(M1130-02-10) Understanding How UHPLC System Characteristics Effect “Going Green” with Regulated Methods

Purpose: Liquid chromatographic analysis with the use of optical detectors are usually not considered green due to the substantial amounts of solvent waste generated, especially in high-throughput laboratories. Most regulated methods have been validated using High-Performance Liquid Chromatography (HPLC) separation techniques. However, when compared to Ultra High-Performance Liquid Chromatography (UHPLC) methods, the difference in solvent consumption can be significant. To develop or scale methods that use fewer solvents, a shift to UHPLC is necessary. By using columns that are less than half the length of standard HPLC columns with decreased column internal diameter, analyses can be completed more quickly, thereby reducing solvent usage. To ensure the success of these UHPLC separation methods, a thorough understanding of an instrument's capabilities is necessary. This includes knowledge of system dwell volume and dispersion, and how these factors may affect chromatography. This study was conducted using various UHPLC systems with different dwell volumes to analyze the same United States Pharmacopeia (USP) method, demonstrating techniques to mitigate potential system issues

Methods: Following the Amlodipine Besylate Organic Impurities USP method, a bulk mobile phase was prepared and spit among four UHPLC systems with a range of different system characteristics. This method is an 11-minute gradient with 1.36 g/L of potassium phosphate monobasic in water as Solution A and methanol as Solution B. Each system used the same 1.7 µm, 2.1 × 50 mm L1 column that was kept at 40° for the analysis. With the flow rate set to 0.5 mL/min, 2 µL of each sample was injected and analyzed with UV detection at 240 nm. Using a diluent of 50:50 (methanol:water), the system suitability solution was prepared containing 0.5 mg/mL Amlodipine Besylate and 1.5 µg/mL Amlodipine Related Compound F. The sensitivity and  standard solution contained 0.25 µg/mL and 0.5 µg/mL of Amlodipine Besylate respectively. Acceptance criteria for the organic impurities method includes resolution of NLT 3.0 between amlodipine related compound F and amlodipine, tailing factor of NMT 2.5 for amlodipine, RSD of NMT 5.0%, and signal-to-noise ratio of NLT 10.

Results: All four systems exhibited some chromatographic differences due to their dwell volume. The retention time for the amlodipine besylate peak is observed to increase with the increase in system dwell volume. As shown in Figure 2, System 1, which has the lowest dwell volume (91 µL), exhibits an earlier eluting amlodipine besylate peak while System 4, with the largest characteristics, exhibits the latest eluting amlodipine besylate peak. However, the large dwell volume (1008 µL) for System 4 made analysis of the sensitivity solution impossible due to poor resolution of the amlodipine besylate peak. Since the initial analysis also exhibited insufficient re-equilibration time at the end of the gradient due to the lack of return to the baseline, an extra 4-minutes was added to extend the re-equilibration time. As shown in Figure 1 panel B, the 4-minute extension shows a much more resolved amlodipine besylate peak as well as evidence of proper re-equilibration at the end of the run. It was then determined that a delay of 2 minutes for the next injection was enough for re-equilibration (Figure 1, panel C). With this modification, the amlodipine besylate peak was detected in the sensitivity solution and thus all four systems successfully met system suitability requirements.

Conclusion: UHPLC systems provide the opportunity to have analysis with shorter run times and well as low injection volumes and have shown to be more efficient. With understanding of how a system with a larger dwell volume may affect chromatography, adjustments need to be made to ensure a successful method development and method migration as shown with System 4. However, these adjustments like using the next injection delay would affect the greenness of a method due to the use of more solvents during the delay. All systems successfully met acceptance criteria.


Figure 1: Stacked overlay of Amlodipine Besylate Sensitivity Solution under three conditions on System 4. (A) Original 11-minute method; (B) Final isocratic hold extended to 15 minutes; (C) Original 11-minute method with a 2-minute next inject delay applied. (1) Amlodipine Besylate



Figure 2: Stacked overlay of Amlodipine Besylate Sensitivity Solution on the four systems showing Amlodipine Besylate (1) for all four systems


Table 1: Results for Amlodipine Besylate Organic Impurities system suitability om all systems tested. (1) Results from System Suitability Solution; (2) Results from Sensitivity Solution; (3) Results from Standard Solution