(T1030-11-62) Impacts of Method Conditions and Autosampler Characteristics on Method Migration

Purpose: Method migration from one HPLC instrument to another is routinely performed by analytical laboratories, in moving a method within the organization or to external contractors, often among instrumentation from different vendors. This process can encounter complications if method conditions and instrument characteristics are not considered and may lead to failure to meet system suitability requirements for a given method. For instance, methods with high organic sample diluent and low injection volumes may present challenges for injection precision of an autosampler due to possible evaporation over the course of a run and added injection variability for small volumes. As such, high performance is required of an autosampler, but this may differ due to autosampler characteristics. In this study, we examined two compendial HPLC methods using high organic sample diluent and low injection volumes and investigated the impact of method conditions on autosampler performance of different flow-through-needle HPLC systems. The autosamplers in this study utilize either a sampling syringe or metering device as the mode of sample delivery. The sampling syringe is low-pressure and isolated from the flow path while the metering device is high-pressure and located in-line with the flow. The chromatography was evaluated using system suitability requirements defined by the compendial methods, such as retention time reproducibility, peak area reproducibility, and resolution.

Methods: USP Ketoconazole Organic Impurities and CHP Ketoconazole Related Substances were performed following monograph guidance across three different HPLC systems. Mobile phases, standard solutions, and samples were prepared once and split across the three systems to keep preparations consistent. A 4.6-mm × 100-mm; 3.5-µm C18 column was used on all HPLC systems for both methods. The USP method required adjustments to accommodate a larger column particle size; corresponding changes to flow rate and gradient segments were made following USP < 621 > guidance. The CHP method required no adjustments per CHP 0512.

Results: Compendial methods may use highly organic sample diluents when compound solubility is an issue, but if highly organic sample diluents evaporate of the course of a run, the sample concentration and repeatability is impacted. System suitability criteria were evaluated, but peak area was examined as a proxy for autosampler performance. Both compendial methods used 100% methanol as the sample diluent with an injection volume of 10 µL, which are challenging method conditions for the autosamplers. Individual HPLC characteristics are defined in Table 1. HPLC 1 uses a sampling syringe, while HPLC 2 and 3 use metering devices. System suitability criteria for USP Ketoconazole Organic Impurities were met for all three HPLC systems (Table 2). Peak area %RSD was higher on HPLC 2 and 3 than HPLC 1 but did not exceed criteria (NMT 5.0%). For HPLC 2 and 3, no trend for sample evaporation could be determined from tracking the absolute peak area, see Figure 1A for chromatographic overlays. System suitability criteria for CHP Ketoconazole Related Substances were more stringent (NMT 2.0%). HPLC 1 failed to meet peak area %RSD while HPLC 2 and 3 met criteria (Table 2). When tracking the absolute peak area on HPLC 1, the ketoconazole peak area decreased with consecutive injections, see Figure 1B for chromatographic overlays.

Conclusion: Method migrations may encounter complications due to method conditions and autosampler characteristics. Two compendial HPLC methods with challenging method conditions were evaluated on three HPLC systems with different autosampler characteristics. In testing these methods, we did not see significant impacts of sample evaporation to peak area reproducibility. This could be due to the low number of injection replicates (n=6; n=5) as well as environmental conditions (e.g. refrigerated sample compartments), where evaporation is mitigated. However, we did observe that an instrument characteristic impacted the CHP system suitability on HPLC 1. The CHP reference solution requires 5 consecutive injections, but the absolute area of ketoconazole decreased with each injection, which can indicate accumulation of air in the sampling syringe. A further complication is low injection volume (10 µL) coupled with the default syringe size of HPLC 1 (250 µL), which can add variability. In cases where low injection volumes must be drawn, a smaller sampling syringe size could be more suitable.

References: P. Hong, Improved Injection Precision of USP Methods with an Arc HPLC System (Waters Corp., 2020).
F. Steiner and P. Carsten, HPLC Autosamplers: Perspectives, Principles, and Practices (LCGC North America, 2019).
A. Dlugasch, et al., Alliance Carryover Performance (Waters Corp., 2018)
United States Pharmacopeia (2020). USP Monographs, Ketoconazole. USP-NF. Rockville, MD.
United States Pharmacopeia (2023). General Chapter: Chromatography, USP-NF, Rockville, MD.
Chinese Pharmacopeia (2020). CHP Monographs (Volume II), Ketoconazole. PPRC, Beijing, China.
Chinese Pharmacopeia (2020). General Requirements (Volume IV), 0512 High Performance Liquid Chromatography. PPRC, Beijing, China.


Table 1: Instrument Characteristics of HPLC Systems.


Table 2: System Suitability Results. Standard solutions, system suitability solutions, and reference solutions were assessed following each monograph.


Figure 1: Chromatographic Overlays. Overlays show ketoconazole peak in A) standard solution for USP Ketoconazole Monograph (n=6 replicate injections) and in B) reference solution for CHP Ketoconazole Monograph (n=5 replicate injections).