UPLC Technology can provide tremendous benefits to routine testing of generic drug products, including increased sample throughput and decreased solvent consumption (including subsequent disposal).The goal of this document is to educate the reader on how to properly transfer HPLC methods found in the USP-NF (and other Pharmacopeias) to UPLC Technology in order to realize the full benefits of higher throughput, lower costs, and faster time-to-market for routine analysis of generic drugs.
Manufacturers of generic drugs must be able to prove that the compounds they produce are comparable to the brand-name drug while maintaining and/or improving the cost effectiveness of the drug’s production. While the analytical methodologies used to characterize many drugs can be both difficult and costly to develop, generic drug producers can refer to the United States Pharmacopeia-National Formulary (USP-NF),1 as well as the British, European, and Japanese Pharmacopeias for information on how to analyze drug substance, drug product, and associated excipients using various analytical techniques such as high performance liquid chromatography (HPLC).
UPLC Technology enables the use of sub-2-μm particle columns with smaller inner diameters (≤ 3.0 mm) and short lengths (50 to 100 mm) on low dispersion, high pressure instrumentation. As a result, lower flow rates and shorter run times are used, which reduces solvent and sample consumption and disposal. The same resolution can be achieved in less time with UPLC, which saves the time and money associated with method development and routine analysis of drug substances and drug products.2
The goal of this document is to educate the reader on how to properly transfer HPLC methods found in the USP-NF (and other Pharmacopeias) to UPLC Technology in order to realize the full benefits of higher throughput, lower costs, and faster time-to-market for routine analysis of generic drugs. While each application focuses on a particular drug product (tablets, capsules, ointments, and oral suspensions), the process for transferring HPLC methods found in the USP-NF to UPLC follows a standard procedure, which be reviewed and explained so that the reader can adapt the approach for any USP monograph in existence today. The reader will understand how to select a modern chemistry equivalent based on the “L” designation in the USP-NF, transfer the HPLC separation to UPLC, and understand the practices necessary to ensure that the transferred method can be used routinely in a production quality control (QC) laboratory. The main goals of this document are to demonstrate that compendial methods can be transferred to UPLC Technology in three easy steps, and supply the reader with the best practices for using UPLC for method development and routine analysis of generic drug formulations.
The following three steps are used to transfer an existing USP method to UPLC Technology (Figure 1):
The next three sections will briefly describe the details of these steps to ensure successful implementation of UPLC Technology for generic drug analysis.
Parameters such as base particle chemistry, pore size, and ligand density can make selecting an “equivalent” column difficult for routine analysis. All of the HPLC columns specified for use in the USP monographs can be converted to equivalent Waters columns due to the availability of Waters columns in both HPLC and UPLC particle sizes. This facilitates transfer of HPLC methods to UPLC without a change in selectivity of the separation.
In order to properly choose a Waters HPLC column equivalent to the column type specified in the USP-NF monograph, the Waters Reversed-Phase Column Selectivity Chart can be used at (http://www.waters.com/waters/promotionDetail.htm?id=10048475). This chart plots the hydrophobicity of a column on the x-axis and the relative retention for bases (e.g., selectivity) on the y-axis using a controlled set of conditions. It can be used to compare columns from many different vendors to determine which columns have similar selectivity to each other.
For example, in the USP monograph for ziprasidone HCl,3 the suggested column designation is “L7” which is a C8 ligand bonded to porous silica particles. The column used to develop the original USP monograph was an Agilent Zorbax Rx-C8 column. Using the Waters Reversed-Phase Column Selectivity chart, the Waters column with the closest selectivity to the Zorbax column in the L7 category is XBridge C8 (Figure 2).
Once this is determined, the Waters column is used to run the methodology specified in the USP monograph to ensure that the separation on the Waters column meets the specified system suitability criteria.
Once the USP monograph for each generic drug is run with a Waters equivalent HPLC column on an HPLC system and the system suitability criteria are met, the separation is then transferred to an equivalent UPLC column on a UPLC system.
In order to properly transfer a method between systems, the following requirements must be met:
To address the first two requirements above, Waters offers Method Transfer Kits that contain an HPLC column and UPLC column that have the same resolving power and chemistry but different particle sizes. The advantage to using smaller particles is that shorter column lengths can be used to achieve the same efficiency (or peak capacity in a gradient).2 As a result, shorter run times and faster linear velocities can be used (Figure 3) to increase throughput and decrease costs in a routine analysis laboratory. The following column dimensions have the same resolving power, which is calculated from the ratio of the column length (L) to particle size (dp):
HPLC - 150 mm, 5 μm
HPLC - 100 mm, 3.5 μm
HPLC - 75 mm, 2.5 μm
UPLC - 50 mm, 1.7 μm
The third requirement can be achieved using the ACQUITY UPLC Columns Calculator. This calculator allows the transfer of methods from HPLC to UPLC, UPLC to HPLC, and between different HPLC and/or UPLC systems. The original method conditions are entered into the calculator by the user, and the conditions for the new method are automatically displayed into a results window. The resulting method can also be directly exported to Empower without the need for manual transcription. For gradient separations, the dwell volumes of the two systems should be measured and entered for proper method transfer.
The isocratic method transfer calculations for the USP assay method for levonorgestrel and ethinyl estradiol tablets4 using the ACQUITY UPLC Columns Calculator are shown in Figure 4. Figure 4A shows the original method conditions on HPLC, and it also indicates the UPLC system to which the method will be transferred. There is a field to select the column dimensions of the UPLC column to ensure that the resolving power (L/dp ratio) is maintained. Figure 4B shows the resulting UPLC method options. One of the options accounts for the change in particle size, which calculates the optimum flow rate based on both the changes in column dimensions and particle size (smaller particles can be operated at faster flow rates without loss in performance). The second option disregards particle size, and only accounts for the column geometry. It is recommended to use the method that accounts for particle size to take full advantage of UPLC for optimal separations.
For gradient separations, the dwell volume of each system should be specified to ensure that the gradient reaches the head of the column at the same time for both the HPLC and UPLC methods, allowing the selectivity of the original separation to be preserved. Figure 5 shows an example of a gradient method transfer from HPLC to UPLC for mometasone furoate ointment.5 The original HPLC conditions are shown in Figure 5A and the resulting UPLC method is shown in Figure 5B. The resulting UPLC method suggests a pre-injector volume of 62 μL. This number can be entered into the UPLC instrument method in Empower to ensure the sample is injected at the same time the gradient reaches the head of the column, ensuring that the separation selectivity of the HPLC method does not change. If this field indicates that a hold time is needed, the user has the option to incorporate this into the gradient table for UPLC. Additional information on using the ACQUITY UPLC Columns Calculator can be found in a previously published application note.6
There are some general precautions and best practices that will ensure successful implementation of UPLC for routine analysis of generic drug formulations:
UPLC Technology can provide tremendous benefits to routine testing of generic drug products, including increased sample throughput and decreased solvent consumption (including subsequent disposal). Analysis times can be reduced by 70 to 90%, and solvent consumption can be decreased by over 90%. This offers significant cost benefits associated with running more samples in less time, and can decrease the overall operating expenses in development and quality control laboratories. Most of all, it helps provide a competitive advantage to generic drug companies who leverage UPLC Technology to bring drugs to market faster and cheaper.
720004251, February 2012