Drug Formulations Development and Validation of a Method for Trace Level Zinc Quantification in Pharmaceutical Zinc Supplements Using a Carboxyl Functional Group Packed Column and Refractive Index Detector Nagavardhana Reddy Vanga, 1,  Venkata Ratnam Kesamsetty, 1 Kumaraswamy Ummiti , 2 Venkata Nadh Ratnakaram, 1 and Hanimi Reddy Bapatu 3 1 Industrial Chemical Product Development and Analysis Centre, Department of Chemistry, GITAM School of Science, GITAM Deemed to be University-Bengaluru Campus, Nagadena Halli, Bengaluru, Karnataka 561203, India 2 Koneru Lakshmaiah Education Foundation, Department of Chemistry, College of Engineering, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh 522502, India 3 JNT University, Department of Chemistry, Hyderabad, Telangana 500085, India  Corresponding author’s e-mail: nagavardhanar@gmail.com Abstract Background: Zinc helps with cell division, growth, wound healing, and carbohydrate breakdown. Humanbeings have to obtain zinc from food or supplements because our bodies do not produce it naturally. In view of the greater advantages (such as low cost, time of analysis, and stability-indicating) compared to other quantification methods (titration, ion chromatography, Atomic absorption spectroscopy) proposed in the literature, a refractive index detector coupled with HPLC has been used in quantification of zinc. Objective: The goal of this research is to develop and validate a sensitive, low-cost, high-resolution, and stability- indicating method for detecting and quantifying zinc levels in zinc supplement pharmaceutical products (injectables, tablets, and capsules). Methods: A novel isocratic reverse-phase HPLC method with a refractive index detector (with sensitivity 64, detector temperature 35  C, and positive polarity) was developed using a carboxyl functional group packed column and 0.8% (v/v) formic acid as the mobile phase to detect and quantify zinc content. Results: The reported method has a good optimal sensitivity (LOQ: 0.006 mg/mL and LOD: 0.002 mg/mL). The correlation coefficient (r) obtained from the zinc calibration plot was greater than 0.998, indicating that the method was linear and that there was a strong correlation between zinc concentration (0.006 mg/mL to 0.375 mg/mL) and peak response. The accuracy at LOQ level was found to be 95–105% and 97–103% at the remaining levels (50%, 100%, and 150%). Conclusion: The proposed method was successfully developed and validated as per International council for harmonisation (ICH) guidelines. Therefore, this method can be used for the quantitative testing of zinc in the QC laboratory. Highlights: A novel method was developed for zinc levels determination in pharmaceutical products using HPLC with a refractive index detector. The present approach has a quick run time of 10 min and is inexpensive. Zinc is an essential nutrient that serves a variety of functions in our bodies. It is essential for protein synthesis, cell growth, DNA formation, tissue repair, and immune system maintenance (1, 2). During pregnancy, infancy, and childhood, zinc is required for the body to properly grow and develop. Zinc additionally upgrades insulin action (3). There are several dosage forms avail- able to prevent zinc deficiency, including tablets, capsules, and injectables. Zinc supplements include zinc acetate tablets, zinc sulphate tablets, zinc sulphate capsules, zinc sulphate injections, zinc chloride injections, and so on (4, 5). Accurate determination of zinc levels in pharmaceutical compounds is required. Titration (6, 7), ion chromatography (8, 9), and atomic absorp- tion spectroscopy (10–12) can all be used to determine zinc levels. Each method, however, has limitations. To determine zinc, edetate sodium is used as a titrant in the titration method. Because it involves manual titration, there is a possibility of vari- ation in end point determination from person to person (13). Furthermore, using titration analytical methods, trace amounts of zinc are difficult to measure. All ion chromatography problems can be traced back to the water used to prepare eluents, stand- ards, and samples. With aqueous eluents, total organic carbon (TOC) in water concentrates on all polymeric column surfaces. This concentrated TOC results in unstable conductivity and UV baselines, loss of column efficiency, which results in broad peak shapes, and loss of column capacity, which results in decreased retention times (14). TOC can serve as potential ion exchange sites in a few cases and hence the zinc estimation is influenced. These problems will not be evident in HPLC. For determining zinc Received: 14 February 2023. Revised: 17 June 2023. Accepted: 28 August 2023 # The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com Journal of AOAC INTERNATIONAL, 2023, 1–12 https://doi.org/10.1093/jaoacint/qsad101 Advance Access Publication Date: 7 September 2023 Research Article Downloaded from https://academic.oup.com/jaoac/advance-article/doi/10.1093/jaoacint/qsad101/7263166 by guest on 26 September 2023