Eco-friendly based stability-indicating RP-HPLC technique for the determination of escitalopram and etizolam by employing QbD approach

Figures & data

Figure 1. Structure of ESC and ETZ.

Figure 2. Standard ESC and ETZ chromatograms.

Table 1. CCD experimental design and measured responses.

Runs	Factors			Responses
	A: Ethanol (%, v/v)	B: Flow rate (mL/min)	C:pH	Capacity factor (k)	Retention time (Rt2)	Resolution (RS12)
1	68.40	1.00	3.50	0.65	4.30	4.60
2	55	0.80	3.00	1.21	7.43	11.80
3	60	1.00	2.65	1.01	8.34	9.30
4	60	1.00	3.50	1.01	6.50	10.56
5	65	0.80	3.00	0.72	4.60	6.80
6	60	0.66	3.50	1.02	4.70	11.80
7	55	1.20	3.00	1.10	8.50	12.20
8	65	0.80	4.00	0.91	4.80	6.80
9	60	1.00	3.50	1.01	6.58	10.82
10	65	1.20	4.00	0.90	4.50	7.00
11	60	1.33	3.50	0.70	4.30	8.80
12	55	1.20	4.00	1.23	9.70	12.60
13	60	1.00	3.50	1.01	6.80	10.72
14	60	1.00	4.34	1.54	8.90	13.40
15	60	1.00	3.50	1.01	6.58	10.24
16	60	1.00	3.50	1.09	6.80	11.76
17	60	1.00	3.50	0.98	6.30	10.84
18	65	1.20	3.00	0.89	4.40	8.60
19	51.59	1.00	3.50	1.69	11.00	12.60
20	55	0.80	4.00	1.85	8.67	15.00

Table 2. ANOVA and Regression summary of models.

Response	Model	Y (Equation model)	R^2 (Adjusted)	P-value	% CV	Precision (Adequate)
Capacity factor (k)	Quadratic	+1.02-0.27* A-0.081* B+0.14* C+0.11* AB-0.071* AC-0.086* BC+0.052* A^2-0.057* B^2+0.090* C^2	0.9779	<0.0001	5.87	27.11
Retention time(Rt2)	Quadratic	+6.61-2.00* A+0.067* B+0.26* C-0.33* AB-0.28* AC-0.015* BC+0.31* A^2-0.82* B^2+0.64* C^2	0.9862	<0.0001	4.86	29.25
Resolution (RS12)	Quadratic	+10.81-2.64* A-0.38* B+0.64* C+0.50* AB – 0.64* AC-0.56* BC-0.76* A^2-0.17* B^2+0.20* C^2	0.9385	<0.0001	8.58	17.90

Figure 3. (a) Drug effects capacity factors – ESC, ETZ – shown in a perturbation graph. (b) Diagram displaying the Retention time of drugs – ESC, ETZ. (c) Perturbation graph showing the Retention of drugs – ESC, ETZ.

Figure 4. Graphical representation of the overall desirability function.

Table 3. Compared predicted and experimental values.

Optimum conditions	A: Ethanol (%, v/v)	B: Flow rate (mL)	C:pH	Capacity factor (k)	Retention time(Rt2)	Resolution (RS12)
1	59	0.8	3.5
		Predicted		1.09	6.56	9.45
		Experimental		1.09	6.41	9.12
		Predicted Error %			1.82	2.88

Figure 5. Sample chromatogram of ESC and ETZ.

Table 4. Validation of analytical parameters using HPLC.

Parameters	ESC	ETZ
Linearity range (µg/mL)	5–30	2–12
Correlation coefficient	0.9975	0.9987
Slope	3456.8	1564.8
Intercept	567.7	678.3
Limit of Detection (µg/mL)	0.176	0.326
Limit of Quantification (µg/mL)	0.456	0.987
System precision (% RSD)	1.342	1.256
Assay of marketed formulation (Etizola plus)
Label claim	10mg	0.5mg
Amount found	9.88mg	0.49mg
% of Assay	99.55	99.94
% Recovery (w/w)	98.93	99.21

Figure 6. (a) Blank. (b) Acid Hydrolysis. (c) Alkali Hydrolysis. (d) Oxidation. (e) Photo Degradation. (f) Dry Heat. (a-f) Typical Chromatogram of ESC and ETZ (10 mg/mL) with Stress test.

Table 5. Forced degradation studies of ESC and ETZ.

Description	ESC				ETZ
	% Recovery	% Total degradation	Mass ratio	Rt	% Recovery	% Total degradation	Mass ratio	Rt
STD	99.95	0.05	99.95	3.5	99.23	0.77	99.23	6.5
Acid (0.1 M HCl) – 24 Hrs	44.52	55.48	100	3.5	98.86	1.14	99.86	6.3
Alkali (0.1M NaOH) – 24 Hrs	97.02	2.01	99.03	3.3	29.28	70.01	99.29	6.2
Peroxide (3% H2O2) – 24 Hrs	98.69	1.31	98.69	3.5	98.70	1.30	98.70	6.2
Photo degradation – UV light	28.43	71.02	99.45	3.6	98.63	1.37	98.63	6.4
Thermal degradation	98.29	1.71	98.29	3.5	98.39	1.61	98.39	6.5

Figure 7. (a) NEMI, (b) GAPI, (c) AMGS, and (d) AGREE with metric Results of the suggested green assessment method.