Achieving Method Modernization with the New Liquid Chromatographic Gradient Allowances Provided by USP General Chapter <621> Chromatography and the Alliance™ iS HPLC System

The extent to which the various parameters of a chromatographic test may be adjusted without fundamentally modifying the pharmacopeial analytical procedures is defined in U.S. Pharmacopeia (USP) General Chapter <621> Chromatography. In this application brief, we combine the gradient method adjustments described in this chapter with the Alliance™ iS HPLC System to achieve both column dimension and system modernization for the USP monograph separation of antiviral drug, abacavir sulfate. 

Benefits

• When paired with the Alliance iS HPLC System, U.S. Pharmacopeia General Chapter <621> Chromatography gradient method allowances generate quality data that meet regulatory requirements
• The Alliance iS HPLC System’s extended chromatographic backpressure limits provide high-efficiency separations using an array of modern column dimensions resulting in run time, injection volume, and solvent savings

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 December 1, 2022 a harmonized standard for General Chapter <621> Chromatography was released. This standard incorporates the USP with 2.2.46. Chromatographic Separation Techniques European Pharmacopoeia (EuPh) and 2.01 Liquid Chromatography Japanese Pharmacopeia (JP) texts. Harmonization of these regulatory guidance provides increased method flexibility through the employment of modernized of chromatographic tools without the need for full monograph re-validation.

Chromatographic separations are affected by both column hardware and system hardware. These parameters are critical to method performance and limitations can restrict flexibility after monographs are validated. For example, modern HPLC column hardware is commonly offered in 4.6 mm diameter for an array of new stationary phase substituents, while 5 µm HPLC particle sizes have been substituted with ≤3.5 µm particle sizes for comparable separations in less time, and with less solvent. A modern HPLC system, such as the Alliance iS HPLC System (Figure 1), which features high chromatographic separation efficiency, an intuitive touchscreen interface, tool-free column fittings, and extended HPLC operating backpressure limits, provides dependable flexibility when adjusting monograph methods to suit modern column hardware dimensions.

In this application note, we employ the gradient method allowances described in General Chapter <621> Chromatography to achieve both column and system modernization for a representative USP monograph (Table 1).

The impurities separation of antiviral compound, abacavir sulfate, was selected for this exercise because the validated gradient method generates a challenging, partially resolved, peak critical pair from which the chromatographic system suitability criteria are based. After performing several of the gradient system adjustments provided in USP <621> that facilitate the use of modernized hardware, resulting chromatograms were examined for the ability to meet original monograph system suitability requirements.

A systematic approach was employed for 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 diameter column hardware, equipped with L1 stationary phase substituent was selected in lengths (L) of 100 mm and 150 mm to perform method modernization. The stationary phase substituent for the columns was of 5 µm or 3.5 µm particle size (d_p). In all instances, the USP <621> guidance L/d_p ratio allowance was met at -25% to +50% of the monograph ratio.

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 1 maintained the linear velocity of the monograph separation by adjusting the flow rate. Second, the injection volume was adjusted according to Equation 2 to maintain the ratio of the analyte to column volume. Finally, gradient start times were adjusted in Equation 3, Table 2 according to the calculated target column flow rate, length, and particle size. The start time adjustment preserved the gradient slope to column volume ratio reported in the monograph separation. The USP <621> guidance provided Equation 4 to allow adjustment for the system dwell volume, if specified during the monograph validation. System dwell volume was not reported in the abacavir sulfate monograph, and the separation does not include an initial isocratic hold time. As a result, a dwell volume adjustment was not applied to the calculated gradient start times during method modernization.

Manual gradient calculations for the target columns were confirmed using both the Waters Preparative OBD Column Calculator, and the Columns Calculator 2.0 (Figure 2). Online calculators were especially important because they provided an estimated maximum gradient backpressure for the adjusted gradients. This estimation, although computed for 100% organic mobile phase composition, rather than 85% organic composition in the monograph, added confidence that the adjusted methods would not exceed the 10,000-psi backpressure limit of the Alliance iS HPLC System.

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 unresolved, abacavir critical pair was successfully achieved (Figure 3). 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 4). As noted in USP <621>, peak deletions and/or inversions, may be observed using various substituents, therefore chromatographic peak identity was confirmed after method adjustment by PDA spectral analysis on an alternate system.

The Alliance iS HPLC System, in combination with modernized column hardware dimensions, provided unique benefits for the validated monograph separation. The LC system’s modernized tubing diameters facilitated the use of column hardware generating separation backpressures up to 10,000-psi, compared to conventional HPLC systems with backpressure limits of only 5,000-psi. Additionally, the monograph method particle size of 5 µm was successfully reduced to 3.5 µm while retaining USP <621> guidance L/d_p ratio requirements. These hardware combinations resulted in significantly reduced run time, injection volume, and solvent consumption for the validated monograph separation (Table 3).

USP monograph gradient separations can be successfully adjusted to maintain system suitability acceptance criteria on the Alliance iS HPLC System, as per the General Chapter <621> Chromatography (December 1, 2022). The simple-to-use Alliance iS HPLC System, with intuitive touchscreen interface, enables column modernization without compromising the validated monograph system suitability. The hardware provided an operating backpressure limit that supported a variety of modern HPLC column diameters, from which unique chromatographic benefits such as run time, injection volume and solvent savings were observed. 

1. General Chapter <621> Chromatography, Official Date: 01-Dec-2022, www.USP.org, referenced 01/06/2023.
2. Abacavir Sulfate Monograph official 01-May-2020, www.uspnf.com, referenced 01/06/2023.
3. Preparative OBD Column Calculator, www.waters.com, accessed 01/06/2023.
4. Columns Calculator 2.0, www.waters.com, accessed 01/06/2023.