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A Well-Written Analytical Procedure for

Regulated HPLC Testing
October 15, 2024
By Michael W. Dong
News Article

LCGC International
October 2024
Volume 1 Issue 9
Pages: 22–29

This paper describes the content of a well-written

analytical procedure for regulated high-performance liquid
chromatography (HPLC) testing. A stability-indicating
HPLC assay for a drug product illustrates the required
components for regulatory compliance, including
additional parameters to expedite a laboratory analyst’s
execution.

High-performance liquid chromatography (HPLC) plays a

significant role in the quality control of pharmaceuticals, and
the development of stability-indicating assays is often the first
key task for separation scientists in the pharmaceutical
industry. The intricated method development process of these
analytical procedures and regulatory expectations have been
described in books (1–3), journal articles (4), and regulatory
guidelines (5). This column focuses on the recommended
contents of the analytical procedure as outlined in a United
States Food and Drug Administration (FDA) guidance
document published in 2015 (6). An HPLC assay method for a
small-molecule drug product is used here as an illustrative
example of the required regulatory compliance elements and
suggested parameters that help the analyst for more
straightforward method execution with better accuracy.

The Content of an Analytical Procedure

This section is extracted from the US FDA guidance document
(6) on the expected content of an analytical procedure used in
regulated testing:
“You should describe analytical procedures in sufficient detail to
allow a competent analyst to reproduce the necessary conditions
and obtain results within the proposed acceptance criteria. You
should also describe aspects of the analytical procedures that
require special attention. The analytical procedure may be
referenced from FDA-recognized sources ([such as the] United
States Pharmacopeia/National Formulary [USP/NF], [or the]
Association of Analytical Communities [AOAC] International) if
the referenced analytical procedure is not modified beyond what
is allowed in the published method. You should provide in detail
procedures from other published sources. The following is a list
of essential information you should include for an
analytical procedure.”

Principle/Scope

A description of the basic principles of the analytical

test/technology (for example, separation or detection); target
analyte(s) and sample(s) type (for example, drug substance
[DS], drug product [DP], or impurities or compounds in
biological fluids).

Apparatus/Equipment

All required qualified equipment and components (including

instrument type, detector, column type, dimensions, alternative
column, and filter type).

Operating Parameters

Qualified optimal settings and ranges (include allowed

adjustments supported by compendial sources or development
or validation studies) critical to the analysis (such as flow rate,
components temperatures, run time, detector settings, and
gradient). A drawing with experimental configuration and
integration parameters may be used as applicable.

Reagents/Standards

The following should be listed where applicable:

Description of reagent or standard

Grade of chemical (for example, USP/NF, American
Chemical Society, HPLC-grade and preservative-free)
Source (for example, USP reference standard, qualified in-
house reference material)
Purity (for pure chemicals only), state (for example, dried or
undried), and concentration
Potencies (where required by Code of Federal Regulations
[CFR], USP)
Storage conditions
Directions for safe use (as per current Safety Data Sheet)
Validated or documented shelf life

Sample Preparation
Procedures (such as extraction method, dilution or
concentration, desalting procedures, and mixing by sonication,
shaking, or sonication time) for the preparations for individual
sample tests. A single preparation for qualitative and replicate
preparations for quantitative tests with appropriate units of
concentrations for working solutions (for example, µg/mL or
mg/mL) and information on the stability of solutions and
storage conditions.

Standards Control Solution Preparation

Procedures for the preparation and use of all standard and

control solutions with appropriate units of concentration and
information on stability of standards and storage conditions,
including calibration standards, internal standards, system
suitability standards, etc.

Procedure

A step-by-step description of the method (equilibration times,

and scan/injection sequence with blanks, placebos, samples,
controls, sensitivity solution [for impurity method] and
standards to maintain the validity of the system suitability
during the span of analysis), and allowable operating ranges
and adjustments, if applicable.

System Suitability

Confirmatory tests procedures and parameters to ensure that

the system (equipment, electronics, and analytical operations
and controls to be analyzed) will function correctly as an
integrated system at the time of use. The system suitability
acceptance criteria applied to standards controls and samples,
such as peak tailing, precision and resolution acceptance
criteria, may be required as applicable. For system suitability of
chromatographic systems, refer to the FDA guidance for
industry on Validation of Chromatographic Methods and USP
General Chapter <621> Chromatography.

Calculations

The integration method and representative calculation

formulas for data analysis (standards, controls, samples) for
tests based on label claim and specification (such as assay,
specified and unspecified impurities, and relative response
factors). This includes a description of any mathematical
transformations or formulas used in data analysis and a
scientific justification for any correction factors used.

Case Studies from an Early-Phase Small-

Molecule Development
Project: Background Information
A case study from an early-phase small-molecule oncology
drug development project was used to illustrate a regulated
HPLC method’s content and operating parameters. The new
chemical entity (NCE) is a multi-chiral molecule with a complex
synthetic scheme to ensure chiral purity, requiring the
development of 40+ HPLC achiral and chiral methods to
support process chemistry development (7,8). The NCE is a
hygroscopic basic compound developed as a monochloride
salt with partial crystallinity. The Phase I clinical trial material
(CTM) DP was the powder in a capsule (PIC) dosage form.
Refrigeration and storage with a desiccant were required for
DS and DP to eliminate moisture absorption of the hygroscopic
active pharmaceutical ingredient (API).

Case Study: A Stability-Indicating Early-Phase

HPLC Method
In this case study, a stability-indicating early-phase HPLC
method illustrates the content and parameters of a well-written
analytical procedure used in regulated testing. Comments are
included as explanations, clarifications, or justifications for the
inclusion of additional information or parameters.

Principle/Scope

To determine the assay (% Label Claim), related substances,

and identity in G-1234 drug product capsules by HPLC.

Comments: This method serves as an assay procedure for three

critical quality attributes of the DP: % Label Claim (potency or
the amount of the API), related substances (levels of impurities
and degradants), and identification (by matching retention time
of the main peak with that of a qualified reference standard).
The sample is a drug product (5-mg capsule) and the technique
is HPLC using reversed-phase LC with ultraviolet (UV) detection.

Apparatus/Equipment

HPLC system equipped with a binary or quaternary pump,

auto-sampler, temperature-controlled column
compartment, UV-detector, and electronic integrator or
computer system capable of peak integration or equivalent
HPLC Column: ACE 3 C18,150 mm x 4.6 mm, 3 µm (P/N
ACE-111-1546), or equivalent
Analytical balance capable of accurately measuring to 0.01
mg
Top loading balance capable of measuring to 0.1 g
pH meter
Vortex mixer
Sonicator
Class A volumetric glassware
Automatic delivery pipettes, or Class A volumetric pipettes
Syringe Filter, 0.45 μm nylon.
Disposable syringes.
Comments: For better method portability or applicability of the
analytical procedure across laboratories and countries using
equipment from different manufacturers, their requirements
should be kept “generic” if possible. The HPLC system must be
qualified for Good Manufacturing Practice (GMP) applications
(9,10). The exception is the HPLC column, whose description
should be detailed and specific, including the part number,
manufacturer, dimension, bonded phase, and particle size. An
alternate column is often not found in stability-indicating assays,
as column equivalency is challenging to demonstrate in complex
separations.

Operating Parameters

See Table I.

Comments: The operating parameters should have sufficient

details to allow duplication by another analyst, including
parameters such as the composition of the needle wash solution
and spectral bandwidth of the diode array detector (DAD). The
full gradient program should be listed, including the post-run
equilibration time. Including the expected initial column pressure
is highly recommended to help in method troubleshooting. The
maximum absorbance wavelength of the API is often used as the
detection wavelength, as most related substances have the same
chromophoric properties as the API. Far ultraviolet (UV)
wavelengths may occasionally be selected to provide higher
sensitivity to the API and its impurities. Including system dwell
volume may be helpful for ultrahigh-pressure liquid
chromatography (UHPLC) methods for complex samples.

Reagents/Standards

Purified water, suitable for HPLC analysis, or equivalent.

Acetonitrile (ACN): HPLC grade.
Formic acid: ≥97%, or equivalent.
 Ammonium formate: LC/MS grade (e.g., high-purity grade
from Sigma-Aldrich, P/N 516961, ≥99.995%).
G-1234 reference standard.

Comments: Reagents and their specified grades or purity should

be listed. A qualified reference standard (2,8) is generally
required in regulated pharmaceutical analysis to calibrate the
testing system in the potency assay of the API and its
identification.

Sample Preparation

Preparation for 5 mg capsules: Approximately 0.50 mg/mL in

diluent. Prepare in duplicate. For example, gently open and
drop five capsules into a dry 50 mL wide-mouth volumetric
flask. Add diluent and sonicate for at least 5 min to dissolve.
Once the sample is dissolved, dilute to volume with the diluent
and mix well. Pass an aliquot of the solution through a 0.45 µm
nylon filter into an HPLC vial, discarding the first 0.5 mL.

Comments: For DS and DP analysis, a simple “dilute-and-shoot”

method is generally adopted, with an extra filtration step for
tablets and capsules using a disposable 22-mm i.d. membrane
filter (2).

Mobile Phase and Standards Control Solution

Preparation

Mobile Phase A Preparation (MPA): 20 mM ammonium

formate buffer, pH 3.7 (For example, weigh 2.52 g ± 0.2 g
of ammonium formate on a balance and transfer into 2 L
of purified water and mix well. Mix in 1.3 mL of formic acid
to arrive at the target pH 3.7 ± 0.1. If required, adjust the
pH using additional formic acid. Do not filter.
Mobile Phase B Preparation (MPB): 0.05% Formic acid in
ACN For example, pipette 500 mL of formic acid into 1 L of
acetonitrile. Mix well.
Diluent: 20 mM ammonium formate buffer, pH 3.7
Reference Standard Solution: Approximately 0.5 mg/mL G-
1234 Reference Standard in diluent. Prepare in duplicate.
For example, accurately weigh 25 mg of G-1234 Reference
Standard and transfer to a 50 mL volumetric flask. Add
sufficient diluent to dissolve, and mix thoroughly using a
vortex mixer and/or sonication, if needed. Dilute to volume
using diluent and mix well.
System Suitability Sensitivity Check Solution: 0.05%
Reference Standard Solution prepared in diluent. For
example, pipette 50 mL of Reference Standard Solution
into a 100 mL volumetric flask containing diluent. Dilute to
volume with diluent and mix well.
Retention Time Marker Solution: Approximately 0.5 mg/mL
G-1234 toxicology lot prepared in diluent.

Comments: Detailed procedures for the preparation of MPA (the

weaker aqueous MP) and MPB (the stronger organic MP) are
included in this procedure. No MP filtration is required when a
high-purity reagent such as an LC/MS grade ammonium
formate is used (2). The Retention Time Marker Solution is
prepared using a toxicologic DS lot (often called the Good
Laboratory Practice toxicological evaluation lot) (10) containing
some of the expected process impurities and degradation
products. The solution is part of the System Suitability Testing
(SST) Solution to ensure the executed method can provide
adequate resolution between key analytes. Alternatively, this
solution can be made by spiking the reference solution with
synthesized reference standards of related substances. The use of
this solution helps with the accurate identification of key
analytes in release testing and stability studies. Note that
standard and sample solution stability data may be included if
available.

Procedure

Before analysis, equilibrate the system and column by pumping

the mobile phase at the set flow rate. Test injections may be
performed until a stable baseline and/or acceptable response is
obtained. Flush the column with a water-acetonitrile mixture or
another suitable solvent if a clean baseline is not obtained with
blank injections.

The suggested injection sequence is as follows:

Re-inject Reference Standard A (Bracketing Reference) after not

more than 9 sample injections and again at the end of the
sequence.

Comments: The injection sequence table of nine initial injections

followed by sample analysis and bracketed standards is typical
in most regulated testing (2).

System Suitability Test (SST)

Evaluate the blank chromatograms for the presence and

impact of any peaks that elute in the region corresponding
to G-1234 or known related substances. There should be
no significant interference from the blank.
The signal-to-noise ratio (S/N) for the G-1234 peak in the
Sensitivity Check Solution is ≥ 10.
 The % RSD of the G-1234 peak area in all Reference
Standard A injections (including bracketing injections) is ≤
2.0%.
The % Recovery of the G-1234 peak in Reference Standard
B against the average G-1234 peak area in the first 5
Reference Standard A injections is 100.0% ± 2.0%
Report the USP tailing factor (Tf) of the G-1234 peak in the
first Reference Standard A injection.
Evaluate resolution using the Retention Time Marker
Solution. USP Resolution (Rs) is ≥ 1.2 between the SRS
peak and the G-1234 peak, and Rs is ≥ 1.5 between the G-
1234 peak and the RRR diastereomer peak.
The % RSD for the retention time of the G-1234 peak in all
Reference Standard A injections, including bracketing
injections, is ≤ 2.0%.

Comments: The SST acceptance criteria are typical of those

found in USP <621> (11). A tighter acceptance criterion of ≤
0.73% RSD is more useful and realistic for the performance
expectations of modern HPLC systems. The specified tailing
factor is “report” in this method, even though most assays
specify an acceptance criterion of ≤ 2.0. Note that for many
NCEs with multiple basic functional groups (for example,
amines), a Tf of 3.0 is not unusual.

Calculations

Label Claim Determination

Calculate the % Label Claim using equation 1:

where PF equals Purity Factor of the Reference Standard

(expressed as a decimal); SDF equals Sample Dilution Factor
(Volume of Sample [mL] / Number of Capsules; SF equals salt
conversion factor (for example, for mono-HCl = 1.08); and RFall
ref std A equals the mean peak area of G-1234 peak in all Ref Std
A inj. / Std A Conc. (mg/mL).

Calculate the average % Label Claim from n = 4 injections per

test sample.

Report the average % Label Claim for all samples.

Related Substances Determination

For each sample injection, integrate all peaks ≥ DL (0.02%),

excluding those due to the blank and any matrix-related peaks.
Calculate the amount of each individual related substance in
the G-1234 drug product using equation 2:
Calculate the average % individual related substances from n =
4 injections per sample.

Report related substance levels (Area %) for each individual

related substance ≥ Quantitation Limit (QL. 0.05%), where each
individual related substance is identified by its relative
retention time (RRT) or compound name, if available. Report
individual-related substances less than the QL but greater than
or equal to the Detection Limit (DL) as “<QL.”

Determine the Total Related Substances by summing each

injection’s individual related substances (≥ QL). Calculate and
report the average (n = 4) as Total Related Substances.

Comments: The reporting and calculations of the Label Claim

and related substances were discussed and explained in more
detail in an earlier paper on Certificate of Analysis (CoA)(8).
Note that a relative response factor (RRF) is used for late-phase
methods if the molar absorptivity of the related substance varies
more than 80-120% of that of the API.

Example Chromatograms
Example chromatograms of the various solutions (in
normalized and expanded scales of the retention time marker,
reference, sensitivity, and sample solutions) should be included,
such as those shown in Figures 1–3.

FIGURE 1: The full-scale overlaid chromatograms of the reference standard, retention time
marker, sensitivity check, and diluent blank solutions.The full-scale chromatograms show the
overall separation, including the API’s peak shape and height.The optimum peak height of the
API is kept at 1.0 to 1.5 absorbance units to prevent UV detector saturation while maximizing
method sensitivity to ensure a QL of 0.05% can be reached.
 FIGURE 2: The expanded-scale overlaid chromatograms of the reference standard, retention
time marker, sensitivity check, and diluent solutions are helpful to the analyst in providing a
high-sensitivity view of the expected resolution of the retention time marker solution, the S/N
ratio of the sensitivity check, and the presence of “blank” peaks in the diluent blank.

FIGURE 3: The expanded-scale overlaid chromatograms of the reference standard spiked with
impurities (retention time marker) and several representative sample solutions of the drug
products of different strengths.

Conclusions and Summary

The analytical procedures for quality assessment and control of
pharmaceuticals are critical tools, and these documents must
be appropriately developed and written to allow for more
straightforward implementation and transfer by analysts for
release testing and stability studies. Well-written procedures
are clear and include mandatory compliance components and
sufficient details to minimize the risk of analyst errors caused
by misinterpretation. Here, the 2015 US FDA guidance
document’s recommendations for the basic elements of an
analytical procedure for regulated testing are described. In this
installment, a DP method illustrates the best practice by
documenting the mandatory regulatory components and
additional operating parameters/chromatograms to aid the
analyst in successfully performing the regulated assay.

Disclaimers
This paper discusses the recommended content of analytical
procedures for regulated testing from the 2015 FDA guidance
document and cites an actual case study example of an early-
stage stability-indicating method (phase II) to illustrate the
expected contents. The reader is referred to textbooks,
reference articles, regulatory guidelines, and company-specific
standard operating procedures for considering the specific
stage-appropriate content and details of the analytical
procedures.

Acknowledgments
The authors thank the reviewers for providing timely technical
and editorial inputs to this article: He Meng of Sanofi, Leon
Doneski of Arcutis Biotherapeutics, Alice Krumenaker of
Hovione, Kate Evans of Longboard Scientific Consulting, David
VanMeter of Proteome Sciences, Archana Bahuguna of
Raptakos Brett & Co, Vaheh Martyr of Person and Covey,
Ahmed Keed of British Pharmacopoeia Commission, Christina
Vanhinsbergh of AstraZeneca, Wayne Way of MilliporeSigma,
Frank Wu of Neurocrine Biosciences, and Mike Shifflet of
Kenvue.

References
(1) Snyder, L.R.; Kirkland J. J.; Glajch, J. L. Practical HPLC Method
Development, 2nd Ed.; Wiley-Interscience, 1997.

(2) Dong, M.W. HPLC and UHPLC for Practicing Scientists,2nd

Ed.; Wiley, 2019. Chapters 7, 9, and 11.

(3) Rasmussen, H. T., Li, W., Redlich, D., al el., HPLC Method
Development in Handbook of HPLC in Pharmaceutical Analysis;
Elsevier, 2005, Chapter 6.

(4) Dong, M. W.; Huynh-Ba, K.; Ayers, J. T., Development of

Stability-Indicating Analytical Procedures by HPLC: An
Overview and Best Practices. LCGC North Am. 2020, 38 (8),
440-455.

(5) ICH Q14, Analytical Procedure Development. ICH, 2022,

https://database.ich.org/sites/default/files/ICH_Q14_Document
_Step2_Guideline_2022_0324.pdf

(6) Analytical Procedure and Method Validation for Drugs and

Biologics: Guidance for Industry, US FDA, CDER and CBER, 2015.

(7) Remarchuk, T.; Dong, M.; Askin, D. et al., Synthesis of Akt

Inhibitor (Ipatasertib). Part II. Total Synthesis and First Kilogram
Scale-up, Organic Process Research and Development, 2014, 18
(12), 1652–1666.
DOI: 10.1021/op500270z

(8) Dong, M. W. Certificates of Analysis and Calculations for

Small-Molecule Drugs, LCGC Intl.2024, 1 (6), 10-13. DOI:
10.56530/lcgc.int.nr5488j7

(9) CFR Title 21, Part 211, Good Manufacturing Practice for
Finished Pharmaceuticals, Government Publishing Office.
https://www.ecfr.gov/current/title-21/chapter-I/subchapter-
C/part-211.

(10) Doneski, L.; Dong, M. W. Pharmaceutical Regulations: An

Overview for the Analytical Chemist, LCGC North Am. 2023, 41
(6), 211-215. DOI: 10.56530/lcgc.na.ua3181v7

(11) United States Pharmacopeia (USP), https://www.usp.org/

About the Column Editor

 Michael W. Dong is a principal of MWD Consulting, which provides training and consulting
services in HPLC/UHPLC, CMC, method development, pharmaceutical analysis, and drug
quality. He was formerly a Senior Scientist at Genentech, a Research Fellow at Purdue Pharma,
and a Senior Staff Scientist at Applied Biosystems/PerkinElmer. Michael holds a Ph.D. in
Analytical Chemistry from the Graduate Center of the City University of New York. He has over
130 publications and two best-seller books in HPLC from Wiley. He is an advisory board
member of LCGC International.

[email protected]

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