Hydrophilic Interaction Liquid Chromatography (HILIC) Method Migration Part 1: From Legacy HPLC Systems to the Alliance™ iS HPLC System

This application note helps illustrate a successful hydrophilic interaction liquid chromatography (HILIC) method migration from legacy HPLC systems found across industries to the Alliance iS HPLC System. Due to differences in HPLC instrument designs, HILIC method migration could be challenging since results could potentially be impacted. A well-designed system like Alliance iS HPLC System, helps overcome those challenges to allow for seamless migration. For this study, the United States Pharmacopeia (USP) monograph for cetirizine hydrochloride assay and organic impurities were analyzed on two legacy HPLC systems and migrated to the Alliance iS HPLC System. All three systems met suitability requirements and demonstrated comparable results.

Benefits

• Seamless HILIC method migration from standard HPLC systems to Alliance iS HPLC System
• Improved signal-to-noise (s/n)

Most regulated laboratories, such as pharmaceutical quality control (QC) labs, utilize high performance liquid chromatography (HPLC) systems for analysis of intermediate and final products. When considering a new HPLC system, the ability to migrate existing validated methods is critical. Specific attributes of the method may add complexity to this process. For example, hydrophilic interaction liquid chromatography (HILIC) separations use high organic mobile phases, and different washes than used for the more typical reversed-phase chromatography (RP). This results in specific considerations and challenges associated with HILIC methods.

In this study, the Cetirizine Hydrochloride assay and organic impurities monograph, from the USP, is used to assess HILIC method migration from legacy HPLC systems to the Alliance iS HPLC System.¹ The performance of the method will be evaluated by analysis of the system suitability requirements: Tailing factor, resolution, signal-to-noise (s/n), peak area %RSD, and retention time %RSD.

Assay

The standard solution was prepared at 0.5 mg/mL Cetirizine Hydrochloride (CAS No.: 83881-52-1) in mobile phase, as described in the USP monograph.

Organic Impurities

• The System Suitability Solution was prepared at 4.0 µg/mL Cetirizine Hydrochloride and 4.0 µg/mL Cetirizine Related Compound A (CAS No.: 246870-46-2) in mobile phase
• The Sensitivity Solution was prepared at 0.1 µg/mL Cetirizine Hydrochloride in mobile phase
• The Standard Solution was prepared at 0.5 µg/mL Cetirizine Hydrochloride in mobile phase

For the Cetirizine Hydrochloride Monograph, both the assay and organic impurities methods require the same mobile phase and column, enabling sequential analysis. In this study, the mobile phase and samples were prepared prior to each analysis, and a new column was used for each system. Each new column was conditioned for 12 hours prior to analysis. All HPLC systems followed a similar sample sequence. The mobile phase and the assay standard were used for the assay analysis while the system suitability solution, sensitivity solution, organic impurities standard solution, and mobile phase were used for the organic impurities analysis.

The assay results, which include injection of the standard, demonstrated the ability to successfully move the method across HPLC systems. The system suitability requirements (Table 1) associated with the assay standard include relative standard deviation (RSD) for peak area and retention time of Not More Than (NMT) 0.73% and tailing factor of NMT 2.0 (Table 1). For this isocratic method, retention time and tailing factor may be impacted by the column and method conditions, as HILIC methods typically require long equilibration times. Alternatively, the area %RSD is most likely to be impacted by the injection precision of the system and can be challenging to meet with the combination of the high organic sample diluent (>93% acetonitrile) and a low Area %RSD requirement (NMT 0.73%). While all three systems met the system suitability criteria, the Alliance iS HPLC System, with newly designed sample metering pump, produced the lowest overall area RSD of 0.042%.

The organic impurities method included a standard, a system suitability solution and a sensitivity solution, the latter to ensure the system is suitable to quantify low levels of impurities. The suitability requirements were retention time and peak area of NMT 2.0% (standard), signal-to-noise of Not Less Than (NLT) 10 (sensitivity solution), USP tailing ≤2, and USP resolution ≥2 for cetirizine and cetirizine related compound A (system suitability solution). As shown (Figure 1) all systems produced comparable chromatography. While all systems met the suitability requirements (Table 2), the s/n on the Alliance iS HPLC System was greater than the legacy systems while also achieving the lowest peak area RSD.

Calculating Signal-to-noise Using Custom Calculations: Impact of Carryover

With recent changes to the USP General Chapter <621>, current guidelines specify signal-to-noise (s/n) calculated using a blank injection, with the noise region centered–if possible-on the peak retention time using a half height multiplier of 5.² However, with the need of a blank, any extraneous peaks in the blank can impact s/n calculations, including solvent peaks or carryover.

In this analysis, s/n was calculated from the blank at 5x the peak width at half height, centered around the peak. While all systems met system suitability criteria, Legacy System 2 produced the lowest sensitivity. Upon review of the data, a peak was observed in the blank chromatogram of Legacy System 2, near the elution of the analyte of interest (Figure 2). The retention time alignment with the peak of interest, suggest it is likely due to carryover. While modern HPLC methods have various needle washing designs, many legacy HPLC systems have few options to wash any previous samples from the injection needle. In contrast, during an injection cycle of the Alliance iS HPLC System, the sample needle and puncture needle are washed before and after injections with the wash solvent, continually being aspirated out. Thus, the Alliance iS HPLC System showed no observable peaks in the blank, providing improved s/n (Figure 3).

While Legacy System 2 met system suitability criteria, an alternative methodology to calculate s/n results without any carryover interference was evaluated. This methodology used a different region of the blank not affected by carryover. To perform the calculation, a custom field was used. The custom field was created as follows:

1. Label “Blank” in the Sample Set Method (Figure 4).

2. Create the custom field using intersample syntax with the label definition for the blank injection in the formula field box (Figure 5). The full formula is as follows:

2*(Height-(0.5*Blank.1.(Peak to Peak Noise/Scale to µV)))/Blank.1.(Peak to Peak Noise/Scale to µV).

3. Set the Noise Value for s/n specified in suitability tab of the processing method. For this custom field, the Noise value for s/n must be Peak to Peak Noise (Figure 6).

4. Next set the appropriate segment to use for the blank in the Noise and Drift tab of the processing method (Figure 7).  

Using this intrasample custom calculation, the s/n results increased from 21.32 to 85.53 for Legacy System 2 after re-processing, demonstrating the impact of extraneous peaks in a blank s/n value.

Modern HPLC systems such as the Alliance iS HPLC System offer many benefits such as successfully running methods from legacy HPLC systems and producing similar and even improved results as observed with s/n. A HILIC method, which can be present issues in routine analysis, was able to be successfully migrated from legacy HPLC systems to the Alliance iS HPLC System. All suitability criteria were met for migration of the Cetirizine Hydrochloride Assay and Organic Impurities USP method with the Alliance is HPLC System having comparable chromatography as Legacy systems with slightly improved area %RSD’s and sensitivity. In addition, due to the improved needle wash mechanism on the Alliance iS HPLC System, no carryover was observed.

1. Monograph: USP. Cetirizine Hydrochloride. In: USP-NF. Rockville, MD: USP; 01 Sep 2021.DOI: https://doi.usp.org/USPNF/USPNF_M2902_06_01.html
2. General Chapter: USP. 621 Chromatography. In: USP-NF. Rockville, MD: USP; 01 Apr 2023. DOI: https://doi.usp.org/USPNF/USPNF_M99380_07_01.html
3. “How to Create an Intersample Custom Field - Tip93.” Waters, 11 Nov. 2022, https://support.waters.com/KB_Inf/Empower_Tips_of_the_Week/WKB68884_How_to_create_an_Intersample_Custom_Field