Sig=220,4 Ref=550,50 (F:\3\500_B...SETS\50_RESULTS_MATERIALS\03_RE\LC-UV\013-1101_REF3_NHEX_RE.D) 

mAU 

400 

300 

200 

100 

9 . 2 6 7 

1 0 . 4 4 3 

1 0 . 8 9 9 

1 1 . 8 2 7 

.1 2 4 

1 2 . 4 4 7 

1 2 . 7 5 5 

1 3 . 23 17 80 

1 3 .9 2 6 

1 4 . 4 7 2 

2.5 5 7.5 10 12.5 15 17.5 20 22.5 

1 5 . 5 0 2 

1 6 . 6 1 7 

1 7 . 9 2 1 

min 

0 


Practices for 


• In some cases we did not observe known 

additives. Understand you may need to alter 

extraction conditions in some cases to 

facilitate extraction. Or realize that you may 

never see them even through due diligence 

(e.g. consumed processing agent) 

-100 

5 10 15 20 

min 




Where did my 

trees (peaks) go? 

36


Working Group 



Observations 

How low could we go? 

• By applying state-of-the-art analytical procedures and 

instrumentation, the limits of identification for single chemical 

entities (extractables) observed were in the 

range of 0.1 – 100 µg/g of material… 



Practices for 


Phthalic 

anhydride 

3 µg/g 




GC/MS – Chromatogram: Isopropanol (Reflux) extract of PVC 

37


Working Group 



Observations (and Last Tree Analogy) 

In the end, materials extractables characterization can produce a complex 

scene, difficult to interpret or understand but full of information and when 

done correctly, paints a great picture. See the posters for the full picture 

(warning: not quite as nice as the one below). 



Practices for 





Maroon Bells, Aspen Colorado 

38


Working Group 




Recommended 








Part 3: Recommended Best 

Practices for Extractables Testing 

Christopher T Houston 

Principal Scientist, Bausch + Lomb 




February 22-23, 2011


Working Group 

Best Practices 




Recommended 



• In September 2006, PQRI issued prior 

guidance for OINDP: 

• “Safety Thresholds and Best Practices for 

Extractables and Leachables in Orally Inhaled and 

Nasal Drug Products” 

• Contained 10 best practices with respect to 

controlled extraction studies 

• Most of these are quite relevant to PODP 

• PODP nuances result from aqueous drug product 

formulations 




40


Working Group 




Recommended 






Review of OINDP Best Practice 

Recommendations 

1. Controlled Extraction Studies should employ vigorous extraction with multiple 

solvents of varying polarity 

2. Controlled Extraction Studies should incorporate multiple extraction techniques 

3. Controlled Extraction Studies should include careful sample preparation based on 

knowledge of analytical techniques to be used 

4. Controlled Extraction Studies should employ multiple analytical techniques 

5. Controlled Extraction Studies should include a defined and systematic process for 

identification of individual extractables 

6. Controlled Extraction Study “definitive” extraction techniques/methods should be 

optimized 

7. During the Controlled Extraction Study process, sponsors should revisit supplier 

information describing component formulation 

8. Controlled Extraction Studies should be guided by an Analytical Evaluation 

Threshold (AET) that is based on an accepted safety evaluation threshold 

9. Polyaromatic hydrocarbons, N-nitrosamines, and 2-mercaptobenzothiazole (MBT) are 

considered to be “special case” compounds, requiring evaluation by specific 

analytical techniques and technology defined threshold 

10. Qualitative and quantitative extractables profiles should be discussed with and 

reviewed by pharmaceutical development team toxicologists so that any potential 

safety concerns regarding individual extractables, i.e. potential leachables, are 

identified early in the pharmaceutical development process 

41


Working Group 




Recommended 



“Controlled Extraction Studies should 

employ vigorous extraction with multiple 

solvents of varying polarity” 

• Prior OINDP recommendation: 

• Advocated use of water in extraction studies for 

aqueous products 

• Discouraged using water as the sole extraction 

solvent for components from aqueous drug products 

• PODP Experience: 

• Aligned with prior recommendation (Examples 1 - 3) 

• Relevance of pH (Examples 4 and 5) 




42


Working Group 

Example 1: 

LDPE / Aqueous Drug Product 




Recommended 



• In PODP protocol, extracted LDPE with known 

additive package by multiple solvents / 

techniques including water 

• In ophthalmology, significant prior art for 

extractables used water as the only solvent 

• Although aqueous solvent extracts may be 

useful for gauging significant extractables, they 

may not promote understanding of the material 




43


Working Group 

Example 1: 




Known 

Additive 

Sonication 

pH 2.5 

Sonication 

pH 9.5 

Sealed 

Vessel 

IPA/Water 

Irganox 1010 


Irgafos 168 

Recommended 






BHT 

Ca Stearate 

(as stearic acid) 

Erucamide 

Red = Not detected / Green = detected by GC and/or HPLC 44


Working Group 

Example 1: 





Recommended 



• Sonication in aqueous solvents successfully detects 

erucamide, but no other anticipated additive 

• Solvents of different polarity provide better 

understanding of the material 

• Relevant to an aqueous product? 

• An aqueous extraction profile lacking extractables does 

not necessarily impart material knowledge 

• Though non-polar additives such as Irganox 1010 are less 

likely to migrate into aqueous product, its identification 

alerts the researcher to look for more polar degradation / 

transformation products 




45


Working Group 

Example 2: 

Extraction of DEHP from PVC 



• PVC material contained 30% bis(2-ethylhexyl) 

phthalate (DEHP) 

• Sealed vessel extracts: 

DEHP 


Recommended 



IPA/Water 

pH 9.5 

pH 2.5 




46


Working Group 




Recommended 



Example 2: 

Extraction of DEHP from PVC 

• DEHP is a significant extractable from PVC 

• Aqueous (pH 2.5, 9.5) solvent detect 

significantly less DEHP than IPA/water 

• Many “aqueous” formulations are not purely 

aqueous, but contain surfactants or other 

excipients that enhance solubility 

• Using water as the only extraction solvent runs 

the risk of not observing a key extractable that 

is poorly water soluble but nonetheless 

soluble in drug product 




47


Working Group 

Example 3: 

Solvent Polarity and Polycarbonate 




• The strongest solvent does not always yield 

the worst-case extraction profile 

• Consider this example of isopropanol and n- 

hexane extracts generated by Soxhlet and 

reflux 

Recommended 






48


Working Group 

Example 3: 

Solvent Polarity and Polycarbonate 




Recommended 



Extractable 

IPA 

Reflux 

Estimated Quantity (μg/g) 

IPA 

Soxhlet 

Hexane 

Reflux 

Hexane 

Soxhlet 

4-t-butylphenol 65.7 13.7 2.2 2.9 

2,4-di-t-butylphenol 56.0 24.3 2.0 29.4 

Bisphenol A 77.1 9.4 9.4 12.7 

Irgafos 168 35.9 13.6 0.3 0.8 

Irgafos 168, oxidized 25.1 17.2 0.0 1.6 

Irganox 1076 21.8 8.3 0.1 0.5 




IPA is a superior solvent for many of these 

extractables, despite being “weaker” 

49


Working Group 

Example 4: 

pH Effects and Polycarbonate 



• Polycarbonate extraction at pH 2.5 and 9.5 

BPA 


IPA/ 

Water 

Recommended 



pH 9.5 

pH 2.5 




50


Working Group 

Example 4: 

pH Effects and Polycarbonate 




Recommended 



• Bisphenol A detected from polycarbonate 

• pH 2.5: 0.12 mg/L 

• pH 9.5: 1.7 mg/L 

• Not detected in isopropanol/water extract 

• At alkaline pH, an order of magnitude more 

BPA is observed than at acidic pH 

• Implications for product pH and packaging 

compatibility 




51


Working Group 




Recommended 



Example 5: 

pH and Solvent Effects / PVC 

• Diethylhexyl phthalate (DEHP) and monoethylhexyl 

phthalate (MEHP) detected 

• HPLC semiquantitative results: 

Extractable pH 2.5 

(mg/L) 

pH 9.5 

(mg/L) 

IPA/water 

(mg/L) 

DEHP 0.05 0.79 930 

MEHP 0.02 0.57 0.09 




• DEHP and MEHP are extracted in significantly 

higher quantities at basic versus acidic pH 

• No single solvent system is sufficient for all analytes 

52


Working Group 




Recommended 






“Controlled Extraction Studies 

should incorporate multiple 

extraction techniques” 

• Prior OINDP recommendations: 

• Justification needed for low temperature techniques 

such as sonication with respect to extraction efficiency 

• Sonication found to be less efficient than Soxhlet 

and reflux by OINDP working group 

• Extraction profiles from higher temperature extraction 

techniques should be carefully examined for extraction 

artifacts. 

• PODP Experience 

• Aqueous extraction by sonication versus sealed vessel 

(Example 6) 

• Thermal methods, headspace GC (Example 7) 

53


Working Group 

Example 6: 

Sonication v. Sealed Vessel / Polycarbonate 




Recommended 



• Sealed vessel (121 °C, 1 hr) at pH 2.5 or 9.5 

• Bisphenol A detected 

• 0.12 mg/L @ pH 2.5 

• 1.7 mg/L @ pH 9.5 

• Sonication (2 hr, 0 °C) at pH 2.5 or 9.5 

• No bisphenol A detected, even at high pH 

• Sealed vessel extraction was more efficient than 

sonication in this example 

• Sonication required significant effort to standardize 

temperature relative to sealed vessel 




54


Working Group 

Example 7: 

Thermal Methods / Headspace GC 




Recommended 






• Headspace GC (80 °C, 2 hours) on all materials 

• Observed extractables generally included low levels 

of hydrocarbons 

• Multiple hydrocarbons for PVC ranging from 0.5 to 4.6 

μg/g 

• Butyl rubber related oligomers in the rubber/elastomer 

sample 

• Low parity with other extraction approaches, 

including Soxhlet and reflux with IPA or hexane 

• However… 

55


Working Group 

Example 7: 

Thermal Methods / Headspace GC 




Recommended 



• Headspace GC holds significant value in some 

PODP dosage forms 

• Certain dosage forms in semipermeable 

packaging systems are susceptible to volatile 

leachables from secondary packaging systems 

• Examples to be provided in Ophthalmology 

session 

• Stay tuned for further evaluation of 

headspace… 




56


Working Group 




Recommended 



“Controlled Extraction Studies should 

include careful sample preparation 

based on knowledge of analytical 

techniques to be used” 


• Promoted the notion of solvent exchange when extraction 

solvent not suitable for use with analytical method 


• Solvent exchange (Example 8) 

• Extract concentration, 100X (Example 9) 




57


Working Group 

Example 8: 

Solvent Exchange for HPLC 




Recommended 






• The HPLC method developed for the PODP 

protocol intended to span a broad polarity. 

• Caprolactam included in the system suitability 

mixture as the most polar compound 

• With low retention, caprolactam 

exhibits poor peak shape 

if injection solvent is too strong 

• Underscores necessity for 

solvent exchange from nonpolar 

extracts 

58


Working Group 

Example 9: 

100x Extract Concentration 




Recommended 



• Aqueous extracts were back-extracted into 

methylene chloride for GC analysis 

• A 100x concentration step was performed to 

increase sensitivity 

• In such concentration schemes, care must be 

taken to ensure that background contaminants 

are not concentrated with samples… 




59


Working Group 

Example 9: 

100x Extract Concentration 




Recommended 






GC-MS, total ion chromatograms 

60


Working Group 

“Controlled Extraction Studies 

should employ multiple analytical 

techniques” 




Recommended 




• No single analytical technique can detect and 

identify all possible extractables 

• Techniques used should be “compound specific” 

• Detector response proportional to extractable 

quantity 


• Aligned with prior recommendation (Example 10) 




61


Working Group 




Recommended 



Additives 

Example 10: 

Extractables Detected in LDPE 

Irganox 1010 

Irgafos 168 

BHT 

Stearic acid 

Erucamide 

Irganox 1010, related 

Irgafos 168, related 

Detected by HPLC 

(UV,MS) 

Detected by GC 

(FID,MS) 

DEHP* 




Oleamide* 

* Not included in resin composition information 62


Working Group 




Recommended 






Example 10: 

Extractables Detected in LDPE 

• Strength of multiple analytical survey techniques for organic 

extractables: 

• Although overlap exists, HPLC and GC contain some 

complementary data 

• Where overlap exists, the different techniques provide confirmation 

and may aid in identification 

• Multiple survey methods allows for broader characterization, 

including the identification of unanticipated extractables (DEHP and 

oleamide in Example 9) 

• Inorganic analyses 

• The PODP protocol also included ICP-MS for elemental analysis 

• If metals are a concern, the added orthogonality of atomic 

spectroscopy is critical 

63


Working Group 




Recommended 






“During the Controlled Extraction Study 

process, sponsors should revisit 

supplier information describing 

component formulation.” 


• Check list of experimentally-derived extractables 

against supplier information 

• Are anticipated extractables observed? 

• Are other extractables observed? 

• Supplier information can serve as a starting point 

for the development of analytical methods 


• Experimental work can reveal extractables that 

were not anticipated based on supplier information 

(Example 11) 

64


Working Group 

Example 11: 

Extraction Studies of COC 




Recommended 



• Additives known to be formulated into the COC: 

• Irganox 1010, ultramarine blue (pigment) 

• Noteworthy examples of extractables observed during the 

PODP study of COC: 

• Irganox 1010, monoethylhexyl phthalate, DEHP, cis/transdecahydronaphthalene, 

oleamide, hexadecanoic acid, octadecanoic 

acid 

• Additional extractables observed from COC material beyond 

those anticipated from supplier information 

• Sponsors cannot claim to understand critical component 

chemistry simply on the basis of supplier information 




65


Working Group 




Controlled Extraction Study “definitive” 

extraction techniques/methods should 

be optimized 

• Prior OINDP Recommendations: 

• After first pass extraction studies, development 

team should choose a “definitive” method / 

technique to optimize. 

• Complete validation is not recommended or 

expected for Controlled Extraction studies 

• Method should be demonstrably fit for purpose 

with respect to accuracy and precision 


• Some extractables may require unique or 

dedicated methods to meet this objective 

(Example 12)


Working Group 

Example 12: 

“Borderline” Analytes (ESBO) 



ESBO „Pattern“ in LC/MS 

( not quantifiable) 


O 

O 

O 

Recommended 



Epoxidized trilinolein 

C 57 H 98 O 12, MW 975.45 g/mol 

O 

O 

O 

O 

O 

O 

O 

O 

O 




Epoxidized soybean oil from PVC 

67


Working Group 

Conclusion 




Recommended 



• The PODP protocol underscores the value of 

extrapolating the OINDP best practice 

recommendations to other dosage forms 

• For many PODPs, water is an essential solvent and 

suggests nuances on OINDP recommendations 

• Although the OINDP recommendations discuss water, that 

work focused on more non-polar solvents (isopropanol, 

hexane, methylene chloride) 

• PODP work demonstrates the importance of pH as a 

consideration for extraction solvent selection 




68

PODP Approach to Acquire Extractable Profile Data - PQRI

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