The Good, the Bad, and the Ugly of HPLC in Pharmaceutical Analysis

This article is a personal reflection on the “good” and the “not-so-good” aspects of high performance liquid chromatography (HPLC) applications used in pharmaceutical analysis and regulated testing.

The Good

High performance liquid chromatography (HPLC) is indispensable in pharmaceutical development and quality control. It enables precise quantification of both active pharmaceutical ingredients (APIs) and their impurities or degradation products, offering excellent resolving power, accuracy, precision, sensitivity, and reliability (1-3).

Reversed-phase liquid chromatography (RPLC) aligns well with the hydrophobic nature of small-molecule drugs, providing sufficient retention and mass balance for purity assays. Ultraviolet (UV) detection—whether variable wavelength ultraviolet-visible (UV-vis) or photodiode array (PDA)—is ideal because most drugs are chromophoric. Since the entire injected sample passes through the flow cell, it delivers exceptional precision (RSD < 0.2%) for quality control (QC).

The five-order magnitude of linear UV response (from limits of quantitation to about two absorbance units) allows for convenient single-point calibration in stability-indicating assays. Monitoring at the API’s λ_max (wavelength of maximum absorbance) yields both specific and sensitive quantitation of related substances, including process impurities and degradation products, which often maintain similar chromophores with the API.

The Bad

Despite its strengths, the standard RPLC/UV method has notable limitations.

Firstly, HPLC is inherently complex. The technique involves multiple modules—pump, autosampler, column oven, and detector—that must work in harmony with appropriate mobile phases and columns. These are orchestrated by chromatography data systems (CDSs), which manage sample sequences and data integration. However, mastering CDS software requires extensive training, often taking months for new analysts to become proficient in instrument control, sequence setup, post-analysis integration, calibration, and reporting (4).

Secondly, the cost of instrumentation—often exceeding $100,000—is prohibitive for many organizations. The market is dominated by large manufacturers that collectively hold ~85% of the market share (2). High marketing and service costs, along with the need for CDS compatibility, have historically discouraged new entrants.

Thirdly, UV detection depends on baseline resolution of all key analytes as a regulatory requirement! This peak resolution requirement makes method development for assays, such as stability-indicating tests, particularly challenging. Reference standards are also necessary for the identification of peaks based on retention time. These limitations can be mitigated by incorporating mass spectrometry (MS) into method development, using mobile phases with volatile additives.

Fourthly, sample preparation remains labour-intensive. While straightforward for drug substances and products, procedures such as weighing, grinding, and extraction (filtration) are manual and time-consuming, requiring Class A volumetric flasks for accuracy. Attempts to automate these steps with robotics have largely failed (5).

Finally, improvements in HPLC speed, resolution, and cost have been modest, perhaps three- to five-fold over the past six decades. In contrast, DNA sequencing has advanced by five orders of magnitude in under fifty years. While the comparison may be imperfect, regulatory requirements for method and equipment validation have undeniably slowed innovation in HPLC.

The Ugly

For many laboratory scientists, the most frustrating part of their routine is navigating regulatory compliance in quality control (QC) testing. The term “ugly” may be too harsh; “bureaucratic” or “inefficient” are more accurate.

Regulations such as 21 Code of Federal Regulations (CFR) Parts 210 & 211 (GMP) are legislated by Congress and written by lawyers. Each company’s quality assurance (QA) team interprets these into internal quality systems and standard operating procedures (SOPs). New analysts must read and understand hundreds of SOPs before beginning lab work, followed by qualification or on-the-job training (6,7). These often become “check-box” exercises, as laboratory managers and analysts are eager to begin testing. SOPs tend to expand over time without holistic reassessment.

Most scientists support clear, enforceable, and reasonable regulations—such as Good Documentation Practices (GDocP) using Accurate, Legible, Contemporaneous, Original, Attributable, Complete, Consistent, Enduring, and Available. (ALCOA+) principles Unfortunately, the rise of electronic data systems has made internal quality procedures increasingly convoluted.

Pharmaceutical regulations are supplemented by the International Council on Harmonization (ICH) guidelines, which provide detailed compliance expectations. These are widely followed during late-stage development and production. While many ICH guidelines—such as those on stability (Q1A-F), validation (Q2), and impurities (Q3A-D, Q5A-E)—are clear and practical, they are also demanding. For example, analytical procedures must be validated per ICH Q2(R2) and USP <1225> and <621>. System suitability testing must be performed before regulated testing, typically by injecting ten samples to confirm system reliability.

In contrast, newer ICH guidelines—such as those on Life Cycle Management, Quality Risk Management, Quality by Design (QbD), and Analytical Procedure Development (ICH Q8, Q9, Q10, Q12, and Q14)—are more conceptual and less prescriptive in nature. While their intent is commendable, implementation details are often vague, leaving organizations and individuals to interpret them. More practical guidance and case studies would enhance their impact.

Despite these challenges, the regulatory framework does function as intended: products are released, and new drugs are approved. To foster a more collaborative compliance culture, educational programs should emphasize the intent behind regulations, the critical role of QC chemists in patient safety, and the importance of mentorship in analytical chemistry and method development.

Disclaimer

The title of this article was inspired by the 1966 Clint Eastwood Western classic, “The Good, the Bad, and the Ugly.” These reflections stem from my two decades of experience supporting drug development as a Chemistry, Manufacturing and Control (CMC) analytical lead. While regulations are essential for ensuring drug quality and preventing fraud, many of us would prefer a simpler, more efficient system. Since sweeping changes are unlikely, the most realistic path forward is to foster a culture of participatory compliance at the laboratory level.

Acknowledgments

Thanks to the reviewers for their timely technical and editorial input: Alice Krumenaker (retired from Hovione), David VanMeter (Proteome Sciences plc), Kate Evans (Longboard Scientific), and Leon Doneski (Arcutis Biotherapeutics).

References

1. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis, 7th Ed., Cengage Learning, 2017.
2. Dong, M. W. HPLC and UHPLC for Practicing Scientists, 2nd Ed., Wiley, 2019, Chapters 4, 9, 11, and Foreword.
3. Guillarme D.; Dong, M. W. Newer Developments in HPLC Impacting Pharmaceutical Analysis: A Brief Review. Amer. Pharm. Rev. 2013, 16 (4), 36-43.
4. Mazzarese, R.; Bird, S. M.; Dong, M. W. Chromatography Data Systems: Perspective, Principles and Trends. LCGC North Am. 2019, 37 (12), 852-866.
5. Dong, M. W. Sample Preparation of Drug Substances and Products in Regulated Testing: A Primer. LCGC Intl. 2025, 2 (4), 28-32. DOI: 10.56530/lcgc.int.bm8278b4
6. 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
7. Doneski, L.; Dong, M. W.Good Manufacture Practice (cGMP): An Overview for the Analytical Chemist. LCGC North Am. 2023, 41 (10), 416-421. DOI: https://doi.org/10.56530/lcgc.na.qh7467g7