Pharmaceutical Reference Standards

PHARMACEUTICAL REFERENCE
STANDARDS
11th International Symposium

organised by the
European Directorate for the Quality of Medicines
& HealthCare (EDQM), Council of Europe
3-4 September 2012, Strasbourg, France
Session 2b - Presentations
Addressing the Challenges in the

Characterisation of Small Molecules
Characterization of Pharmaceutical
reference standards
by NMR
_______________________________
Ian McEwen
Medical Products Agency, Sweden
When establishing a reference standard, its identity

and purity has to be ascertained.
1D and 2D NMR spectra can be used to establish

the identity. It can be done by:
1. comparing the spectra with spectra obtained
earlier from the same kind of substance
2. elucidating the identity with only the help of
NMR spectra
1
Data from different NMR experiments are like the pieces of a jigsaw
puzzle that can be put together to obtain the identity of a substance
without a reference
Quantitative NMR (qNMR)
Before determining the purity of a substance, 1H-NMR

signals of the spectrum has to be assigned.
The integral of an NMR signal is proportional to the number
of protons and concentration of the solute.
For qNMR, a suitable NMR signal is compared with the

NMR signal of an internal standard. Knowing the
concentration of both substance and internal standard, and the
number of protons contributing to the NMR signals, the
purity of the substance can be assessed.
2
Quantitative NMR (qNMR). The use of internal standard
Fumaríc acid
(internal standard)
arginine Hα
To obtain reliable qNMR results, the NMR signals to be integrated

have to be well resolved, in classical chromatography terms R >30
(because the NMR signals are Lorentzian, broadened at the
bottom). The baseline and the ends of integral curves have to be
horizontal. The peaks have to be well in phase.
3
Excel sheet used for qNMR
Sample: Arginine
Internal standard: fumaric acid
Standard Sample
Constants: MW (g/mol) 116.07 174.2
Purity (%) 99.8
NMR signal (ppm) 6.74 6.34
No of protons 2 1
Standard Sample Purity(%)

Analysis 1
m (mg) 1.9657 3.7979
Integral area 200 128.248
99.62
Analysis 2
m(std) 1.1001 1.1423
100.25
Analysis 3
m(std) 1.5614 1.2521
99.43
Results Mean value, purity (%) 99.77

Standard deviation 0.43
RSD % 0.43
Other ways of performing quantitative NMR (qNMR)?
For practical reasons, 13C-NMR signals cannot be used for qNMR,

19F-NMR signals can on the other hand be used for qNMR.
2D NMR signals can be used for quantification, after calibration and

validation.
4
Comparison of qNMR with other quantitative
analytical methods. Example: OSCS in heparin.
% OSCS
3,50
3,00
2,50
2,00
1,50
1,00
0,50
0,00
W14 W12 W1 W3 W6 W13 W11 W9 W7 W10 W2 W4 W5 W8 W0
spiking NMR SAX CE
Fondaparinux sodium .
An anticoagulant medication chemically related to low
molecular weight heparins
5
1H-NMR spectrum of LMW heparin
1H-NMR spectrum of Fondaparinux
COSY (proton-proton correlation) spectrum of Fondaparinux
6
Proton spectrum
Carbon spectrum
Anomeric protons
HSQC (proton-carbon correlation) spectrum of Fondaparinux
Proton spectrum
Carbon spectrum
HSQC spectrum (red) and

superimposed HMBC
spectrum (blue) of
Fondaparinux
7
The peptide Terlipressin
H-alfa proton signals of Terlipressin
8
AA sequencing of Terlipressin with HMBC
C=O carbon signals H-alfa proton signals
Human insulin
Human insulin has 51 amino acids. Six insulin

molecules assemble in a hexamer with Zn.
Insulin does not dissolve in neutral water.
9
H-C correlation signals (HSQC) of insulin (not isotope labelled)
carbon spectrum Proton spectrum
H-N correlation signals (HSQC) of insulin (not isotope labelled)
Proton spectrum
Nitrogen spectrum
10
Human insulin. 6 mg in 0.6 ml D2O. Spectrum obtained at 20 °C. Spectra
obtained at pH=1.5 and pH=2.5
Human insulin. 3 mg in 0.6 ml D2O, pH=1.5.

Spectra obtained at 50 °C and at 25 °C
11
Human insulin. 6 mg in 0.6 ml D2O at pH 1.5 and 12.
qNMR on human insulin

(qNMR)
Sample: Human Insulin
Internal standard: Sulfolen

Solvent: D2O
Instrument Bruker Avance 600 MHz
Standard Substance
Constants: MW (g/mol) 118.16 5808
Purity (%) 99.7
NMR signal (ppm) 6.11 8.86
No of protons 2 2
Standard Sample (%)

Analysis 1 m (mg) 0.09836 5.9
89.62
Analysis 2 m(std) 0.09836 5.599
91.66
Analysis 3 m(std) 0.09836 3.267
90.63
Results Mean value, purity (%) 90.64

Standard deviation 1.02
RSD % 1.12
12
04/09/2012
International Symposium on Pharmaceutical

Reference Standards, 3-4 September 2012
CHARACTERIZATION OF
SECONDARY STANDARDS: KEY
CHALLENGES ADDRESSED
Dr. Matthew Borer
Mr. David Lytle
Mr. Vaughn R. Stultz
Talk Outline
Definitions
Costs, Risks, and Benefits
How to Establish
• Characterization Approaches
• Equivalency with Official Standards
Conclusions
Definitions Costs and Benefits Characterization Equivalency Conclusions

M. Borer, et al., 2012 IRSS Copyright © 2012 Eli Lilly and Company 2
1
04/09/2012
International Conference on
Harmonization - Q7
11.19 Secondary reference standards should be
appropriately prepared, identified, tested,
approved, and stored. The suitability of each batch
of secondary reference standard should be
determined prior to first use by comparing against
a primary reference standard. Each batch of
secondary reference standard should be
periodically requalified in accordance with a written
protocol.

International Conference on
Harmonization - Q7
Reference Standard, Primary
A substance that has been shown by an extensive set of analytical
tests to be authentic material that should be of high purity. The
standard can be: (1) obtained from an officially recognized source, or
(2) prepared by independent synthesis, (3) obtained from existing
production material of high purity, or (4) prepared by further
purification of existing production material.
Reference Standards, Secondary

A substance of established quality and purity, as shown by
comparison to a primary reference standard, used as a reference
standard for routine laboratory analysis.

2
04/09/2012
Handling and Use of Reference Standards

in the OMCL Network PA/PH/OMCL (11) 204 3R
2.7 Primary standard

Standard that is designated or widely acknowledged as
having the highest metrological qualities an whose value is
accepted without reference to another standards of the same
quantity, within a specified context.
2.8 Secondary standard

Standard whose value is assigned by comparison with a
primary standard of the same quantity.

Handling and Use of Reference Standards

in the OMCL Network PA/PH/OMCL (11) 204 3R

3
04/09/2012
US FDA Draft Guidance for Industry:

Manufacturing, Processing, or Holding
Active Pharmaceutical Ingredients

Guidelines for the Establishment, Handling,

Storage and Use of ASEAN Reference Substances

4
04/09/2012
WHO General Guidelines for the Establishment,

Maintenance, and Distribution of Chemical Reference
Substances
Part B. Secondary chemical reference substances

• B.1 Assessment of need
• B.2 Obtaining source material
– B.2.1 Selection of candidate substance
– B.2.2 Documentation to be supplied with the candidate reference substance
– B.2.3 Initial testing for compliance with the requirements of the monograph
• B.3 Packaging
• B.4 Interlaboratory testing to establish the assigned content
– B.4.1 Competence of the participating laboratories
– B.4.2 Dispatch of the candidate materials
– B.4.3 Test protocol
– B.4.4 Evaluation of test results
– B.4.5 Traceability
• B.5 Adoption of the secondary reference substance
• B.6 Retesting programme
• B.7 Information to be supplied with secondary chemical reference substances
• B.8 Period of use
• B.9 Distribution and supply

WHO General Guidelines for the Establishment,

Maintenance, and Distribution of Chemical Reference
Substances
primary chemical reference substance
A designated primary chemical reference substance is one that is widely

acknowledged to have the appropriate qualities within a specified context,
and whose assigned content when used as an assay standard is accepted
without requiring comparison with another chemical substance.
secondary chemical reference substance
A secondary chemical reference substance is a substance whose

characteristics are assigned and/or calibrated by comparison with a
primary chemical reference substance. The extent of characterization and
testing of a secondary chemical reference substance may be less than for a
primary chemical reference substance. Although this definition may apply
inter alia to some substances termed “working standards”, part B of these
guidelines is intended to apply to secondary reference substances supplied
as “official”, e.g. regional/national standards, and not to manufacturers’ or
other laboratories’ working standards.

5
04/09/2012
Recent USP Publication

International vocabulary of metrology –

Basic and general concepts and associated terms (VIM) JCGM 200:2012
5.4 primary standard

measurement standard established using a primary reference
measurement procedure, or created as an artifact, chosen by
convention
5.5 secondary measurement standard

measurement standard established through calibration with respect to a
primary measurement standard for a quantity of the same kind

6
04/09/2012
Secondary RS in Summary
It is acceptable to establish a Secondary RS versus a
compendial or in-house Primary RS
Full characterization, including comparison to a

Primary RS, is performed according to intended use
(although not as extensive as a Primary RS)
Robust procedures must exist for all aspects of

managing a Secondary RS
Periodic requalification is required
For routine use in the laboratory (a “working standard”)

What are the Costs?

Cost to properly manage Secondary RS
• Procurement of source material
• Packaging/labeling
• Characterization and periodic re-evaluation testing
• Storage facilities
• Processing and shipping customer orders
• Customer service
• Project management
• PROCEDURES AND DOCUMENTATION
With economy of scale, costs can be comparable to

commercial standards, especially if an in-house Secondary
RS is equivalent to more than one Official RS
7
04/09/2012
What are the Risks?

Risk of Regulatory findings. For example, eight Warning Letters issued
by the US FDA since 2010 include reference standard problems:
• Insufficient documentation
“you failed to have documentation describing how the standards used for
the testing of the APIs are obtained, prepared or qualified”
• Insufficient justification of dating intervals
“your firm failed to follow your SOP for expiration dating of "in-house"
reference standards”
• Insufficient understanding of handling properties
“there is no data to demonstrate that these "as-is" purity factors will remain
accurate over the one year expiry period of the standard”
• Lack of material control
“observed 31 expired standards (i.e., ) in a laboratory drawer
next to a separate drawer containing unexpired standards”

Why Establish an In-house RS?

Pre-compendial support
• Official RS is not available during development and initial marketing
Reliable supply
• It is unacceptable to halt manufacturing if an Official RS is out of stock
Usage rate
• Agencies cannot necessarily supply the volume of RS required by the
pharmaceutical industry
Intended use
• An in-house RS can be shown suitable for intended uses beyond
monographs
Site-to-site consistency
• When global manufacturing sites use the same RS, there is more
assurance of consistency

8
04/09/2012
Why Establish a Secondary RS?

Allows the Primary Reference Standard to last for the
maximum time span
• Long-term consistency in the measurement system
• Line-of-site to original clinical studies
Global supply chain

• An in-house RS can be Secondary to more than one official Primary
Reference Standards
Cost
• In-house RSs can be less expensive to maintain, especially when
there are multiple Official Standards and reference standard material
consumption is high (e.g., thousands of vials per year)

Characterization Approach
Determine what property values must be certified
Determine which Official Standards are relevant
Design a protocol
• Include comparison to Official Standards in a way that assure the
Secondary RS will generate equivalent test results as compared to
applicable Official Standards
• Include orthogonal testing to substantiate certified property values
• Include testing to demonstrate suitability for any non-compendial
intended uses
• Include other testing to prove identity and assure stability

9
04/09/2012
Traceability to the Primary Standard

Metrological Traceability (VIM 2.41)
• Property of a measurement result whereby the result can be related to a
reference through a documented unbroken chain of calibrations, each
contributing to the measurement uncertainty
By Comparison
• By “comparing against a primary reference standard” (ICH Q7)
• “as shown by comparison to a primary reference standard” (ICH Q7)
• “value is assigned by comparison with a primary standard” (OMCL Network)
• “as shown by comparison to a primary reference standard” (FDA)
• “characteristics are assigned and/or calibrated by comparison to a primary reference
standard (ASEAN)
• “characteristics are assigned and/or calibrated by comparison with a primary chemical
reference substance” (WHO)
• “established through calibration with respect to a primary measurement standard”
(VIM)
Ways to Demonstrate Equivalency

Comparative Assay
Official RS Standard
Secondary RS Sample
Certified
Property Value
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04/09/2012

Mass Balance
Assign the in-house RS by another means (e.g., mass balance) and

show that this assignment is equivalent to comparative assay results
versus the compendial standard(s) (e.g,. mass balance is within the
95% confidence interval)
Certified Official RS Standard

Property Value
equivalent
Secondary RS Sample
Mass
Balance Secondary RS


In-House Primary
Demonstrate that the in-house Primary RS is equivalent to the Official

RS(s). In-house standards that are Secondary to the Primary will also
be equivalent
equivalent
In-House Standard
Official RS Primary RS
Secondary RS Sample
Certified Property Value

11
04/09/2012

Same Batch and Assignment
If two reference standards are from the same batch and

have equivalent assignments, the standards can be
deemed equivalent
Same Batch
Official RS Equivalent Assignment In-House RS
OR
In-House Same Batch In-House
Primary RS Equivalent Assignment
Secondary RS

Why Not Only Assign by Comparative

Assay (“unbroken chain of comparisons”)?
For highly pure pharmaceutical materials, it is very
reasonable to assume that all components of mass
balance are known
• Sourced from a fully validated manufacturing process
• Fully validated methods exist for impurities
If the above statement is true, mass balance is more

accurate, precise, and efficient to measure
• Typical HPLC comparative assay (n=9) has an uncertainty of 0.5%
(the true value is within +/- 0.5% at a 95% C.I.)
• A typical mass balance has an uncertainty of 0.05%
– 99.0% pure material, using impurity methods with intermediate precision
of 10%
12
04/09/2012
Conclusions
There is a general agreement between agencies and guidelines
regarding the definition of a Secondary Reference Standard
There can be significant benefits associated with establishing in-

house Secondary Reference Standards, but you must consider
the cost and the risks
There are clear requirements for comparability to official

standards such as reference standards provided by compendial
agencies
There are options for demonstrating comparability that are

scientifically sound and can be more accurate and precise than
assignment by direct comparative assay

Thank you!

13
World Health Organization 04/09/2012
Role of WHO International

Chemical Reference
Substances (ICRS)
Dr Herbert Schmidt
Quality Assurance and Safety: Medicines
Technical Officer
Dr Herbert Schmidt
1| International Symposium on Pharmaceutical Reference Standards
03 – 04 September 2012, Strasbourg, France
Outline of presentation
 World Health Organization
– setting and promoting norms and standards
 The International Pharmacopoeia (Ph. Int.)
– main features
 International Chemical Reference Standards (ICRS)
– main features
– efforts to keep the number of ICRS at a minimum that are
necessary to perform analytical tests described in the Ph. Int.
 Conclusion
Dr Herbert Schmidt
Dr Herbert Schmidt 1
World Health Organization

 ICRS are established and distributed under the aegis of
the World Health Organization
– WHO is a specialized agency
of the UN
– directing and coordinating authority
for international health matters
– mandated by 194 Member States
– principal objective:
the attainment by all people of the
highest possible level of health
• i.a. by setting norms and standards
and promoting their implementation
Dr Herbert Schmidt
World Health Organization

 WHO is setting and promoting norms and standards
concerning the quality of medicines by
– International Nonproprietary Names (INN)
– guideline development
– workshop and training courses
– coordination and promotion of anti-counterfeiting measures
– prequalification of medicines for priority diseases
• activities: evaluation, inspection, capacity building, monitoring
– pharmacovigilance for global medicines safety
– regulatory and other information exchange
– The International Pharmacopoeia
Dr Herbert Schmidt
The International Pharmacopoeia

 contains analytical methods
and specifications for
– active pharmaceutical
ingredients (API)
– excipients
– dosage forms
 focuses on
– Model List of Essential
Medicines
– medicines recommended by WHO
specific disease programmes
Dr Herbert Schmidt
Ph. Int. – Main Features

 provides public standards for major public health needs
– Ph. Int. monographs are often the only publicly available
compendial standards for priority medicines
 is part of a graduated approach to fight SSFFC medicines
– (SSFFC: substandard/spurious/falsely-labelled/falsified/counterfeit)
– basic tests
• devised merely to confirm identity and gross degradation
• may be used at peripheral levels of distribution (wholesale, pharmacies)
– pharmacopoeial tests
• for final evaluation of compliance
• by skilled pharmacists at fully-equipped pharmaceutical laboratories
Dr Herbert Schmidt
Ph. Int. – Main Features

 aims at global applicability of its specifications
– when complex, technically demanding methods are proposed by
the manufacturer, less sophisticated analytical methods are
usually developed and primarily considered (if equally satisfying)
 is ready to use
– “The Ph. Int. (…) is intended to serve as source material for
reference or adaptation by any WHO Member State wishing to
establish pharmaceutical requirements. The pharmacopoeia, or
any part of it, shall have legal status, whenever a national of
regional authority expressly introduces it into appropriate
legislation.”
 is free for use by WHO Member States
Dr Herbert Schmidt
International Chemical Reference

Substances (ICRS)
 primary reference standards
 established by
European Directorate for the
Quality of Medicines and
Healthcare
 under the authority of
WHO Expert Committee on
Specifications for
Pharmaceutical Preparations
Dr Herbert Schmidt

Substances (ICRS)
 are intended
– to serve as a benchmark for physical and chemical
tests and assays described in
– to be used for establishing
secondary reference substances
• supplied as official,
e.g. regional/national standards
• to satisfy all local needs
(also in remote areas)
• to reduce cost and delay
in receiving reference material
Dr Herbert Schmidt

Substances (ICRS)
 are accompanied by
– leaflets with
• information necessary to use ICRS as intended
• additional analytical data obtained at the time of
establishment (for unintended use of ICRS the Ciprofloxacin ICRS
intended use:
responsiblity for suitability rests with user or other
identification by IR
authority)
Dr Herbert Schmidt
10 | International Symposium on Pharmaceutical Reference Standards

Substances (ICRS)
 Ph. Int. keeps the number of ICRS necessary for testing at
a minimum
– by providing equally satisfying analytical procedures as
alternatives that do not require reference standards
– by providing spiked reference standards and mixtures thereof
– by in situ preparation of impurities
– by providing International Infrared Reference Spectra (IIRS)
– by referring the response of possible impurities to the parent
compound (if appropriate)
 to facilitate - in particular - post-marketing surveillance
testing of medicines in low- and middle-income countries
Dr Herbert Schmidt
Minimum number of ICRS – in detail

 by proposing, e.g. stoichiometric titrations or spectro-
photometric methods (plus absorptivity values) as equally
satisfying alternatives
– example: ARTESUNATE TABLETS
• ASSAY
• Either method A or method B may be applied.
• A: Carry out the test as described under 1.14.4 High-performance
liquid chromatography, using …
• B: Weigh and powder 20 tablets. To a quantity of the powder
containing about 0.5 g of Artesunate, accurately weighed, add ...
Titrate 25 ml of the filtrate with sodium hydroxide (0.05 mol/l) VS,
using 2 drops of phenolphthalein/ethanol TS as indicator.
Dr Herbert Schmidt

 Are titrations "equally satisfying" as chromatographic
methods for content determinations?
– condition: test for related substances "Estimates of the variability of HPLC
is performed with discriminating method assays from our data and from the
literature ranged from 0.6 to 1.1%
– conclusion: titrations are often more R.S.D. This variability is an
precise that chromatographic methods appreciable portion of a typical
acceptance range (e.g., 98.0–102.0%)
and frequently exceeds the variability
of the manufacturing process.
Therefore, the results of the HPLC
assay are questionable at best to
determine the acceptability of the
drug substance batch."
Journal of Pharmaceutical and Biomedical Analysis, 44 (2007) 906-913
Dr Herbert Schmidt

 by providing spiked reference standards and
mixtures thereof
– for system suitability testing or identification of impurities
Monograph on Lumefantrine
Test for related substances by HPLC:
Lumefantrine for SS ICRS

contains lumefantrine and
impurities A, B and C
Dr Herbert Schmidt

 by in situ production of impurities
– with simple chemical reactions performed by the user of Ph. Int.
– for system suitability testing or identification of impurities
Monograph on tenofovir disoproxil

fumarate, test for related substances
by HPLC:
• formation of tenofovir monoester
by heating a solution of tenofovir
in a boiling water bath for 20 min
• SST: resolution between tenofovir
and its monoester > 25
(see also: Ashenafi et. al., J. Sep. Sci. 2010,

33, 1708-1710)
Dr Herbert Schmidt

 by providing International Infrared Reference Spectra (IIRS)
IIRS for Pyrimethamine

for comparison with
IR spectra from samples
Dr Herbert Schmidt

 by referring the response of possible impurities to the
parent compound – if appropriate
– to evaluate compliance with a test for related substances, the
response of an impurity is preferably compared with the
response of the parent compound (rather than with the response
of an impurity standard)
– in cases where the response factors of a parent compound and
the impurity are not close, a correction factor is introduced
– approach acknowledges the fact that candidate material for
impurity ICRS are often difficult to procure
– establishment of impurity ICRS shall not be the limiting step in
the development of Ph. Int. monographs
Dr Herbert Schmidt
WHO Guidance on chemical RS

 The International Pharmacopoeia
– New chapter on Reference substances and Reference Spectra
in the supplementary information section (under elaboration)
 Quality Assurance of Medicines
– General guideline for the establishment, maintenance and
distribution of chemical reference substances
 Prequalification of Medicines Programme
– Guidelines on submission of documents for a multisource
(generic) finished pharmaceutical product for the WHO
Prequalification of Medicines Programme: quality part
Dr Herbert Schmidt
Summary
 The role of ICRS
– ICRS are intended
• for physical and chemical tests and assays as described in
• for establishing official secondary standards
– The International Pharmacopoeia aims to keep the number of
ICRS needed to perform analytical tests at a minimum by
different means
• to facilitate and promote - in particular - effective post-marketing
surveillance in low- and middle-income countries
• to promote the global applicability of the specifications and methods of
Dr Herbert Schmidt
Thank you very much

for your kind attention !
"Turn off the tap of unsafe medicines!"

Dr Margaret Chan – WHO Director-General
Abbreviations
 ICRS International Chemical Reference Standards
 IIRS International Infrared Reference Spectra
 Ph. Int. The International Pharmacopoeia
 SSFFC Substandard/spurious/falsely-labelled/ falsified/
counterfeit (medical products)
 UN United Nations
 WHO World Health Organization
Dr Herbert Schmidt
Further reading
 New chapter on Reference substances and Reference
Spectra in the supplementary information section
– under elaboration
– To be published as part of the supplementary section of The
International Pharmacopoeia
 General guideline for the establishment, maintenance and
distribution of chemical reference substances
– Annex 3, WHO Technical Report Series 943, 2007
– http://www.who.int/medicines/areas/quality_safety/quality_assur
ance/control/en/index.html
Dr Herbert Schmidt
Further reading
 Guidelines on submission of documents for a multisource
(generic) finished pharmaceutical product for the WHO
Prequalification of Medicines Programme: quality part
– Annex 15, WHO Technical Report Series 961, 2011
– http://www.who.int/medicines/areas/quality_safety/quality_assur
ance/regulatory_standards/en/index.html
Dr Herbert Schmidt
Matrix Reference Materials
Hendrik Emons
Joint Research Centre –

Institute for Reference
Materials and
Measurements
Serving society
Stimulating innovation
Supporting legislation
The Topic
Reference Materials
classified according to ‘complexity‘ of composition
Pure substances Matrix materials

(& their solutions)
‘Natural’matrix Synthetic
materials ‘mixtures’
usually certified: usually certified:

- identity - content of specified
- purity trace/minor component(s)
1
Establishing matrix CRMs
Reliable characterisation of candidate matrix RMs
 identical subsamples (at the level of ‘minimum sample intake’)

 appropriate stability during RM transport
 appropriate stability during RM storage
(also for matrix!)
 certified values for components-of-interest with adequate
metrological traceability & fit-for-use measurement uncertainty
Specify the ‘component’
“trivial” for:
Total elements
(atoms) & ions in
“active” Ca2+
in blood 
water (e.g. Ca2+)
Complex molecules (functions)

whole molecule ? functional
(identity, number…) properties
activity/reactivity
part(s) of the
molecule ? (identity…)
2
Molecules in a matrix
sample
weighing,
milling
CAP?
? clean-up
extraction
Traceability
‘chain’ ?
?
quantification
e.g., by LC-MS fill to
volume
for operationally
Which reference? defined measurands
to be
definition of
internationally
(SI) unit agreed & accepted
CRM
primary calibrator primary reference
measurement procedure
secondary reference
CRM measurement procedure
laboratories
working calibrator
laboratories
measurement procedure
routine sample
RESULT  3D plots?
(identities)
3
Traceability statements
on ERM®
Operationally
Certificates
defined
Identity
(measurand)
Structurally
Traceability is defined
split into
SI reference
Quantity
value
(number and unit)
Artefact
reference
Structurally defined measurands

ERM®-CD281 ‘Rye Grass’
Mass Fraction
Element Certified value 1) Uncertainty 2)
[mg/kg] [mg/kg]
As 0.042 0.010
B 5.5 0.5
Cd 0.120 0.007
Cr 24.8 1.3
Cu 10.2 0.5
Hg 0.0164 0.0022
Mn 82 4
Mo 2.22 0.12
Ni 15.2 0.6
Pb 1.67 0.11
Sb 0.042 0.007
Se 0.023 0.004
Sn 0.062 0.011
Zn 30.5 1.1
1)The value is the unweighted mean of accepted sets of data, each set being obtained in a different
laboratory and/or with a different method. The certified values are reported on dry mass basis and
are traceable to the SI.
2)Expanded uncertainty with a coverage factor k = 2 according to the Guide to the Expression of
Uncertainty in Measurement (GUM), corresponding to a level of confidence of about 95 %.
4
Operationally defined measurands
ERM®-DA474/IFCC
‘CRP in Human Serum’
Mass concentration
Certified value 2) Uncertainty 3)
[mg/L] [mg/L]
C-reactive protein 41.2 2.5
(CRP)1
1) CRP as measured by immunonephelometry and immunoturbidimetry using ERM-DA470 as calibrant
(Baudner et al., EUR reports 15423 and 16882 European Communities, Luxembourg (1993)), applying the
procedures described for the certification of ERM-DA472/IFCC, ERM-DA470 and 1st Int. St. for CRP Code
85/506.
2) The value is the unweighted mean of 6 accepted mean values, independently obtained by 4
laboratories. The certified mass concentration is traceable to the SI, via ERM-DA470.
3) The certified uncertainty is the expanded uncertainty with a coverage factor k = 2 corresponding to a
level of confidence of about 95 % estimated in accordance with ISO/IEC Guide 98-3, Guide to the
Expression of Uncertainty in Measurement (GUM 1995), ISO, 2009.
Characterisation principles
Approaches:
 via gravimetric preparations (not for matrix RMs)
 by measurements
 Structurally defined measurands: at least two

independent methods (ideally, completely different
measurement principles, incl. preparations, should be applied)
 Operationally defined measurands: all have to use the

same method/procedure
 all important input factors must be properly calibrated
5
Measurement method
 Must give relevant information about the ‘true’ value

 Results traceable to a useful reference
 therefore: Method must be fully validated in a way that a
complete uncertainty statement can be drawn up
 All requirements of ISO/IEC 17025 must be fulfilled
 Usually high performance methods & labs required
PT study
RSD = 55 % RSD = 11 %
RM
characterisation
study
Value assignment
Model: cCRM  cchar  cbb  clts  csts

cchar: average value for the batch
cbb: deviation of a bottle average due to heterogeneity
clts: deviation of a bottle average due to degradation during storage
csts: deviation of a bottle average due to degradation during
transport
Goal:
Obtaining the best estimate of the measurand’s ‘true value’ for
the batch of material within the scope of intended traceability
Outcome:
Average property value of the batch and its uncertainty
6
Certified value & uncertainty
IRMM policy
 if normally distributed values from characterisation
study: unweighted mean of means
 if blank value is to be certified: LOQ of the most
sensitive method(s)
 any other case: find a sound concept and describe it in
the Certification Report
 describe clearly the traceability chain
Report the expanded combined uncertainty:
UCRM  k uchar
2
 ubb
2
 ults
2
 usts
2
Take-home Messages
For reliable characterisation of candidate matrix RMs:

 specify the relevant measurand
 agree on adequate measurement procedures for
operationally defined measurands (e.g., molecules in a matrix,
functional properties)
 design properly the traceability chain & realize the
corresponding calibrations and uncertainty estimation
 operate rigorous quality assurance & documentation
“You may think you know a subject, but until you can measure it and
calculate it your knowledge is of a vague and unsatisfactory kind”
Ernest Rutherford
(1871-1937)
7
Acknowledgements
IRMM’s RM Unit
Collaborators all over
the world
Various international
organisations
€€€ our funding bodies
and customers
8
How to monitor Reference
Substances for continuous fitness for
purpose: novel approaches or
solutions to challenges?
Jane Weitzel
Quality Analysis Consultants
USP Expert Committee Member
3rd September 2012
USP’s Vision
• USP’s vision is to produce

cutting-edge USP
Reference Standards,
including CRMs, based on
sound, scientific,
metrological principles. This
includes the use of
metrological concepts such
as Measurement
Uncertainty.
• The Continues Suitability for

Use (CSU) program is
being examined as part of
this metrological approach.
Metrological Approaches Being Adopted
• The USP has introduced the metrological principles in several stimuli
articles and journal articles during the 2005 – 2010 cycle.
• In these articles the impact of the use of Measurement Uncertainty
was presented, discussed and evaluated. Many of these uses impact
the CSU program.
• For example, the stimuli article “Uncertainty Statements Regarding
USP Reference Standards” in PF Vol. 34(2) includes a section on
Stability.
Certified Reference Materials (CRM)

• CRMs are reference
materials accompanied
by a certificate of
analysis.
• A CRM is rigorously
tested for one or more
chemical or physical
properties (such as
purity).
• A property value with
associated uncertainties
certified to be true based
on these tests is assigned
to the CRM after a
thorough statistical
treatment of the data.
USP Work Plan
• USP is working towards its vision

following a Work Plan for its Expert
Committees.
• The work plan includes
– Recommendations on a Stability Program for
USP Reference Materials
and
– Recommendations for expiry dating of
Pharmacopeial Reference Materials.
CSU
• Continued Suitability for Use (CSU) is the

program at USP that ensures that the
Reference Standards maintain the
properties determined at the initial
evaluation.
• The retesting intervals and protocols are a
function of the uses and properties of the
standard and of the information available
about its stability.
Review & Recommend
• The charge to the Reference Standards

Expert Committee (RSEC) subcommittee
is to review the practices, procedures, and
policies in the present CSU program and
compare them against best practices and
sound, scientific, metrological principles.
• Some of the challenges and novel

approaches are presented here.
The Analytical Requirement
• In all cases the maximum permissible size of

the uncertainty should form part of the
definition
of the analytical requirement.
• The uncertainty needs to be specified clearly.
• Specifying the measurement uncertainty
(MU) can be a challenge.
How do you set this

“Maximum
Permissible Size”?
Fitness-for-Purpose
Test Accuracy Ratio (TAR)
• A criterion for maximum uncertainty of the CRM

assigned value can then be considered in terms
of the relationship between the expanded
uncertainty and the acceptance criteria.
Rule of Thumb (1 to 4 ratio)
 A 4:1 ratio means the
expanded uncertainty (U)
of the RM needs to be
less than one-fourth of ½
the acceptance interval.
This ratio is sometimes
referred to as the test
accuracy ratio (TAR).
 Different associations
use different terms, so
one must be careful
when defining the TAR.
Relationship of U to Specifications
Expanded Uncertainty (U)
½ Acceptance Zone
Lower Acceptance Zone Upper

specification Specification
Example Acceptance Interval
• If the acceptance interval is 4% or ± 2 %, then a

4:1 ratio means the expanded uncertainly of the
RM needs to be less than 0.5% (one-fourth of
2%).
U < 0.5 %
½ Acceptance Interval 2 %
TAR & MU
• Use of the TAR
approach allows the
required uncertainty,
or target
measurement
uncertainty, to be
stated clearly.
• The end user of the

CRM knows how the
fitness-for-purpose is
determined.
Continued Testing
• How is the continued suitability of the reference

standard determined and monitored? This is
another challenge.
2012 2013 2014 ???
ICH Q1E
• This guideline
provides
recommendations
on how to use
stability data.
• BUT...
Decision Tree for Data Evaluation
There is a useful
decision tree to
assist in
analyzing the
data.
BUT ...
Significant? Little?
Does long-term data

Is there a show little or no
Significant change over time?
change at
accelerated
condition within
6 months?
What is Little?
Measurement Uncertainty
• Measurement Uncertainty can define what is “little”

and what is “significant”.
• The European Measurement Approach (ERM) is
used to determine if a monitoring measurement
Rmeas is deemed to remain within specification.
– Application Note 1, T. Linsinger, European Commission, Joint
research Centre, Institute for reference Materials and
Measurements, January, 2010
│ CCRM - Rmeas │ ≤ 2 (u2CRM + u2meas)1/2

Trends
• Another
challenge to
CSU is how to
deal with
trends from
changes in
the CRM.
• To evaluate
this a novel
approach has
been taken.
Video Game Data Simulator
• A data simulator was

developed to see how
well the approach
would work.
• An EXCEL
spreadsheet was
created that would
plot the data over
time and calculate if
the data passed the
requirement of the
EMR equation.
Variables
Variables Value
True content (%) 99.5
Between‐lab component SD 0.2
Within‐lab SD 0.1
Slope of time trend (per year) ‐0.1
u CRM 0.03
u meas 0.255
The Display
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
EMR Equation: Pass Pass Pass
|CCRM‐ Rmeas| ≤ 0.513 0.293 0.283 0.133
│ CCRM - Rmeas │ ≤ 2 (u2CRM + u2meas)1/2
Scenario 1
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
|CCRM‐ Rmeas| ≤ 0.513 0.261 0.408 0.399
Scenario 2
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
|CCRM‐ Rmeas| ≤ 0.513 0.195 0.440 0.416
Scenario 3
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
EMR Equation: Pass Pass Fail
|CCRM‐ Rmeas| ≤ 0.513 0.176 0.319 0.809
Scenario 4
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
|CCRM‐ Rmeas| ≤ 0.513 0.080 0.102 0.296
Scenario 5
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
EMR Equation: Pass Pass Fail
|CCRM‐ Rmeas| ≤ 0.513 0.318 0.162 0.792
Scenario 6
102.0
TAR U = 0.23 % which is < 0.5 %
101.5 Det'n 1
Det'n 2
101.0
Average
A
+ 2U
s 100.5
s ‐2U
a 100.0
y
99.5
%
99.0
98.5
98.0
Time (Year)
0 1 2 3 4 5
Year: 1 3 5
|CCRM‐ Rmeas| ≤ 0.513 0.186 0.303 0.393
How to Win the Video Game
• Make the slope of the time trend over the

years 0.
Variables Value
True content (%) 99.5
Between‐lab component SD 0.2
Within‐lab SD 0.1
Slope of time trend (per year) ‐0.1
u CRM 0.03
u meas 0.255
0
Usefulness of the Data Simulator
• The uncertainty needs to be estimated

carefully.
• The patterns that appear, and still pass,
are surprising.
• The data simulator is a useful tool for
visually seeing the effect of varying
different uncertainty components on the
fitness-for-purpose.
Operating Characteristic Curve
• The formulas used in the EXCEL

spreadsheet could be presented as
Operating Characteristic Curves (OCC).
The Metrological Approach
• Using measurement uncertainty allows

specifications to based on sound, scientific,
metrological principles.
• By using the TAR decision rule, the initial
value established for the CRM is shown to
be fit-for-purpose.
• The European Measurement Approach
(EMR) allows the assessment of continued
suitability for use to be based on the
metrological principle of measurement
uncertainty.
Data Simulation
• The data simulator is

a useful tool that
clearly shows the
effects of various
102.0
101.5 Det'n 1
Det'n 2
uncertainty
101.0
Average
A
+ 2U
s 100.5
‐2U
components.
s
a 100.0
y
99.5
%
• It even allows people

99.0
98.5
to have fun and 98.0

0 1 2 3 4 5
Time (Year)
Year: 1 3 5
achieve the EMR Equation: Pass Pass Pass
|CCRM‐ Rmeas| ≤ 0.289 0.000 0.000 0.000
impossible!
Conclusion
• There are challenges in monitoring reference

standards for continued suitability for use;
however, the metrological approach allows us to
use novel approaches to overcome these
challenges.
And Fun!

Pharmaceutical Reference Standards

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Pharmaceutical Reference Standards

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PHARMACEUTICAL REFERENCE

11th International Symposium

3-4 September 2012, Strasbourg, France

Addressing the Challenges in the

When establishing a reference standard, its identity

1D and 2D NMR spectra can be used to establish

Quantitative NMR (qNMR)

Before determining the purity of a substance, 1H-NMR

For qNMR, a suitable NMR signal is compared with the

To obtain reliable qNMR results, the NMR signals to be integrated

Standard Sample Purity(%)

Results Mean value, purity (%) 99.77

Other ways of performing quantitative NMR (qNMR)?

For practical reasons, 13C-NMR signals cannot be used for qNMR,

2D NMR signals can be used for quantification, after calibration and

1H-NMR spectrum of Fondaparinux

COSY (proton-proton correlation) spectrum of Fondaparinux

HSQC (proton-carbon correlation) spectrum of Fondaparinux

HSQC spectrum (red) and

H-alfa proton signals of Terlipressin

C=O carbon signals H-alfa proton signals

Human insulin has 51 amino acids. Six insulin

Insulin does not dissolve in neutral water.

carbon spectrum Proton spectrum

H-N correlation signals (HSQC) of insulin (not isotope labelled)

Human insulin. 3 mg in 0.6 ml D2O, pH=1.5.

qNMR on human insulin

Internal standard: Sulfolen

Standard Sample (%)

Results Mean value, purity (%) 90.64

International Symposium on Pharmaceutical

Costs, Risks, and Benefits

Definitions Costs and Benefits Characterization Equivalency Conclusions

Definitions Costs and Benefits Characterization Equivalency Conclusions

Reference Standards, Secondary

Definitions Costs and Benefits Characterization Equivalency Conclusions

Handling and Use of Reference Standards

2.7 Primary standard

2.8 Secondary standard

Definitions Costs and Benefits Characterization Equivalency Conclusions

Handling and Use of Reference Standards

Definitions Costs and Benefits Characterization Equivalency Conclusions

US FDA Draft Guidance for Industry:

Definitions Costs and Benefits Characterization Equivalency Conclusions

Guidelines for the Establishment, Handling,

Definitions Costs and Benefits Characterization Equivalency Conclusions

WHO General Guidelines for the Establishment,

Part B. Secondary chemical reference substances

Definitions Costs and Benefits Characterization Equivalency Conclusions

WHO General Guidelines for the Establishment,

primary chemical reference substance

A designated primary chemical reference substance is one that is widely

secondary chemical reference substance

A secondary chemical reference substance is a substance whose

Definitions Costs and Benefits Characterization Equivalency Conclusions

Recent USP Publication

Definitions Costs and Benefits Characterization Equivalency Conclusions

International vocabulary of metrology –

5.4 primary standard

5.5 secondary measurement standard

Definitions Costs and Benefits Characterization Equivalency Conclusions

Full characterization, including comparison to a

Robust procedures must exist for all aspects of