(W1130-02-11) Adapting USP Monographs to the Capillary Scale using USP 621

Purpose: The United States Pharmacopeia (USP) offers numerous pre-validated monographs of pharmaceutically relevant compounds which are used in regulated environments around the world. With environmental concerns impacting major pharmaceutical manufacturers, labs are working to reduce their consumption of harmful mobile phases in LC analysis. One method of solvent reduction is the use of capillary-scale liquid chromatography, where columns with an inner diameter (i.d.) between 0.075 and 0.5 mm are operated between 0.1 and 10 µL/min. The considerable cost and time required for method revalidation has previously limited the adoption of capillary-scale LC in regulated environments. By using guidelines established in USP < 621 > [1], monographs can be adapted to the capillary scale without the need for full revalidation. This work demonstrates the adaptation of a monograph for hydrochlorothiazide [2] on both analytical and capillary scale. Both scales were able to meet the system suitability criteria of the monograph. The capillary-scale system was able to reduce the total solvent consumption by 99.6% when compared to the initial monograph. This work marks a significant step in the integration of capillary-scale LC into regulated environments.

Methods: The USP monograph for hydrochlorothiazide was adapted to both the analytical and capillary scale using guidance from USP < 621 >. A diluent solution of 70:30 water to acetonitrile with 16 mM sodium phosphate (pH 2.7) was prepared as directed in the monograph. Samples were then prepared in this solution, with a system suitability test mixture containing 0.32 mg/mL of hydrochlorothiazide and 0.0032 mg/mL of chlorothiazide and benzothiadiazine-related compound A was prepared. Additionally, a quantitation limit solution of 0.16 µg/mL hydrochlorothiazide was also prepared.

For the analytical scale a 2.1 x 50 mm column packed with 2.6 µm L1 material was selected, and for the capillary scale, a 0.3 x 50 mm column packed with 3.0 µm L1 material was used. Mobile phase A was a 3:1 mixture of acetonitrile/methanol, and mobile phase B was a 5 in 1000 solution of formic acid/water. Detection was performed at 275 nm. The flow rates and injection volumes of both systems were scaled proportionally based on USP guidelines, as well as the programmed gradient methods.

Results: A system suitability test for hydrochlorothiazide was performed using a capillary scale LC as well as using an analytical scale LC across 6 injections. The resulting chromatograms are shown in Figure 1 and Figure 2, respectively. To meet system suitability criteria, the system suitability sample must meet performance metrics for resolution, tailing, and repeatability, as well as verification of quantitation limit solution run in triplicate to be considered an acceptable adaptation of the monograph. A table of these limits, and the performance of each system are given in Table 1. Performance differences were observed between the two scales, particularly with the sensitivity of UV detection due to differences in flow cell length; however, both systems were capable of meeting system suitability requirements, making this a valid adaptation of the monograph. Due to the reduced solvent consumption of capillary scale LC, the monograph separation generates only 85 µL of solvent waste. This represents a 98% reduction in solvent consumption from the translated analytical scale method and a 99.6% reduction compared to the original monograph conditions.

Conclusion: A USP monograph for hydrochlorothiazide was adapted to capillary and analytical scales using USP < 621 > guidelines. A capillary scale LC demonstrated performance within the acceptable ranges provided by the monograph as well as its ability to generate comparable data to existing analytical scale instrumentation. Although there were differences in performance across the two scales, both systems were able to meet system suitability criteria, making them valid platforms for the monograph. During a single run, the capillary scale LC generates 99.6% less waste when compared to the original monograph conditions. This demonstrates the viability of using capillary scale separations as a greener, cheaper, and more compact option within regulated environments.

References: [1] USP General Chapter Chromatography. United States Pharmacopoeia Convention Inc., Rockville, MD.
[2] United States Pharmacopeial Convention. Hydrochlorothiazide. United States Pharmacopoeia National Formula (USP 40-NF 35) 2017, 3421–3422.

Figure 1: Capillary scale chromatograms obtained following the USP monograph: (A) full test mixture separation and (B) zoomed-in time window of the separation focusing on benzothiadiazine-related compound A, and chlorothiazide.

Figure 2: Analytical scale chromatograms obtained following the USP monograph: (A) full test mixture separation, and (B) zoomed in time window of the separation focusing on benzothiadiazine-related compound A and chlorothiazide.

Table 1