Modernize Your Pharmaceutical Methods With Confidence

Useful Strategies for Modernizing USP Methods Application Compendium 2 The pharmaceutical industry is undergoing a transformative shift, driven not only by the demand for more efficient and sustainable analytical methodologies but also by evolving regulatory expectations. Central to this evolution is the U.S. Pharmacopeia (USP), which, in collaboration with the United States Food and Drug Administration, European Pharmacopoeia, and British Pharmacopoeia, is leading a global initiative to modernize monographs and general chapters to reflect current scientific standards. This modernization effort aims to replace outdated, nonspecific tests with advanced techniques such as high-performance liquid chromatography (HPLC) and ion chromatography (IC), enhancing the accuracy, safety, and reliability of pharmaceutical testing. As these standards become enforceable and increasingly adopted worldwide, laboratories must align with these changes to maintain compliance and ensure product quality. In this complex regulatory landscape, organizations are seeking reliable partners who can provide both technical expertise and strategic guidance to help navigate modernization requirements and uphold the integrity of their analytical practices. Foreword Modernization of USP Methods Application Compendium USP monograph for an analytical method adjustment USP <621> Allowed adjustments Make one change Make multiple changes Make assessment of the method followed by verification Do a protocol based risk assessment Additional verifications Non-allowed adjustments Revalidation is required Assessment of Analytical Methods 3 Introduction 4 LC method modernization 5 Sustainability and cost-saving enhancements 6 Revisions to USP General Chapter <621> 7 Excipient and API impurities analysis 8 Modernize your methods with confidence 9 Contents This application compendium showcases Agilent's commitment to supporting the modernization of small-molecule analytical methods. Each application note demonstrates practical, real-world strategies for improving method performance, sustainability, and regulatory compliance, utilizing our innovative portfolio of instruments, columns, and software. Agilent is your partner in progress. Bring your scientific vision to life with our expert support and cutting-edge solutions. Together, we can work to streamline and elevate your method modernization, achieving the precision, robustness, and reliability your laboratory demands. 4 As the pharmaceutical industry continues to evolve, so to must the analytical techniques that support the development, quality, and safety of small-molecule therapeutics. This application note compendium presents eight application notes that exemplify Agilent's innovative approaches to updating and enhancing analytical methods, serving as a practical guide for laboratories seeking to align with USP modernization efforts while embracing sustainability, efficiency, cost-savings, and analytical excellence. Explore our range of modernization strategies, including: optimizing LC methods to superficially porous particle (SPP)-based columns for improved throughput and cost savings; updating the use of legacy instrumentation to more future-ready technology; adopting greener practices like reduced solvent consumption and the use of helium as a carrier gas in GC; and many more. For more information on USPs modernization initiatives and resources, visit Agilent's Revisions per USP 621. Introduction Return to contents 5 LC method modernization LC remains a cornerstone of pharmaceutical analysis, yet many small-molecule testing methods still rely on outdated column technologies, legacy instrumentation, and environmentally or LC-damaging mobile phases. Not only does this impact accuracy and reproducibility of testing results but also leads to workflow inefficiencies, higher costs, and lack of sustainability. Modern LC technologies—such as SPP columns and UHPLC systems—enable scientists to significantly reduce analysis time, solvent consumption, and operational costs while achieving robust analytical performance, data quality, and regulatory compliance. The following application notes illustrate practical solutions for implementing LC method modernization. The examples presented here include: Combining the analysis of two APIs into a single, efficient testing method with LC-friendly mobile phases and smaller modernized particle column technology Read application note Seamlessly transferring a USP monograph method from a legacy LC to a new, future-ready UHPLC system Read application note Application Note Biopharma/Pharma Authors Monique Paré Speirs, PhD and Megan K. Bean Rhyz Analytical Labs Rongjie FU Agilent Technologies (Shanghai) Co., Ltd, China Abstract Phenylephrine HCl and pramoxine HCl are active pharmaceutical ingredients (APIs) used independently or combined in a broad range of multisymptom relief over the counter (OTC) products. These APIs are generally tested separately using different extraction techniques, HPLC methods with outdated column technologies, and LC‑damaging salt buffers. Combining the separate techniques into a single extraction and acquisition method with LC‑friendly mobile phases and smaller modernized particle technology is critical to improve the efficiency and sustainability of product quality control (QC) testing. An LC method to determine both compounds from a single, simple sample extraction was developed. This method was performed with a gradient of 0.05% trifluoroacetic acid (TFA) and methanol in lieu of traditional salt buffers. The analysis was conducted on an Agilent 1260 Infinity II LC system with an Agilent InfinityLab Poroshell 120 EC‑C8 (3.0 × 150 mm, 2.7 μm) column and diode array detection at 224 nm. Modernizing LC Methods for USP Phenylephrine HCl and Pramoxine HCl in OTC Products Return to contents 6 Sustainability and cost-saving enhancements The first step in enhancing sustainability and cost-savings is through reduction of time, resources, and waste. By adopting streamlined workflows with shorter run times, laboratories can use significantly less mobile phase per analysis. This not only cuts down the volume of costly and often environmentally hazardous organic solvents but also reduces energy consumption and instrument wear. In turn, this leads to lower operational costs, less hazardous waste, and more efficient use of staff time—delivering both environmental and economic benefits without compromising performance. The following application notes illustrate practical solutions for: Improving gradient separations by transferring to smaller columns to reduce solvent use and analysis time Read application note Simplifying GC method transfer of residual solvent analysis from using helium to the more sustainable and cost-effective hydrogen carrier gas Read application note Application Note Pharma/Biopharma Author Rongjie Fu Agilent Technologies (Shanghai) Co., Ltd Abstract The original USP monograph HPLC method of related-compounds analysis for iohexol was transferred to smaller particle size 3.5 and 1.8 μm Agilent ZORBAX columns following the newly revised U.S. Pharmacopeia (USP) General Chapter <621> guidelines. The original method uses a gradient separation with a 4.6 × 250 mm, 5 μm column and requires 60 minutes for the analysis. The analysis time was reduced from 60 to 25 minutes when the method was transferred to the Agilent ZORBAX SB-C18, 3.0 × 150 mm, 3.5 μm column (58% reduction in analysis time and 75% reduction in solvent consumption). Furthermore, analysis time was reduced from 60 to 8.6 minutes when the method was transferred to the Agilent ZORBAX RRHD SB-C18 column, 2.1 × 100 mm, 1.8 μm (86% reduction in analysis time and 92% reduction in solvent consumption), without method revalidation. All system suitability requirements were met while achieving significant reductions in both analysis time and solvent consumption. Gradient Method Transfer of the Iohexol USP Monograph HPLC Method for Related Compounds to Smaller Particle Size ZORBAX Columns Application Note Pharma and Biopharma Author Jie Zhang Agilent Technologies (Shanghai) Co. Ltd. Abstract The manufacturing process for active pharmaceutical ingredients (APIs) may contribute to residual solvents remaining in the final product. Due to regulatory requirements, producers need to monitor and control the levels of residual solvents. In this work, an Agilent 8850 gas chromatography (GC) system coupled with an Agilent 8697 headspace sampler was applied to the residual solvents analysis following the USP Method <467>. The system performance was verified in terms of detectability, resolution, qualification accuracy, and quantitation precision using both helium and hydrogen carrier gas. Residual Solvents Analysis for the Pharmaceutical Industry Using the Agilent 8697 Headspace Sampler and 8850 GC-FID System Return to contents 7 Revisions to USP General Chapter <621> Historically, strict method parameters limited the ability of laboratories to optimize efficiency, sustainability, or newer instrumentation. The revision of USP <621> Chromatography now allows for greater flexibility in method adjustments, including changes to column dimensions, particle sizes, flow rates, and gradient conditions, as long as system suitability is maintained. With this new revision, laboratories are empowered to modernize their legacy methods without the need for full revalidation, helping them adopt more efficient and environmentally conscious techniques without compromising on performance or compliance. The following application notes illustrate how these new revisions can be applied in practice, including: Refining a compendial method for an API with modern LC equipment, reduced analysis time, and less solvent consumption Read application note Applying USP <621> revisions to modernize the acetaminophen and caffeine tablets HPLC assay with smaller particle size columns for improved performance without revalidation Read application note Application Note Pharmaceutical Author Rongjie Fu Agilent Technologies (Shanghai) Co. Ltd. Abstract The original United States Pharmacopeia (USP) Acetaminophen and Caffeine Tablets HPLC assay was modernized to take advantage of smaller particle columns, including totally porous particle (TPP) and superficially porous particle (SPP) columns, following the newly revised USP <621> guidelines. The updated methods replaced conventional 5 μm TTP columns with 3.5 μm Agilent ZORBAX Eclipse Plus C18 columns and 4 μm Agilent InfinityLab Poroshell 120 EC-C18 columns without revalidation. All system suitability requirements were met and significant reductions in both analysis time and solvent consumption were achieved. Modernizing the USP Acetaminophen and Caffeine Tablets HPLC Method Following the Revised USP <621> Guidelines Realizing the benefits of smaller particle size columns without revalidation Return to contents 8 Excipient and API impurities analysis Excipients and impurities play a critical role in influencing the safety, stability, and performance of pharmaceutical products. Therefore, their accurate analysis is essential throughout drug development and quality control processes. As formulations become increasingly complex and regulatory standards continue to evolve, traditional analytical strategies—particularly those involving LC—often lack the ruggedness, speed, and sensitivity required to meet modern expectations. Modernization of LC methodologies enables analysts to detect and quantify excipients and impurities with significantly improved precision. In addition to enhanced analytical performance, these updated methods offer benefits such as reduced analysis time, improved sustainability, and cost savings, making them highly valuable in today’s regulatory and operational landscape. The following application notes present practical solutions for: Modernizing a USP assay for an API by replacing outdated 10 μm columns with smaller particle columns without revalidation, enabling significant improvements in efficiency. Read application note Easily converting a USP impurity analysis to SPP columns with faster analysis and higher resolution Read application note Application Note Pharmaceutical Author Rongjie Fu Agilent Technologies (Shanghai) Co. Ltd. Abstract The original United States Pharmacopeia (USP) Ceftizoxime Sodium HPLC assay was modernized to take advantage of smaller particle columns, including totally porous particle (TPP) and superficially porous particle (SPP) columns, following the newly revised USP <621> guidelines. The updated methods replaced conventional 10 μm TTP columns with 5 and 3.5 μm Agilent ZORBAX Eclipse Plus C18 columns and Agilent InfinityLab Poroshell 120 EC-C18 columns without revalidation. All system suitability requirements were met and significant reductions in both analysis time and solvent consumption were achieved. Modernizing the USP Ceftizoxime Sodium HPLC Method Following the Revised USP <621> Guidelines Realizing the benefits of smaller particle size columns without revalidation Application Note Pharma & Biopharma Author William J. Long Agilent Technologies, Inc. Abstract The transfer of the USP Impurities method for diphenhydramine hydrochloride is demonstrated using Agilent ZORBAX Eclipse Plus C8 and Agilent InfinityLab Poroshell 120 EC-C8 columns. The initial method uses a 5 μm 4.6 × 250 mm column and requires 40 minutes for the analysis. When InfinityLab Poroshell 120 EC-C8 columns (4.6 × 100 mm, 2.7 μm) are used, analysis time is reduced from 40 to 33% of the original method time, without need for revalidation using the InfinityLab Poroshell 120 EC-C8 column. Pressure is monitored and considered a factor in instrument transfer. This transfer is consistent with allowed adjustments under USP37-NF32S1 (official August 1, 2014), and USP Stage 4 Harmonization, to be official December 1, 2022. A Simple Conversion of the USP Method for Diphenhydramine HCl Impurities to the Agilent InfinityLab Poroshell 120 EC-C8 Column Return to contents DE-009808 This information is subject to change without notice. © Agilent Technologies, Inc. 2025 Published in the USA, October 1, 2025 5994-8682EN For more information, visit: www.agilent.com/en/solutions/biopharma-pharma/pharmaceutical-small-molecules www.agilent.com/en/solutions/biopharma-pharma/pharmaceutical-small-molecules/pharmaceutical-method-development/usp-621-chromatography Modernize your methods with confidence The USP’s initiative to modernize monographs and the revised USP Chapter <621> provides greater flexibility for method adjustments, empowering scientists to adopt these innovations while remaining within compendial guidelines. By modernizing your current USP methods, you can not only prepare your laboratory for evolving regulatory requirements but also enhance operational efficiency—saving you time, money, and resources. Providing easy access to real-world strategies for method modernization, Agilent offers a roadmap for transforming outdated methods into high-performance, sustainable, and future-ready workflows. As your reliable partner, we can provide the expertise, technologies, and solutions to update your small-molecule analytical methods with confidence. Return to contents