Future-Proofing Validated Pharmacopeia Methods: A Case Study of Improving Throughput and Decreasing Operational Cost

Older pharmacopeia monographs are methods that are validated and ready to be used in the analysis of formulated pharmaceutical products or bulk material. Most of these monographs use older column technology and instrumentation, which were cutting-edge at the time they were created. However, with the creation of newer technology, such as smaller particle size stationary phases, there is potential to reduce operational costs of these older methods. This article shows one example of modernizing an older pharmaceutical monograph from a high performance liquid chromatography (HPLC) column to an ultrahigh-pressure liquid chromatography (UHPLC) column along with calculated time and solvent savings.

Validated liquid chromatography (LC) pharmacopeia methods, whether from the United States Pharmacopeia (USP), European Pharmacopoeia (Ph. Eur.), or Japanese Pharmacopoeia (JP), are normally associated with specific assays for quantifying the amount of an active ingredient, or even the level of impurities present in a product. These monograph methods are convenient for generic pharmaceutical manufacturers who don’t have the time or capacity to develop a new method. However, these methods are far from ideal, often using large column configurations packed with 5 μm particles, which require both lengthy analysis times and large quantities of solvent to test a single batch.

Fortunately, all three pharmacopeias permit certain aspects of a monograph to be altered, allowing for modernization of these older legacy methods to smaller column configurations and particle sizes. This yields methods that require considerably less analysis time and solvent usage, reducing overall operational cost while improving cycle time (1–3). This article will show one example of modernizing a USP monograph method from older high performance liquid chromatography (HPLC) column technology to more modern ultrahigh-pressure liquid chromatography (UHPLC) column technology on the same chromatographic system. The monograph method—for ketoprofen organic impurities—was modernized from a 4.6 × 250 mm, 5-μm column to a 2.1 × 100 mm, 2.5-μm C₁₈ column in inert specially-modified surface hardware. The modernized conditions for the isocratic analysis yielded a 65% reduction in analysis time, and 85% reduction in solvent usage. These benefits would be cumulative with other modernized monograph methods in each laboratory, adding to the cost and time savings.

Experimental

An Acquity UPLC H-Class Plus System equipped with a photodiode array detector and a four‑column manager (all Waters) was used for all tests. The original USP monograph conditions required the use of a 4.6 × 150 mm, 5-μm L1 column, which has a calculated length‑to‑particle-diameter ratio (L/dp) of 30,000 (4). A 4.6 × 250 mm, 5-μm XTerra MS C₁₈ column (Waters) was selected for this analysis. A mobile phase composition of 43:55:2 acetonitrile–water–potassium phosphate buffer was also specified. Phosphate buffer was prepared as 68 g/L of monobasic potassium phosphate titrated to pH 3.5 with phosphoric acid. Other specifications for the monograph included flow rate (1.0 mL/min), detection wavelength (233 nm), and injection volume (20 μL). A sample run time of 7× the retention of ketoprofen was specified as well.

The modernized test conditions used a 2.1 × 100 mm, 2.5-μm column (Waters), which has an L/dp of 40,000. USP <621> guidelines specify that L/dp can be adjusted by -25% to +50% of the original conditions, which this configuration meets. For the modernized test conditions, a 2.1 × 100 mm, 2.5-μm XBridge Premier BEH C₁₈ column (Waters) was selected. Both columns contain hybrid base particles, with trifunctionally bonded C₁₈ ligands. The modernized test conditions require a flow rate of 0.417 mL/min and an injection volume of 2.8 μL. The sample composition, outlined below, stayed constant between the two test conditions.

System suitability and standard solutions were created from stock solutions. The system suitability sample contained 5 μg/mL of ketoprofen and 1.5 μg/ mL of ketoprofen‑related compound D, 3-acetylbenzophenone. The standard solution contained only ketoprofen at 2 μg/mL. All samples and stock solutions were created in freshly prepared mobile phase.

Results and Discussion

Prior to any modernization experiments, the original test conditions must be shown to be accurate and reliable. For generic pharmaceutical manufacturers who create this product, the data are readily available in the form of previous testing. However, in this case, the original monograph conditions were tested on a UHPLC system to not only ensure the system and column were appropriate for the assay, but also to determine the run time of the separation. Figure 1 shows the results for the original monograph conditions using a 4.6 × 250 mm, 5-μm column.

The run time of the analysis was 7× the retention of ketoprofen, and on this column that meant a 40.2-min method was needed. Table 1 presents the system suitability criteria and the results obtained for the ketoprofen impurity assay using the above conditions.

As shown, the results passed the system suitability criteria for the monograph conditions. The efficiency was almost 5× higher than required, and the tailing factor and relative standard deviation were well below the limits. As mentioned previously, laboratories that use this monograph method will often have considerably more data to compare with these results. Once that data has been generated, the modernization can be evaluated. USP <621> outlines all the equations and calculations needed for modernization; however, these are complicated and prone to human error. Tools, such as column calculators and method scalers, are available to simplify the modernization process. These downloadable calculators take an existing method and scale it to a new column with minimal inputs from the user. Figure 2 shows a column calculator being used to modernize the ketoprofen impurity assay from the original monograph column to the more modern column. When using a column calculator for the described modernization activity, a longer run time was inputted into the calculator than was seen on the original method column. This was done to ensure that enough time was allocated to elute all compounds off of the modern column. A new flow rate was calculated at 0.417 mL/min and an injection volume of 2.8 μL was needed. The indicated run time of 17.33 min was scouted with the modernized column and adjusted accordingly. Figure 3 shows the results obtained using the 2.1 × 100 mm, 2.5-μm column on the system used previously.

The results obtained on the new column were comparable to those obtained with the monograph column. All system suitability criteria were met while requiring only a 14.3‑min run time (Table 2).

The column that was selected is among the most well-documented stationary phases in peer-reviewed publications. The monograph method—for ketoprofen organic impurities—was modernized from a 4.6 × 250 mm, 5-μm column to a 2.1 × 100 mm, 2.5-μm C₁₈ column in inert surface hardware modified with a hybrid organic-inorganic ethylene-bridged siloxane chemistry (5–9). This technology mitigates secondary interactions between analytes, or mobile phases, with the stainless-steel hardware of the column.

The modernization of this assay led to an almost 35-min reduction in run time per injection. For a single batch, the original monograph conditions required ~723 min (about 12 h), while the modernized conditions only required ~257 min (about 4.5 h). This is approximately a 65% reduction in cycle time for the analysis, increasing throughput and allowing faster release of batches. When calculating solvent usage based on injection run time and flow rate, the monograph conditions required ~723 mL of mobile phase per batch of material tested, not including any equilibration or blank injections. The modernized conditions only required ~107 mL of mobile phase—an ~85% reduction in solvent usage. This drastically reduced operational cost, not only to purchase but also to dispose of the solvent used for this analysis.

Modernizing monograph methods may seem intimidating and a challenging task scientifically, but with the proper tools and guidance of the pharmacopeias, these methods can be future‑proofed to ensure they stay accurate and reliable for years to come.

Conclusion

This article has shown one example of method modernization and the benefits that can be obtained from modernizing older monograph methods. Results obtained using both sets of conditions and columns passed all system suitability criteria for the analysis; however, the modernized column improved the cycle time and reduced solvent usage considerably. These benefits, when combined with those from other modernized assays, can provide significant financial savings for a laboratory. In addition, by modernizing these methods to a 2.1 × 100 mm, 2.5-μm C₁₈ column in inert surface hardware modified with a hybrid organic-inorganic ethylene-bridged siloxane chemistry, a laboratory can eliminate any potential secondary interactions. This elevates confidence in the results and reduces out‑of‑specification or failing results due to these deleterious interactions.

References

(1) USP, USP General Chapter <621>, “Chromatography”. https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf (accessed 2023-04-13).

(2) EDQM, EurPh, Chapter 2.2.46 Revisions (EDQM, Strasbourg, France, 2022). https://extranet.edqm.eu/4DLink1/pdfs/addon/20246.pdf (accessed 2023- 04-13).

(3) Japanese Pharmacopeia, 18th Edition Supplement I. https://www.mhlw.go.jp/content/11120000/001022457.pdf (accessed 2023- 04-13).

(4) Ketoprofen USP monograph. https://www.drugfuture.com/Pharmacopoeia/usp35/PDF/3621-3622%20Ketoprofen.pdf (accessed 2023-04-13).

(5) Delano, M.; Walter, T.H.; Lauber, M.; et al. Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93, 5773–578. DOI: 10.1021/acs.analchem.0c05203

(6) Walter, T.H.; Alden, B.A.; Belanger, J.; et al. Modifying the Metal Surfaces in HPLC Systems and Columns to Prevent Analyte Adsorption and Other Deleterious Effects. Recent Developments in HPLC and UHPLC, LCGC Supplement 2022, 40 (s6), 28–34. DOI: 10.56530/lcgc.na.pm7270x6

(7) Layton, C.; Rainville, P. Advantages of Using MaxPeak HPS Technology for the Analysis of Non-Steroidal Anti-Inflammatory Drugs. Waters Application Note. https://www.waters.com/nextgen/us/en/library/application-notes/2022/advantages-of-using-maxpeak-hps-technology-for-the-analysis-of-non-steroidal-anti-inflammatory-drugs.html (accessed 2023-04-13).

(8) Alkhateeb, F.; Rainville, P. Batch-to-Batch Robustness of MaxPeak Premier Columns for the Analysis of Dexamethasone Phosphate and Related Compounds. Waters Application Note. https://www.waters.com/nextgen/us/en/library/application-notes/2021/batch-to-batch-robustness-of-max-peak-premier-columns-for-the-analysis-dexamethasone-phosphate-and-related-compounds.html (accessed 2023-04-13).

(9) Gaye, M.; Knowles, C.; Addepalli, B.; Lauber, M. CRISPR Single Guide RNA Characterization by IP-RP-LC-MS with a Premier Oligonucleotide BEH 300Å C₁₈ Column. Waters Application Note. https://www.waters.com/nextgen/in/en/library/application-notes/2023/crispr-single-guide-rna-characterization-by-ip-rp-lc-ms-with-a-premier-oligonucleotide-beh-300-a-c18-column.html (accessed 2023-04-13).

Kenneth Berthelette works as a senior scientist in the Consumables and Lab Automation Group of Waters Corporation where he focuses on marketing support and collateral generation. Ken’s areas of expertise include reversed‑phase LC method development and troubleshooting and HILIC method development.

Kim Haynes is the director of chemistry marketing and product management in the Small Molecule Portfolio of the Consumables and Lab Automation Group at Waters Corporation. Her group is responsible for managing many of the columns that Waters offers.