A Well-Written Analytical Procedure for Regulated HPLC Testing

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 RF_{all 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.

chroncich@mjhlifesciences.com