INTRODUCTION:

Olanzapine is an antipsychotic drug used in the treatment of patients with schizophrenia and bipolar disorder¹. Its IUPAC name was 2-methyl-4-(4-methylpiperazin-1-yl)-5H-thieno [3, 2-c] [1, 5] benzodiazepine was revealed in Figure 1. According to the literature review a few methods was reported for the quantification of Olanzapine which includes UV-Spectroscopic^2-6, HPLC^7-17, and LCMS methods^{18, 19}. This research was intended to establish a new and simple RP-HPLC validated method for quantification of Olanzapine as per ICH guidelines²⁰.

Fig 1: Olanzapine Structure

MATERIALS AND METHODOLOGY:

Chemicals:

Olanzapine API was attained from Kekule Pharma., Hyd, India. Triethylamine, Water and Acetonitrile (Merck Chemical Company, HPLC-Grade) were used as solvent system. Oliza® tablet contain Olanzapine 20mg is procured from Intas Pharmaceuticals, India.

Instruments:

Agilent LC-1220 HPLC, attached with VWD detector and EZ Chrome software. Inertsil ODS C18 (4.6mm × 250mm, 5µm) column was employed. Sample weighing, pH detection and sonication were performed by Contech electronic balance, Global pH Meter and Life care Sonicator.

Chromatographic conditions:

Stationary phase was Inertsil ODS C18(4.6mm × 250mm, 5µm) column with mobile phase contains Acetonitrile: Water: Triethylamine (60:40:0.1v/v/v) be selected and passed with 1.0mL/min flow rate at 270 nm was delivered. 20μl Inj. volume was used and run time observed 3mins and shows 2.143min retention time.

Mobile phase preparation:

HPLC grade 1000mL water was added with 0.1ml of 2% triethylamine and sonicated followed by vacuum filtration through 0.45µm filter. HPLC-Grade Acetonitrile were mixed with above solvent in a ratio 60:40 (v/v) and sonicated for 10minutes followed by vacuum filtration through 0.45µm filter which produce diluent.

Standard Stock Solution preparation:

100mg Olanzapine were moved to 100mL vol. flask and dissolved upto mark with diluents to attain concentration 1000μg/mL.

Preparation of Sample Solution:

Twenty tablets of Oliza^®(20mg Olanzapine) were mashed to fine powder and 100mg Olanzapine equivalent weight powder was kept in 100ml vol. flask and dissolved with diluents. Further sonicated 30 minutes followed by vacuum filtration through 0.45µm Whatmann filter paper. Diluents were added upto mark to achieve concentration 1000μg/mL. Then 10mL be transferred to 100mL volumetric flask and diluents were added upto mark to attain concentration of 100 µg/mL. Then 5mL was transferred to 10mL volumetric flask and diluents were added upto mark to attain concentration of 50µg/mL. 20µl from above solution was taken for injection into HPLC to obtain the chromatogram. From chromatogram, peak areas were measured and revealed in Fig 2 and 3.

Fig 2: Standard chromatogram-Olanzapine

Fig 3: Sample chromatogram-Olanzapine

The % Assay be calculated by comparing sample with standard chromatogram peak areas and the outcome be revealed into Table 1.

Table 1: Olanzapine Assay

Drug

Oliza®

Labeled Claim in (mg)

Amt. Found in (mg)

% Labeled Claim ±%RSD (n=3)

Olanzapine

19.99

99.98±0.2

RESULTS AND DISCUSSION:

Numerous mobile phase mixtures were approached for method optimisation. Suitable separations with excellent peak symmetry for Olanzapine were acquired at 270nm. Olanzapine was found to be 2.143min retention time with 10-125µg/ml linearity range having R²=0.999. Accuracy of proposed method was established to 99.47-100.01% with %RSD values <2%. Method precision, system precision and reproducibility %RSD value was 0.71%, 1.26% and 0.07% respectively. LOD and LOQ for Olanzapine be 0.35µg/ml and 1.05µg/ml correspondingly. Robustness studies %RSD values was < 2%.

System Suitability:

50µg/ml six replicates of Olanzapine were injected for system suitability check and outcomes were revealed on Table 2.

Table 2: Olanzapine System-suitability

Parameters (n=6)	Olanzapine
Retention-time in minutes	2.143
Theoretical-plates number	2652
Tailing/asymmetry-factor	1.02

Specificity:

Blank solutions were injected into HPLC and chromatogram was revealed in Fig 4 correspondingly.

Fig 4: Blank chromatogram

Linearity:

Volume of 0.1, 0.25, 0.5, 0.75, 1, 1.25mL were acquire from 1000µg/ml standard solution and moved into 10ml vol. flasks individually and diluents were added upto mark to obtain concentrations range from 10-125µg/ml. Injection of each 20µl above solution were injected to HPLC to obtain the chromatogram. From the chromatogram, retention time and peak area be recorded and a graph be plotted against concentrations is revealed in Fig 5 and Table 3.

Fig. 5: Olanzapine linearity range graph

Table 3: Olanzapine linearity

Conc. (µg/mL)	Peak-Areas
10	4098129
25	11976087
50	24838733
75	35714569
100	46685641
125	59154416

Accuracy:

It was find out by standard-addition method at a level of 50%, 100% and 150% and the outcomes were revealed on Table 4.

Table 4: Olanzapine accuracy

Level	Added Amount (µg/ml)	Found Amount (µg/ml)	Recovery %	Statistical Data
50%	25	24.8	99.20	Mean-99.77 S.D-0.58 %RSD-0.58
50%	25	24.94	99.76
50%	25	25.09	100.36
100%	50	49.84	99.68	Mean-99.47 S.D-0.21 %RSD=0.21
100%	50	49.73	99.46
100%	50	49.63	99.26
150%	75	75.07	100.09	Mean-100.01 S.D-0.11 %RSD-0.11
150%	75	74.92	99.89
150%	75	75.04	100.05

Precision:

Precision Method:

50µg/ml of Olanzapine homogenous sample were injected 6 times and %RSD meant for peak areas of 6 repeated injections were determined as reported on Table 5.

Table 5: Olanzapine precision method

Conc. in μg/mL	Assay (%)
50	99.02
50	100.65
50	99.06
50	100.2
50	99.05
50	100.44
Avg.	99.74
S D	0.71
% RSD	0.71

Precision System:

50µg/ml of Olanzapine was injected 6 times and %RSD meant for peak areas of 6 repeated injections were determined as reported on Table 6.

Table 6: Olanzapine precision system

Conc. in μg/mL	Peak Areas
50	24569874
50	24579783
50	25189675
50	24569767
50	24569781
50	25167821
Avg.	24774450
S D	313267.83
% RSD	1.26

Intermediate Precision (ruggedness):

50µg/ml of Olanzapine was injected six times in different laboratory through different analyst and days. %RSD meant for peak areas of 6 repeated injections were determined as mentioned on Table 7.

Table 7: Olanzapine precision intermediate

Lab-A (% Assay)-HPLC-A					Lab-B (% Assay)-HPLC-B
	A-Analyst		B-Analyst		A-Analyst		B-Analyst
Conc. in μg/ml	Day:1	Day:2	Day:1	Day:2	Day:1	Day:2	Day:1	Day:2
50	100.74	101.85	100.32	100.24	100.74	99.75	98.7	99.18
50	100.24	100.74	100.74	99.25	99.15	101.32	101.85	99.75
50	100.74	100.24	101.32	100.24	100.24	100.24	99.15	101.32
50	99.15	100.74	101.85	101.85	101.32	98.25	100.24	100.74
50	101.32	100.32	99.75	99.15	100.24	101.32	99.25	101.85
50	100.24	99.15	99.15	98.7	100.74	100.24	100.24	99.75
Avg.	100.41	100.51	100.52	99.91	100.41	100.19	99.91	100.43
S D	0.73	0.88	1.00	1.14	0.73	1.14	1.14	1.04
% R.S.D	0.73	0.87	0.99	1.14	0.73	1.14	1.14	1.03
Precision intermediate within laboratories variations (n-24)
Lab-A (% Assay)-HPLC-A				Lab-B (% Assay)-HPLC-B
Avg.	100.33			Avg.		100.23
S D	0.29			S D		0.24
% RSD	0.29			% RSD		0.24
Between the labs (n-48) Assay % (Reproducibility)
Avg.	100.28
S D	0.07
% R.S.D	0.07

Table 8: Olanzapine validation data

Parameters	Olanzapine
Linearity (µg/mL) with range	10-125
Slope (m)	47278
Intercept (y)	63531
Correlation Coefficient (r²)	0.999
LOD µg/mL	0.35
LOQ µg/mL	1.05
Precision Method (% R.S.D;n-6)	0.71
Precision System (% R.S.D; n-6)	1.26
Precision Intermediate (% R.S.D; n-24)	Lab-A	Lab-B
	0.29	0.24
Reproducibility (% R.S.D;n-48)	0.07
Accuracy in %	99.47-100.01
Robustness (% R.S.D;n-6)	Flow Rate less	Flow Rate more
	0.07	0.01
	Organic phase less	Organic phase more
	0.02	1.01

Table 9: Olanzapine Robustness by modify in flow rate and mobile phase

Factors	Avg. Peak Areas (n-3)	Std. Dev.	% RSD	Retention Time	Theoretical Plates
0.9ml/min Flow rate	25524021	16762.6	0.07	2.49	3142
Actual flow rate 1ml/min	24974798	533247	0.471	2.143	2652
1.1ml/min Flow rate	22304511	1839.43	0.01	1.973	2984
10% less organic (54:46)	24307996	4200.9	0.02	2.17	2158
Actual mobile phase (60:40)	24974798	533247	0.471	2.143	2652
10% more organic (66:34)	22777565	230269	1.01	2.273	2984

Limit of Quantification and Limit of Detection:

LOQ and LOD be estimated through formula LOD=3.3×SD/S and LOQ=10×SD/S correspondingly. Where, SD=Std. deviation of response i.e. Y-intercept and S=Slope of Calibration curve and revealed inside Table 8.

Olanzapine Robustness:

A deliberate change in mobile phase proportion and flow rate of ±10% were made to evaluate robustness of method and the outcomes are presented on Table 9.

CONCLUSION:

The quantification and the validation for Olanzapine in pharmaceutical formulation was performed through HPLC as stated by the ICH guidelines. 10-125µg/mL linearity having correlation coefficient value 0.999 linearity was achieved for Olanzapine. 99.47%-100.01% recovery of drug was achieved which was within acceptance criteria. Less than 2% RSD of precision were achieved which confirmed the method be precise, simple and accurate for the application of analytical research development of Olanzapine and its pharmaceutical formulations.

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Received on 07.04.2024 Revised on 11.12.2024

Accepted on 04.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5621-5625.

DOI: 10.52711/0974-360X.2025.00811

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