Improvement of characteristics of HPLC-UV methods for determining trace amounts of substances in rinses during cleaning validation

In the pharmaceutical industry, cleaning of technological equipment and laboratory glassware plays a crucial role in ensuring the quality and safety of pharmaceutical products. In this study, we investigated the possibilities for reducing the limit of quantification using HPLC-UV for determining small amounts of substances in washings from technological equipment. In the research Knauer Azura chromatograph was used, equipped with the Azura DAD 6.1 diode-array detector with a 50mm fiber-optic flowcell. The chromatographic columns used was the Discovery HS F5 250×4,6 (5 µm) and Discovery HS C18 250×4,6 (5 mm). It was shown that the use of aqueous-ethanol mixtures in combination with pentafluorophenyl stationary phase (Discovery HS F5) achieves better retention of the model substance – caffeine, compared to octadecylsilane (Discovery HS C18). Using Discovery HS F5 and a mobile phase containing 40% ethanol in water, the retention factor was 0,7, while using Discovery HS C18 under the same conditions, the retention factor was 0,2. The advantages of using ethanol compared to acetonitrile were also demonstrated, as using 40% acetonitrile in the mobile phase resulted in a lower retention factor of 0.4. The effect of increasing the injection volume on the chromatographic peak characteristics was investigated, and it was shown that when a sample solvent with a lower eluting strength than the mobile phase is used, the injection volume can be increased up to 250 ml, which leads to a lower limit of quantification. By increasing the injection volume, a low limit of quantification is achieved, which is commensurate with the limit of quantification observed for the HPLC-MS method. Thus, when injecting 250 ml of the test solution into the system, it was possible to achieve a limit of quantification of 0,27 ng/ml. The results of the study expand the scope of the HPLC-UV method as a potential alternative to the high-cost HPLC-MS method for analyzing residue quantities of pharmaceuticals during the cleaning validation of technological equipment or laboratory glassware.