Central Composite Design Aided Method Development and Validation of a Reverse Phase HPLC Method for Escitalopram in Synthetic Mixture

 

Kajal Vable Deshmukh*, Priti Kale, Madhuri Deshmukh, Minal Ghante

Department of Pharmaceutical Quality Assurance, Smt. Kashibai Navale College of Pharmacy,

Sinhgad Institute of Pharmacy, Kondhwa, Savitribai Phule, Pune University.

 

ABSTRACT:

The goal of this study was to use high-performance liquid chromatography with an Analytical Quality by Design (AQbD). The experiment was performed on a Methanol: Water HPLC system equipped with a Raptor C-18 column. To achieve chromatographic separation, a mobile phase of Triethylamine buffer (0.05%; pH 3.0) and Ortho Phosphoric acid in an 80:20 v/v ratio was used with a flow rate of 0.7mL/min for 10 minutes at 235 nm. AQbD was used to optimize chromatographic conditions using a central composite design (CCD). A factorial design of 17 (three factors and three responses) was used with StatEase Inc.'s Design Expert 10.0.1 software. The HPLC method was validated using ICH, and it met all acceptance criteria. In the dilution range of 10to60µg/mL, the procedure was shown to be linear. HPLC method development and validation for tablet dosage form qualitative determination of antidepressants and antipsychotic drugs in a respective manner.          

 

 

 

INTRODUCTION:

Selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs) are first-line pharmacological treatments for people with major depressive disorder (MDD) and anxiety disorders. However, SSRIs and SNRIs are associated with a range of side effects, including loss of appetite, weight loss, drowsiness, dizziness, fatigue, headaches, increased suicidal thoughts, nausea/vomiting, sexual dysfunction, and increased risk of cardiovascular and cerebrovascular events.

 

While specific antidepressant side effects are diverse, they also occur generally with a high frequency, intensity, and burden. Previous studies suggest that frequency, intensity, and burden of side effects are impacted by antidepressant treatment type, anxiety symptoms, and the presence of anxious depression.^{1- 4}

 

 

Figure 1: Structure of Escitalopram (ESCI)⁵

 

MATERIALS AND HPLC METHOD DEVELOPMENT:^6,7

Selection of Analytical Technique

Chromatographic conditions:

 

 

Table 1: Chromatographic condition HPLC method development

 

Table 2: Selected Optimised Mobile Phase

Column used	Mobile phase, Flow Rateand Wavelength	Inj. Vol.	Observat ion	Conclus ion
Cosmosil C17 (250 ×4.6mm, 5.0μ)	Methanol+ 0.05% TEA (OPA pH3.0) Water (80+20% v/v) pH 6.6 at 235nm 0.7 ml/min	20μl	Sharp and well resolved Peaks were obtained	Hence selected

 

 

 

Figure 2: Chromatogram Trial 1 ESCI

 

 

Figure 3: Chomatogram Trial 2 ESCI

 

 

Figure 4: Chromatogram Trial 3 ESCI

 

 

 

Figure 5: Chromatogram Standard ESCI

 

 

VALIDATION PARAMETER^8-10:

Linearity:

Table 3: Regression equation data for ESCI

Conc µg/ml	Peak area (µV.sec)		Average peak area (µV.sec)	SD	% RSD
10	2079.947	2058.125	2069.036	15.43	0.75
20	4259.418	4238.584	4249.001	14.73	0.35
30	6138.83	6172.834	6155.832	24.04	0.39
40	8322.29	8344.15	8333.22	15.46	0.19
60	12618.55	12442.61	12530.58	124.41	0.99
Equation		y = 208.58 x + 7.1372
R2		= 0.999

 

Accuracy:

Table 4: Result of Recovery data for ESCI

Level (%)	Amt. taken	Amt. Added	Amt.found (Mean±SD)	Amt. recovered (Mean±SD)	%Recovery (Mean±SD)
80%	20	16	36.42±0.10	16.42±0.10	102.64±0.62
100%	20	20	40.41±0.37	20.58±0.37	102.04±1.85
120%	20	24	44.59±0.15	24.59±0.15	101.58±0.62

 

Repeatability:

Table 5: Repeatability studies on ESCI

 

Precision:

Table 6: Intraday and Interday Precision ESCI

Conc. (µg/ml)	Intraday Precision		Interday Precision
	Area (mean±SD)	%RSD	Area (mean±SD)	%RSD
20	844.69±5.95	0.70	835.69±6.77	0.81
30	1084.±5.58	0.51	1083±4.0	0.37
40	1373±8.78	0.64	1379.69±0.7	0.06

 

Robustness:

Table 7: Result of Robustness Study of ESCI

Parameters	Conc. (µg/ ml)	Area (mean±SD)	%RSD
MP composition(89ml+11ml) Methanol + 0.05%(TEA)water with OPA	40	1238.5±19.91	1.61
MP composition(91ml+09ml) Methanol + 0.1% (OPA)water withTEA	40	1422.99±2.03	0.14
Wavelength change 234 nm	40	1342.7±2.78	0.21
Wavelength Change 236 nm	40	1411.41±1.63	0.12
Flow rate change (0.6 ml)	40	1395.76±13.51	0.97
Flow rate change (0.8 ml)	40	1340.25±5.20	0.39

 

LOD:                    

LOD = 3.3 (SD)/S

= 3.3 X 3.49 /27.67

= 0.416

 

LOQ:    

LOQ  = 10 (SD)/ S

= 10 X 3.49 / 27.67

= 1.261

 

Analysis of tablet Formulation:

Weigh 20 Escitalopram tablets and calculated the average weigh 4.076mg accurately weigh and transfer the sample equivalent to 50.95mg Escitalopram into 10 ml volumetric flask. Add about 10ml of diluent and sonicate to dissolve it completely and make volume up to the mark with diluents. Mix well and filter through 0.45 µm filter. Further pipette 0.2ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluents. (20µg/ml).   the amounts of Escitalopram per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for % Lable claim for %RSD Calculated.

 

Table 8: Tablet for Marketed Formulation ESCI

Sr. no	Amount (mg)	Area(I)	Amount found in mg	% Label claim
1	20	818.6	19.81	99.05
2	20	824.39	21.02	100.10
Mean	821.50 4.09 0.50		19.91	99.58
SD			0.15	0.27
%RSD			0.75	0.27

Tablet Assay for % Label Claim:

Table 9: Tablet for Assay % label claim ESCI

Sample	Label claimed	%Label claimed. ± SD	%RSD
Escitalopram	Cilentra 10 mg	99.58±0.27	0.27

 

METHOD OPTIMIZATION BY CCD:

Table 10: Independent variables recommended by the DoE and the associates responses.

Std	Run	Factor 1: Flow Rate (mL/min)	Factor 2: Concentration (%)	Factor 3: Detection (nm)	Response 1 (R1) mL/min	Response 2 (R2) %	Response 3 (R3) nm
13	1	1.0	80	226.591	3.5	88.2	245
3	2	0.5	85	230	2.8	84.3	240
11	3	1.5	85	230	4.0	90.1	247
15	4	1.0	80	233	3.7	86.5	243
12	5	0.5	75	225	2.5	80.2	238
4	6	1.5	75	225	3.9	85.4	244
10	7	0.5	75	235	2.6	81.3	239
1	8	1.5	75	235	4.1	86.7	248
7	9	0.5	85	235	3.0	85.6	241
14	10	1.5	85	235	4.2	91.5	250
5	11	1.0	90	230	3.8	89.1	246
8	12	1.0	70	230	2.9	82.0	239
9	13	1.0	80	230	3.6	87.3	244
6	14	1.0	80	230	3.6	87.5	244
2	15	1.5	85	230	4.0	90.2	247
17	16	0.7	80	235	3.2	85.0	247
16	17	1.3	80	235	3.9	89.0	241

 

The quadratic equation and ANOVA analysis revealed the independent factors

 

The table includes: Factors (Independent Variables): A: Flow Rate (mL/min) – How fast the mobile phase flows, B: Concentration (%) – Likely the mobile phase or solvent concentration, C: Detection Wavelength (nm)  UV detection wavelength. Responses (Dependent Variables):R1: Retention Time (mL/min) – Time the drug takes to elute, R2: % Assay Indicates the accuracy of the method (% label claim), R3: Wavelength (nm) Peak wavelength detected.

 

 

Figure 6: 3D Surface response graphic illustrating how independent factors have an impact on the flow rate

 

Figure 7: 3D Surface response graphic illustrating how independent factors have an impact on the Concentration

 

Figure 8: 3D Surface response graphic illustrating how independent factors have an impact on the Detection Wavelength

 

Purpose of CCD in This Case: CCD is used here to study the effect of flow rate, concentration, and detection wavelength on method performance. Identify the optimal combination of factors to ensure: Accurate retention time, High assay percentage (close to 100%), Consistent wavelength detection. CCD method optimization helps refine the analytical method for Escitalopram by providing a systematic way to understand and adjust the parameters for best performance with minimal experimental runs. This leads to a robust and validated method.

 

CONCLUSION:

Attempts were made to develop a reverse-phase HPLC method for the estimation of Escitopram from tablets. For the RP-HPLC method, Younglin (S.K) Gradient System UV Detector and C18 column with 235nm x4.6 mm i.d and 5μm particle size, methanol: 0.05% TEA with OPA (80:20 %v/v) pH 3.0 was used as the mobile phase for the method. The detection wavelength was 235 nm, and the flow rate was 0.7ml/min. In the developed method, the retention time of Escitopram was found to be 5.5min. The developed method was validated according to the ICH guidelines. The linearity concentration 10to60µg/ml and %RSD found to be less than 2%, Accuracy level used 80%, 100% and 120%, precision concentration used to 20, 30 and 40µg/ml and %RSD found to be less than 2%, range 30µg/ml and %amount found to be 98.97%, and robustness concentration 40µg/ml was within the limits as specified by the ICH guidelines. Hence, the method was found to be simple, accurate, precise, economic, and reproducible.

 

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Received on 11.02.2026      Revised on 16.03.2026 Accepted on 06.04.2026      Published on 16.04.2026 Available online from April 18, 2026 Asian Journal of Pharmaceutical Analysis. 2026; 16(2):114-118. DOI: 10.52711/2231-5675.2026.00017 ©Asian Pharma Press All Right Reserved
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