Simplifying Methods Transfer: Novel Tools for Replicating Your Established Methods on an ACQUITY Arc System

In this application note, a method transfer from another manufacturer’s HPLC System to an ACQUITY Arc System was performed using the Path 1 of the Arc Multi-flow path technology.

This flow path, which was designed for methods transfer, allows the ACQUITY Arc System to mimic the dwell volume of a typical HPLC system. The results produced retention times within 5% of the original method. Additional fine-tuning of retention times was accomplished using Gradient SmartStart Technology, which adjusts the initial hold of the method without requiring any changes to the gradient table. The combination of these two features enables methods transfer to an ACQUITY Arc System in just two injections for a simplified methods transfer approach, including the following:

1. Transfer method to an ACQUITY Arc using appropriate Flow Path (1 or 2).
   
2. Compare chromatograms and evaluate retention.
3. If retention does not meet method criteria, adjust gradient start using Gradient SmartStart. Re-run method.

Benefits

• Ability to select between two flow paths with different dwell volumes using Arc Multi-flow path technology

• Ability to adjust gradient start relative to injection with a single setting using Gradient SmartStart

• Adjustment of gradient start using units of time rather than volume for intuitive methods transfer adjustment 

Retention shifts in methods transfer are commonly observed. Retention can be impacted by differences in system and pump characteristics such as dwell volume and mixing characteristics. Many laboratories require there be no adjustments to a method, particularly for validated methods. If adjustments are necessary, consideration is generally made to be in accordance with regulatory guidelines. With reference to dwell volume, chapter <621> of the USP monograph states “If adjustments are necessary, change in... the duration of an initial isocratic hold (when prescribed), and/or dwell volume adjustments are allowed.”¹ While adjustments for dwell volume can be manually entered into a gradient table, this approach requires calculations and manual changes to the gradient table; both of which require additional time and effort. Using a feature in the software to adjust the time between the start of the gradient and the point of injection, the duration of the gradient hold can be adjusted. This is accomplished without the need to make changes to the gradient table allowing systems – such as the ACQUITY Arc System – to mimic systems with different dwell volumes.

By being able to enter the value directly in time, methods transfer can be streamlined by measuring the difference in retention time and adjusting the initial hold accordingly. 

In a scintillation vial, 20 μL of Preparative Chromatography Mix Standard (p/n 186006703) and 100 μL of ACQUITY UPLC MS Start-Up Solution 2 (p/n: 700002741) were combined with 880 μL of 30:70 H₂O: acetontrile. The final concentration of each component is listed below. 

A sample containing two major components and five minor components was analyzed on an Agilent 1260 Quaternary LC System. The method was transferred from the Agilent 1260 System to the ACQUITY Arc System with no changes in method conditions or column. Since retention time shifts for gradient separations can, in part, be attributed to the dwell volume differences of the instruments, the ACQUITY Arc System was configured using Flow Path 1 of the Arc Multi-flow path technology. This instrument feature uses of a six-port valve to enable switching of the flow from the pump to the injector between two different paths, each with a different physical volume (Figure 1). Selection of Path 1 results in a dwell volume of approximately 1.1 mL on the ACQUITY Arc System – a value that is typical of many HPLC systems. Path 2, which was not selected for this study, results in a system dwell volume of 0.76 mL. 

Using Flow Path 1, the ACQUITY Arc System produced a separation with similar retention times to that of the Agilent 1260 Quaternary system (Figure 2, Table 1). The retention times for all the analytes were within 3% of the initial separation observed on the Agilent 1260 Infinity System. A noticeable shift, however, was observed for peak 3, which had a retention time difference of 0.10 min. This represented a shift 5–10 times greater than observed for all the other peaks. Since this peak was shifted more than the peaks that eluted earlier or later, the difference could not be entirely explained by dwell volume. Inspection of the programmed gradient revealed the peak eluted in the middle part of the first step of the gradient. While there are many factors that can impact a separation, differences in pump characteristics, such as the gradient delay, mixing and shape of the gradient can impact retention. Additional studies, not described here, indicate the shift in retention of peak 3 is influenced by gradient shape. 

One approach to adjust retention includes adjusting the initial hold of the separation. On the ACQUITY Arc System, the gradient can be adjusted to occur “at injection”, “after injection” or “before injection”, thereby allowing the system to emulate other chromatographic systems with larger or smaller dwell volumes (Figure 3). This dwell volume adjustment (Gradient SmartStart), which does not require any changes to the gradient table, can be entered in either time or volume, allowing  for the flexibility required to emulate different  HPLC systems. 

We can use this feature for the methods transfer example illustrated earlier. While the method transfer results produced retention times within  5% of the original separation – which represents a typical retention time window for peak identification – more stringent retention time criteria can be met by adjustment of the dwell volume. An overlay of the analyses on the Agilent 1260 and the ACQUITY Arc Systems illustrate the noticeable shift in retention time, particularly for peak 3. Measuring the retention time of each peak we can determine the shift for peak 3 is 0.1 min, while the remaining peaks have a much smaller shift of 0.01–0.02 minutes (Figure 4). Using the aforementioned approach, the gradient start was adjusted to begin 0.05 min after injection on the ACQUITY Arc System to improve the retention  time correlation for peak 3. This value was  selected since it is the mid-point of the retention time shifts observed.

The effect of adjusting the initial hold of the method produced retention time shifts of between 0.01 to  0.05 minutes for the analytes (Table 2). The change in retention times of the first two peaks (acetaminophen and caffeine) was minimal (< 0.02 minutes), while the later eluting peaks had retention time shifts much  closer to the gradient adjustment of 0.05 min. For the analyte of interest (Peak 3) the retention times shifted 0.06 min earlier, resulting in a retention time deviation of 1.5% relative to the Agilent 1260 Infinity System (Figure 5). The effect of the adjustment on retention may vary depending on the mobile phase composition at elution. When analytes elute in an isocratic step or under constant mobile phase conditions, retention times will be shifted similar to the change in gradient start. When peaks elute under gradient conditions, changing the timing of the solvent delivery can have a more complex effect on retention. 

Nevertheless, the adjustment of the gradient start enabled fine-tuning of the retention. The change of the gradient start resulted in a maximum retention time deviation of 1.4% compared to the Agilent 1260 separation. As compared to the first analysis on the ACQUITY Arc System, adjustment of the gradient start produced similar average deviations with lower maximum deviation or difference. 

One of the prime capabilities of the ACQUITY Arc System is the ability to easily transfer traditional/legacy HPLC methods (e.g. USP, or NF) from other HPLC systems. In this example, a method transfer from another manufacturer’s HPLC System to an ACQUITY Arc system was performed using the Path 1 of the Arc Multi-flow path technology. This flow path, which was designed for methods transfer, allows the ACQUITY Arc System to mimic the dwell volume of a typical HPLC system. The results produced retention times within 5% of the original method. Additional fine-tuning of retention times was accomplished using Gradient SmartStart Technology, which adjusts the initial hold of the method without requiring any changes to the gradient table. The combination of these two features enables methods transfer to an ACQUITY Arc System in just two injections for a simplified methods transfer approach, including the following:

1. Transfer method to an ACQUITY Arc using appropriate Flow Path (1 or 2).
2. Compare chromatograms and evaluate retention.
3. If retention does not meet method criteria, adjust gradient start using Gradient SmartStart. Re-run method.

In United States Pharmacopeia and National Formulary (USP 37-NF 32 S2).; United Book Press, Inc.: Baltimore, MD, 2014; Vol., p 6376.