Stability Indicating Method Development & Validation for The Concurrent Analysis of Olanzapine and Samidorphan

Salla Pujitha^*, Maddela Dhanunjay, Bashamoni Anjaneyulu, Md Arif ,

Arva Mahesh, Pachimadla Mallikharjun

Surabhi Dayakar Rao college of Pharmacy, Rimmanaguda(V), Gajwel (M), Siddipet(D),Telangana(S), 502312.

*Correspondence: sallapujitha@gmail.com;

DOI: https://doi.org/10.71431/IJRPAS.2026.5609

INTRODUCTION

Pharmaceutical analysis plays a pivotal role in ensuring the safety, efficacy, and quality of drug products. Stability-indicating methods (SIMs) are considered essential because they not only quantify the active pharmaceutical ingredients (APIs) but also detect and separate degradation products formed under stress conditions. A stability-indicating method is defined as a validated analytical procedure that accurately and precisely measures the active ingredients without interference from degradation products, impurities, or excipients.[1-3] The main objective of a stability indicating method is to monitor results during stability studies in order to guarantee safety, efficacy and quality. It represents also a powerful tool when investigating out-of-trend (OOT) or out-of-specification (OOS) results in quality control processes. These methods ensure that the therapeutic agent maintains its intended pharmacological activity throughout its shelf life. In modern pharmaceutical practice, combination therapies are increasingly common and offer synergistic effects, improved patient compliance, and reduced pill burden. However, the simultaneous presence of multiple APIs in a single dosage form poses analytical challenges. The concurrent analysis of two drugs requires methods that can separate, identify, and quantify each component accurately, even in the presence of degradation products. [4-5] Olanzapine molecular formula is C17H20N4S (2-Methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b] [1,5] benzodiazepine) and used as Antipsychotic used in the treatment of Schizophrenia, Bipolar disorder, Major depressive disorder by acting on Dopamine and serotonin receptors.[6-9] Samidorphan molecular formula C21H26N2O4 (5-Chloro-3-[(1R)-1-[(2S)-2-(3,3-dimethyl-1-piperidinyl) propanoyl] pyrrolidin-3-yl]-1,2-benzisoxazole) and used as Antipsychotic (Second-generation) in the treatment of Schizophrenia, bipolar disorder, major depressive disorders by acting on Dopamine D2 and serotonin 5-HT2A receptors.[10-12] The combination of olanzapine and samidorphan have several analytical challenges like difference in chemical structure, potential degradation pathways, matrix interference and sensitivity requirements. These challenges necessitate the development of a robust, validated stability-indicating method that can separate and quantify both APIs and their degradation products.[13-15]

MATERIALS AND METHODS

Optimization of chromatographic conditions

Instrument used          : High performance liquid chromatography equipped with Auto Sampler and            PDA or         UV detector

Temperature                   :  Ambient

Column                           : Platisil C18 Column, (150×4.6mm, 3µm)

Buffer                              :  Potassium dihydrogen orthophosphate PH 3.5

Mobile phase                  : 30% KH2PO4 PH 3: 70% MEOH

Flow rate                         : 1.0ml /min

Wavelength                     :       225 nm

Injection volume         :           10 µl

Run time                     :           8 min.

Standard Solution Preparation of olanzapine& Samidorphan:

Accurately weighed and transferred 20mg of olanzapine and 10mg of samidorphan working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.6ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Preparation of buffer and mobile phase:

Preparation of KH2PO4 Buffer pH 3.5:

Prepared KH2PO4 Buffer solution, by adding 6.8Grams of Potassium dihydrogen orthophosphate in 1000ml water. Adjust this solution to pH 3.5 by using sodium hydroxide.

Preparation of mobile phase:

Mixed a mixture of Methanol 700ml (70%), 300 ml KH2PO4 (30%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration. Mobile phase is also used as Diluent.

Wave length selection:

UV spectrum of 10µg/ml Olanzapine and Samidorphan in diluents (mobile phase composition) was recorded by scanning in the range of 200nm to 400nm and the isobestic λmax of both the drugs obtained at 225 nm.

Validation Parameters

ASSAY:

Sample Solution Preparation:

Accurately weighed and transferred 20 mg of Olanzapine and 10 mg Samidorphan working standard into 20 ml clean dry volumetric flasks add Diluents and sonicate  make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.6ml of each of the above stock solutions into a 10ml volumetric flasks and dilute up to the mark with Diluents. (60ppm Olanzapine and 30ppm Samidorphan). Inject 20 µL of the standard, sample into the chromatographic system and measure the areas for the Olanzapine and Samidorphan peaks and calculate the % Assay by using the formulae.

LINEARITY:

Preparation of Level – I (20ppm of Olanzapine and 10 ppm Samidorphan):

0.2 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – II (40ppm of Olanzapine and 20 ppm Samidorphan

0.4 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – III (60ppm of Olanzapine and 30 ppm Samidorphan

0.6ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – IV (80ppm of Olanzapine and 40 ppm Samidorphan):

0.8ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – V (100ppm of Olanzapine and 50 ppm Samidorphan):

1ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Inject each level into the chromatographic system and measure the peak area.

Precision:

The standard solution was injected for six times and measured the area for all six injections in HPLC.

Intermediate precision/ruggedness:

The standard solution was injected for six times and measured the area for all six injections in HPLC.

Accuracy

Preparation 50%, 100%, 150% Sample solutions:

Accurately weigh and transfer 10 mg, 20mg, 30mg of Olanzapine and 5mg, 10mg,15mg of Samidorphan working standard respectively into a 20 ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

Further pipette 0.6ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents. Inject the solutions calculate the individual recovery and mean recovery values.

Limit of detection:

Preparation of 0.05µg/ml Olanzapine and 0.36µg/ml Samidorphan solution:

Accurately weighed and transferred 20 mg of Olanzapine working standard into 20 ml clean dry volumetric flasks add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.6ml of each of the above stock solutions into a 10ml volumetric flasks and dilute up to the mark with Diluents. Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent. Further pipette 0.85 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Accurately weighed and transferred 10 mg Samidorphan working standard into 20 ml clean dry volumetric flasks add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.6ml of each of the above stock solutions into a 10ml volumetric flasks and dilute up to the mark with Diluents.  Further pipette 0.12 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Limit of Quantification:

Preparation of 0.18µg/ml Olanzapine and 1.22µg/ml Samidorphan solution:

Accurately weigh and transfer 20 mg of Olanzapine working standard into 20 ml clean dry volumetric flasks add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.6ml of each of the above stock solutions into a 10ml volumetric flasks and dilute up to the mark with Diluents.  Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent. Further pipette 3 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Accurately weigh and transfer 10 mg Samidorphan working standard into 20 ml clean dry volumetric flasks add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.6ml of each of the above stock solutions into a 10ml volumetric flasks and dilute up to the mark with Diluents.  Further pipette 0.81 ml of the above stock solution into a 20ml volumetric flask and dilute up to the mark with Diluent.

Robustness:

Standard solution 60 µg/ml of Olanzapine and 30 µg/ml Samidorphan prepared and analyzed at  varied flow rate of 0.8 ml/min to 1.2 ml/min using varied mobile phase from 56% to 84% along with the actual mobile phase composition in the method.

Forced Degradation Studies

Acid degradation:

Pipette 3 ml of Olanzapine and Samidorphan of the stock solution into a 10ml volumetric flask added about 3 ml of 0.1N Hcl and sonicated for 10minutes and kept it in darkness for 24 hours then refluxed under heat at 60 degrees in a heating mantle for 1 hours. Neutralized the sample solution using 0.1N NaOH and diluted up to the mark with diluents. The Final Sample was filtered through 0.45-micron Injection filters and injected into HPLC system.

Base Degradation:

Pipette 3 ml of Olanzapine and Samidorphan of the above stock solution into a 10ml volumetric flask added about 3 ml of 0.1N NAOH and sonicated for 10minutes and kept it in darkness for 24 hours then refluxed under heat at 60 degrees in a heating mantle for 1 hours. Neutralized the sample solution using 0.1N HCL and diluted up to the mark with diluents. The Final Sample was filtered through 0.45-micron Injection filters and injected into HPLC system.

Thermal Degradation:

Pipette 3 ml of Olanzapine and Samidorphan of the above stock solution into a 10ml volumetric flask and kept in oven under heat at 105 degrees for 24 hours and diluted up to the mark with diluents. The Final Sample was filtered through 0.45-micron Injection filters and injected into HPLC system.

Peroxide Degradation: -

Pipette 3 ml of Olanzapine and Samidorphan of the above stock solution into a 10ml volumetric flask added about 3ml of 3% Hydrogen Peroxide (H₂O₂) and sonicated for 10 minutes and kept in darkness for 24 hours and refluxed under heat at 60 degrees in a heating mantle for 1 hours. The Final Sample was filtered through 0.45-micron Injection filters and injected into HPLC system.

Photo Degradation:

Pipette 3 ml of Olanzapine and Samidorphan of the above stock solution into a 10ml volumetric flask added about and kept in darkness for 24 hours. The Final Sample was filtered through 0.45-micron Injection filters and injected into HPLC system.

RESULT AND DISCUSSION

Assay:

Table.1: Results of Assay for Olanzapine and Samidorphan

Linearity:

Table.2: Results of linearity of different concentration of Olanzapine and Samidorphan

Figure.1: Calibration Graph of Olanzapine      Figure.2: Calibration Graph of Samidorphan

Table.3 Analytical performance parameters of Olanzapine and Samidorphan

Precision

Table.4: Results of Precision for Olanzapine and Samidorphan

Intermediate Precision (Ruggedness)

                    Table. 5: Results of Intermediate precision for Olanzapine and Samidorphan

Limit of Detection

Table.7: Results of LOD

Limit of Quantification

Table.8: Results of LOQ

Robustness

Table.9: Results for variation in flow for Olanzapine and Samidorphan

Table.10: Results for variation in mobile phase composition for Olanzapine and Samidorphan

Degradation Studies

Table.11: Results of Degradation Studies

CONCLUSION
The developed RP-HPLC method proved to be robust, reproducible, and suitable for routine quality control analysis of Olanzapine and Samidorphan in combined dosage forms. Robustness studies indicated that minor variations in chromatographic conditions did not significantly affect the results, confirming the reliability of the method. Forced degradation studies demonstrated that the method is stability-indicating, as it effectively separated degradation products under various stress conditions such as acidic, basic, oxidative, thermal, and photolytic environments. Therefore, the proposed method can be confidently applied for assay determination, stability studies, and pharmaceutical analysis of these drugs in bulk and formulation.

REFERENCES

1.      International Council for Harmonisation (ICH). ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology. Geneva: ICH; 2005.

2.      International Council for Harmonisation (ICH). ICH Q1A(R2): Stability Testing of New Drug Substances and Products. Geneva: ICH; 2003.

3.      United States Food and Drug Administration (FDA). Guidance for Industry: Analytical Procedures and Methods Validation. Rockville (MD): FDA; 2000.

4.      Swartz ME, Krull IS. Analytical Method Development and Validation. New York: Marcel Dekker; 2012.

5.      Blessy M, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs—A review. J Pharm Anal. 2014;4(3):159–65.

6.      PubChem. Olanzapine (CID: 4585): Chemical and physical properties. Bethesda (MD): National Center for Biotechnology Information; Available from: https://pubchem.ncbi.nlm.nih.gov

7.      ChemSpider. Olanzapine (ChemSpider ID: 10442212): Structure and physicochemical data. Cambridge (UK): Royal Society of Chemistry; Available from: http://www.chemspider.com

8.      Brunton LL, Hilal-Dandan R, Knollmann BC. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 14th ed. New York: McGraw-Hill Education; 2023.

9.      Katzung BG, Vanderah TW. Basic and Clinical Pharmacology. 16th ed. New York: McGraw-Hill Education; 2021.

10.  DrugBank. Samidorphan: Drug information, pharmacology and mechanism of action. Version 5.0. Available from: https://go.drugbank.com

11.  PubChem. Samidorphan (CID: 11949646): Chemical and physical properties. Bethesda (MD): National Center for Biotechnology Information; Available from: https://pubchem.ncbi.nlm.nih.gov

12.  ChemSpider. Samidorphan (ChemSpider ID: 64858193): Structure and physicochemical data. Cambridge (UK): Royal Society of Chemistry; Available from: http://www.chemspider.com

13.   Blessy M, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs—A review. J Pharm Anal. 2014;4(3):159–65.

14.  Bakshi M, Singh S. Development of validated stability-indicating assay methods—critical review. J Pharm Biomed Anal. 2002;28(6):1011–40.

15.  Gorog S. The importance and the challenges of impurity profiling in modern pharmaceutical analysis. TrAC Trends Anal Chem. 2006;25(8):755–63.