USP Analysis of Glimepiride on an Alliance HPLC System: Modernization of a USP Method

In this application, the assay for glimepiride was performed according to the USP monograph.

Using the ACQUITY Columns Calculator, the USP assay for glimepiride was scaled to a column with a smaller particle size and a shorter length column. Additional parameters, including flow rate and injection volume, were also scaled accordingly. These modifications allowed for a reduction in the run time of over 50% and a significant reduction in the mobile phase consumed, while still meeting system suitability criteria – thereby illustrating the potential for method modernization on traditional HPLC systems.

Benefits

• Alliance system flexibility enables method modernization for USP methods
• Scalable columns allow for method adjustment
• ACQUITY UPLC Columns calculator provides tools to ensure proper method scaling, including column configuration and conditions (flow rate and injection volume)

Many United States Pharmacopeia (USP) monographs consist of legacy methods that were developed on older and/or obsolete HPLC instrumentation. While these analyses meet the pre-defined system suitability criteria, the conditions often have long runtimes and higher flow rates, typical of HPLC column dimensions. These conditions often require significant resources such as, instrument and chemist time and solvent consumption. The USP, however, has outlined allowable method adjustments¹ thereby enabling laboratories to modernize legacy methods. These adjustments can include scaling particle size and column dimensions to maintain a constant L/d_p, where L is the length of the column and dp is the particle size of the packing material. In this application, the assay for glimepiride was performed according to the USP monograph.² Following the allowable adjustments, the isocratic method was also scaled to a smaller particle size and column dimensions to decrease run time and improve throughput.

All standards and samples were prepared following the USP monograph.² Glimepiride standard (p/n 1292303), related compound B (p/n 1292325), related compound C (p/n 1292336) were purchased from United States Pharmacopeia. For the glimepiride standard preparation, 20 mg was weighed and dissolved in 80/20 acetonitrile/water (v/v) and brought to volume in a 100 mL volumetric flask yielding a final concentration of a 0.2 mg/mL. Stock solutions containing related compound B and related  compound C were prepared in 80/20 acetonitrile/water at 0.1 mg/mL. The system suitability standard was then prepared by combining 1 mL of related compounds stock and adding 49 mL of the 0.2 mg/mL glimepiride standard solution, yielding final concentrations of 0.196 mg/mL (glimperide) and 2 µg/mL (related compound B and C). The glimepiride drug substance sample was prepared by transferring 20 mg of sample into a 100 mL volumetric flask and bringing to volume with 80/20 acetonitrile/water (v/v).

The assay for glimepiride was performed following the USP monograph on an Alliance HPLC System with a 2998 Photodiode Array Detector and a 30 cm column heater equipped with a passive preheater (Figure 1, top chromatogram). The samples were prepared as previously described according to the USP monograph.² Five injections of the system suitability standard and the API standard were analyzed.

The system suitability criteria for the assay states, “the resolution, R, between glimepiride related compound B and glimepiride related compound C is not less than 4.0… [and] the relative standard deviation (RSD) for replicate injections is not more than 2.0%.”¹ Based on the specified RSD requirements and USP Chapter <621>, the results generated using both methods met system suitability criteria (Table 1). In addition, the relative standard deviation for glimepiride was 0.11% for the retention time and less than 0.3% for peak area. Both of which are well within the system suitability criteria for this assay of NMT 2.0%. The USP resolution for related compound B and related compound C of 4.6 meet the system suitability criteria of not less than 4.0.

The HPLC assay for glimepiride, while meeting system suitability criteria, requires a 4.6 x 250 mm column and a 30 minute run  time. Method adjustments were desired to decrease the overall run time. The method was scaled according to the USP guidelines,1 which permits scaling column dimensions, particle size, and flow rate for isocratic separations. Keeping the L/dp ratios within -25% to +50% of the original method, the 4.6 x 250 mm, 5 µm column was scaled to a 4.6 x 150 mm, 3.5 µm column (Figure 1, bottom chromatogram). The flow rate was adjusted based on particle size of the column as per USP Chapter <621> based on F₂=F₁ (d₂²/d₁²) where F= flow rate and d= diameter of the column. The injection volume was also adjusted for the column volume. All calculations were performed using the ACQUITY UPLC Columns Calculator (Figure 2).

As with the original HPLC method, the scaled method met all the system suitability requirements (Table 1). The glimepiride retention time RSD was 0.05% while the peak area was 0.27%. In addition, the USP resolution, while slightly lower, met the system suitability criteria with a value of 4.0 for all injections. 

To determine the stability of the scaled method, the drug substance samples were bracketed by five replication injections of the standard, aliquoted into two separate vials. Each analysis was 13 minutes, therefore the series of injections occurred over 3.25 hours. Analysis of the standards show the results meet the USP system suitability criteria, with a glimepiride peak area RSD of 0.31 and a retention time RSD of 0.22 (Table 2).

As mentioned previously, the active pharmaceutical substance of glimepiride was analyzed using the scaled assay for glimepiride (Figure 3). The percentage of glimepiride was found to be 99.67%. This value was calculated by using the formula found in the USP monograph.

By taking advantage of the guidelines in USP Chapter <621>, many legacy methods can be updated for higher throughput while still maintaining current system suitability criteria on legacy HPLC instrumentation. Using the ACQUITY Columns Calculator, the USP assay for glimepiride  was scaled to a column with a smaller particle size and a shorter length column. Additional parameters, including flow rate and injection volume, were also scaled accordingly. These modifications allowed for a reduction in the run time of over 50% and a significant reduction in the mobile phase consumed, while still meeting system suitability criteria – thereby illustrating the potential for method modernization on traditional HPLC systems.

1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP38-NF33 S1) Baltimore, MD: United Book Press, Inc.; 2015. p. 424.
2. Glimepiride Official Monographs, USP38–NF33. United States Pharmacopeia and National Formulary (USP 38-NF 33 S1) Baltimore, MD: United Book Press, Inc.; 2015. p. 3671.