Development and validation of a novel HPLC-DAD method for simultaneous quantification of active pharmaceutical ingredients in fixed-dose combination tablets.

Project Abstract

A robust and sensitive high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed and validated for the simultaneous quantification of multiple active pharmaceutical ingredients (APIs) in fixed-dose combination (FDC) tablets, addressing the growing demand for quality control efficiency in pharmaceutical manufacturing. The study focuses on selecting appropriate chromatographic conditions, including column type, mobile phase composition, gradient program, flow rate, and detection wavelengths, to achieve adequate separation, peak symmetry, and minimal co-elution of APIs and potential excipients. Method development employed a systematic approach using design of experiments (DoE) to screen critical factors and optimize robustness, followed by meticulous validation per ICH guidelines (Q2(R1)) across specificity, linearity, range, accuracy, precision, detection and quantitation limits, robustness, solution stability, and system suitability parameters. The chosen mobile phase comprised a gradient of acetonitrile and water with volatile modifiers to ensure compatibility with downstream mass balance considerations while maintaining MS-free operation for routine QC workflows. Detection was performed at multiple wavelengths corresponding to the UV-Vis absorbance maxima of the constituent APIs, enabling reliable simultaneous quantification and reducing cross-talk between spectral signals. The validated method demonstrated good specificity against placebo and excipients, with no interference identified at the retention times of the APIs. Linearity was established over clinically relevant concentration ranges with correlation coefficients (r) exceeding 0.999 for all analytes. Recovery studies yielded accuracy within 98–102% across low, medium, and high concentration levels, and intra- and inter-day precision demonstrated relative standard deviations below 2.5% for all APIs. The limits of detection (LOD) and quantitation (LOQ) were sufficiently low to quantify trace levels in dissolution studies and to meet regulatory requirements for assay and impurity profiling. System suitability tests indicated stable theoretical plate numbers, tailing factors close to unity, and consistent retention times under varied injection loads. The method was applied to commercially available fixed-dose combination tablets containing two to three APIs with varying physicochemical properties. Robustness tests, including deliberate small variations in column temperature, pH of the mobile phase, and flow rate, confirmed the method’s reliability under routine manufacturing conditions. Forced degradation studies under acidic, basic, oxidative, thermal, and photolytic stress conditions demonstrated the method’s stability-indicating ability, with API degradation products well resolved from the intact drugs. The study also explored sample preparation strategies, including direct dissolution and solid-phase extraction, to ensure compatibility with diverse tablet matrices and batch sizes. Overall, the developed HPLC-DAD method provides a validated, cost-effective, and high-throughput analytical tool for the quality control of FDC tablets, enabling accurate quantification of multiple APIs in a single run, reducing analysis time, solvent consumption, and operational complexity while maintaining compliance with international guidelines and ensuring patient safety through rigorous pharmaceutical analysis.

Project Overview

What This Project Is About

---

A straightforward, beginner-friendly look at how scientists measure several active ingredients in fixed-dose combination tablets using a single analytical method. The project investigates making a reliable, simple test that can quantify multiple drugs at once, ensuring the pills contain the right amounts of each ingredient.

---

---

The Problem It Addresses

---

In many medicines, more than one drug is combined in one tablet. Traditional methods may be slow, costly, or only good for one ingredient. This project aims to create a faster, cost-effective method that can accurately measure several components at the same time, which helps quality control and patient safety.

---

---

Objectives of the Project

---

1. Develop a single testing method that can quantify multiple active ingredients in fixed-dose tablets.
2. Validate the method to prove it is accurate, precise, and reliable.
3. Check the method’s robustness, ensuring it works under small changes in conditions.
4. Compare the new method with existing standard methods to show any advantages.

---

---

What You Will Do Step by Step

---

1) Learn basic concepts of chromatography and method validation. 2) Choose a representative fixed-dose tablet and plan which ingredients to measure. 3) Develop the testing procedure and optimize conditions for separation of ingredients. 4) Validate the method for accuracy, precision, linearity, detection limits, and range. 5) Test robustness by varying small factors like solvent composition and flow rate. 6) Analyze data to confirm the method performs well. 7) Prepare a report comparing results with existing methods.

---

---

---

---

Expected Outcome

The project should deliver a validated, user-friendly HPLC-DAD method that can quantify multiple active ingredients in fixed-dose tablets with good accuracy and precision, along with a clear protocol for routine quality control and a comparison to conventional single-analyte methods.