New RP-UPLC method for The Simultaneous Determination of Haloperidol and Benzhexol in Tablets

 

Rajesh Pudi*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra pradesh-530045, India.

 

ABSTRACT:

Haloperidol is an anti-psychotic drug and Benzhexol (Trihexyphenidyl) is an anticholinergic drug. A newstability indicating UPLC method has been developed for the simultaneous determination of Haloperidol and Benzhexol and the method was validated. Waters UPLC with Empower software, with Acquity UPLC SB C18 x 1.7m. column and PDA detector was used for the simultaneous determination of Haloperidol and Benzhexol. A mixture of phosphate buffer and Acetonitrile taken in the ratio 56:44 v/v was used as mobile phase with a flow rate of 0.2 ml/min (UV detection at 245 nm) (Injection volume 1ml). Haloperidol and Benzhexol have shown linearregression equations, y=13361x+476.2(R² = 0.9998) and y =10609 x + 899.5 (R² = 0.9998). The LOD and LOQ are found to be 0.03 µg/mL and 0.10 µg/mL for Haloperidol and 0.03µg/mL and 0.09 µg/mL for Benzhexolrespectively. The present liquid chromatographic method is precise, accurate and can be used for the simultaneous estimation of Haloperidol and Benzhexol tablets.

 

 

 

INTRODUCTION:

Haloperidol¹ (Figure 1A) is chemically phenyl-piperidinyl-butyrophenone with antipsychotic, neuroleptic activities. Haloperidol competitively blocks postsynaptic dopamine (D2) receptors in the mesolimbic system of the brain, thereby eliminating the dopamine neurotransmission.Trihexyphenidyl², also known as Benzhexol (Figure 1B) is an anticholinergic drug and it acts by blocking acetylcholine and it helps to decrease muscle stiffness and improve walking ability in people with Parkinson's disease^3-4. Very few liquid chromatographic methods have been developed for the simultaneous estimation of Haloperidol and Benzhexol in tablets. The combination of Haloperidol and Benzhexol was determined earlier in the literature by Srikantha et al ⁵, Amulyaet al ⁶, Wateet al ⁷ using liquid chromatographic techniques and Wate et al⁸ and Manoj Charde et al⁹ using spectro photometric methods.

 

N the present study the authors have proposed a new stability indicating reverse phase UPLC method for the simultaneous estimation of Haloperidol and Benzhexol tablets and the method was validated.

 

 

Figure 1A: Chemical structure of Haloperidol

 

Figure 1B: Chemical structure of Benzhexol

 

 

 

MATERIALS AND METHODS:

The combination of Benzhexol and Haloperidol is available with brand names Carbidol (Intas Pharmaceuticals Ltd, India) (Label claim: Haloperidol10 mg and Benzhexol 2 mg; Haloperidol 5 mg and Benzhexol 2 mg); Halopidol Plus-10 (Label claim: Haloperidol 10 mg and Benzhexol 2 mg); Halopidol Plus-5 (Haloperidol 5 mg and Benzhexol 2 mg) (Johnson and Johnson); Hexidol (Label claim: Haloperidol 1.5 mg and Benzhexol 2 mg) (Torrent Pharmaceuticals Ltd, India). Benzhexol and Haloperidol were obtained as gift samples from Torrent Pharmaceuticals Ltd, India and used as it is without further purification.

 

HPLC instrument and chromatographic conditions:

Waters UPLC with Empower software, with Acquity UPLC SB C18 x 1.7m. Column and PDA detector was used for the simultaneous determination of Haloperidol and Benzhexol. A mixture of phosphate buffer and Acetonitrile taken in the ratio 56:44 v/v was used as mobile phase with a flow rate of 0.2 ml/min (UV detection at 245 nm) (Injection volume 1ml).

 

Method validation¹⁰

Stock solution containing both Haloperidol and Benzhexol was prepared by accurately weighing about 10 mg of Haloperidol (1000 μg/mL) and 2 mg of Benzhexol (200 μg/mL) in a 50 ml volumetric flask with the mobile phase containing 0.01N KH₂PO₄ buffer: Acetonitrile (56:44 v/v) and a series of Haloperidol (25-150 µg/mL) and Benzhexol (5-30 µg/mL) solutions were prepared from the stock solution with diluent (Water: Acetonitrile (50:50 v/v)). The method was validated by performing different studies–linearity, system precision, method precision, accuracy and robustness. Robustness study was performed by modifying the chromatographic conditions slightly and there by measuring the percentage relative standard deviation of the method developed. 1μl of all these diluted solutions were injected in to the UPLC system and the peak area (n=3) was noted at the retention time for the linearity study and a calibration curve was plotted (Concentration vs mean peak area) separately for both Haloperidol and Benzhexol. The limit of quantification (LOQ) and limit of detection (LOD) were calculated from the calibration curve response.

 

Assay of Haloperidol and Benzhexol tablets:

Tablets of three different brands containing both Haloperidol and Benzhexol were procured from the local pharmacy store, weighed and extracted with mobile phase and then further diluted with the diluent. These solutions were injected in to the UPLC system and the peak area was noted. The quantities of Haloperidol and Benzhexol were determined from the linear regression equations and the percentage of recovery was calculated.

 

RESULTS AND DISCUSSION:

A new stability indicating UPLC method has been developed for the simultaneous determination of Haloperidol and Benzhexol in tablets. The previously established liquid chromatographic methods were compared with the present UPLC method in Table 1. A mixture of 0.01N KH₂PO₄ buffer and Acetonitrile (56:44 v/v) was used as mobile phase and a mixture of water and Acetonitrile (50:50 v/v) was used as diluent.

 

Table. 1. Review of the reported methods with the present method

Mobile phase (v/v)	Flow rate (ml/min)	Column	Method	λ (nm)	Linearity (mg/ml)	Observations	Reference
Methanol: Acetonitrile: Water (50:40:10)	1.2	Zodiac C18 (250 x 4.6 mm, 5 μm)	HPLC	221	5-50 (HAL) 2-20 (BNZ)	Very low linearity	5
Methanol: TEA buffer pH 4.5: Acetonitrile (50:25:25)	1	Altima C18 (4.6 x 150mm, 5μm)	HPLC	225	37.5-187.5 (HAL) 15-75 (BNZ)	Very low linearity	6
Methanol: Water (70:30)	1	C18 (250 x 4.6 mm, 5 μm)	HPLC	220	2.5-12.5 (HAL) 1-5 (BNZ)	Very low linearity	7
0.01N KH2PO4 buffer : Acetonitrile (56:44)	0.2	Acquity UPLC SB C18 , 1.7 µ	UPLC	245	25-150 (HAL) 5-30 (BNZ)		Present work

 

Method validation:

Many trials were made and finally the chromatographic conditions were chosen where a good resolution was observed. Haloperidol was eluted at 0.754 min. and Benzhexol was eluted at 0.966 min (Figure 2). Haloperidol has shown linearity 25-150 µg/mland Benzhexol shown linearity 5- 30 µg/ml (Table 2) with linear regression equations y=13361x-+476.2 (R²=0.9998) for Haloperidol and y=10609x+899.5 (R²= 0.9998) for Benzhexol respectively (Figure 3). The LOD and LOQ are found to be 0.03 µg/mL and 0.10 µg/mL for Haloperidol and 0.03 µg/mL and 0.09 µg/mL for Benzhexol respectively. The % RSD in precision and accuracy studies were found to be less than 2.0 proving that the method is precise and accurate.

 

Table 2: Linearity of Haloperidol and Benzhexol

Conc (µg/mL)		*Mean peak area		% RSD
HAL	BNZ	HAL	BNZ	HAL	BNZ
25	5	331506	52931	0.13	0.15
50	10	668815	106903	0.13	0.04
75	15	1018382	163511	0.05	0.07
100	20	1327671	213073	0.40	0.37
125	25	1657944	266522	0.14	0.04
150	30	2013748	317303	0.15	0.22

*Mean of three replicates

 

Figure 2: Typical chromatograms of Haloperidol (Rt0.754 min) & Benzhexol (Rt0.966 min)

 

Figure 3: Calibration curve of A) Haloperidol B) Benzhexol

 

Table 3: Intraday and Intraday precision study of Haloperidol and Benzhexol

Conc (µg/mL)		Statistical analysis
		*Mean peak area ± SD (% RSD)
HAL	BNZ	HAL	BNZ
100	20	1328890 ± 8452.5 (0.64)	213917 ± 1296.2 (0.61)	Intra day
100	20	1317174 ± 6341.8 (0.48)	223479 ± 2447.5 (0.10)	Inter day

*Mean of six replicates

Table 4: Accuracy study of Haloperidol and Benzhexol

Conc. (µg/mL)				*Total found		% Recovery		% RSD
Formulation		Pure drug		Conc. (µg/mL)
HAL	BNZ	HAL	BNZ	HAL	BNZ	HAL	BNZ	HAL	BNZ
10	5	5	2.5	15	7.5	101.55	99.76	0.90	0.13
10	5	5	2.5	15	7.5	101.28	99.91
10	5	5	2.5	15	7.5	99.86	99.65
10	5	10	5	20	10	100.07	99.79	0.09	0.80
10	5	10	5	20	10	99.89	100.81
10	5	10	5	20	10	99.96	101.38
10	5	15	7.5	25	12.5	99.30	99.08	1.12	1.18
10	5	15	7.5	25	12.5	101.20	101.36
10	5	15	7.5	25	12.5	101.31	99.67

*Mean of three replicates

 

Assay of Haloperidol and Benzhexol (Tablets):

The combined dosage form containing Haloperidol and Benzhexol has shown 98.95-99.9 and 99.54-99.79 recovery respectively (Table 7) in the tablet formulations. Haloperidol has shown 98.7-99.8 and Benzhexol has shown 98.8-99.6.

 

Table 5: Assay of Haloperidol and Benzhexol tablets

Formulation	Label claim (mg)		*Amount found (mg)		*Recovery (%)
	HAL	BNZ	HAL	BNZ	HAL	BNZ
Brand I	10	5	9.87	4.98	98.7	99.6
Brand II	10	5	9.93	4.94	99.3	98.8
Brand III	10	5	9.98	4.96	99.8	99.2

*Mean of three replicates

CONCLUSIONS:

This new validated UPLC method for the simultaneous determination of Haloperidol and Benzhexolin tablets can be applied for the determination of pharmaceutical formulations and also for the kinetic studies also in vitro as well as in vivo.

 

ACKNOWLEDGEMENT:

The authors are grateful to Torrent Pharmaceuticals Ltd, India for providing the gift samples of Haloperidol and Benzhexol. There is no conflict of interest.

 

REFERENCES:

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4.       Dorje F, Wess J, Lambrecht G, Tacke R, Mutschler E, Brann MR: Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther. 256(2); 1991: 727-733.

5.       Srikantha D and Ramesh Raju R.A RP-HPLC method for simultaneous determination of Haloperidol and Trihexyphenidyl hydrochloride in tablet dosage form. Asian Journal of Pharmaceutical and Clinical Research. 7; Suppl 7; 2014: 14-18.

6.       Amulya E, Naveen Kumar N, Mounika CH, Kowmudi V, Supriya N, Ramya Madhuri K. Development and validation of RP-HPLC method for the simultaneous estimation of Haloperidol and Trihexyphenidyl in API and combined tablet dosage form. International Journal of Applied Pharmaceutical Sciences and Research. 3 (3); 2018: 36-40.

7.       Wate SP, Borkar AA. RP-HPLC estimation of Haloperidol and Trihexyphenidyl HCl in tablets. International Journal of Chem Tech Res. 1(3); 2009: 675-676.

8.       Wate SP, Borkar AA. Simultaneous spectrophotometric estimation of Haloperidol and Trihexyphenidyl HCL in tablets. Indian Journal of Pharmaceutical Sciences. 72(2); 2010:265-267.

9.       Manoj Charde, Imran Sheikh, Sarika Dhole and Avinash. V. Kasture. Simultaneous Estimation of Benzhexol hydrochloride and Haloperidol by absorbance correction method. Journal of Pharmacy Research. 3(9); 2010: 2099-2101.

10.     ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization, 2005.

 

 

 

 

Received on 12.04.2019          Modified on 23.05.2019

Accepted on 10.06.2019        © RJPT All right reserved