Reference Standards

Presented By
Dr. Pramod Kumar
Assistant Professor
Department of Pharmaceutical Analysis
National Institute of Pharmaceutical Education and Research,
Guwahati-781125
 Introduction
Reference Standards (RSs) are the chemical substances or drug
substances with high purity.
Employed as standard in a assay, identification or purity test
Characterized by additional procedures other than used in routine testing
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.
2
 Sources of Reference Materials
Primary
sources Secondary
sources
United States Pharmacopeia(USP)
European Pharmacopoeia (EP) Custom Manufactures
Japanese Pharmacopoeia (JP) Contract Manufacturers
Swiss Pharmacopoeia (SP) Chemical Suppliers
French Pharmacopoeia (FP)
Mexican Pharmacopoeia (MP)
In India,
Indian Pharmacopoeia Commission,
Ghaziabad
United State Pharmacopoeia, Hyderabad
Brach 3
Contd
…

RSs are generally obtained and prepared from United States of

Pharmacopeia-National Formulary (USP-NF) and Indian Pharmacopoeia
Commission
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 (Indian Pharmacopoeia Commission) is

responsible for making available IP RSs.
NIST provide a wide variety of standard reference materials (SRMs) for
validating and calibrating analytical methods

4
 Definition As per USP-NF

USP Reference Other organizations

Standards are highly- also involved in
characterized physical establishment of RSs,
specimens used to like IPC, Ghaziabad,
ensure the identity, Japanese & European
strength, quality, and pharmacopeias, WHO,
purity of medicines Reference Materials
(drugs, biologics, and Committee of ISO
excipients), dietary (REMCO), etc.
supplements, and food
ingredients.
5
 Other
Definitions:
FDA: Specifically ICH (Q6A): A reference standard
prepared drug from new drug substances
substance batch and intended for assays, its impurities
an authentic should be adequately
material of the identified and/or controlled and
highest purity used purity should be measured by a
for structural quantitative procedure
elucidation and
benchmark for
working standards

6
Indian Pharmacopoeia Reference Substance
(IPRS) & Certied Reference Material (CRM)
I.P. Reference Substance is a primary
Reference substance that has the
appropriate quality within a specied
context and is accepted without
requiring comparison to another
substance

Certied Reference Material (CRM)

is characterized by a
metrologically valid procedure
for one or more specied
properties, accompanied by a
certicate that provides the
value of the specied property,
its associated uncertainty and a
statement of metrological
traceability. 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
Qualitative Analysis of materials Quantitative Analysis
By comparison of RS and chemical HPLC
substance in aspects Limit test
IR- Functional Group Conformation
Melting point & HPLC- Purity
identification
U.V.- identification, etc.

Specific uses
Method validation
Calibration of instruments

9
Qualitative and 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 1
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
DSC Indium
NMR TMS
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
 Classificatio
n
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 ❖ For Laboratory purpose & As visual images to
Drug development process in QC compare certain test
R&D articles to meet
Calibration of apparatus, requirements
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
 Types of Reference
Standards:
Reference Solution Related Substance System Suitability is
Compounds which include the related a mixture of the
are difficult to impurity, By- compound with a
handle like very products, small amount of the
hygroscopic or degradation impurity is required;
moisture sensitive, a products and used mixtures of
stable solution of to prove the system impurities or a
drug substance can suitability, mixture of impurities
be manufactured qualitative or and the compound
and filled into tight quantitative analysis as reference
glass ampule for standard. SST test
once use only with a based on the
detail of storage unadjusted RRT, RT
condition and shelf and the resolution.
life
17
 Classification According To ISO & USP

Certified Reference
Reference Material
Material
• A (CRM)
reference material (RM)
• It is a Working level
whose property values Reference Material
are certified by a used for the calibration
technically valid of an apparatus, the
procedure assessment of a
accompanied by or measurement method,
traceable to a or for assigning values
certificate or other to materials.
documentation which
is issued by a certifying 18
body.
 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.

19
 Production and Planning
Selection of Candidate Material
Measurement/Testing Procedure: (IP/BP/EP/USP/In-house method)
Validation of measurement Procedure (Q2R1)
Verification and calibration of measuring equipment
Assessment of homogeneity study
Assessment of stability study
Assigning property value based on the results of measurement
Uncertainties of assigned value
Ensuring appropriate packaging and labelling
Transport arrangements
Post production stability

2
 Selection of Candidate Material

▪ Materials should be highly pure and complies as per Pharmacopoeial specification

▪ Candidate material received from WHO-GMP, USFDA, EDQM, CDSCO
approved or equivalent industry
▪ Purest form of candidate material by synthesis/purification and Prep HPLC
▪ Candidate material is purchased as per government procedure

2
 Storage and Containerization
▪ Candidate material stored at 2⁰C to 8⁰C
▪ Otherwise specified in the monograph.
▪ Store in a tightly closed amber colored container
▪ If the candidate material is hygroscopic in nature should be stored in
vacuum desiccator.

Vacuum Desiccator

2
Introduction to Homogeneity, Stability and Characterization of a
CRM

Introduction to-
▪ Homogeneity
• Within-unit vs. between-unit

▪ Stability
• Short term vs. long term

▪ Characterization
 Homogeneity
▪ Always required (CRMs and RMs) as the definition states “sufficiently homogeneous”.

▪ Reference Material (RM) is sufficiently homogenous and stable with respect to one or more
specified properties, which has been established to be fit for its intended use in a
measurement process.

▪ Certified Reference Material (CRM) is characterized by a metrologically valid procedure for

one or more specified properties, accompanied by a certificate that provides the value of the
specified property, its associated uncertainty and a statement of metrological traceability.

▪ The role of CRM’s in chemical analysis is to provide “measurement benchmarks” that

chemists can use to calculate or assess the accuracy of their analysis. When several
laboratories can achieve the same analytical results for a given CRM, they demonstrate
comparability of their measurements.
 Between-unit Homogeneity
To ensure sufficient homogeneity, the composition of
the units needs to be as similar as required (as
possible)

Dispensin Distribution
g
Bulk Individual
Homogenization units
 Stability
▪ Always required (CRMs and RMs) as the definition states “sufficiently stable”.
▪ Covered in ISO Guide 34 (requirements) and ISO Guide 35 (experiments and data
analysis).
▪ All property value(s) to be certified must be assessed.
▪ The stability of the reference material shall be assessed. Testing, calibration,
measurement, sampling and other activities performed for the assessment of stability
shall be carried out in compliance with ISO/IEC 17025. Stability testing can be
performed only if sufficient homogeneity is demonstrated.
▪ Two different types of stability-
Long-term stability
Short-term stability
Transport stability
 Long-term stability versus short-term stability

▪ Long-term stability (LTS): must predict degradation

⚫
⚫
As long as minimum (at least 1 year)

⚫
At relevant temperature (2ºC to 8ºC)
The uncertainty for long-term stability must be quantified (uLTS).
▪ Short-term stability/Accelerated (STS): must predict degradation
⚫
⚫
At accelerated temperature (40ºC/ 75% RH) for 6 months
Performed at 1, 3, and 6 months interval.
▪ Transported stability (TS): Stability of the material under transport conditions
⚫
⚫
Temperature can reach from 25 ºC to 50 °C during dispatch.
Any evidence of degradation at that temperature.
 Standardization of IPRS
There can be three different situations depending upon the availability of
reference material

Situation
When any pharmacopoeia reference standard is not available then potency
shall be assigned on the basis of chromatographic purity, LOD/Water,
Residual solvents, and Sulphated ash.
Followed by complete Identification/Molecular evidence (characterization) of
the API as per the protocol .

2
 Assigned value determination by mass balance method

Potency (% w/w) = 100 – [Related substances + Water + Residual solvent +

Sulphated ash]
(on as is basis)
Or

= [Chromatographic purity – (water + Residual solvent + Sulphated ash)]

Or

= 100 – [Related substances + LOD + Sulphated ash]

Or

= [Chromatographic Purity – (LOD + Sulphated ash)]

Note: Sulphated ash will not be done on salts such as Sodium, Potassium, etc. and in such cases were salts content
such as Sodium, Potassium will be done additionally.

2
 Protocol for IPRS standardization
Parameters Experiment
Description Physical examination by naked eye
By Infrared Spectroscopy
Identication By chemical /TLC analysis / UV
By HPLC
Molecular mass conrmation by mass spectrometry
Complete structure elucidation of sample by Nuclear Magnetic
Resonance Spectroscopy
Characterization
Percentage conformation of C,H,N,S & O by CHNSO Analyzer

Other technique
As per IP monograph
Sulphated Ash
Should be done in duplicate
As per IP monograph
Loss on drying Should be done in duplicate
Develop new method for LOD
Water content As per IP monograph

3
 Protocol for IPRS standardization
Parameters Experiment Acceptance Criteria
Related substances as per IP monograph by
HPLC

Related substances by other

pharmacopoeia monograph by HPLC
Chromatographic purity
should not be less than
Related substances by developing new
purity method by HPLC
99.0 %
Difference between
Inject blank in duplicate from two different three CP values of test
Chromatograph
vials should not be more than
ic purity(CP)
Inject test solution in triplicate by three 1 %
different weighings

Inject test solution so that absorbance

response should lie between 0.8 to 1.2 AU

Perform system suitability as per monograph Pass as per monograph

Peak purity test should

Check peak purity by PDA
pass

3
 Protocol for IPRS standardization
Acceptance
Parameters Experiment
Criteria
As per IP monograph

Inject blank solution in duplicate Pass as per IP

monograph
Inject system suitability as per IP
monograph
Assay Correlation between two
(By Titration/UV/ Inject ve injections of standard-1 and standard solutions
HPLC) should be between 99.5 -
duplicate injections of standard-2
100.5 %

Difference between
Prepare two sample solutions by injections of test should
duplicate weight. not be more than 1%

As per ICH Q3B

In-house method
Guideline
Analyse as per manufacturer organic
Residual Solvent volatile impurities method
Should comply
Unknown impurity shall be calculated by
nearby known standard volatile impurity

3
 IPRS- Process Flow
Preparation & Sampling as per
Candidate
approval of test designed protocol
material
protocol
received
Lab-1
(IPL)
Lab -2
Analysis of Issuing of candidate material to
candidate Lab -3
concerned laboratories
material Lab -4

Lab -5

Data evaluation by Quality Control Laboratory Head

Review of data by IPRS Expert committee

Ready
to
dispatch
Final approval of Filling, labeling &
Results of Analysis Final check of IPRS

33
 Assignment of property value(s) and their
Uncertainty
⚫ Assessment of uncertainties of property value(s) in accordance with
the GUM

⚫ Stability

⚫ Homogeneity

⚫ Value assignment
Term Standard
uncertainty due to
uchar Characterization
ubb Between-unit
(in)homogeneity
ults Long-term
(in)stability
uCRM Property value

3
Certicate of CRM

3
3
 Reference Standards available at IPC

Reference Standards available at

NIPER-G
IP Reference StandardImpurity Reference
Standard
1. Para hydroxy prenylamine
2. Nor-Ethylmorphine
3. Carboxy toremifene
4. Octopamine sulfate
5. Etilfrine sulfate
6. Norfenefrine sulfate
Phytochemical Reference
Standard
Botanical Reference
Standard

3
IP Prednisone Tablet (Dissolution Apparatus) Calibrator)

API: 537
Impurity: 117
(Available)

API: 50
Impurity: 40
(Under Process)

3
Instruments in Reference standard
development Laboratory
Instruments in Reference standard
development Laboratory

4
IPRS Filling Machine

4
Steps involved
1) Bulk material procured from manufacturer will be characterized &
purified (i.e., WS)
2 From above 5-50 grams collected and stored in large glass ampoules

3) Analytical tests will be done according to pharmacopoeial requirements

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

42
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

43
 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

44
 Basic requirements for qualifications
❖ 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

45
 Steps Involved In Characterization
Physical Description Proof of structure
Elemental analysis
Visual Inspection
Optical microscopy U.V. spectroscopy
I.R. Spectroscopy
Mass Spectrometry
NMR Spectroscopy

46
 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

47
Proof Of Structure:

Mass spectrometry

IR
spectroscopy NMR 48
 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
TGA: Measures the change in the mass of sample as the temperature is
changed
Gives information regarding moisture and solvent levels, an indication if the material
is a hydrate or solvate, and the rate of reaction
DSC: Measures the difference in energy (heat flow) between reference and
sample.
Used to accurately measure the melting point and purity of the reference
49
material.
Contd…

• Gives information on presence of inorganic impurities

ROI • 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

TLC amounts of separated components & impurities

GC • Determines presence of Organic Solvents as impurities

HPLC • Impurities, relative substances detection

50
 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.

51
 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

Availabilityof 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

52
 Prerequisite for analytical standard
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 such as amorphous,

crystalline or polymorphic

A material safety data sheet

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

53
 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
54
 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.

55
 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 and Heat

Type of Container Used for

Moisture resistant Hygroscopic Drugs

Amber colored Light Sensitive Drugs

Heat proof Thermo Labile Drugs

56
 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,
57
5
 Research articles published from NDTL Project : 02

Impact factor 3.345

Impact factor 3.345

Patent filed so far from the NDTL Project
1. Process to prepare carboxy-toremifene.
Gangasani Jagadeesh Kumar, Sachin pawar
Pullapanthula Radhakrishnanand , Upadhyayula
Suryanarayana Murty, Puran Lal Sahu, Sachin
Dubey, Kapendra Sahu, Awanish Upadhyay and
Pramod Kumar Application No. : 202131058419.
2. Process to prepare octopamine sulfate.
Subarna Jyoti Kalita, Sachin pawar Prachi
Vernekar , Mayur Arun Pawar , Veena K. S. , KM
Abha Misra, Kalyan Kumar Sethi, Pullapanthula
Radhakrishnanand , Upadhyayula Suryanarayana
Murty, Puran Lal Sahu, Sachin Dubey, Kapendra
Sahu, Awanish Upadhyay and Pramod Kumar
Application No.: 202231000058
THANK YOU