USP Updates on Extractables and

Leachables (E&L)

Ravi Kiran Kaja, Ph.D.,

Senior Principal Scientist,
Complex Generics,
General Chapters – USP

Aug 26, 2024

USP Open Forum
USA
Background

USP E&L System Suitability Standards Proposal:

 Headspace GC-MS
 GC-MS
 LC-MS, APCI with positive and
negative ionization
 LC-MS, ESI with positive and
negative ionization

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 Recap: USP interest on E&Ls?
 Qualitative Survey (2021)
– Small survey (14, global)
 Quantitative Survey (2023)
– Large study (356, global)
 Email communication to the USP staff
• Feedback:
-USP should prioritize the E&Ls topic due to growth and uncertainty.
-USP should offer E&L reference standards and/or mixtures, including system suitability
standards.
-USP should offer a digital library to help identify relevant E&Ls.
-USP should provide more guidance on extractable and next steps after they have been
identified, including providing AET limits.
-USP offering training and educational courses related to E&Ls.

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 Respondents see value in USP developing extractables system
suitability standards (62% rate it highly/very valuable); supported by
similar scores for system suitability standards in the E&L 2023 survey
2024 Survey: Do You See Value In USP Developing Extractables System Suitability Standards?

23% 39% 28% 7% 4%

Highly valuable Very valuable Neutral Not very valuable Not valuable at all

Opinions on system suitability standards tested in

Feedback Survey 2023
E&L Stimuli Article

previous E&L study System

suitability
➢ Over 7 in 10 feel that there is value in having a reference standards
standards solution to facilitate system suitability testing
during regulatory submissions tested in
2023
➢ Over 7 in 10 agree that there is a need for a universal set
of standards for system suitability testing.

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Q13. In your opinion, do you see value in USP developing extractables system suitability standards? Base, n =181 © 2019 USP
Background: Stimuli article published USP-PF
49(4), July 2023

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 Major Comments on the Stimuli Article:
 Relevance of proposed standards:
– Concerns for the relevance of some of the proposed standards, ex., Toluene is not an extractable
 Compounds selection:
– Selection of compounds should be material (packaging or manufacturing component) specific
 Round-robin testing:
– Majority mentioned to perform a round-robin testing at multiple labs for a wider acceptability
– Results presented from only one lab
 Cost-effectiveness of standards:
– Use of vast number of standards for “routine use” may not be cost effective or scientifically justifiable
 Evaluation of non-MS and other methods:
– GC-FID, CAD, use of Acetonitrile and Methanol, multiple columns, etc.,
 Flexibility in method parameters:
– Flexibility regarding the instruments specified, like using other similar instruments from other vendors
 Premixed standards:
– Lot of interest for pre-mixed standards than individual
 Clarity on linking to documentary standards:
– Will this be added to any current USP chapters?
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Headspace GC-MS Method Parameters
Instrument: SHIMADZU -TQ8050NX triple quad mass spectrometer with AOC-20S
autosampler

Column: DB-624 60-m x 0.25-mm x 1.4 µm (Agilent part number 122-1364)

Instrument Operating Parameters:

Headspace Parameters: Vial Temp: 75°C, Sample Line Temp: 85°C Transfer Line Temp: 95°C
Vial Equilibration Time: 20 min, GC Cycle Time: 65 mins

GC Parameters: Carrier Gas and Flow: He in constant flow 1.5mL/min, Total Flow: 19.5mL/min,
Total Program Time: 45 mins

Injection Mode: Interface Temp: 250 °C Ion source Temperature: 250 °C

Oven Program: Initial 3 min at 45 °C, to 90 °C (4 min) at 8°C/min
to 200 °C ( 3 min) at 5 °C/min to 220 °C (5.37 min ) at 10 °C/min

MS Parameters: Ionization Mode: Electron Impact

Acquisition Mode: Full Scan
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Mass Range (m/z): 35 – 300 © 2019 USP
 Revised List of Compounds
[Procedure-1: Head Space GC/MS]
Composition of the HS-GC/MS Suitability Mixture: Solvent = UPW

Compound CAS Number Peak Number Retention Time (min) Concentration (mg/L)

Acetaldehyde (New) 75-07-0 1 4.81 1

Dimethoxymethane 109-87-5 2 6.87 0.5

Trimethlysilanol (New) 1066-40-6 3 9.21 1

Methyl cyclopentane (New) 96-37-7 4 9.39 1

Hexamethyl cyclotrisiloxane 541-05-9 5 16.70 1

(New)
Cyclohexanone 108-94-1 6 23.15 1

2-Octanone (New) 111-13-7 7 26.08 1

n-Tetradecane 629-59-4 8 38.05 0.05 8

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Headspace GC-MS Chromatogram

* Blank Peaks
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 GC-MS Direct Injection Method Parameters
GC Parameters:
Instrument: SHIMADZU -TQ8050NX triple quad mass spectrometer with AOC-20S autosampler.
Column: HP-5MS Ultra Inert 30-m × 0.25-mm × 0.25-μm
(Agilent part number 19091S-433UI)
Carrier gas: Helium
Constant flow: 1 ml/min
Injection mode: Splitless
Inlet temperature: 270 °
Injection volume:1 µL

- Rate°/min Value° Hold time (min)

Column oven
conditions Initial - 50 4
Ramp-1 8 300 12

MS parameters:
Ionization mode: Electron impact
Acquisition mode: Full scan
Mass range (m/z): 35-700
Ion source temperature: 250°
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Interface temperature:250° © 2019 USP
Procedure-2: GC-MS (Direct) Compound
Selection
Composition of the GC/MS Suitability Mixture: Solvent = DCM

Compound CAS- Peak Number Retention Time (min) Concentration (mg/L)

number

Methyl methacrylate (New) 80-62-6 1 2.84 10

Cyclohexanone 108-94-1 2 6.78 20

2-Ethyl-1-hexanol 150-13-0 3 9.97 10

Caprolactam 150-60-2 4 14.45 20

n-Nonadecane 629-92-5 5 24.14 5

2-Heptadecanone 2922-51-2 6 24.19 5

n-Nonacosane (New) 630-03-5 7 34.44 10

Irgafos 168 31570-04-4 8 40.45 10

18-Pentatriacontanone 504-53-0 9 45.66 50

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GC-MS Chromatogram

* Blank Peaks

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LC/MS-APCI Method Parameters
Instrument: Shimadzu / LCMS triple quad-8045
Column: Waters Acquity CSH-C18 100-mm × 3.0-mm × 1.7-μm (Waters part number 186005301)
Colum temperature: 40°
Autosampler temperature 5°
Injection volume: 5 µL Time Flow UPW Methan
(min) rate ol
Acquisition mode: Full Scan APCI +/APCI- (mL/mi
Mass range:100-1500 n)

MS conditions: 0 0.5 80 20

Nebulizing gas flow: 3 L/min 7 0.5 0 100

25 0.5 0 100
Interface temp: 350°
26 0.5 80 20
DL temperature:250°
29 0.5 80 20
Heat block temperature: 250°
Drying gas flow : 5 L/min
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 Procedure-3: LC/MS-APCI Compound Selection
Composition of the LC/MS-APCI Suitability Mixture: Solvent = MeOH

Compound CAS-number Peak Number Retention time Mode Concentration (mg/L)

(min)

4-Aminobenzoic acid 150-13-0 1 2.4 +/- 10

Caprolactam 150-60-2 2 2.9 + 1

*N,N-Diethyl cyclohexylamine 91-65-6 3 3.04 + 10

2-Mercaptobenzothiazole 149-30-4 4 5 +/- 1

Propyl paraben 94-13-3 5 6.3 +/- 1

Hostanox 03 32509-66-3 6 8.5 - 1

Erucamide 112-84-5 7 9.9 + 1

Irgafos 168 31570-04-4 8 14.1 +/- 1

1,3 Distearin (New) 504-40-5 9 16.2 + 1

Irganox PS802 693-36-7 10 24.6 + 1

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 LC/MS-APCI Chromatogram

Total Ion Chromatogram (TIC) and Extracted Ion Chromatograms(EIC) of standard mixture in +Ve mode

TIC of standard mixture 5 EIC of Peak-5

1 7 EIC of Peak-7
EIC of Peak-1
Intensity

2 EIC of Peak-2 8 EIC of Peak-8

3
EIC of Peak-3 Negative9 EIC of Peak-9

10
4
EIC of Peak-4 EIC of Peak-10

Time in minutes Time in minutes

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 LC/MS-APCI Chromatogram
Total Ion Chromatogram (TIC) and Extracted Ion Chromatograms(EIC) of standard mixture in -Ve mode

TIC of standard mixture

1
EIC of Peak-1
Intensity

4
EIC of Peak-4

5
EIC of Peak-5

6
EIC of Peak-6

8
EIC of Peak-8

Time in minutes
Note: Peak-1, 4, 5, 6 and 8 are expected in both +Ve and –Ve modes.
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 LC/MS-ESI Method Parameters
Instrument: Shimadzu / LCMS triple quad-8045
Column: Waters Acquity HSS-C18 100-mm × 2.1-mm × 1.8-μm (Waters part number 186003533)
Colum temperature: 40°
Autosampler temperature 5°
Time (min) Flow rate 0.05% formic 0.05% formic
Injection volume: 5 µL (mL/min) acid +UPW acid+
Methanol
Acquisition mode: Full Scan ESI+/ESI- 0 0.25 98 2
Mass range:100-1500 15.5 0.25 35 65
17.5 0.25 2 98
MS conditions:
18 0.25 0 100
Nebulizing gas flow: 3 L/min 19.5 0.25 0 100
Heating gas flow: 10 L/min 29.5 0.25 0 100
32.0 0.25 98 2
Interface temp: 300°
37.0 0.25 98 2
DL temperature:250°
Heat block temperature: 400°
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Drying gas flow : 10 L/min © 2019 USP
 Procedure-4: LC/MS-ESI Compound Selection
Composition of the LC/MS-ESI Suitability Mixture: Solvent = UPW/MeOH 98/2 v/v

Compound CAS-number Peak Number Retention time Mode Concentration (mg/L)

(min)*
Guanine 73-40-5 1 1.7 +/- 1
L-Phenylalanine-15N 29700-34-3 2 5.3 +/- 0.1-1
4-Aminobenzoic acid 150-13-0 3 5.6 + 1
*Nitrosopyrrolidine (New) 930-55-2 4 5.7 + 1

Adipic acid 124-04-9 5 7.3 - 0.1-1

2-Mercaptobenzothiazole 149-30-4 6 14.3 +/- 0.1-1
Propyl paraben 94-13-3 7 17.1 +/- 0.1-1
*N-Lauryl diethanolamine (New) 1541-67-9 8 18.9 + 0.1-1

Stearic Acid (New) 2975-39-3 9 22.5 - 0.1-2

Octadecyl 3-(3,5-di-tert-butyl-4- 2082-79-3 10 27.3 + 0.1-1

hydroxyphenyl)propionate
(Irganox 1076)
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 LC/MS-ESI Chromatogram
Positive Negative
TIC of standard mixture (+Ve mode)
TIC of standard mixture (-Ve mode)

1
EIC of Peak-1
1
EIC of Peak-1

2
EIC of Peak-2

2 EIC of Peak-2
3

Intensity
Intensity

EIC of Peak-3

4
EIC of Peak-4 EIC of Peak-5

5
6 EIC of Peak-6
6
EIC of Peak-6

7 EIC of Peak-7

7
EIC of Peak-7
8 EIC of Peak-8

9
10 EIC of Peak-9
EIC of Peak-10

Time in minutes Time in minutes 19

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 Next Steps
 Round Robin Study:
– 10-12 Labs shown interest to participate for the study
– Create a flexible approach [Not to mention instrument Make/Model/column/Methods etc.,]
– Appropriateness of compounds in the mixtures
– Adequacy of the selection of standards
– Instrument sensitivity
– Column efficiency (critical pair, anchor compounds, etc.,)
– Orthogonal approach
– Method Parameters like Specificity, Precision, Accuracy and Linearity (Acceptance Criteria’s will be proposed
based on the round robin study)
– Timelines may vary depending upon the completion of round robin study [Target: July-Aug 2024]
 Why this proposal?
– Intended only for the system suitability
– To make sure the Instrument is working appropriately prior to the sample analysis
– Not intended for either Extractable or Leachable analysis
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– Revised Stimuli Article to clarify and achieve consensus © 2019 USP
 Rubber Oligomers
 Rubber Oligomers
 Oligomers: C13H23Br/ C13H23Cl and C21H39Br/ C21H39Cl

– Halogenated Cyclic Aliphatic Hydrocarbons (allyl halides)

– Alkylating Agents
– One double bond
– Structure Activity Relationship: Human carcinogenicity is plausible
– High concern

– Often arises due to Polymerization, the rubber curing at high temperatures and an interaction of elastomeric
material with drug product.
– The butyl and halo butyl rubbers are among the most common elastomers used as components for injectable
and other delivery systems
– Difficult synthesize/currently the low availability of both rubber oligomer physical standards and spectra in
most commercial mass spectrometry libraries makes analysis challenging
– Known to draw concern from regulatory authorities about an effective leachable assessment for drug products
– USP is launching a new line of E&L Rubber Oligomer Analytical Reference Materials (ARMs) soon
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 Goal: MOC based E&L Standards

 Packaging or Manufacturing component based standard proposals:

– Currently, Lack of guidance for selection of Reference Standards for E&L Study
– Goal is to design reference standards mixtures suitable for different material of
construction (MOC) based on a priori knowledge
– Based on the specific packaging & manufacturing components (HDPE bottles,
rubber stoppers, IV bags, and laminated pouch’s & Filters, Gaskets, printed label,
tubing, )
– These Standards can be used either for Extractable study, Method Suitability and
Leachable study purposes
Targeted
– Necessary to use authentic standard to measure concentration
– Method becomes highly accurate and more sensitive, selective and precise than
screening…validation 22
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 USP General Chapter Updates:

 USP <1663> and <1664>

– Conducted a survey to identify stakeholder needs (Apr 2024)
– Intended to receive inputs and revise chapters
– Total 190 respondents

 New Chapter Proposals:

– To address special considerations for Leachable assessment in various drug products
– USP Subcommittee is currently working on
• <1664.2> Leachable Chapter for Parenteral Drug Products [Tentative Timeline: FY 25 Q2]
• <1664.3> Leachable Chapter for Ophthalmic Drug Products [Tentative Timeline: FY 25 Q3]
• <1664.4> Leachable Chapter for Topical and Transdermal Drug Products [Tentative Timeline: FY
25 Q4]
• <1664.5> Leachable Chapter for Oral Dosage forms [Tentative Timeline: FY 25 Q3]
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USP E&L Contacts

USP E&L Contacts

 Dr. Ravikiran Kaja, Ph.D,
– Senior Principal Scientist, Complex Generics, General Chapters
– Email: [email protected]

 Dr. G. Prabhakar Reddy, Ph.D.,

– Director-Pharmaceutical Sciences, General Chapters
– Email: [email protected]

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