Detection and identification of potential bioactiveExtractables and leachables from Pharmaceutical Products by using LC-MS analytical technique.

1. Detection & identification of
Potential Bioactive Leachables and
Extractables from Pharmaceutical
Products By Using LC-MS
Presented By: Ashitosh Panchal
M Pharm Sem-I
Pharmaceutical Chemistry
Dr. D.Y. Patil College Of PharmacyAkurdi, Pune

2. Contents
• What are Extractables and Leachables?
• Why do we needs E & Ls Testing?
• Guidelines
• Confident identification
• Different analytical techniques
• Conclusion
• References

3. Introduction
Extractables:
These are chemical compounds and inorganic elements
that can be forced to migrate from the contact material
(container, syringe, tubing etc.) under aggressive
conditions such as extreme surface exposure, elevated
temperature and/or strong solvents.
Leachables:
These are chemical compounds and inorganic elements
that migrate from the contact material into the product
of concern under normal conditions of use. Leachables
are usually a subset of extractables.

4. Who Needs E&L Study?
Biomedical devices Food Packaging Pharmaceutical Packaging

5. Plastic Materials: Source of Contamination
Sources of extractables are plastic and elastomeric components
(monomers, polymeric initiators, plastizicers, etc.) ink and adhesives
(label) and degradation products (processing, storage, sterilization)
GLASS
₋ If glass is coated (with silicone),
a study may need to look
specifically for extractables that
are coming from coating. Needs
to be determined based on the
drug product formulation and
the potential for it to interact
with the coating.

6. Harmful Compounds Identified as
Extractable/Leachables:

7. Case Study Extractables and leachables:
In 2010, Kellogg’s product recall was launched for 28 million boxes of breakfast cereal after
consumers reported a strange taste and odour, with some complaining of nausea and
diarrhoea. These symptoms were subsequently found to be caused by elevated levels of
methylnaphthalene in the product packaging.
Also in 2010, a product recall was launched for a medical product, Tylenol (paracetamol),
used for pain relief and the relief of cold and flu symptoms, after consumer reporting of a
strange odour coming from the bottles in which the product was stored. The odour was
later linked to 2,4,6-tribromophenol, a wood preservative, that was present within wooden
pallets that were used to transfer the product.
These examples and many like them therefore highlight the need to understand potential
sources of leachables and to proactively look for and control them.

8. Risk-Based Approach To Evaluating E&L
Safety Considerations
•Toxicity, immunogenicity etc.
Efficacy considerations
•Leachables interacting with a product
•Loss of activity
•Leachable may induce development of neutralising activity
Quality considerations
•Impact on manufacturing process, product stability etc

10. Guidelines:
• 1993 CDRH - Reviewer Guidance for Nebulizers, Metered Dose Inhalers,
Spacers and Actuators
• 1998 FDA - MDI/DPI Draft Guidance
• 1999 FDA - Guidance for Industry: Container Closure Systems for Packaging
Human Drugs and Biologics
• 2002 FDA – Guidance on Inhalation solution, suspension, spray and nasal
spray products
• 2005 CHMP, CVMP - Guideline for Plastic Immediate Packaging Materials
• 2006 PQRI – Safety Thresholds & Best Practices For Extractables &
Leachables

11. ICH Guidelines:
•ICH Q3A: Chemical Impurities in new drug substances
•ICH Q3B: Impurities in new drug products
•ICH Q3C: Impurities: Residual solvents
ICH Q3E: Guideline for Extractables and Leachables (E&L)
•ICH Q5E: Comparability of biotechnology/biological products subject to
changes in their manufacturing process 2005
•ICH Q7A: GMP of active pharmaceutical ingredients
•ICH Q8: Pharmaceutical Development 2006
•ICH Q9: Quality Risk Management 2006

12. The Analytical Techniques For E & L Analysis

13. Liquid Chromatography-Mass Spectrometry
• Liquid chromatography-Mass spectrometry ( LC-MS ) is a powerful
analytical technique that combines the resolving power of liquid
chromatography with the detection specificity of mass spectrometry.
• Liquid chromatography ( LC ) seperates the sample components and then
introduces to the mass spectrometer (MS). The MS creates and detects
charged ions.
• The LC-MS data used to provide information about the molecular weight,
structure, identity and quantity of specific sample components.
• In LC-MS we are removing the detector from the column of LC-MS and
fitting the column to interface of MS.
• In most of the cases the interface used in LC-MS are ionization source.

14. High Pressure Liquid Chromatography (HPLC)
• Liquid Chromatography generally utilizes very small particles packed
and operating at relatively high pressure, and is referred to as high
performance liquid chromatography.
• Modern LC-MS methods use HPLC instrumentation essentially for
sample introduction.
• In HPLC, the sample is forced by a liquid at high pressure ( the mobile
phase ) through a column that is packed with a stationary phase
generally composed of irregularly or spherically shaped particles
chosen or derivatized to accomplish particular types of separations.

15. Mass Spectrometry
• Mass Spectrometry (MS) is an analytical technique that measures the
mass-to-charge ratio of charged particles.
• MS works by ionizing chemical compounds to generate charged
molecules fragments and measuring their mass to charge ratio.
• In a typical MS procedure, a sample is loaded onto the MS instrument
and undergoes vaporization. The components of the sample are
ionized by one of a variety of methods (eg. By impacting them with an
electron beam) which results in the formation of charged particles
ions. The ions are separated according to their mass to charge ratio in
an analyser by electromagnetic fields.

16. Instrumentation of LC-MS
1. HPLC Constitutes the LC Part:
a) Solvent System ( Mobile Phase )
b) Pumps
c) Mixer
d) Injector
e) Column
2. Mass Spectrometer
A) Ion sources
i) Electrospray ionization
ii) Atmospheric pressure
Chemical ionization
iii) Photoionization

17. B) Mass Analyzers
1. Quadrupole
2. Time-of-flight
3. Ion Trap
4. Fourier transform-ion cyclotron resonance ( FT-ICR )

18. Sample Preparation
Sample extracts were prepared by cutting packaging materials into 5
mm2 pieces and sonicating 1 g of the pieces in 10 mL of 2-propanol for
nine hours at an average temperature of 45 °C.

19. LC Conditions
LC system: ACQUITY UHPLC I-Class FTN
Column(s): CORTECS C18 Column, 90Å, 1.6 µm,2.1 mm X 100 mm
(p/n: 186007095)
Column temp.: 40 °C
Injection volume: 1 µL
Flow rate: 0.4 mL/min
Mobile phase A: Water + 1 mM ammonium acetate +0.1% acetic acid
Mobile phase B: Methanol

20. MS Conditions
MS system: Vion IMS QTof
Ionization mode: ESI+, ESI-
Acquisition mode: HDMSE
Acquisition range: 50–1500 m/z
Scan time: 0.2sec
Source temp.: 120°C
Desolvation temp.: 500 °C
Desolvation gas: 1000 L/hr
Cone gas: 50 L/hr
Reference mass: Leucine enkephalin [M+H]+ m/z 556.276

23. •LC-MS identification of E&Ls

24. Conclusion
• The study has been shown that the LC-MS is the valuable tool
for detection and identification of extractables and leachables.
• Properly conducted controlled extractables and leachables
studies, when accomplished early in the pharmaceutical
development process, permit a pharmaceutical development
team to begin early evaluation of potential drug product
leachables. This evaluation can alert the pharmaceutical
development team to potential leachables with toxicological
concern, allowing adequate time to begin appropriate safety
qualification studies.

25. References:
1. https://www.agilent.com/cs/library/eseminars/Public/Analysis_of_
Extractables_Leachable_Compounds_in_Pharmaceutical_Products
2. www.wikipedia.com_extractables_&_leachables
3. USFDA,CodeofFederalRegulations,21CFR211.94http://www.accessd
ata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=211.94
4. Guidance for industry: Container closure systems for packaging
humandrugsandbiologicshttp://www.fda.gov/downloads/Drugs/Gui
dances/ucm070551.pdf
5. https://www.pall.com/content/dam/pall/biopharm/lit-library/non-
gated/scientificandtechnicalreports/12.5361_USTR2859_Extractabl
es_Leachables_Single-Use_Systems_TR_EN.pdf