Design and Validation of a RPHPLC Method for Concurrent Determination of Haloperidol and Trihexyphenidyl in API and Combined Tablet Formulations

  Prathyusha Barla, Mangamma Kuna*, Abeda Begum Shaik, Nikhil Deep Gunnada

Department of pharmaceutical analysis, school of pharmaceutical sciences and technologies JNTUK, Kakinada, India- 533003

*Correspondence: kunamanga@gmail.com, mobile no: +91 8465039394

DOI: https://doi.org/10.71431/IJRPAS.2025.4601  

INTRODUCTION

High pressure liquid chromatography is a sophisticated technique used to detect, separate and measure substances in a liquid mixture. It is a type of chromatography, a method used to break down mixtures into individual substances. High-pressure Liquid Chromatography is a refined analytical method derived from liquid chromatography, designed to separate and identify components within complex chemical mixtures. It is extensively used in scientific and industrial applications.[1] It is extensively used in scientific and industrial applications. It is commonly employed to examine complex samples in areas like chemistry, biology and the pharmaceutical industry. The method relies on a physical separation process where compounds are divided as they pass through a narrow column filled with stationary material, while being pushed along by a liquid (or sometimes gas) called the mobile phase.[2] This technique can provide detailed information about substances, such as their size, shape, charge, polarity (hydrophilic or hydrophobic nature), structure, & molecular weight. The High pressure liquid chromatography system is made up of several parts, including a sample injector, mobile phase, stationary phase, and a detector. Common types of detectors used in High pressure liquid chromatography include UV-visible, fluorescence, & electrochemical detectors. These detect compounds based on how much light they absorb, how they fluorescence, or the current they generate. Mass spectrometry can also be used for more detailed or complex analysis.[3]

                     High pressure liquid chromatography has a wide range of applications; In pharmaceuticals, its used to detect active ingredients, check for impurities, develop new drugs, & test biological samples. In biotechnology, it helps in studying & purifying proteins & enzymes. In forensics, it is used to identify substances found at crime scenes. The agrochemical industry uses it to check pesticide levels in crops, food, & water. In environmental analysis, High pressure liquid chromatography helps detect pollutants like metals & chemicals in air, soil, & water.[5] High pressure liquid chromatography is known for its high sensitivity, precision, & reproducibility. It is considered a gold standard for analytical methods because it delivers fast, accurate, and cost-effective results, making it one of the most reliable techniques for analyzing a wide range of substances.[4]

MATREIALS AND METHODOLOGY

MATERIALS

Haloperidol, trihexyphenidyl hydrochloride reference standards, combined tablet formulations purchased from nearby medical stores, high-purity acetonitrile, analytical grade methanol, H3PO4.[5]

Formulation of buffer solution and mobile phase for analysis

Formulation of triethylamine buffer solution:

In a volumetric flask, combine 1 liter of HPLC- grade solvent with 1ml of triethylamine to obtain a 0.1% v/v concentration. Stir the solution well. Bring the pH to the desired level, usually around 2.5 to 3.0, by adding dilute phosphoric acid. Add acid dropwise while monitoring check the pH using a standardized PH meter. Once it reaches the target value, move the solution into a 1000 mL volumetric flask (if not already in it) and make up the amount to 1 liter with water. Pass the buffer solution through a 0.45-micron membrane filter and degas by sonication (typically 5–10 minutes) before use.[6]

Making the mobile phase solution:

Mobile Phase Composition:

Methanol:0.1% Triethylamine Buffer (the pH was set to 3.0 using H3PO4) and the mixture was prepared in a 70:30 volume-to-volume ratio.[7]

Preparation Procedure:

1. Formulating the buffer (0.1% Triethylamine, pH 3.0):

Take 1 mL of Triethylamine and dilute it to 1 liter of HPLC-grade solution. Adjust the pH to 3.0 ± 0.05 using orthophosphoric acid (approximately 1–2 mL of 10% v/v solution) pass the buffer through a 0.45 micro meter membrane filter, then remove dissolved gases by sonicating it for 10 minutes.

2. Mixing the Mobile Phase:

Combine 700 milliliters of solvent-grade methanol used in HPLC procedures with 300 milliliters of the prepared buffer. Filter the final mixture pass through a 0.45 micrometer membrane. Then subject it to degassing the mobile phase using ultrasonic treatment for 10–15 minutes before use.[8]

Diluent preparation:

Accurately measure 500mL of acetonitrile and place it in a clean, and moisture free beaker. Mix 500mL of purified water with acetonitrile. Mix thoroughly using a magnetic stirrer until a homogenous solution is obtained. Ensure the diluent is passed through a 0.45- micron filter before it is utilized. Store the prepared diluent in an amber bottle at room temperature and use within a few days to avoid contamination or degradation.[9]

PREPARATION OF STANDARD STOCK SOLUTIONS:

HALOPERIDOL STANDARD STOCK SOLUTION (1000µg/mL)

Measure 10mg of haloperidol with precision and transfer it to a 100mL flask. Introduce approximately 70mL of the suitable diluent. Sonicate to dissolve completely. Adjust the final volume to 100mL with the mobile phase. Final concentration is 1000 µg/mL.

TRIHEXYPHENIDYL STANDARD STOCK SOLUTION (100µg/mL)

Accurately weigh 10mg of trihexyphenidyl (API). Transfer it to a 100mL measuring flask. Roughly 700mL of mobile phase should then be added. Sonicate to dissolve thoroughly. Top up the solution to 100mL with the previously used mobile phase. Final concentration is 1000 µg/mL.

PREPARATION OF WORKING STANDARD SOLUTION:

Withdraw 1mL of each standard stock and transfer it to a 10mL volumetric container. Dilute to volume with the mobile phase. A working standard solution was made with a final concentration of;

Haloperidol is 10 µg/mL

Trihexyphenidyl is 10 µg/mL[10]

RESULTS AND DISCUSSION

The proposed RP-HPLC method was systematically validated according to the ICH protocols. The methods performance was evaluated based on system suitability, linearity, precision, accuracy, sensitivity (LOD & LOQ), and robustness.

SYSTEM PERFORMANCE CHECK

To validate the consistency & reliability of the chromatographic system, system suitability tests were conducted. A total of five injections of the standard were made, and the relative standard deviation of the peak areas was evaluated. The %RSD values were 1.09% for trihexyphenidyl & 0.73% for haloperidol, confirming the systems adequacy for analysis.[11]

LINEARITY

To check linearity, each analyte was tested at five different concentrations. Trihexyphenidyl showed a strong linear response within the concentration range of 15 to 75 microgram per milliliter, the method showed a correlation coefficient of 0.999 and a regression equation of y= 12066x + 13756. Similarly, haloperidol exhibited linearity from 37.5-187.5 µg/mL, also with an r² value of 0.999, & its regression equation was y=18768x + 33265. These findings demonstrate that the method produces a proportional response across the studied concentration ranges.

SENSITIVITY (LOD & LOQ)

The methods sensitivity was evaluated by calculating the LOD & LOQ. Trihexyphenidyl had an LOD of 0.8 µg/mL, while the LOD for haloperidol was 6.9 µg/mL. The respective LOQ values were found to be 2.6 µg/mL and 21.1 µg/mL. These results demonstrate that the method can accurately detect and measure low concentrations levels of both drugs.

PRECISION

INTRA-ASSAY PRECISION

Intra-assay precision was assessed through examination of the standard solution 5 times under identical conditions. The %RSD values were 0.42% for trihexyphenidyl and 0.18% for haloperidol, confirming good precision.

INTERMEDIATE PRECISION

Intermediate precision, which evaluated the methods reproducibility under varied conditions (such as different days), yielded %RSD values of 0.37% for trihexyphenidyl and 0.13% for haloperidol. These findings demonstrate that the method consistently produces reliable and reproducible results.

ACCURACY

To verify accuracy, recovery experiments were conducted at 3 concentration levels; At levels of 50%, 100%, & 150%, known quantities of the standard drugs were spiked into previously analyzed samples and the samples were reanalyzed. The percentage recoveries were within acceptable ICH limits for both analytes, confirming the methods accuracy in estimating drug content in combined tablet formulations.

DURABILITY (ROBUSTNESS)

To assess the robustness of the RP-HPLC method, small deliberate changes were made to the parameters like the flow rate and mobile phase composition. These changes did not significantly affect the peak area, retention time, or other system suitability parameters, showing that the method remains consistent and dependable under slight operational deviations. The findings validated that the method remains stable and reliable for regular analytical use.[12]

FORMULATION ASSAY

To determine the concentration of the drug within the market solid formulation, the assay was performed using the developed method. Both the sample and standard solution concentrations were determined using a conventional calculation formula. Purity analysis of the tablet dosage form revealed 99.6% for trihexyphenidyl and 99.7% for haloperidol. These values reflect the accuracy and efficiency of the method in estimating the active pharmaceutical ingredients in commercial products.

%ASSAY=

(Area of sample / Area of standard) x (Concentration of standard / Concentration of sample) x (Purity of standard)[13]

CONCLUSION

A robust, dependable and exact reverse-phase HPLC method was successfully formulated and confirmed for the combined analysis of haloperidol and trihexyphenidyl in both active drug substance (API) form and combined tablet dosage formulation. The method demonstrated excellent validation parameters, in compliance with ICH protocols. The retention times for both analytes were well-resolved, unaffected by excipients or breakdown compounds, confirming the methods specificity. The proposed method is therefore appropriate for regular quality testing & stability examination of haloperidol and trihexyphenidyl within medicinal preparations.

ACKNOWLEDGMENT

I would like to express my sincere gratitude to Dr. k. Mangamma Assistant professor Department of Pharmaceutical Analysis, Jawaharlal Technological University Kakinada. For her valuable guidance, encouragement, and support throughout the work.

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