INTRODUCTION

1. INTRODUCTION

1.1. Pharmaceutical Packaging

Pharmaceutical packaging plays a vital role in ensuring product's safety, stability, and
efficacy, with primary, secondary, and tertiary packaging addressing various needs.
Modern packaging such as the container closure system for the ophthalmic
formulations, prefilled syringes, and dual-chamber device designs enhance safety,
accuracy, and convenience. Pharmaceutical packaging is critical because it protects
medical components (devices/drugs) from the environment and keeps them safe.
Packaging materials protect items by reducing possible harm from external elements
such as light, temperature, moisture, bacteria, particles, and airborne gases.

Pharmaceutical packaging materials or modern drug delivery systems and some

medical devices are meant to protect drug products inside, from environmental
contamination and effects. It plays a crucial role in storage, transport, dispense, sale,
etc. Hence pharmaceutical packaging materials must have compatibility with
pharmaceutical drug products.

Packaging materials should be selected according to the specific characteristics of the

product to avoid issues such as moisture transfer, evaporation of volatile components,
and leaching of contents. Beyond environmental protection, the packaging must also
shield the product from physical damage, including pressure, impact, friction, punctures,
and shocks. It is essential that the packaging does not interact with the product and
maintains its integrity throughout the product’s shelf life. Additionally, it must not trigger
any immune reactions in the patient. Overall, the packaging must be compatible with the
drug formulation.
 1.2. Understanding of Extractable and Leachable

Extractable is any organic or inorganic chemical substance that migrates

from pharmaceutical packaging in laboratory conditions or harsh
conditions
Extractable : Potential Compound Migration

Leachable is both organic or inorganic chemical species released from

manufacturing or pharmaceutical packaging in the actual storage condition
of the drug product. Is called leachable.

Leachable studies are an assessment performed on the drug product.

EXTRACTABLE
EXTRACTABLE

LEACHABLE
LEACHABLE

Figure: Comparison of Extractable and leachable in accelerated and real condition

Extractable is a large set of chemical species that can be leached in drug products after
exaggerated conditions.
It's considered that all extractable can potentially be leachable. But in actual condition,
all extractable could not be leachable.

1.3. Regulatory Guidance

FDA Cosmetic Act Section 501 (a)(3) states that a drug is deemed to be adulterated "if
its container is composed, in whole or in part, of any poisonous or deleterious
substances that may render the contents injurious to health. 510(k) of the US Food and
Drug Administration Act details the experimental design for the medical devices based
on the route of entry of leachable (Moyer, n.d.).

FDA guidance for industry packaging components or container closure systems for
packaging human drugs and biologics states that the possibility of component-dosage
form interaction is high for injectable, parenteral and inhalational products.

USP (United States Pharmacopeia)

• USP<1663> Assessment of Extractables Associated with Pharmaceutical Packaging /

Delivery Systems Revised.

• USP<1664> Assessment of Drug Product Leachable Associated with Pharmaceutical

Packaging / Delivery Systems Revised

EP (European Pharmacopeia)

• EP 3.1 Materials used for the manufacture of containers

• EP 3.2 Containers for pharmaceutical use

ICH (International Conference of Harmonization)

ICH Q3E, Specifications: Test procedures and acceptance criteria for new drug
substances and new drug products: chemical substance.
ISO (International Organization of Standardization)

• ISO10993 Part 12- For sample preparation and reference materials &

Biological Evaluation of Medical Devices

• ISO 10993 Part 17- Establishment of allowable limits for Leachable substances

• ISO 10993 Part 18- Chemical characterization of materials

• ISO 15747 Plastic containers for intravenous injections (62305-2, 2003).

• ISO 14971 Application of Risk Management to Medical Devices (Speer and Rish,
2016).

• ISO 10993 Part16- Toxicokinetic study design and for degradation products and
Leachable.

ELSIE (Extractable and Leachable Safety Information Exchange)

• Assessing the safety of extractable and leachable is an essential element of the

research and development process.

PQR/ (Product Quality Research Institute)

• PQRI for parenteral and ophthalmic drug products (PODP) working group has
developed this experimental protocol for Extractable and Leachable and issued a
recommendation regarding safety thresholds and best practices for Oral Inhalation Drug
Products (OINDP).

• Some advisory boards like BPSA, BPOG, and ELSIE also aid in the designing of
extractable and leachable studies. (Norwood et al., 2008).

1.4 Sources of Extractable and Leachable

• Plastic Components- Polymers like Polyvinyl Chlorides (PVC), Polymer Additives like
Plasticizers Phthalates, Lubricants, Fatty Acids, Nitrosamines, etc.
• Elastomer/Rubber- Vulcanizing Agent, Poly Aromatic Hydrocarbons (PAHs),
Accelerators, Antioxidants, Carbon Black, etc.

• Inks/Labels- Azo Dyes, Aromatic Amines etc.

• Adhesives- Antioxidants (AO), Catalyst Residues, Heavy Metals, Silicones,Surfactants

• Pigments/Cyclic Oligomers- Inorganic (TiO2, FeO's), Organic Pigment, Polybutylene

terephthalate (PBT) Polyester Silicone Cyclic Oligomers etc.

1.5 Key Principles for Extractable and Leachable Study

• Evaluating the Interaction Risk of Packaging With the Dosage form

• Extractable Study- Apply Extraction conditions and Analytical techniques

• Toxicological Assessment- Define the Evaluation Threshold for Leachable.

• Detection, Identification, and Quantification of Leachable

Degree of Concern Likelihood of Packaging Component-Dosage Form Interaction

Associated with the
Route of HIgh Medium Low
Administration

Highest Inhalation Aerosols and Sterile powders and

solutions; Powders for
Injections and Injection;
Injectable solutions

High Ophthalmic Solutions and

Suspensions;
Transdermal Ointments and
Patches; Nasal Aerosols and
Sprays

Low Topical Solution and Topical Powders, Oral Tablets and

Suspension; Topical and Oral Powders oral (Hard and
Lingual Aerosols, Oral Soft Gelatin)
Solutions and Suspensions Capsules)

Table : Evaluating interaction risks of packaging with dosage form .

1E&L Evaluation Methodology:

To assure the integrity and quality of pharmaceutical items, the assessment of

extractables and leachable (E&L) entails many crucial approaches and procedures.
Experimental design should evaluate the following factors.

Appropriate solvent selection for extraction.

❖ Extraction time points and temperature.

❖ Type of material tested
❖ Surface area to volume ratio of selected packaging material.
❖ Mass of the selected component.
❖ Time of contact of the component.

Figure : Fishbone diagram for extractable study

HYPOTHESIS

● We hypothesized to develop a method for the determination of in situ generated

drug-impurity complex in marketed formulation by liquid chromatography.
● We hypothesized that major leachable product forming in marketed formulation
will be identified and characterized by using LC-HRMS.

OBJECTIVE

● Screening material of construction for marketed formulations.

● Optimization of HPLC Method with Different columns and developed to
optimize the extraction protocol.
● In-situ generation of drug-monomer complex (impurity) and its
assessment in different marketed formulations.
● Identification & characterization of leachable (impurity) by LC-MS.
MATERIAL AND METHODS
 1. MATERIAL AND METHODS
1.1. CHEMICALS AND REAGENT

Sr. No Chemicals Grade Make

Table 1: Chemical used for HPLC analysis and sample preparation

Ophthalmic formulations of Ciprofloxacin and Dexamethasone eye drop solution

were purchased from the four different brands as follows.

Sr. No Formulation (Brand Name) Batch No Manufacturer Qty.

1 CIPLOX - D Cipla 3

2 ZOXAN - D FDC 3

3 CASTOR - D Leeford 3

4 CEFLOX DEE Laborate 3

Table 1: Vendors for the ophthalmic formulation

Sr. No Instruments/ Apparatus Model No. Manufacturer

1 Analytical Weighing Balance

2 HPLC

3 Hot Air Oven

4 Sonicator

5 pH Meter

Table 2: Instrument and apparatus used in the experiment

 1.1. Methods
1.1.1. Extractable Study
Extraction is the process of subjecting the test article (packaging material or medical
device) to stress using various solvents at a particular temperature and time conditions
so that the components from the test article migrate into the extraction medium till the
equilibrium is established. This analysis is conducted on packaging materials with the
extraction process which is typically performed at elevated temperatures, and various
pH to get the worst-case profile of Leachable by using techniques like reflux, soxhlet,
sonication, etc. The purpose is to create a worst-case scenario by subjecting the
packaging components to extreme conditions that force the release of any potential
extractables.

[Link]. Selection of Solvents

Solvents are selected based on the physical-chemical properties of pharmaceutical packaging

materials or medical devices. These solvents are typically designed to mimic formulation or
blood conditions to generate representative extractable capable of predicting leachable with
minimal interference with analytical test methodology. The solvent should cover a wide range of
polarity. (Li et al., 2015)

Typical laboratory solvents used to cover wide range of solvents

• Non-polar: Heptane, cyclohexane, and hexane

• Mid-polar: Dichloromethane, ethanol, or isopropanol

• Polar: water, pH adjusted buffers

[Link] Stoichiometry

It depends on the ratio of the surface area of the test article to the volume of extracting media. It
is done to reduce the sample-to-sample variability and increase the surface area of the test
article which will ultimately increase extraction efficiency.
 Thickness (mm) Extraction Ratio Examples
3
< 0.5 6 cm /ml Film Sheet, tubing wall
0.5 to 1.0 3 cm3/ml Tubing wall, slab small molded items
> 1.0 3 cm3/ml Larger molded items
> 1.0 1.25 cm3/ml Elastomer closures
Irregular Shaped Solid 0.2 g/ml Powder, pellets, foam, non-absorbent
Devices molded items
Irregular Shaped porous 0.1 g/ml Membranes, textiles
Devices
(low density material)
Table: ISO Guidance Selection of Standard surface areas and extract liquid volumes

[Link] Time and Temperature

The extractable study is performed under a simulation condition where a higher temperature is
used to decrease the extraction time and simulate the scenario of a long-term leachable study.
ISO 10993-12 mentions 4 extraction conditions.

• (37+ 1) °C for (72 + 2) hours

• (50+ 2) °C for (72 + 2) hours
• (70 + 2) °C for (24 + 2) hours
• (121 + 2) °C for (1 + 0,1) hours

[Link] Leachable Study

It is mostly conducted during the late stage of drug product development or during stability
studies. It must also consider real-time assessment along with accelerated conditions. The
leachable study can be performed in two ways-
• On actual drug product and with actual packaging in which it is commercialized and no worst-
case scenario is considered.
• On the product which is fabricated simulating the conditions of the actual commercial
processes of pharmaceutical filling, sterilization process,distribution, and storage conditions.

2.2.3 Analytical techniques used for E&L Study

As the extractable and leachable compounds are present in very low concentrations,
hyphenated techniques are required for its analysis (.(Singh et al.2021)
• GC-MS/MS- Analysis of semi-volatile and volatile organic compounds.
• LC-MS/MS- Analysis of non-volatile organic compounds (Norwood et al., 2009)
• ICP-MS- analysis of elemental impurities or components

2.2.4 Analytical Evaluation Threshold

This approach is for the development of analytical testing and safety evaluation threshold
leachable for PODP and OINDP.

Safety Concern Threshold: (SCT) 0.15g for OINDP or 1.5 ug per day for PDP, which is defined
as the threshold below which an individual leachable would have a dose so low as to present
negligible safety concerns from carcinogenic and non-carcinogenic toxic effects.

Qualification Threshold: (QT) 5 ug/day, Threshold below which a given leachable is not
considered for safety qualification (toxicological assessments) unless the leachable presents
structure-activity relationship (SAR) concerns.

Analytical Evaluation Threshold: (AET) Threshold at or above which the pharmaceutical

development team should identify and quantify a particular extractable and/or leachable and
report it for potential toxicological assessment.(PQRI, Washington DC, US). Safety concern
threshold for Geno-toxicant compound in Parenteral and Ophthalmic Drug Products (PODP) is
1.5 ug/day and based on daily deliver volume of drug product; estimated analytical evaluation
threshold is calculated as below.

PODP: SCT = 1.5 ug/day or total daily intake; If not mutagenic , QT= 5 ug/day,
UF - Uncertainty Factor
2.2.6 Compound Identification

One of the most extensively used techniques for component identification is mass spectrometry.
Because of its relatively fast scan speed (many scans per second), ease of connection to
chromatographic techniques, high level of specificity, and extremely low detection limit, it's a
suitable tool for finding and analysing low-level contaminants in a complex matrix. Large floor-
standing magnetic sector systems were quickly supplanted by bench-top high-resolution
systems (both orbital trap and time-of-flight [TOF] based). The characterization of extracted
components is described in full in USP <1663>. As seen below, the recommended approach is
a four-step process.
Scouting: Provide information on the packaging materials' bulk properties (Fourier-transform
infrared spectroscopy [FT-IR], pH, total organic carbon [TOC]).
Discovery: Analyses the sample extracts using a variety of analytical techniques.
Separation techniques and individual compound reaction evaluation are frequently included in
these procedures.
Identification: looking for an answer to the question "What is the compound?" It is a difficult
task to identify Expertise and advanced instrumentation are required.
Quantitation: identifying specific target components, sometimes at extremely low amounts, is
required to answer the question "How much of the compound is in the sample?" Semi
quantitation is frequently performed against a single or several individual surrogate standards
when authentic reference material is not available for quantification either by using external
standard or internal standard calibration.

The USP general chapter <1663> describe the category of identification are as follows
Tentative: A tentative identification means that data have been gathered that are only consistent
with a specific class of molecule, which is normally achieved only using data obtained from
automated library searches or by expert interpretation of fragmentation behavior.

Confident: Confidence identification can be aided by molecular weight confirmation, accurate

mass elemental composition confirmation, or orthogonal spectral confirmation, such as nuclear
magnetic resonance.
Confirmed: In order to validate identification, the mass spectrum and chromatographic retention
index must match an authentic reference.
Selected Packaging Material

Figure : Marketed Ophthalmic formulation container closure system

Ophthalmic formulation container closure offers a convenient and easy to use dosage form. Self
administration and less contamination of the drug product is the key advantage of this type of
container closure.

The leachable study is carried out on Ciprofloxacin & Dexamethasone eye drop formulation
Which is mostly used in eye irritation. Ciprofloxacin is from Quinolone class and dexamethasone
is from Steroid Class. These two drug products are available as a fixed dose combination in
eye drop solution formulation.

For the leachable study we have selected this drug product formulation and procured from four
different brands of this formulation.

Brand - 1 - CIPLOX-D which is manufactured by CIPLA Pharmaceuticals.

Brand -2 - ZOXAN-D which is manufactured by FDC Pharma

Brand - 3 - Castor -D which is manufactured by LEEFORD Pharma

Brand -4 - CEFLOX DEE - which is manufactured by LABORATE Pharmaceutical

 Drug Profile

Figure: Chemical Structure of Ciprofloxacin

Figure: Chemical Structure of Dexamethasone

Selection of Packaging material

Figure : Part of Ophthalmic formulation Container closure system –

1. Fluid Reservoir
2. Nozzle
3. Cap

Preparation of Reaction mixture

Preparation of Control
For the preparation of control, the same procedure was followed as a reaction mixture without
the heating step.

Preparation of Sample
Ciprofloxacin + Dexamethasone eye drop solution contains 3000 PPM Ciprofloxacin + 1000
ppm Dexamethasone. To make a test concentration 750 ppm for Ciprofloxacin and 250 ppm of
Dexamethasone in 0.5 ml solution 0.375 ml of Methanol is added

Figure: Preparation of Sample

2.3 HPLC Method Parameter for CIPROFLOXACIN AND DEXAMETHASONE -

SIMULTANEOUS METHOD Optimization

MOBILE PHASE PREPARATION:

0.1% FA in Deionized Water : Take 500 ml of Deionized water and add 500 µl of formic acid.
 RESULT
AND
DISCUSSION
3. Result and Discussion

3.1 Objective 1 -

Screening material of construction for marketed formulations.

Leachable that can be Leach from Material

Polystyrene , Styrene

Diethyl phthalate

2-methoxy-benzylamine
Linalyl acetate

Polytetrafluoroethylene

2,6-di-tert-butyl-P-benzoquinone

Diisobutyl glutarate

Polyethylene

After Exhaustive Screening Search of different materials of constructions we have

finalized the screening material of low density polyethylene which is mostly used in
the eye drop container closure system.

Fluid Reservoir is mostly made up from the low density polyethylene(LDPE); While cap
is made up of High density polyethylene(HDPE); top and nozzle is made up of
polystyrene.
For this material of construction we have decided to take the Combination of
Ciprofloxacin and Dexamethasone eye drop solution.

We have taken FOUR different brands.

Objective - 2

Method Optimization