Reference standards in Pharmaceutical Industries

SOURCES,
PREPARATION,
CHARACTERIZATION
AND STORAGE OF
REFERENCE
STANDARDS
Presented By,

Bhavana Vedantam,
I/II M.Pharmacy,
Dept. Of Pharmaceutical Analysis.

CONTENTS
Introduction
Sources
Uses
Preparation
Characterization
COA, Handling, Maintenance, Records
Storage
Dept. Of Pharmaceutical Analysis

2

INTRODUCTION
Reference Standards (RSs) are the chemical substances or drug substances with high

purity.
These are not intended to use as drugs.
Reference Standards (RS) plays vital role in all phases of Drug Development process.

RS serves as basis for evaluation of both Process and Product performance &
serves as bench marks for drug Safety.


3

Sources of Reference Materials
Primary Sources
United States Pharmacopeia(USP)
European Pharmacopoeia (EP)
Japanese Pharmacopoeia (JP)
Swiss Pharmacopoeia (SP)
French Pharmacopoeia (FP)
Mexican Pharmacopoeia (MP)
In India,
National Institute for Standards and
Testing (NIST),
Central Drug Laboratory, Calcutta.

Secondary Sources
Custom Manufactures
Contract Manufacturers
Chemical Suppliers

4

Contd…
RSs are generally obtained and prepared from United States of Pharmacopeia-National

Formulary (USP-NF)
USP RSs collection consists of more than 3,000 items ranging from drug substances,
related impurities, residual solvents, biologics, excipients, botanicals, polymers and

melting point standards.
In India, Central Drug Laboratory is responsible for making available IP RSs.

NIST provide a wide variety of standard reference materials (SRMs) for validating and
calibrating analytical methods


5

Definition As per
USP-NF
USP Reference Standards are
highly-characterized physical
specimens used to ensure the
identity, strength, quality, and
purity of medicines (drugs,
biologics, and excipients), dietary
supplements, and food ingredients.


Other organizations also
involved in establishment of
RSs, like Japanese &
European pharmacopeias,
WHO, Reference Materials
Committee of ISO (REMCO),
etc.

6

Other Definitions:
FDA: Specifically prepared drug substance
batch and an authentic material of the
highest purity used for structural
elucidation and benchmark for working
standards

ICH (Q6A): A reference standard from
new drug substances intended for assays,
its impurities should be adequately
identified and/or controlled and purity
should be measured by a quantitative
procedure

7

Uses of Reference Standards (RSs)
• Reference standards are used to support measurements concerned with
chemical composition, biological, clinical, physical and miscellaneous
areas.
• Substances characterized for chemical purity and/or trace impurities.
• Standard solutions and gas mixtures, often prepared gravimetrically from
pure substances and used for calibration of gases.
• Physico-chemical reference materials characterized for properties such as melting
point, viscosity.
• Reference objects or artefacts characterized for functional properties such as taste,
odour, octane number, and hardness.

8

Contd…

Qualitative analysis of materials by comparison of RS & chemical substance in aspects
• IR- Functional Group Conformation
• Melting point & HPLC- Purity identification
• U.V.- identification, etc,

Quantitative analysis
• HPLC
• Limit tests

Specific Uses
• Method validation
• Calibration of Instruments

9

Qualitative

&

Quantitative Analysis

Includes following aspects,
1) Identification
2) Purity analysis
3) Assay of sample.
Results are compared with RS & sample should meet standards of RS.
These can be performed by using spectroscopic & chromatographic methods.


10

Calibration Of Instruments
When an instrument is giving irrelevant results then it can be calibrated by using
RSs. If results are close to acceptance criteria then referred as instrument is working
properly.
Some examples are given below,

Instrument

RS Used for Calibration

UV-Vis Spectrophotometer

Potassium dichromate

IR Spectrophotometer

Poly Styrene film

Karl Fischer Titrimeter

Di Sodium Tartrate

Flame Photometer

Potassium dichromate

Gas Chromatography

1.0% v/v Toluene solution in n-Hexane


11

Method validation & Uncertainty of Method

 Generally in a method validation estimation of Bias is most critical aspect .

 RSs will have limited values of Uncertainty.
 Uncertainty associated with RS should be NMT 1/3rd of sample measurement.


12

Classification
Analytical Reference
Standards (ARS)/ Primary
Standard

Working / Secondary
Standard (WS)

Authentic Materials (AM)


13

Analytical Reference Standards
(ARS)
Definition:
A material or substance with well established properties, used for the
calibration of apparatus the assessment of a measurement method or for assigning values
to materials.

USES:
 Mainly for Research & Drug development purpose
 To characterize WS


14

Working Standard
(WS)
Definition:
A drug substance of established quality and purity as shown by
comparison to the reference standards material and used as reference working
substance for routine quality control.
USES:
 Mainly for Laboratory purpose.


15

Comparison Among ARS, WS & AM
CATEGORY

ARS

WS

AM

Synonym

Primary (1˚) / Gold standard

Secondary(2˚) standard

Authentic Visual Reference

Purity

NLT 99.5%

NLT 95%

NLT 80%

Quality

Highly Characterized

Not so as ARS

Not so as WS

Characterization

Acceptable without comparison

Requires comparison with 1˚
standard

Purpose

For industrial purpose in
 Drug development process
 R&D
 Calibration of apparatus,
methods and materials
 Calibration of 2˚ standard

 For Laboratory purpose & in
QC

As visual images to
compare certain test articles
to meet requirements

Not Used

As drug or cosmetic for
consumption

For research purpose

Not for chemical analysis

Availability

Synthesized independently &
available in very less quantity.

Procures from Bulk
manufacturer or prepared in
laboratory itself

Available in small quantity

Cost

Very expensive

Low cost

Low cost


16

Types of Reference Standards:


Reference Solution
Compounds which are
difficult to handle like
very hygroscopic or
moisture sensitive, a
stable solution of drug
substance can be
manufactured and filled
into tight glass ampule
for once use only with a
detail of storage
condition and shelf life

Related Substance
include the related
impurity, By-products,
degradation products
and used to prove the
system suitability,
qualitative or
quantitative analysis

System Suitability is a
mixture of the
compound with a small
amount of the impurity
is required; mixtures of
impurities or a mixture
of impurities and the
compound as reference
standard. SST test based
on the unadjusted RRT,
RT and the resolution.
17

Classification According To ISO & USP

Certified Reference
Material (CRM)
• A reference material whose
property values are certified
by a technically valid
procedure accompanied by or
traceable to a certificate or
other documentation which is
issued by a certifying body.

Reference Material
(RM)
• It is a Working level
Reference Material used for
the calibration of an
apparatus, the assessment of a
measurement method, or for
assigning values to materials.
18

Reference Standards

ARS Preparation:

Synthesized independently by the Industry for their use.

Preparation of ARS from WS:
INCASE OF:
 Analytical laboratories facing difficulty to procure ARS from Official sources
 For daily analysis
CONDITIONS:
 Prepared ARS from WS should meet properties of RS.


19

Contd..

Bulk material procured from manufacturer will be
characterized & purified (i.e., WS)
From above 5-50 grams collected and stored in large
glass ampoules
Analytical tests will be done according to

pharmacopoeial requirements
Standardize the content by 3 analysts, if results are
close
Transfer Standardized WS into amber colored
ampoules of capacity 50-100 mg and store properly

20

WS Preparation
Bulk material should satisfy following tests

•
•
•
•

Evidence of chemical structure
Assay
Purity
Accelerated stability testing, etc.

Then material can referred as Working standard


21

Characterization Of Reference
Standards

Qualification of RSs is a critical parameter in which a thorough characterization
is necessary in aspects of
1) Identity
2) Strength
3) Quality
4) Purity


22

Basic Requirements For Qualification
 Should understand Physico chemical Profile of RS
 ARS should have good chemical stability on wide range of storage
 Requirements for characterization should be cross-checked to avoid Analytical errors.
 The validity (i.e., accuracy, precision, sensitivity, specificity) of the analytical methods used
for purity determination should be demonstrated

 Highly pure (i.e., ≥99.5%)
 In total the degradation and by-products should not be more then 0.5%
 The levels of all impurities found (including those not chemically identified, isolated, and
characterized) should be provided as a summary tabulation
 Content of enantiomer in case of chiral compound is not taken into account as byand degradation products by normalized area percent

products

23

• Visual inspection
• Optical microscopy

Proof of structure

Physical Description

Steps Involved In Characterization

• Elemental analysis

• U.V. spectroscopy
• I.R. Spectroscopy
• Mass Spectroscopy
• NMR Spectroscopy
• Raman Spectroscopy


24

Contd…

Purity
• Loss On Drying (LOD)
• Karl Fisher Titration
• Thermo Gravimetric Analysis (TGA)

• Differential Scanning Calorimetry (DSC)
• Residue On Ignition (ROI)
• Thin Layer Chromatography (TLC)

• High Performance Liquid Chromatography (HPLC)
• Gas Chromatography (GC)

Assay
• Titration

• Phase - Solubility Analysis

25

Proof Of Structure:

Mass spectroscopy
Elemental analysis by

ICP

(inductively-coupled plasma)
detects up to 24 metals, measuring
less than 5μ in size

IR spectroscopy

NMR spectroscopy

26

Purity Determination
Karl Fischer Titration Determines free content & water of hydration trapped into the crystal
• Caution must be taken while doing KF Titration. No external moisture should interrupt.

LOD

Determines amount of volatile matter
• The % of material lost on drying should correlate to the total amount of solvent and
moisture obtained by other measurements, when determining mass balance accountability
for reference standard material
Measures the change in the mass of sample as the temperature is
TGA
changed
• Gives information regarding moisture and solvent levels, an indication if the material is a
hydrate or solvate, and the rate of reaction
Measures the difference in energy (heat flow) between reference
DSC
and sample.
• Used to accurately measure the melting point and purity of the reference material.

27

Contd…

ROI
TLC
GC
HPLC

• Gives information on presence of inorganic impurities
• Method involves charring, digestion and ignition of RS
• ROI can be coupled with spectroscopic methods for identification &
quantification of inorganic impurities.
• TLC coupled with Densitometer will measure relative amounts of
separated components & impurities

• Determines presence of Organic Solvents as impurities

• Impurities, relative substances detection

28

Assay Of RS
Titration
• Titration values determines counter-ions & impurities
present in RS.
• Less selectivity compared to Chromatographic techniques

Phase - Solubility Analysis
• Absolute method that provides a totally independent
assessment of purity and identity of impurities.
• Phase-solubility analysis is applicable to all species of
compounds that are crystalline solids and that form stable
solutions.

29

Working Factor And Drug Content Calculation
Contents for Reference standard can be calculated as follow100- (%Loss on drying + %Total related substance + % Residue on ignition*)
or

100- %Water + %Residual solvent +% Total related substance + % Residue on ignition*)

Working factor for Reference standard can be calculated as follow(100- Volatile correction#+ % Total related substance + % Residues on ignition*)/ 100
Working factor and content are
correlated by a factor of 100, e.g.
Drug content= 100xWF. If WF is
0.994 then content= 99.4%. But
working factor should not be used in
the documentation part of Reference
Standard.

*This is project specific correction and depends
again on the information available in the
specification
#Depends on the product specification, require to
consider WC or residual solvents or LOD
If Value of WC+Residual solvents or LOD or DP
and By products is <0.1% , can be disregarded
30

Stability, Expiry & Retest
Expiration and Retest Date:
• For any new chemical entity an initial expiry or retest date decided as 6 months if
stock material stored at -20⁰C
• Availability of stability data for 3 months at 40⁰C/75%RH demonstrates the retest
period of 24 months if stored at -20⁰C
• Expiry date is periodically extended on the basis of retest performed and stability
proved
• Reference standard may have a maximum 5 years expiry period

31

Prerequisite for analytical
• Essential for bulk drug testing, should be safeguarded by proper storage
• No purification required if purity >99.0% for RS and for Impurities >95.0%
• Supplied with a CoA including complete identification testing
• Stability data indicates the storage conditions
• Information of its hygroscopicity and solid-state properties, e.g. amorphous,
crystalline, polymorphic form etc..
• A material safety data sheet

• A list of potential impurities (if an active substance) with response factors

32

Certificate Of Analysis
Information needed for COA:
• Name and unique batch number
• Manufacturer
• Manufacturing date
• Retest date
• Expiry date

• Transport instructions
• Storage condition
• Content of reference substance
• Note on calculation of the contents
• Special remark on handling requirement if any
• All the required tests with their specifications and results

33

Usage and Maintenance
Handling during analysis:
Use and maintenance:
• Must be equilibrated to room temperature for 30 minutes before use
• Protect from light and very tight packaging

• For NCEs packaging recommended in amber color bottle with tight sealed cap
• Moisture sensitive and hygroscopic reference substance are packed into once use
packaging material only and discarded after the use if any remaining quantity
available


34

..HANDLING
• Hands should be thoroughly washed before handling.
• Transfer should be done with clean stainless steel spatula. The spatula used should
be cleaned after use.
• While weighing clean spatula has to be used.

• Containers should be immediately closed as soon as the sample is taken out.
• Containers has to be labelled.

35

STORAGE
Qualified Reference standard should be stored in Sealed Ampoules of Single or multiple
dose at proper storage conditions.
Should be protected from
Light
Moisture
Heat
Type of Container

Used for

Moisture resistant

Hygroscopic Drugs

Amber colored

Light Sensitive Drugs

Heat proof

Thermo Labile Drugs


36

Records
• Only authorized personnel of the laboratory should have an access to reference standards.
• Codes should be given to reference standards for easy identification and handling.
• A log book should be maintained in order to keep the record of the reference drugs used.
• The personnel handling reference standards should take utmost care while maintaining these
records.
• The log book should have compulsorily have the details like date, name of standard taken,
purpose
for which it is issued, amount issued, amount remaining, signature.

37

Conclusion
RS is an Important Analytical Tool in industry & in QC labs.
Result of any analytical procedure or any instrument and drug safety
depends on RS.


38

Referen
ces

Hand Book Of Isolation And Characterization Of Impurities In Pharmaceuticals, By
Satinder Ahuja, Elsevier Publications, Volume 5, Pg No: 15, 119-143.
http://www.usp.org/reference-standards
http://www.ipapharma.org/events/IPA%20-%20EDQM%20pdf/Antony%20Gomes%20%20reference%20standards.pdf
http://www.discoverysciences.com/uploadedFiles/Library/Technical_Literature/Brochures/B505A_Drug_Cat

alog_LR_04_17_09.pdf
http://www.fda.gov/downloads/Drugs/Guidances/ucm122858.pdf
39


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Reference standards in Pharmaceutical Industries

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