(M0930-08-52) Modern Liquid Chromatographic Gradient Method Allowances Provided by General Chapter, USP Chromatography < 621 >

Purpose: The U.S. Pharmacopeia (USP) portfolio of solutions addresses quality assurance, enhances regulatory predictability, and helps manufacturers distribute quality medicines, dietary supplements and foods. On Dec 1, 2022, a harmonized standard for General Chapter < 621 > Chromatography was released. This standard incorporates < 621 > Chromatography (USP), 2.2.46. Chromatographic Separation Techniques (EuPh) and 2.01 Liquid Chromatography (JP) texts. Additions to this guidance provide limits of flexibility for liquid chromatographic gradient method separation parameters such particle size, flow rate, gradient slope, and injection volume. In this poster, we utilize the new gradient method adjustments described in USP Chapter < 621 > to demonstrate method modernization provided for determination of impurities using an antiviral drug monograph.

Methods: A systematic approach was employed for gradient method modernization of the USP, abacavir sulfate monograph separation. First, the monograph column was identified as an L1 stationary phase substituent with a 5 µm particle size and 3.9 x 150 mm column hardware. Modern 4.6 mm and 2.1 mm diameter column hardware, equipped with L1 stationary phase substituent was selected in lengths (L) of 75 mm, 100 mm, and 150 mm to perform method modernization. The column stationary phase substituent was of 5 µm, 3.5 µm, or 2.5 µm particle size (dp). In all instances, the USP < 621 > guidance L/dp ratio allowance was met at -25% to +50% of the monograph ratio for the particle size and column dimension combinations employed. The flow rate, injection volume and gradient start times were mathematically adjusted for the modern target columns according to the equations provided in the USP < 621 > guidance. First, Equation A was used to maintain the linear velocity of the monograph separation by adjusting the flow rate. Second, the injection volume was adjusted according to Equation B to maintain the ratio of the analyte to column volume employed in the monograph. Finally, gradient start times were adjusted using Equation C, and the calculated target column flow rate, length, and particle size. The dwell volume of the instrument used during validation was not reported in the monograph, and therefore the information was not utilized for the gradient adjustment. Equation D and E (FIO, not included in USP < 621 >) were applied to the gradient to adjust for the difference in system volume when moving between HPLC and UHPLC column and system dimensions.

Results: The monograph system suitability impurities mixture (SST) was analyzed with the adjusted gradient and modernized chromatographic hardware. For all modern column dimensions, the monograph system suitability resolution requirement of NLT 1.5 for the abacavir impurity critical pair was successfully achieved. When the SST relative retention times (RRTs) were compared to those generated with the stationary phase utilized for the initial monograph separation, they were most similar for columns of the same L1 stationary phase substituents, while RRTs varied with different L1 stationary phase substituents (Figure 1). As noted in USP < 621 >, peak deletions and/or inversions, may be observed using various substituents, therefore chromatographic peak order was confirmed after method adjustment by PDA spectral analysis. Modern columns provided up to a 16-fold increase in the number of samples analyzed per day, demanded up to 100-fold less sample volume for injection, and resulted in up to a 5-fold decrease in the volume of mobile phase waste generated by the analysis (Table 1).

Conclusion: The data presented shows that separation flexibility, within the parameters of the new, USP adjustment guidance, successfully promoted the employment of modern column diameters and innovative stationary phase substituents. The newly, allowable USP gradient adjustments successfully maintained the original, validated, monograph system suitability testing criterion, while providing greater user flexibility and overall benefit.

References: 1. General Chapter Chromatography, Official Date: 01-Dec-2022, www.USP.org, referenced 1/7/2023.
2. Abacavir Sulfate Monograph official 01-May-2020, www.uspnf.com, referenced 4/15/2022
3. Preparative OBD Column Calculator, www.waters.com, accessed 1/15/2023
4. Columns Calculator 2.0, www.waters.com, accessed 1/15/2023

Equation 1: (A) Flow rate, (B) Injection volume (C) Gradient time (D) Gradient time offset when converting between HPLC and UHPLC (Not included in USP < 621>) (E) Final UHPLC adjusted gradient with offset.

Comparison of relative retention time (RRT) obtained with modern stationary phase substituents and column dimensions.

Chromatographic savings when the new chromatographic gradient adjustments are employed with the abacavir sulfate monograph.