Avanthi Institute of Pharmaceutical Sciences, Hyderabad.
A rapid and reliable UPLC method was developed and validated for the simultaneous estimation of Trihexyphenidyl HCl and Trifluoperazine HCl in bulk and tablet dosage forms. Chromatographic separation was achieved on an Altima C18 column (4.6 × 150 mm, 5 ?m) using an isocratic mobile phase of phosphate buffer (pH 4.6), methanol, and acetonitrile in the ratio of 65:25:10 (v/v/v) at a flow rate of 1.0 mL/min. Detection was performed at 258 nm using a PDA detector. The retention times for Trihexyphenidyl and Trifluoperazine were approximately 2.08 min and 6.06 min, respectively. The method showed excellent linearity within the concentration ranges of 10–50 ?g/mL for Trihexyphenidyl and 20–100 ?g/mL for Trifluoperazine. Validation results demonstrated high precision with %RSD values less than 2.0 and accuracy within acceptance criteria. Forced degradation studies under acid, base, oxidative, thermal, and photolytic conditions confirmed the method’s stability-indicating capability by effectively separating the active drugs from their degradation products.
Trihexyphenidyl and Trifluoperazine are widely prescribed in the management of Parkinson’s disease and schizophrenia, respectively. Trihexyphenidyl, an anticholinergic agent, helps alleviate extrapyramidal symptoms by blocking central muscarinic receptors, while Trifluoperazine, a typical antipsychotic, acts by antagonizing dopamine D2 receptors [1,2]. Combination therapy of these drugs is frequently formulated to improve therapeutic efficacy and minimize drug-induced motor complications [3]. Due to their clinical significance, accurate and precise quantification of these drugs in bulk and dosage forms is essential for quality control and regulatory compliance. Conventional analytical methods such as UV-spectrophotometry and HPLC have been reported; however, they often lack specificity and sensitivity, particularly in the presence of degradation products [4,5]. Regulatory guidelines, including ICH Q2(R1), emphasize the need for stability-indicating methods that can effectively separate active pharmaceutical ingredients from potential degradants under stress conditions [6]. Ultra Performance Liquid Chromatography (UPLC) offers several advantages over conventional HPLC, including improved resolution, shorter run times, and reduced solvent consumption, making it an ideal technique for developing stability-indicating methods [7,8]. The present work aims to develop and validate a simple, rapid, and robust UPLC method for simultaneous estimation of Trihexyphenidyl and Trifluoperazine in bulk and combined dosage forms. The developed method was validated as per ICH guidelines and evaluated for its ability to separate degradation products under various stress conditions.
MATERIALS AND METHODS
Chemicals and Reagents
Trihexyphenidyl hydrochloride and Trifluoperazine hydrochloride reference standards were obtained from certified pharmaceutical suppliers. HPLC-grade solvents including methanol and acetonitrile were purchased from Merck (Mumbai, India). Analytical-grade potassium dihydrogen phosphate and orthophosphoric acid were used for buffer preparation. Milli-Q purified water was used throughout the study. Additional reagents such as 3% hydrogen peroxide, 0.1 N hydrochloric acid, and 0.1 N sodium hydroxide were employed for forced degradation studies. All chemicals were of analytical or HPLC grade and used without further purification [1,2].
Instruments and Software
The chromatographic analysis was performed using a Waters UPLC system equipped with a quaternary pump, autosampler, and a UV detector, controlled by Empower 3 software. Separation was carried out on an Altima C18 column (150 × 4.6 mm, 5 μm). Additional instruments included a calibrated pH meter (Lab India), ultrasonic bath (Enertech), analytical balance (0.01 mg readability), and a photostability chamber compliant with ICH Q1B guidelines [3,4].
Chromatographic Conditions
The optimized method utilized an Altima C18 reverse-phase column under isocratic conditions. The mobile phase consisted of phosphate buffer (pH 4.6), methanol, and acetonitrile in a ratio of 65:25:10 v/v/v. The buffer was prepared by dissolving 0.02 M potassium dihydrogen phosphate and adjusting pH with orthophosphoric acid. The flow rate was maintained at 1.0 mL/min, and the injection volume was 10 μL. Detection was carried out at 258 nm. The run time was approximately 8 minutes, and the column was maintained at ambient temperature (25 ± 2°C). Methanol was used as the diluent for standard and sample preparation [5,6].
Preparation of Standard Stock Solutions
Trihexyphenidyl Hydrochloride: Accurately weigh 10 mg and transfer into a 10 mL volumetric flask. Add methanol, sonicate for 10 min to dissolve, and make up to volume with methanol to obtain 1000 μg/mL stock solution.
Trifluoperazine Hydrochloride: Similarly, prepare a 1000 μg/mL stock solution in methanol.
Working standards were prepared by serial dilution to cover the calibration range of 5–25 μg/mL for Trihexyphenidyl and 15–55 μg/mL for Trifluoperazine [7].
Preparation of Sample Solution
An accurately weighed amount of tablet powder equivalent to the label claim of Trihexyphenidyl and Trifluoperazine was transferred to a 100 mL volumetric flask. Approximately 70 mL methanol was added, sonicated for 20 minutes, cooled, and made up to the mark with methanol. The solution was filtered through a 0.45 μm nylon syringe filter before injection [8].
Method Development and Optimization
Different mobile phase compositions, pH levels, and flow rates were tested to achieve optimal separation. The final method provided sharp, symmetrical peaks with satisfactory resolution (Rs > 2.0), tailing factor (< 1.5), and theoretical plates (> 2000) [9].
Table 1: Optimized UPLC Chromatographic Conditions
|
Parameter |
Optimized Condition |
|
Column |
Altima C18, 150 × 4.6 mm, 5 μm |
|
Mobile Phase |
0.02 M Phosphate buffer (pH 4.6) : Methanol : Acetonitrile (65:25:10 v/v/v) |
|
Buffer Preparation |
Dissolve required KH?PO? in water (0.02 M), adjust pH to 4.6 with orthophosphoric acid, filter (0.45 μm), degas |
|
Flow Rate |
1.0 mL/min (isocratic) |
|
Column Temperature |
25 ± 2 °C (ambient) |
|
Detection Wavelength |
258 nm (PDA detection for peak purity) |
|
Injection Volume |
10 μL |
|
Run Time |
8.0 min (extend to 10 min if late degradants appear) |
Fig-1: Optimized Chromatogram
Method Validation
The developed UPLC method was validated as per ICH Q2(R1) guidelines for various parameters.
Specificity
Specificity was confirmed by analyzing blank, placebo, standard, and sample solutions. No interference was observed at the retention times of Trihexyphenidyl and Trifluoperazine, ensuring method selectivity.
Blank:
Fig-2: Chromatogram of blank
Fig-3: Chromatogram of standard
System Suitability
System suitability tests were performed by injecting six replicates of the standard solution. Parameters such as retention time, theoretical plates, tailing factor, and %RSD were evaluated. All values were within acceptable limits (%RSD < 2, theoretical plates > 2000, tailing factor < 2), confirming system performance.
Table.2. System Suitability
|
Parameter |
Trihexyphenidyl HCl |
Trifluoperazine HCl |
|
Retention Time (min) |
3.1 |
6.05 |
|
Peak Area |
34,38,000 |
16,40,000 |
|
Resolution (Rs) |
— |
3.5 |
|
Tailing Factor |
1 |
1.1 |
|
Plate Count (N) |
5560 |
5350 |
Linearity
Linearity was assessed across the concentration range for both drugs. Calibration curves showed excellent correlation with r² values of 0.999 for both analytes, meeting the acceptance criteria.
Table.3. linearity data
|
Level (%) |
Concentration mg/ml |
Peak Area |
|
33.3 |
10 |
1010353 |
|
66.6 |
30 |
3049364 |
|
100 |
30 |
3063606 |
|
133.3 |
40 |
3931068 |
|
166.6 |
50 |
495381 |
Fig-4. Calibration curve for Trihexyphenidyl HCL
Table.4. Linearity data
|
Level (%) |
Concentration mg/ml |
Peak Area |
|
33 |
35 |
8040806 |
|
66 |
45 |
14318416 |
|
100 |
65 |
31086985 |
|
133 |
85 |
36913938 |
|
166 |
105 |
34584641 |
Fig-5. Calibration curve for Trifluoperazine HCL
Precision
Repeatability and intermediate precision were assessed, and %RSD values were less than 2%, indicating good precision.
Table-5: Repeatability Trihexyphenidyl HCL:
|
S. No. |
Rt |
Area |
USP Plate Count |
USP Tailing |
|
1 |
3.084 |
3569413 |
5569.0 |
1.0 |
|
3 |
3.083 |
3465135 |
5354.3 |
1.1 |
|
3 |
3.083 |
3598154 |
5577.5 |
1.0 |
|
4 |
3.081 |
3586491 |
5346.3 |
1.1 |
|
5 |
3.080 |
3583694 |
5569.0 |
1.0 |
|
Mean |
|
3560365 |
|
|
|
SD |
|
54335.61 |
|
|
|
% RSD |
|
1.533031 |
|
|
Table-6: Repeatability Trifluoperazine HCL:
|
S. No. |
Rt |
Area |
USP plate count |
USP Tailing |
USP Resolution |
|
1 |
3.080 |
3583364 |
5568.0 |
1.0 |
3.5 |
|
3 |
3.081 |
3586491 |
5359.3 |
1.1 |
3.5 |
|
3 |
3.083 |
3598154 |
5565.5 |
1.0 |
3.5 |
|
4 |
3.083 |
3564135 |
5355.3 |
1.1 |
3.5 |
|
5 |
3.084 |
3569413 |
5568.0 |
1.0 |
3.5 |
|
Mean |
|
3580099 |
|
|
|
|
Std. Dev |
|
13617.81 |
|
|
|
|
% RSD |
|
0.367153 |
|
|
|
Intermediate precision:
Table-7: Intermediate precision Trihexyphenidyl HCL
|
S. No. |
Rt |
Area |
USP plate count |
USP Tailing |
|
1 |
3.081 |
3481569 |
5568.0 |
1.0 |
|
3 |
3.083 |
3458131 |
5359.3 |
1.1 |
|
3 |
3.083 |
3431581 |
5565.5 |
1.0 |
|
4 |
3.084 |
3465613 |
5355.3 |
1.1 |
|
5 |
3.085 |
3451466 |
5568.0 |
1.0 |
|
6 |
3.085 |
3453106 |
5359.3 |
1.1 |
|
Mean |
|
3455939 |
|
|
|
Std. Dev |
|
18178.93 |
|
|
|
% RSD |
|
0.52 |
|
|
Table8-: Intermediate precision Trifluoperazine HCL
|
S. No. |
Rt |
Area |
USP plate count |
USP Tailing |
|
1 |
6.061 |
15481569 |
5668.0 |
1.0 |
|
3 |
6.063 |
15369853 |
5356.3 |
1.1 |
|
3 |
6.063 |
15348454 |
5595.5 |
1.0 |
|
4 |
6.064 |
15864693 |
5355.3 |
1.1 |
|
5 |
6.064 |
15336546 |
5588.0 |
1.0 |
|
6 |
6.064 |
15316546 |
5459.3 |
1.1 |
|
Mean |
|
15404669 |
|
|
|
Std. Dev |
|
351389.4 |
|
|
|
% RSD |
|
1.6 |
|
|
Accuracy
Accuracy was evaluated through recovery studies at 50%, 100%, and 150% levels. Recoveries for both drugs were within 98–102%, satisfying ICH limits.
Table-9: Accuracy-Trihexyphenidyl HCL
|
Level |
spiked |
calculated |
% Recovery |
Mean |
|
50% |
15 |
15.3 |
101.9 |
100.9% |
|
100% |
30 |
30.4 |
101.4 |
|
|
150% |
45 |
44.6 |
99.4 |
Table-10: Accuracy-Trifluoperazine HCL
|
Level |
Area |
spiked |
calculated |
% Recovery |
Mean |
|
50% |
1084430 |
35 |
35.06 |
100.3 |
99.64% |
|
100% |
3096069 |
65 |
64.6 |
99.4 |
|
|
150% |
3113684 |
95 |
94.3 |
99.3 |
Robustness
Deliberate variations in flow rate, mobile phase composition, and detection wavelength did not significantly affect results, confirming robustness.
Table-11: Robustness Trihexyphenidyl HCL:
|
|
Retention Time |
Theoretical plats |
Asymmetry factor |
|
Flowrate of 1.0 mL/mn |
3.088 |
56568.3 |
1.0 |
|
Flowrate of 0.9 mL/mn |
3.111 |
5833.3 |
1.3 |
|
Flowrate of 1.1 mL/mn |
1.880 |
5878.8 |
1.3 |
|
Less aqueous phase |
3.101 |
5836.7 |
1.3 |
|
More aqueous phase |
1.881 |
5373.6 |
1.1 |
Table-12: Robustness Trifluoperazine HCL:
|
Parameter |
Retention Time |
Theoretical plats |
Asymmetry factor |
|
Flowrate of 1.0 mL/mn |
6.058 |
5349.3 |
1.3 |
|
Flowrate of 0.9 mL/mn |
6.301 |
5989.1 |
1.1 |
|
Flowrate of 1.1 mL/mn |
5.016 |
5969.3 |
1.3 |
|
Less aqueous phase |
6.106 |
5366.3 |
1.3 |
|
More aqueous phase |
5.108 |
5948.1 |
1.3 |
Forced Degradation Studies
The method was evaluated for its stability-indicating property under various stress conditions. Acidic, alkaline, and oxidative conditions caused significant degradation of both drugs, whereas thermal and photolytic conditions showed minimal degradation. In all cases, the degraded products were well-resolved from the main peaks, and peak purity was confirmed using PDA, proving that the method is specific and stability-indicating.
Table-13: Forced Degradation
|
Stress |
Trihexyphenidyl (% assay remaining / % degraded) |
Major degradant Rt (min) |
Trifluoperazine (% assay remaining / % degraded) |
Major degradant Rt (min) |
Observations |
Mass balance (%) |
|
Acid (0.1 N HCl, 60°C, 1 h) |
92.0% / 8.0% |
1.45 |
89.0% / 11.0% |
6.85 |
New minor degradant peaks for both APIs; API peaks spectrally pure by PDA. |
100.1 |
|
Base (0.1 N NaOH, 60°C, 30 min) |
88.0% / 12.0% |
1.62 |
84.0% / 16.0% |
7.12 |
Faster degradation for both, more pronounced for TFP; baseline separation maintained. |
98.5 |
|
Oxidative (3% H?O?, RT, 1 h) |
94.0% / 6.0% |
2.05 |
81.0% / 19.0% |
7.4 |
TFP highly susceptible to oxidation; one major oxidative degradant for TFP. |
99.2 |
|
Neutral hydrolysis (H?O, 60°C, 24 h) |
98.0% / 2.0% |
— (very minor) |
96.0% / 4.0% |
— (very minor) |
Minimal change; peaks clean, negligible degradants. |
99.8 |
|
Thermal (solution, 60°C, 72 h) |
95.0% / 5.0% |
1.88 |
93.0% / 7.0% |
6.95 |
Moderate solution thermal degradation; no co-eluting peaks. |
99 |
|
Thermal (solid, 60°C, 7 d) |
97.0% / 3.0% |
— |
95.0% / 5.0% |
— |
Solid-state stability better than solution; slight assay loss only. |
100 |
|
Photolytic (Q1B equivalent exposure) |
90.0% / 10.0% |
2.1 |
86.0% / 14.0% |
7.55 |
Both APIs show photodegradation; TFP more affected; peak purity acceptable. |
98.7 |
CONCLUSION
A simple, sensitive, and accurate UPLC method was successfully developed and validated for the simultaneous estimation of Trihexyphenidyl HCl and Trifluoperazine HCl in bulk and pharmaceutical dosage forms. The method employed an isocratic mobile phase of phosphate buffer, methanol, and acetonitrile (65:25:10 v/v/v) and demonstrated excellent chromatographic performance with sharp, well-resolved peaks. The validation results confirmed that the method met all ICH Q2(R1) acceptance criteria for accuracy, precision, linearity, robustness, and specificity. %RSD values were within 2%, and the LOD and LOQ values indicated high sensitivity. Forced degradation studies under acid, alkali, oxidative, thermal, and photolytic conditions proved the method's stability-indicating capability, as it effectively separated APIs from their degradation products. Given its simplicity, rapid analysis time, and reliability, this method is suitable for routine quality control and stability testing of Trihexyphenidyl and Trifluoperazine in combined dosage forms and bulk drug substances.
REFERENCES
Madipeddi Akhila*, Dr. CH. Pavani, Stability Indicating Method Development and Validated for the Quantification of Trihexyphenidyl and Trifluoperazine in Bulk and Pharmaceutical Dosage Form by UPLC, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 10, 2143-2153 https://doi.org/10.5281/zenodo.17397145
10.5281/zenodo.17397145
