reference standards in pharmaceutical industries

SOURCES, PREPARATION, CHARACTERIZATION AND STORAGE OF REFERENCE

STANDARDSPresented By,

Bhavana Vedantam,

I/II M.Pharmacy,

Dept. Of Pharmaceutical Analysis.

CONTENTSIntroduction

Sources

Uses

Preparation

Characterization

COA, Handling, Maintenance, Records

StorageDept. 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.

Dept. Of Pharmaceutical Analysis 3

Sources of Reference MaterialsPrimary 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


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


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.

Definition As per USP-NF

Dept. Of Pharmaceutical Analysis6

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


• 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.

Uses of Reference Standards (RSs)

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


Contd…

Qualitative &

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.

Quantitative Analysis



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

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.


Classification

Analytical Reference Standards (ARS)/ Primary

Standard

Authentic Materials (AM)

Working / Secondary Standard (WS)


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


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.


Comparison Among ARS, WS & AMCATEGORY 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



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.

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.


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.

Preparation Of

Reference Standards


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

Contd..


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


Characterization Of Reference Standards

Qualification of RSs is a critical parameter in which a thorough characterization is necessary in aspects of

1) Identity2) Strength3) Quality4) Purity



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

Basic Requirements For Qualification

Phys

ical

Des

crip

tion • Visual inspection

• Optical microscopy

Proo

f of s

truc

ture • Elemental analysis

• U.V. spectroscopy

• I.R. Spectroscopy

• Mass Spectroscopy

• NMR Spectroscopy

• Raman Spectroscopy

• X-Ray Diffraction

Steps Involved In Characterization


• 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)

Purity

• Titration

• Phase - Solubility Analysis

Assay

Contd…



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

NMR spectroscopyIR spectroscopy

Mass spectroscopy

Proof Of Structure:

Purity DeterminationDetermines free content & water of hydration trapped into the crystal

Karl Fischer Titration

• Caution must be taken while doing KF Titration. No external moisture should interrupt.

Determines amount of volatile matter LOD

• 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 changedTGA

• 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 and sample.DSC

• Used to accurately measure the melting point and purity of the reference material.


Contd…

ROI• 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 • TLC coupled with Densitometer will measure relative amounts of separated components & impurities

GC • Determines presence of Organic Solvents as impurities

HPLC • Impurities, relative substances detection


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.



Working Factor And Drug Content CalculationContents 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*)

(100- Volatile correction#+ % Total related substance + % Residues on ignition*)/ 100

Working factor for Reference standard can be calculated as follow-

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


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 -20 C⁰

• Availability of stability data for 3 months at 40 C/75%RH demonstrates the retest ⁰period of 24 months if stored at -20 C⁰

• Expiry date is periodically extended on the basis of retest performed and stability proved

• Reference standard may have a maximum 5 years expiry period


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


• 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

Information needed for COA:

Certificate Of 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

Usage and MaintenanceHandling during analysis:

..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.

STORAGEQualified Reference standard should be stored in Sealed Ampoules of Single or multiple dose at proper storage conditions.

Should be protected fromLightMoistureHeat

Type of Container Used for

Moisture resistant Hygroscopic Drugs

Amber colored Light Sensitive Drugs

Heat proof Thermo Labile Drugs



• 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.

Records

Conclusion


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.

References

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%20sta

ndards.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


http://www.usp.org/reference-standards

http://www.ipapharma.org/events/IPA%20-%20EDQM%20pdf/Antony%20Gomes%20-%20reference%20standards.pdf

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_Catalog_LR_04_17_09.pdf

http://www.discoverysciences.com/uploadedFiles/Library/Technical_Literature/Brochures/B505A_Drug_Catalog_LR_04_17_09.pdf



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