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
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 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.

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 Sources of Reference Materials
Primary Sources Secondary Sources

United States Pharmacopeia(USP) Custom Manufactures

European Pharmacopoeia (EP) Contract Manufacturers
Japanese Pharmacopoeia (JP) Chemical Suppliers
Swiss Pharmacopoeia (SP)
French Pharmacopoeia (FP)
Mexican Pharmacopoeia (MP)
In India,
National Institute for Standards and
Testing (NIST),
Central Drug Laboratory, Calcutta.
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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

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

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 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.

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 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
IR Spectrophotometer
Karl Fischer Titrimeter
Flame Photometer
Gas Chromatography
Potassium dichromate
Poly Styrene film
Di Sodium Tartrate
Potassium dichromate
1.0% v/v Toluene solution in n-Hexane

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 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.

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 Classification

Analytical Reference
Standards (ARS)/ Primary Working / Secondary
Standard Standard (WS)

Authentic Materials (AM)

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 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

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 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.

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Dept. Of Pharmaceutical Analysis
 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 As visual images to
 Drug development process compare certain test articles
 R&D  For Laboratory purpose & in to meet requirements
 Calibration of apparatus, QC
methods and materials
 Calibration of 2˚ standard
Not Used As drug or cosmetic for For research purpose Not for chemical analysis
consumption
Availability Synthesized independently & Procures from Bulk Available in small quantity
available in very less quantity. manufacturer or prepared in
laboratory itself
Cost Very expensive Low cost Low cost 16
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 Types of Reference Standards:

Reference Solution Related Substance System Suitability is a

Compounds which are include the related mixture of the
difficult to handle like impurity, By-products, compound with a small
very hygroscopic or degradation products amount of the impurity
moisture sensitive, a and used to prove the is required; mixtures of
stable solution of drug system suitability, impurities or a mixture
substance can be qualitative or of impurities and the
manufactured and filled quantitative analysis compound as reference
into tight glass ampule standard. SST test based
for once use only with a on the unadjusted RRT,
detail of storage RT and the resolution.
condition and shelf life
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 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.
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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.
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 ARS Preparation:
Reference Standards
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.

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 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

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 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

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 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

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 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 by- products
and degradation products by normalized area percent

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 Steps Involved In Characterization

• Visual inspection • Elemental analysis

Proof of structure
Physical Description

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

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 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.
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Contd…

• Gives information on presence of inorganic impurities

• Method involves charring, digestion and ignition of RS
ROI • ROI can be coupled with spectroscopic methods for identification &
quantification of inorganic impurities.

• TLC coupled with Densitometer will measure relative amounts of

TLC separated components & impurities

GC • Determines presence of Organic Solvents as impurities

HPLC • Impurities, relative substances detection

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 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.
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 Working Factor And Drug Content Calculation
Contents for Reference standard can be calculated as follow-

100- (%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.
Dept. Of Pharmaceutical Analysis
*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
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 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

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 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

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 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
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 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

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 ..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.

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 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

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 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.

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 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.

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 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

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