A Scaled USP Method for Fluconazole Using an ACQUITY UPLC I-Class System and CORTECS T3 Column Chemistry

This application note desbribes how we can increase throughput by scaling methods from HPLC to UPLC. 

The Waters Columns Calculator is a useful tool when scaling methods by providing the user with scaled flow rates, injection volumes, and gradient tables (when applicable) while taking into account the particle porosity. Additionally, even within a specified 'L1' column packing, there are a number of C₁₈ columns each differing in selectivity and retention. The CORTECS UPLC T3 Column chemistry proved to be a suitable column for the analysis of fluconazole and related compounds meeting all system suitability requirements of the USP monograph. The solid-core particle of the CORTECS Column provided narrower peak widths and improved efficiencies while reducing the total run time by approximately half when compared to a typical UPLC column and by more than eighty percent when compared to the original HPLC method.

Benefits

Increased throughput by scaling methods from HPLC to UPLC, additional selectivity with CORTECS T3 Columns

Many current USP methods are designed for use with larger particle size (3 μm or greater) HPLC columns and instrumentation, which results in long run times and large volumes of hazardous solvents being consumed. It is possible to scale isocratic methods to sub-2-μm particle columns, which provides the same or improved performance with shorter run times resulting in increased throughput and lower solvent consumption. In order to scale methods appropriately, a number of USP guidelines must be followed^.1 For example, the L/d_p ratio, where L refers to the length of the column and d_p refers to the diameter of the particles packed within the column, must remain within -25% to +50% of the column specified in the original HPLC method. It is critical to be aware of these regulatory requirements when scaling from HPLC to UPLC methodologies.

In addition to method conditions and suitability requirements, USP monographs also specify the type of column packing material to be used for a particular analysis. For example an L1 packing refers to an octadecylsilane or C₁₈ column packing material. However, there are numerous types of C₁₈ columns, with differences in end-capping, the base particle, etc, which can all have an effect on the retention and selectivity of compounds. Therefore, it may be necessary to screen a number of columns when preparing to run a USP method to find one that meets system suitability requirements, which often includes a specified resolution between critical pairs.

Sample description
Fluconazole and fluconazole related compounds A, B, and C were purchased from United States Pharmacopeia. Samples were initially dissolved in acetonitrile, then diluted with 80:20 (v:v) water: acetonitrile per the USP monograph for fluconazole. Samples were vortexed and sonicated to ensure complete dissolution. The final concentration of the sample was of 10 μg/mL for all reference compounds.

The USP method for the analysis of fluconazole and related compounds (Organic Impurities, Procedure I³) was scaled to a UPLC method using the Waters Columns Calculator (Figure 1). Scaling from a 3.5 μm, 4.6 x 150 mm column to a 1.6 μm, 2.1 x 75 mm column results in a L/d_p ratio increase of 9.4%, which is well within the USP criteria (-25% to +50% of the original method L/d_p). The flow rate, scaled accounting for particle size, decreased from 0.500 mL/min in the original method to 0.228 mL/min in the updated method, while the injection volume decreased from the original value of 20 μL to 1.5 μL. Note that the calculator allows the user to specify the porosity factor (porous or superficially porous) to account for the differences in column volumes based on the porosity of the particle.

The appropriately scaled method was then run on the ACQUITY I-Class UPLC System using both CORTECS UPLC C₁₈+ and CORTECS UPLC T3 chemistries. CORTECS Columns utilize solid-core particles (also sometimes referred to as superficially porous), which provide an increase in chromatographic performance and speed when compared to fully-porous particles. The CORTECS C₁₈+ Column has a charged surface, which in addition to giving exceptional peak shape also provides a unique selectivity. The CORTECS T3 Column is designed for improved retention of polar compounds. Both chemistries are classified as L1 packings, so either would be acceptable to use per the USP monograph. Example chromatograms of the separations acquired on each column are shown below (Figure 2). The CORTECS UPLC C₁₈+ Column did not provide baseline separation of fluconazole and related compound C under the scaled USP method conditions. The CORTECS UPLC T3 Column in comparison provided baseline separation for fluconazole and all related compounds A–C.

System suitability requirements for fluconazole and fluconazole related compounds A-C state that resolution between related compound B and related compound C must be NLT (not less than) 1.5. Additionally, relative standard deviation must be NMT (not more than) 5.0% for both retention time and area. The results obtained on the CORTECS T3 are listed in Table 1.

In addition to meeting the system suitability requirements using the CORTECS UPLC T3 chemistry, the run time was decreased from 10 minutes in the original HPLC method to approximately 2 minutes in the scaled UPLC method.

To further highlight the increased efficiency and throughput using solid-core particles, the method was also run on a fully-porous ACQUITY UPLC HSS T3 1.8 μm, 2.1 x 75 mm Column (p/n 186005614) (conditions were scaled accounting for particle size) and shown in Figure 3.

Despite the difference in particle size, use of the solid-core particle resulted in earlier elution of the compounds and a decrease in the run time by nearly 50%. In addition, all peak widths are narrower on the solid-core column. The equation used to calculate efficiency (N), states:

where t_R is the peak retention time, and W1/2 is the peak width at half height.² From this equation, the efficiency is dependent not only on the peak width, but also on the retention time of the peak. The earliest eluting peak (fluconazole related compound A) shows nearly conserved efficiency on the CORTECS UPLC T3 Column vs the ACQUITY UPLC HSS T3 Column. This is because the decrease in retention time effectively cancels out the gains from a decrease in peak width. All other peaks show an increase in column efficiency due to the solid-core particle of the CORTECS Column. Decreased run time, narrower peak widths, and an increase in efficiency are some of the advantages of using a solid-core particle (Figure 4).

The ability to scale traditional HPLC methods to modern UPLC methods provides equivalent or enhanced performance while decreasing run time and solvent consumption. The Waters Columns Calculator is a useful tool when scaling methods by providing the user with scaled flow rates, injection volumes, and gradient tables (when applicable) while taking into account the particle porosity. Additionally, even within a specified 'L1' column packing, there are a number of C₁₈ columns each differing in selectivity and retention. The CORTECS UPLC T3 Column chemistry proved to be a suitable column for the analysis of fluconazole and related compounds meeting all system suitability requirements of the USP monograph. The solid-core particle of the CORTECS Column provided narrower peak widths and improved efficiencies while reducing the total run time by approximately half when compared to a typical UPLC column and by more than eighty percent when compared to the original HPLC method.

1. Chapter <621> CHROMATOGRAPHY. United States Pharmacopeia and National Formulary (USP 37-NF 32 S2) Baltimore, MD: United Book Press, Inc.; 2014. p. 6376–85.
   
2. Snyder, Lloyd R., Kirkland, Joseph J., Dolan, John W., Introduction to Liquid Chromatography, 3rd Edition. Hoboken: Wiley & Sons, 2010. Print.
   
3. Official Monographs, USP 37 NF32 S2. United States Pharmacopeia and National Formulary (USP 37-NF 32 S2) Baltimore, MD: United Book Press, Inc.; 2014. p. 4968.