Absorbance Ratio Method of Vortioxetine and Aripiprazole in Synthetic Mixture by UV Spectrophotometry

Ancy Mathew1, Suresh Kumar1*, Vishnu Sutariya2, Dhara Vashi1

¹Department of Pharmaceutical Quality Assurance, Shree Dhanvantary Pharmacy College, Kim, Surat-394110, Gujarat, India.

2Shree Dhanvantary Pharmaceutical Analysis and Research Centre, Managed by- Ribosome Pharma Pvt.Ltd., Surat- 394110, Gujarat, India.

 

ABSTRACT:

The absorbance ratio method is a modification of the simultaneous equations procedure. In this study, absorbance ratio analytical method is developed in UV spectrophotometry of Vortioxetine and Aripiprazole in synthetic mixture. The measurements were carried out at wavelengths of 225 and 255 nm. The 255nm is the λmax of Aripiprazole & 225nm is the isobestic point. The linear correlation of Isobestic point (r²=0.997) was obtained in the range of 2-20 µg/ml and for Aripiprazole the linear correlation range (r²=0.998) was obtained in the range of 2-32 µg/ml. The method was successfully used for absorbance ratio method of Vortioxetine and Aripiprazole in synthetic mixture form without any interference from excipients and prior separation.

 

 

 

INTRODUCTION:

 

Figure 1: Chemical structure of Vortioxetine Hydrobromide Monohydrate^[9]

 

Vortioxetine is the first antidepressant which was released in the late 1950s. It was officially approved by the FDA on September 30, 2013. Vortioxetine represents another option for the treatment of MDD^[1]

 

Aripiprazole is primarily used in the treatment of schizophrenia and bipolar disorder. It was approved by the FDA for schizophrenia on November 15, 2002^[2]. The combination of Vortioxetine and Aripiprazole is mainly used for the treatment of Obsessive Compulsive Disorder. The combination of both drugs has been approved from 2017^[3]. Obsessive-compulsive disorder (OCD) is a mental health condition characterized by distressing, intrusive, obsessive thoughts and repetitive, compulsive physical or mental acts ^[4]. An abnormality, or an imbalance in neurotransmitters, is thought to be involved in OCD^. It is said that people with OCD are found to have different brain activity^[5]. Serotonin is the chemical in the brain that sends messages between brain cells and it is thought to be involved in regulating everything from anxiety, to memory, to sleep^[6]. Due to the imbalance in the Serotonin level it may lead to OCD. The combination of Vortioxetine and Aripiprazole helps to maintain the serotonin level. Symptoms often appear in teens or young adults^[7]. Stress can make symptoms worse. It has observed that 15 mg of Vortioxetine and 10 mg of Aripiprazole has been shown better result for OCD treatment. The combination of Vortioxetine and Aripirazole is safe for the treatment of OCD. Vortioxetine Hydrobromide Monohydrate is used instead of Vortioxetine^[8]. The IUPAC of Vortioxetine Hydrobromide Monohydrate is 1{2 [2, 4 dimethyl phenyl) sulfanyl] phenyl} piperazine hydrobromide monohydrate. Molecular formula is C₁₈H₂₅BrN₂SO.

 

 

Figure 2: Chemical structure of Aripiprazole^[10]

 

The IUPAC name of Aripiprazole is 7-[4-[4-(2, 3-dichlorophenyl) piperazin-1yl]butoxy]-3,4-dihydro-1Hquinolin-2-one. Molecular formula is C₂₃H₂₇Cl₂N₃O₂.

 

The absorbance ratio is a modification of simultaneous equation procedure. It depends on the property for a substance which obeys Beers Law at all wavelengths, the ratio of absorbance at any two wavelengths is a constant value independent of concentration or pathlength. In USP this ratio is referred as Q value. In this method two wavelength are required. One wavelength is selected as Isobestic point and the second selected wavelength is the maximum absorbance of Aripiprazole^[11].

 

The overlain spectra (zero order) of Vortioxetine (VOR) and Aripiprazole (ARI) at different concentrations exposed that different concentration of VOR and ARI possess isobestic point at 225nm. Seeing the above wavelength 255nm (λ1) and 225nm (λ2) were selected for the assessment of both drugs by absorption ratio method.

 

MATERIALS AND METHODS:

Apparatus and instrumentation:

Spectroscopic analysis was carried out on a UV/ VISIBLE 2450 and UV/VISIBLE 1800 (Shimadzu) double beam UV-Visible spectrophotometer with software of UV- Probe version 2.3.4. The zero order absorption spectra were recorded over the wavelength range of 200-400nm against blank solvent in quartz cuvettes with 1cm diameter. A semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose. All volumetric glassware used was calibrated.

 

MATERIALS:

Vortioxetine Hydrobromide Monohydrate API received as a gift sample from Torrent Pharmaceutical, Dahej. Aripiprazole API received as a gift sample from Akhil health care, Vadodara. Required excipients were provided from Shree Dhanvantary Pharmacy College, Kim.

 

Method development:

Preparation of standard stock solution:

Stock solution was prepared by diluting 10 mg of each drug in sufficient quantity of methanol in separate volumetric flask and volume was made up to 100 ml to get the concentrations of 100μg/ml for each drug. Sonicate for 15 min. Dilutions from stock solution were prepared in the range of 2-20 µg/ml for Isobestic point and 2-32 μg/ml for Aripiprazole. Methanol was used as a blank solution.

 

METHODOLOGY:

Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one which is an isobestic point and other being the λmax of one of the two components. From the overlay spectra of two drugs, it is evident that VOR and ARI show an isobestic point at 225 nm. The second wavelength used is 255 nm, which is the λmax of ARI. Working standards were prepared in methanol and the absorbances at 225 nm (isobestic point) and 255 nm (λmax of ARI) were measured and absorptivity coefficients were calculated using calibration curve. The concentration of two drugs in the mixture can be calculated using following equations.

 

CX = [(QM – QY) / (QX -QY)] × A1 / ax1                 (1)

CY = [(QM – QX) / (QY -QX)] × A1 /ay1                  (2)

 

Where,

A1 and A2 are absorbances of mixture at 255 nm and 225 nm;

ax1 and ay1 are absorptivities of VOR and ARI at 255 nm;

ax2 and ay2 are absorptivities of VOR and ARI respectively at 225 nm;

QM = A2 / A1, QX = ax2 / ax1, QY = ay2 / ay1

 

Calibration Curve for VOR and ARI:

To check linearity of the method, working standard solution having concentration in range of 2-20 μg/ml of VOR and 2-32 μg/ml of ARI were prepared from the standard stock solution of both drugs. The absorbance was measured at 255 nm and at 225 nm. Calibration curves were constructed by plotting absorbance vs. concentration.

 

Preparation of standard mixture solution (VOR+ARI):

There is no any combined dosage formulation of VOR and ARI available in market. A synthetic mixture is been prepared using the excipients. The excipients used to prepare the standard mixture solution (Table: 1) were added to the 100 ml volumetric flask. Which is dissolved with methanol and then it is sonicated for 15 min, the volume is made up to the mark with methanol and then it is been filtered using whatman filter paper. From this solution 1,2,3,4,5 and 6ml aliquot was taken in 10 ml volumetric flask & diluted up to the mark with methanol to make the final concentration of VOR 3,6,9,12,15 and 18 μg/ml and ARI 2,4,6,8,10 and 12 μg/ml respectively which was used for assay.

 

Table 1: Composition of synthetic mixture

Sr No:	Excipients name	Quantity (mg)
1.	Vortioxetine Hydrobromide Monohydrate	30
2.	Aripiprazole	20
3.	Manitol	120
4.	Corn starch	10
5.	Methyl cellulose	10
6.	Sodium Starch	4
7.	Magnesium Stearate	6
	Total	200

 

Spectrophotometric Measurements

 

Figure 3: Overlain of VHM and APZ.

 

Zero-order spectra of standard solutions of Vortioxetine Hydrobromide Monohydrate (12μg/ml) and Aripiprazole (12μg/ml) versus their solvent blank were recorded in the range of 200-400 nm. The absorbance ratio spectra of these solutions were obtained in the same range of wavelength against their blanks. From the overlay spectra of two drugs, it is evident that VOR and ARI show an isobestic point at 225 nm. ARI showed 217 nm and 255 nm λmax from which 255 nm was selected as λmax of ARI in this method due to good linearity (Figure 3).

 

RESULTS AND DISCUSSION:

Absorption maxima:

Absorption maxima of VOR and ARI was found o 226nm for VOR and 255nm for ARI respectively. Individual spectra of both drug is shown in Figure 4.

 

 

 

Figure 4: Determination of maximum wavelength of VOR (226nm) and ARI (255nm).

 

Linearity:

Linear correlation was obtained between absorbance vs. concentration of VOR and ARI in the concentration ranges of 2-20 μg/ml and 2-32 μg/ml respectively and is shown in overlain chromatogram of VOR and ARI, Figure. 5 and 6 respectively.

 

Figure 5: VOR concentration ranges of 2-20 μg/ml

 

Figure 6: ARI concentration ranges of 2-32µg/ml.

Calibration curve data of VOR and ARI shown in Table 2 and 3. Regression parameters are mentioned in Table 4 and the linearity spectra and calibration curves of these two drugs at 255 nm and 225 nm are shown in Fig. 7, 8 and 9, 10 respectively.

 

Figure 7: Calibration curve of VOR at 255nm

 

Figure 8: Calibration curve of VOR at 2-20µg/ml. 225nm 2-20µg/ml.

Figure 9: Calibration curve of ARI at 255nm

 

Figure 10: Calibration curve of ARI at225nm 2-32µg/ml 2-32µg/ml.

 

Table 2: Linearity data for VOR

Sr No	Concentration (µg/ml)	At Wavelength 255 nm Absorbance (Mean±SD)	% RSD	At Wavelength 225 nm Absorbance (Mean±SD)	% RSD
1.	2	0.078 ± 0.0012	1.6551	0.133 ± 0.0015	1.1748
2.	6	0.222 ± 0.0022	1.0173	0.295 ± 0.0017	0.5779
3.	12	0.413 ± 0.00017	0.4288	0.645 ± 0.0023	0.3690
4.	14	0.476 ± 0.00217	0.3765	0.757 ± 0.0012	0.1646
5.	16	0.533 ± 0.0013	0.2595	0.886 ± 0.0009	0.1080
6.	18	0.595 ± 0.0016	0.2744	0.963 ± 0.0010	0.1107
7.	20	0.664 ± 0.00317	0.2570	1.073 ± 0.0015	0.1465

 

Table 3: Linearity data for ARI

Sr No	Concentration (µg/ml)	At Wavelength 255 nm Absorbance (Mean±SD)	% RSD	At Wavelength 225 nm Absorbance (Mean±SD)	% RSD
1.	2	0.073 ± 0.0011	1.5007	0.110 ± 0.0008	0.7422
2.	4	0.172 ± 0.0009	0.5550	0.303 ± 0.0008	0.2694
3.	8	0.287 ± 0.0006	0.2387	0.604 ± 0.0006	0.1136
4.	12	0.392 ± 0.0017	0.4351	0.889 ± 0.0008	0.0918
5.	20	0.665 ± 0.0006	0.1227	1.637 ± 0.0008	0.0498
6.	30	0.996 ± 0.0007	0.0748	2.497 ± 0.0006	0.0275
7.	32	1.069 ± 0.0008	0.0763	2.843 ± 0.0010	0.0351

 

Table 4: Linearity data

Parameters	VOR at 255nm	VOR at 225nm	ARI at 255nm	ARI at 225nm
Linearity range (μg/ml)	2-20 µg/ml	2-20 µg/ml	2-32 µg/ml	2-32 µg/ml
Regression equation	y = 0.0321x + 0.0223	y = 0.0536x + 0.0055	y = 0.0326x + 0.0201	y = 0.0886x - 0.0983
Correlation coefficient (r2)	0.9992	0.9971	0.9988	0.9964
y-intercept	0.0223	0.0055	0.0201	0.0983
Standard deviation of slope	0.0321	0.0536	0.0326	0.0886x

 

Table 5: Recovery data for VOR (225nm)

% Level	Con of VOR  (μg/ml)	Amt.of std. VOR added	Total amt. VOR	Total amt. of VOR found Mean ± SD	% Recovery	% RSD
0 %	9	0	9	9 ± 0.001	99	0.048
80 %	9	7.2	16.2	16 ± 0.002	98.7	0.05
100 %	9	9	18	18.2 ± 0.003	100	0.08
120 %	9	10.8	19.8	19 ± 0.004	97	0.10

 

Table 6: Recovery data for ARI (255nm)

% Level	Con of ARI  (μg/ml)	Amt.of std. ARI added	Total amt. ARI	Total amt. of ARI found Mean ± SD	% Recovery	% RSD
0 %	6	0	6	6 ± 0.00031	100	0.039
80 %	6	4.8	10.8	10 ±0.0003	98	0.018
100%	6	6	12	12.2 ±0.002	97	0.12
120%	6	7.2	13.2	12.7 ±0.001	96.2	0.05

 

Accuracy:

Accuracy method was confirmed by recovery study of the synthetic mixture at three levels of standard additions (80%, 100% and 120%). Percentage recovery for VOR was in the range of 98.7 - 100.00%, while for ARI, it was found to be in range of 96.2–100.0%. The results are shown in Table 5 and 6. Recovery greater than 96% with low SD justifies the accuracy of the method.

 

Precision:

Repeatability (n = 3):

The repeatability was checked by scanning and measurement of the responses of solutions of VOR (2 - 20 μg/ml) and ARI (2 - 32 μg/ml) without changing the parameters of the proposed method. The procedure was repeated three times and % RSD was calculated. The data for repeatability for combined solution of VOR and ARI is presented in Table 7. % R.S.D was found to be 0.24 and 0.15 at 255nm and 225nm of VOR, where of 0.69 and 0.33 of ARI at 255nm and 225nm. % R.S.D was less than 2% complied with the standard limits.

 

Table 7: Repeatability data for VOR and ARI.

Drug	Con (μg/ml)	Abs (Mean ±SD)  at 255nm	% RSD	Abs (Mean ±SD)  at 225nm	% RSD
VOR	12	0.413 ± 0.001	0.1976	0.645 ± 0.0008	0.1265
ARI	8	0.286 ± 0.002	0.2854	0.606 ± 0.0008	0.1347

 

Intra Day and Inter Day Precision (n=3):

The data for intraday precision for VOR and ARI is shown in Table 8 and 9. The % R.S.D. for Intraday precision was found to be 0.08 - 0.1% for VOR and 0.2 - 0.6 % for ARI at 225 nm and 255nm respectively. The data for intraday precision for VOR and ARI is shown in Table10 and 11. The % R.S.D. for interday precision was found to be 0.06 – 0.38 % for VOR and 0.6 -0.7% for ARI.

 

 

 

 

 

Table 8: Intraday precision data for VOR

Condition	Con: of VOR (μg/ml)	Absorbance Mean ± SD	% RSD
At 225nm	6	0.705 ± 0.0017	0.2409
	12	1.064 ± 0.0008	0.0767
	18	1.633 ± 0.0021	0.1322

 

Table 9: Intraday precision data for ARI

Condition	Con: of ARI (μg/ml)	Absorbance Mean ± SD	% RSD
At 255nm	4	0.334 ± 0.0021	0.6467
	8	0.504 ± 0.0029	0.5841
	12	0.781 ± 0.0019	0.2174

 

Table 10: Interday precision data for VOR

Condition	Con: of VOR (μg/ml)	Absorbance Mean ± SD	% RSD
At 225nm	6	0.526 ± 0.0012	0.2369
	12	1.056 ±0.0004	0.0446
	18	1.626 ±0.0008	0.0502

 

Table 11: Interday precision data for ARI

Condition	Con: of ARI (μg/ml)	Absorbance Mean ± SD	% RSD
At 255nm	4	0.301 ± 0.0012	0.4134
	8	0.505 ±0.0021	0.4277
	12	0.778 ±0.0004	0.0605

 

LOD and LOQ:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations as per ICH guidelines,

 

LOD= 3.3 ×SD / slope

LOQ = 10 ×SD / slope

 

The LOD of Vortioxetine hydrobromide monohydrate was found to be 0.059 and of Aripiprazole 0.039 respectively. LOQ of Vortioxetine hydrobromide monohydrate was obtained 0.18 and for Aripiprazole LOQ was 0.12.

 

Robustness and Ruggedness:

The Robustness and Ruggedness data of Vortioxetine hydrobromide monohydrate (15 µg/ml) and Aripiprazole (10 µg/ml) was calculated with changes in the instrument, analyst, solvent and wavelength as shown in the Table 12 & 13.

 

Table12: Robustness data of VOR & ARI Robustness data of Vortioxetine hydrobromide monohydrate (15µg/ml) and Aripiprazole (10µg/ml) (n=3)

Sr No.	Factor	Level	VOR Abs ± SD	% RSD	ARI Abs ± SD	% RSD
1.	Change in instrument	UV- 2450	1.578 ±0.0014	0.0896	0.701 ± 0.0012	0.1777
		UV- 1800	1.574 ± 0.0012	0.0792	0.707 ± 0.0008	0.1154
2.	Change in analyst	Analyst-1	1.576 ± 0.0004	0.0299	0.706 ± 0.0020	0.2909
		Analyst-2	1.573 ± 0.0016	0.1036	0.711 ± 0.0012	0.1753

 

Table13: Ruggedness data of VOR & ARI Ruggedness data of Vortioxetine hydrobromide monohydrate (15µg/ml) and Aripiprazole (10µg/ml) (n=3)

Sr No.	Factor	Level	VOR Abs ± SD	% RSD	Level	ARI Abs ± SD	% RSD
1.	Change in wavelength	223 nm	1.715± 0.0004	0.0274	252 nm	0.787± 0.0016	0.2074
		227 nm	1.431± 0.0008	0.0570	256 nm	0.766± 0.0021	0.2820

 

Table 14: Specificity Data

Sr No:	VOR and ARI 3:2 ratio at 255nm (µg/ml)	Pure sample Abs	Sample spiked with excipients Abs	Difference
1.	3:2	0.133	0.140	4.97
2.	6:4	0.295	0.305	2.69
3.	9:6	0.461	0.450	2.38
4.	12:8	0.626	0.674	4.81
5.	15:10	0.802	0.757	5.7
6.	18:12	0.944	0.922	2.53
Sr No:	VOR and ARI 3:2 ratio at 225nm (µg/ml)	Pure sample Abs±SD	Sample spiked with excipients Abs ± SD	Difference
1.	3:2	0.306	0.314	2.614
2.	6:4	0.637	0.6553	2.664
3.	9:6	0.984	0.959	2.54
4.	12:8	1.325	1.404	5.869
5.	15:10	1.705	1.575	4.775
6.	18:12	2.014	1.936	4.067

 

Specificity:

Specificity is checked by observing the interpretation caused by excipients. Table 14 of Specificity data indicates that there is no interference of excipients.

 

Analysis of Synthetic Mixture:

Here, 9 μg/ml solution of VOR and 6 μg/ml solution of ARI synthetic mixture were prepared in triplicate manner and analyzed. The assay was carried out as per regression equation. The result of assay is shown in Table 15.

 

Table15: Assay of synthetic mixture

Sr. No:	Formulation (Synthetic mixture) (µg/ml)	% Assay ±SD	%RSD	Standard Limit
1.	9	100 ± 0.0021	0.1115	98-102%
2.	6	100± 0.0008	0.0885

 

CONCLUSION:

A new, simple, accurate, and precise UV spectroscopic method was developed for the simultaneous estimation of VOR and ARI in bulk drugs and in the presence of tablet excipients. The recovery studies suggested non-interference of formulation excipients in the estimation. Hence, the proposed method can be used for the quality control of the cited drugs and can be extended for routine analysis of the drugs in their pharmaceutical dosage forms.

 

 

ACKNOWLEDGEMENT:

The authors are thankful to Torrent Pharmaceutical, Dahej for VOR and ARI Akhil health care, Vadodara for providing gift sample. We would like to acknowledge Shree Dhanvantary Pharmacy College, Kim for providing seed grant to support this research work.

 

REFERENCES:

1.     Drug Profile of Vortioxetine Hydrobromide Monohydrate https://www.drugbank.ca/drugs/DB09068

2.     Drug profile of Aripiprazole https://www.drugbank.ca/drugs/ DB01238

3.     Luigi Ollvieri, Francesco Nappi and Gabriella Rapini Vortioxetine and Aripiprazole Combination in treatment Resistant Obsessive Compulsive Disorder J. of Clin. Psychopharmacology.2017; (37) (6): 1-2.

4.     About OCD https://www.medicalnewstoday.com/article/178508.

5.     Causes of OCD https://www.ocduk.org/ocd

6.     Causes of OCD https://www.ocduk.org/ocd

7.     About OCD https://www.webmd.com/mentalhealth/obssesive

8.     Luigi Ollvieri, Francesco Nappi and Gabriella Rapini Vortioxetine and Aripiprazole Combination in treatment Resistant Obsessive Compulsive Disorder J. of Clin. Psychopharmacology.2017; (37) (6): 1-2.

9.     Drug Profile of Vortioxetine Hydrobromide Monohydrate https://www.drugbank.ca/drugs/DB09068

10.   Drug profile of Aripiprazole https://www.drugbank.ca/drugs/ DB01238

11.   A.H. Beckett and J.B. Stenlake. Practical Pharmaceutical Chemistry; fourth edition part two; CBS Publishers and Distributers, New Delhi, 2007, pp286. Daharwal SJ, Saraf Swarnlata and Saraf S. Spectrophotometric method for simultaneous estimation of amoxicillin and tinidazole in tablet dosage form. Indian Journal of Pharmaceutical Education and Research. 2007; 41(1): 35-41.

 

 

 

 

 

 

Received on 06.03.2019       Accepted on 12.04.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(2): 63-68.