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The product's name and purity percentage alone are insufficient for evaluation of research peptides. The Certificate of Analysis (CoA) is the document that presents the analysis performed on a particular batch of the substance. Therefore, it can give a researcher a lot of information on how the sample was analyzed and what the results mean.
In other words, a researcher who works with peptides should
learn how to properly read a CoA as it can be helpful in identifying a batch,
analyzing HPLC results, understanding mass spectrometry results, and making
sure the documentation is informative enough for a research project.
This article will explain the structure of a CoA for peptides, what methods of analysis
allow doing and not allowing, and what questions one should ask while examining
a particular supplier's documentation.
Understanding a Peptide Certificate of Analysis
A Certificate of Analysis is an official documentation for
the analysis done on a specific batch of material. A good CoA will provide
information about analysis results and tie it up with a specific batch/lot as
opposed to making generalized statements about the nature of the products.
This is an important distinction since peptides are chemical
substances composed of amino acids joined together by peptide bonds and which
have certain characteristics such as structure, molecular formula, molecular
weight among others.
Some of the data that might be found in a CoA may include:
• Product/compound
name
• Batch/lot
number
• Date of
manufacture/test
• Measured
amount
• Molecular
formula/molecular weight
• Analytical
technique
• Purity
result
• Identity
result
• Laboratory/testing
organization
• Acceptance
criteria/specification, if available
The exact form will vary between different laboratories and
product suppliers. The quality of the CoA is not based on the appearance of the
document alone but the analytical data provided.
Why a CoA Matters in Peptide Research
Peptide research typically relies on reproducibility. The
difference in identity, purity or impurity of material used for two different
experiments can make it challenging to explain the difference in outcomes of
the experiments.
Batch-specific COA provides scientists with documented
beginning point. It can be used to determine what kind of material was received
and which tests were conducted on the batch of material.
This is especially useful for comparison of batches, problem
solving and recording materials used for research purposes.
COA is not, however, a complete replacement of scientific
assessment of data. Analytical techniques have their own strengths and
weaknesses. ICH recommendations concerning analytical procedures, for instance,
stress characteristics like specificity, accuracy and precision in determining
suitability of the method.
HPLC Purity Is Not the Same as Identity
High-performance liquid chromatography (HPLC) is used to
separate the different components of the sample so that they may be detected
and analyzed using chromatography. According to the USP General Chapter
<621>, chromatography is defined as a method of separation employed for
both qualitative and quantitative analysis.
As regards peptide substances, HPLC may give useful
information on the purity of the substance by means of chromatography. The “99%
HPLC” value that is mentioned usually indicates the percentage of the signal
obtained in the peak.
This doesn’t necessarily mean that the molecular identity is
known.
Mass Spectrometry Helps Address Identity
Mass spectrometry (MS) determines the mass-to-charge ratio
of the ion and gives information about the molecular mass, and, in some cases,
molecular structure. According to USP, MS is an analytical technique which can
be applied to determine the molecular mass and to elucidate the structure. [4]
Thus, the combination of the CoA for the product giving
information about chromatographic purity and the identity by MS may add new
aspects to the information provided.
The difference lies in the fact that the HPLC technique
deals with the separation process and chromatographic purity, while the
mass-spectrometry shows whether the obtained material possesses the expected
molecular mass.
How to Read a Peptide CoA Step by Step
In the process of evaluation of a CoA, go through the
document in a logical order, and not just pay attention to the purity headline.
1. Verify the
identity of the compound.
Make sure that the name of the substance, sequence when
applicable, molecular formula and molecular weight matches the substance you
wanted to have analyzed. Useful for the independent verification of basic
chemical data is the use of online databases like PubChem. [1]
2. Verify the
batch number.
This number must appear on the CoA to match the batch you
want to analyze. Generic certificates that do not refer to your batch are less
helpful in the evaluation of batch-specific results.
3. Find out
when the analysis was conducted.
This may provide a useful background for establishing the
connection between the document and your material.
4. Look for the
analytical method used.
Find information about the methods used, like HPLC, LC-MS or
MS, and not just the general phrase like "laboratory tested."
5. Determine
what purity result means.
Make sure that the result is given either as the percent
area by HPLC, assay, mass fraction or something else. All these terms should
not be confused a priori.
6. CoA identity
verification
Identify if CoA is giving results for MS or LC-MS and also
verify whether the molecular mass reported is of the expected compound.
7. CoA
additional testing
Based on the application of the product in research, there
may be additional tests that were conducted to determine its characteristics
like endotoxins, residual solvents, water content, elemental impurities, or
other parameters.
8. Tested by
whom?
The laboratory conducting the test, laboratory
identification, and accreditation details may provide useful context while
interpreting the report.
9. CoA
additional testing
Based on the application of the product in research, there
may be additional tests that were conducted to determine its characteristics
like endotoxins, residual solvents, water content, elemental impurities, or
other parameters.
10. Tested by
whom?
The laboratory conducting the test, laboratory
identification, and accreditation details may provide useful context while
interpreting the report.
What Researchers Should Look For
A well-written CoA is generally detailed enough so that a
researcher knows what was analyzed and how the result was achieved.
Examples of useful information are the batch number,
analysis date, analytical procedure, chromatogram result, identification
result, laboratory information and acceptance limits.
In addition, researchers should know how to differentiate
between results reported and validated method. A laboratory can report an
analytical result where the document may not contain all the information about
the validation of the procedure used. According to ICH Q2(R2), analytical
procedures should be evaluated based on the purpose of the procedure and the
performance characteristics. [2]
For difficult studies, researchers will have to ask for more
than just the summary certificate; examples are the chromatogram, mass
spectrum, method information and other documents.
Common Misunderstandings
"99% purity means 99% of the material is the target
peptide."
This is not always true; it all depends on the method and
may be a relative peak area rather than absolute mass-based assay.
"An MS result shows that the sample is entirely
pure."
False. MS analysis can give vital identity information, but
identity determination and purity estimation are different issues.
"A CoA guarantees the material is suitable for any
experiment."
Wrong. The suitability of a material depends on the purpose
of the experiment and other criteria.
"A professionally designed PDF is proof of
analytical quality."
How something looks does not make it true. Researchers
should concentrate on batch data, methodology, results, and lab information.
How to Evaluate Quality and Documentation
In relation to peptide documentation, it is important to
take into consideration the overall analytical approach rather than just a
single value.
The documentation kit might comprise of the following
documents:
• Certificate
of Analysis - specifies the batch and provides a summary of analytical data.
• HPLC
analysis – provides chromatographic data relating to purity.
• Mass
Spectrometry or LC-MS – provides data relating to molecular identity and mass.
• Batch data –
correlates analytical data with a specific material batch.
• Date of test
- specifies the period when the analysis was made.
• Analytical
Method - aids researchers in understanding the process of obtaining a specific
analytical result.
• Further
testing - might be necessary in terms of characteristics that are required by a
specific research project.
Publications of the USP on synthetic peptide reference
standards show why peptide characterization might require complementary
analytical approaches such as chromatography and mass spectrometry rather than
a single analytical procedure. [5]
It might be interesting to look at the difference between
CoA of a sample and CoA of the specific batch that is actually being supplied.
PeptidesX Approach to Research-Grade Transparency
PeptidesX claims that its products are manufactured only for
the purposes of in vitro laboratory and scientific research and cannot be used
on people or animals. The company also claims in its terms that its products
are provided with batch-specific Certificates of Analysis and HPLC testing.
As per the current FAQ of the company, its CoA contains
usually batch code, HPLC purity, MS-UPLC identification, molecular formula and
weight, CAS number, LAL test and peptide amount. The company also claims that
its batches are independently analyzed and sample COAs may be obtained on
request.
The point of view for the researcher here is to analyze
those documents on one's own. The policy of the supplier concerning tests
performed should be considered only the first step in analysis, but not the
final one. Researchers are able to see whether the batch number, results of
analyses, methods and lab data are clear enough for their needs.
PeptidesX
also specifically distinguishes its research products from medicines or
therapeutic products. Its Research Use Only policy claims that its products are
provided only for the purpose of laboratory research and cannot be used on
humans or animals.
What to Consider Before Choosing a Research Peptide
Supplier
A checklist of issues to be considered when reviewing
suppliers will include:
• Documentation:
Are CoAs available for checking?
• Lot
information: Does the CoA relate to a particular batch?
• Testing
procedure: Are testing procedures like HPLC and MS used mentioned?
• Product
information: Are the names and amounts of the compounds provided?
• Purpose: Is
it clear that the material is for research only?
• Business
information: Is there any business identification?
• Support
services: Are there ways of getting answers on the CoAs?
• Trackability:
Is it possible to trace the testing results to the material?
In the case of researchers in the UK, it would also be
important to consider regulations separately from analytical aspects of the
research materials. The CoA is just an evidence of analytical testing but not
regulation. UK medicines information and regulatory guidelines are provided by
the Medicines and Healthcare products Regulatory Agency (MHRA).
Frequently Asked Questions
What is a Certificate of Analysis for a peptide?
The Certificate of Analysis (CoA) is an analytical summary
of a batch of the tested material. The information provided can vary from the
supplier to the supplier and compound to compound, however, it usually
comprises such information as purity, molecular identity, molecular weight,
testing method, batch number and test date.
What does HPLC purity mean on a peptide CoA?
By "HPLC purity" we mean the proportion of the
peak of the substance of interest. HPLC purity is an appropriate term for
evaluating the purity of the substance, though it shouldn't be considered an
indicator of total amount of the peptide.
Why is mass spectrometry included on a peptide CoA?
Mass spectrometry provides data about the molecular mass of
the compound and, in some cases, about its structure. Consequently, MS and
LC-MS can help to prove identity of the peptide besides being an alternative to
HPLC.
Should a peptide CoA include the batch number?
Ideally, yes. Batch or lot number ties the analytical data
with the material in question. Otherwise, it becomes complicated for the
researcher to verify whether a certificate pertains to a specific batch of the
tested material.
Is a CoA enough to establish peptide quality?
Not necessarily. A CoA gives useful analytical data, but the
degree of testing required will depend on the research application. In some
cases, other documentation or testing is necessary for aspects of a material
that are not included in the CoA.
What is the difference between a sample CoA and a batch
CoA?
A sample CoA demonstrates the kind of documentation provided
by suppliers. The batch CoA is a CoA giving analytical data for a particular
batch of material. For analyzing the research material, the batch CoA is often
the more useful one since it relates the analytical data to the material
itself.
Does a research peptide CoA mean the product is approved
for human use?
No. Testing and compliance with regulations are separate
issues. A CoA refers to tests carried out on a material, but does not prove
that the research peptide is a medicine or ready for human or veterinary use.
Conclusion
The analysis of a peptide CoA is
much more than checking if there is a sufficiently high purity percentage. The
researcher has to verify the batch identification, know exactly what HPLC is
measuring, consider the possibility of additional identity verification
methods, such as mass spectrometry, and analyze the lab and analytical
information that backs up the report.
The same approach can be used when researching PeptidesX or
any other research-peptide producer; one should evaluate the provided
documentation itself, learn its limitations and correlate the tests performed
with the research requirements. In case of peptides, transparency of the
analytical information is the most valuable when it helps to get a clear idea
of the tests performed and their results.
Sources & References
[1] National Center for Biotechnology Information (NCBI),
PubChem, “Peptide.”
[2] International Council for Harmonisation (ICH), Q2(R2)
Validation of Analytical Procedures, 2023.
[3] United States Pharmacopeia (USP), General Chapter
<621>, Chromatography.
[4] United States Pharmacopeia (USP), General Chapter
<736>, Mass Spectrometry.
[5] United States Pharmacopeia, Reference Standards to
Support Quality of Synthetic Peptide Therapeutics, Pharmaceutical Research,
2023.
[6] PeptidesX, “Research Use Only” and “Terms &
Conditions.”
[7] PeptidesX, “Peptide FAQ – Purity, Testing, CoA & UK
Shipping.”
[8] UK Medicines and Healthcare products Regulatory Agency
(MHRA), official medicines and clinical-trials guidance.