This is an Application Brief and does not contain a detailed Experimental section.
The goal of this study is to transfer a reversed-phase LC gradient method for an API, abacavir, and its related substances from an Agilent 1100 Series LC system to a Waters ACQUITY UPLC H-Class System.
We demonstrate how we preserve the fidelity of the separation, with both retention times and relative retention times within 2%. For this assay, the %RSD’s for injection repeatability were lower on the ACQUITY UPLC H-Class System as compared to the 1100 LC Series System.
In addition, by using Waters’ gradient SmartStart Technology, the differences in system volume were factored into the method – without the need to make any manual adjustments to the gradient table.
These results show that gradient method transfer between different manufacturer’s instruments can be performed with minimal adjustments to the method and/or the system.
The analysis of an active pharmaceutical ingredient (API) and its related substances by HPLC is often conducted throughout the life cycle of a drug to ensure safety and efficacy. These assays are typically performed in regulated laboratories, in which changes to the method are either limited1 or not permitted and may result in the need for a complete revalidation. While many legacy methods were originally developed on traditional HPLC systems, there may be a desire to transfer the method to newer UHPLC instrumentation. This need may be driven by the available resources or by an overall drive to modernize. When transferring the method to a different HPLC/UHPLC instrument, the new instrumentation must typically produce the same separation and meet the system suitability requirements of the original method/instrument.1 However, there are a number of instrument attributes that can affect the success of the method transfer. For gradient separations, the impact of dwell volume can be dramatic. The dwell volume, which is affected by the mixer, valves, and injector, varies from instrument to instrument whether the instrument is from the same or different manufacturers. For gradient method transfer, these instrument characteristics should be considered and compensated for in a gradient table.1
A previously published method for abacavir and related substances2 was transferred from an Agilent 1100 Series LC System to an ACQUITY UPLC H-Class System (Table 1). The Agilent 1100 Series LC System was configured with a passive mobile phase pre-heater (3 μL), while the ACQUITY UPLC H-Class System was configured with an active mobile phase pre-heater. To account for gradient delay differences, each instrument’s dwell volume was measured.3 The measured dwell volume was greater on the Agilent 1100 Series LC System. Therefore, to compensate for the differences in dwell volume between the Agilent 1100 Series LC System and the target ACQUITY UPLC H-Class System, a 915-μL “after injection” delay was used for the analysis on the ACQUITY UPLC H-Class System. This delay was entered directly in units of volume or μLs using gradient SmartStart Technology (Figure 1) in the instrument method.4 This feature compensated for the differences in dwell volume in methods transfer, eliminating the need to make manual adjustments to the gradient table, an action which could trigger a full revalidation of the method. The volume entry into the gradient SmartStart Technology was the only adjustment to the method.
Both the Agilent 1100 Series LC System and the ACQUITY UPLC H-Class System produced comparable separations (Figure 2). Specifically, the retention times for abacavir and related compounds were all within 0.2 min or less than 3% deviation across the two instruments (Table 2). The relative retention times, which were calculated relative to the API, were all within 0.01% deviation for the related substances. The USP resolution for the critical pair (API and compound 3) was ≥2.5 on both systems, indicating no substantial loss of resolution in method transfer. The % area of the related substances and the API were within 0.2% on both instruments.
Many methods use retention time and relative retention time for identification purposes only.5,6 When transferring a method from one manufacturer’s system to another, a generally accepted criterion for retention time variance is within 3–5%.7 The results obtained for the method transfer described meet this criterion.
To evaluate repeatability of the method, five replicate injections were performed on both instruments. The standard deviations and percent relative standard deviations (%RSD) were calculated (Table 3). On both systems, the retention time RSD’s were less than 0.2% and the peak area RSD’s were less than 3% for all the known analytes. In addition, the analysis on the ACQUITY UPLC H-Class System produced lower peak area RSD’s for all the analytes, as compared to that run on the Agilent 1100 Series LC System. While the injection repeatability on both systems was acceptable (<3% RSD), the ACQUITY UPLC H-Class System had slightly lower injection-to-injection variability as measured by the peak area %RSD.
An assay for the analysis of abacavir and related substances was successfully transferred from an Agilent 1100 Series LC System to an ACQUITY UPLC H-Class System. The fidelity of the separation was preserved: the retention times were within 2%, and the relative retention times were within 2%. For this assay, the %RSD’s for injection repeatability were lower on the ACQUITY UPLC H-Class System as compared to the Agilent 1100 LC Series System. In addition, by using gradient SmartStart Technology in the instrument method, the differences in system volume were factored into the method – without the need to make any manual adjustments to the gradient table. These results demonstrate how gradient method transfer from Agilent 1100 Series LC System to an ACQUITY UPLC H-Class System can be performed with minimal adjustments to the method and/or the system.
720005252, December 2014