Development And Validation Of An RP-UPLC Method For Rapid And Simultaneous Analysis Of Samidorphan And Olanzapine With An Integrated Stability Study

The word psychosis was introduced to the psychiatric literature in 1841 by Karl Friedrich Canstatt in his work Handbuch der Medizinischen Klinik. He used it as a shorthand for 'psychic neurosis'. At that time neurosis meant any disease of the nervous system, and Canstatt was thus referring to what was considered a psychological manifestation of brain disease. Ernst von Feuchtersleben is also widely credited as introducing the term in 1845 as an alternative to insanity and mania.

Psychiatric disorders can manifest in a wide range of forms including:

• Mood disorders (e.g. depression, bipolar disorder)
• Anxiety disorders (e.g. generalized anxiety disorder, panic disorder)
• Psychotic disorders (e.g. schizophrenia)
• Personality disorders (e.g. borderline personality disorder)

 Other examples include: obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), attention-deficit/hyperactivity disorder (ADHD), eating disorders. These conditions often result from complex interaction between genetic, biological, environmental, and psychological factors. Diagnosis and treatment typically involve mental  health professionals, such as psychiatrists or psychologists, who use criteria outlined in standardized diagnostic manuals (e.g.,DSM-5 or ICD-10). It’s important to approach mental health with empathy and understanding, as psychiatric disorders are medical conditions that can be effectively managed and treated with appropriate interventions, including psychotherapy, medication, or a combination of both. Seeking professional help is essential for accurate diagnosis and the development of an effective treatment plan tailored to an individual’s specific needs. The combination of samidorphan and olanzapine represents an innovative approach to antipsychotic treatment, aiming to enhance therapeutic outcomes and reduce adverse effects. This study's findings will contribute to the development of optimized treatment protocols and provide a robust analytical framework for future pharmacokinetic and pharmacodynamic studies. By leveraging the advanced capabilities of UPLC, this research will offer high-precision data that can drive clinical decision-making and regulatory approval processes.The integration of samidorphan with olanzapine holds significant promise for addressing the metabolic side effects of antipsychotic therapy, thereby improving patient adherence and overall treatment success. The application of UPLC in this study underscores the importance of advanced analytical methods in pharmacological research, enabling precise and reliable quantification of drug concentrations and interactions. This research aims to pave the way for more effective and safer antipsychotic treatments, ultimately enhancing the quality of life for patients with schizophrenia and bipolar disorder.

MATERIALS AND METHODS

Materials:     

• Samidorphan and Olanzapine pure drugs (API), Combination Samidorphan and Olanzapine tablets (LYBALVI), Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.
• Instruments:
• Electronics Balance-Denver
• pH meter -BVK enterprises, India
• Ultrasonicator-BVK enterprises
• UPLC instrument used was of WATERS Acquity UPLC SYSTEM with Auto Injector and Acquity TUV detector. Software used is Empower 2.
• UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance of Samidorphan and Olanzapine solutions.

Methods:

Diluent:

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50.

Preparation of buffer:

0.1% OPA Buffer:

1ml of Conc Ortho Phosphoric acid was diluted to 1000ml with water.

Buffer:

0.1% OPA Buffer

Accurately take 1.0 ml of OPA in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then added 1ml of Triethylamine then PH adjusted to 4.8 with dil. Triethylamine.

API Preparation

Preparation of Standard stock solutions: Accurately weighed 10 mg of Samidorphan, 10 mg of Olanzapine and transferred to 50 ml volumetric flask separately. 3/4 th of diluents was added to both flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (200µg/ml of Samidorphan and 200µg/ml of Olanzapine)

Preparation of Standard working solutions (100% solution): 1ml from stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (20µg/ml Samidorphan and 20µg/ml of Olanzapine)

Formulation Preparation

Preparation of Sample stock solutions:

10 tablets were weighed and equivalent to 1 tablet is weighed and transferred to 100 ml volumetric flask, to this 50 ml of acetonitrile was added and sonicated. Volume was made upto 100ml with diluents and filtered through 0.45 µm or finer porosity membrane filter (100µg/ml of Samidorphan and 100µg/ml of Olanzapine)

Preparation of Sample working solutions (100% solution):

2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (20µg/ml of Samidorphan and 20µg/ml of Olanzapine)

Validation:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Samidorphan (20ppm) and Olanzapine (20ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The % RSD for the area of six standard injections results should not be more than 2%.

Specificity:

Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Precision:

Preparation of Sample stock solutions:

10 tablets were weighed and equivalent to 1 tablet is weighed and transferred to 100 ml volumetric flask, to this 50 ml of acetonitrile was added and sonicated. Volume was made upto 100ml with diluents and filtered through 0.45 µm or finer porosity membrane filter (100µg/ml of Samidorphan and 100µg/ml of Olanzapine)

Preparation of Sample working solutions (100% solution):

2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (20µg/ml of Samidorphan and 20µg/ml of Olanzapine)

The Repetability was determined by preparing formulation solutions of Samidorphan (20ppm) and Olanzapine (20ppm) and the solutions were injected six times and the % RSD for the area of six standard injections results should not be more than 2%.

Linearity:

Preparation of Standard stock solutions: Accurately weighed 10 mg of Samidorphan, 10 mg of Olanzapine and transferred to 50 ml volumetric flask separately. 3/4 th of diluents was added to both flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (200µg/ml of Samidorphan and 200µg/ml of Olanzapine)

25% Standard solution:

0.25ml from standard stock solution was pipetted out and made up to 10ml. (5µg/ml of Samidorphan and 5µg/ml of Olanzapine)

50% Standard solution:

0.5ml from standard stock solutions was pipetted out and made up to 10ml. (10µg/ml of Samidorphan and 10µg/ml of Olanzapine)

75% Standard solution:

0.75ml from standard stock solutions was pipetted out and made up to 10ml. (15µg/ml of Samidorphan and 15µg/ml of Olanzapine)

100% Standard solution:

1.0ml from standard stock solutions was pipetted out and made up to 10ml. (20µg/ml of Samidorphan and 20µg/ml of Olanzapine)

125% Standard solution:

1.25ml from standard stock solutions was pipetted out and made up to 10ml. (25µg/ml of Samidorphan and 25µg/ml of Olanzapine)

150% Standard solution:

1.5ml from standard stock solutions was pipetted out and made up to 10ml (30µg/ml of Samidorphan and 30µg/ml of Olanzapine)

Accuracy:

Preparation of Sample stock solutions: 10 tablets were weighed and equivalent to 1 tablet is weighed and transferred to 100 ml volumetric flask, to this 50 ml of acetonitrile was added and sonicated. Volume was made upto 100ml with diluents and filtered through 0.45 µm or finer porosity membrane filter (100µg/ml of Samidorphan and 100µg/ml of Olanzapine)

Preparation of Standard working solutions (100% solution):

1ml from stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (20µg/ml Samidorphan and 20µg/ml of Olanzapine)

Preparation of 50% Spiked Solution:

1.25ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 100% Spiked Solution:

2.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 150% Spiked Solution:

3.75ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102

Robustness:

Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there was no recognized change in the result and are within range as per ICH Guide lines. Robustness conditions like Flow minus (0.1ml/min), Flow plus (0.3ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

LOD sample Preparation:

0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.3ml each of Samidorphan, Olanzapine, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents

LOQ sample Preparation:

0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.9ml each of Samidorphan, Olanzapine, and solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent.

Degradation studies:

Oxidation:

To 1 ml of stock solution of Olanzapine and Samidorphan, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For UPLC study, the resultant solution was diluted to obtain 20µg/ml&20µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies:

To 1 ml of stock s solution Olanzapine and Samidorphan, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 20µg/ml&20µg/ml solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali Degradation Studies:

To 1 ml of stock solution Olanzapine and Samidorphan, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 20µg/ml&20µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry Heat Degradation Studies:

The standard drug solution was placed in oven at 105°C for 1 h to study dry heat degradation. For UPLC study, the resultant solution was diluted to 20µg/ml&20µg/ml solution and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability studies:

The photochemical stability of the drug was also studied by exposing the 200µg/ml&200µg/ml solution to UV Light by keeping the beaker in UV Chamber for 1days or 200-Watt hours/m2 in photo stability chamber. For UPLC study, the resultant solution was diluted to obtain 20µg/ml&20µg/ml solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 1hrs at a temperature of 60ºC. For UPLC study, the resultant solution was diluted to 20µg/ml&20µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

RESULT AND DISCUSSION:

System suitability: All the system suitability parameters were within the range and satisfactory as per ICH guidelines

Specificity:

       
           
       
    Figure No. 1 (a) Chromatogram of blank.

       
           
       
    Figure No. 2 (b)Chromatogram of placebo

       
           
       
    Figure No. 3Chromatogram of Standard

Retention times of Samidorphan and Olanzapine were 0.669 min and 0.879 min respectively. We did not found and interfering peaks in blank and placebo at retention times of these drugs in this method. So, this method was said to be specific.

Linearity:  

Six linear concentrations of Samidorphan (5-30µg/ml) and Olanzapine (5-30µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Samidorphan was y = 19497x + 10313 and of Olanzapine was y = 23803x + 7958.2 Correlation coefficient obtained was 0.999 for the two drugs.

Precision:

Repeatability:              

Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 100.41% and 100.26% for Samidorphan and Olanzapine respectively.

Table 8 Robustness data for Samidorphan and Olanzapine.

Robustness conditions like Flow minus (0.1ml/min), Flow plus (0.4ml/min), mobile phase minus (50B:50A), mobile phase plus (60B:40A), temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Assay:

Astra Zeneca pharmaceuticals (LYBALVI), bearing the label claim Samidorphan 10mg, Olanzapine 10mg. Assay was performed with the above formulation. Average % Assay for Samidorphan and Olanzapine obtained was 99.83% and 99.95% respectively

Assay was calculated by:-

Fig No.6 (b) Chromatogram of working sample solution

Degradation data

Acid degradation chromatogram

       
           
       
  Fig.7 acid

Base degradation chromatogram

       
           
       
    Fig.8 base

Peroxide degradation chromatogram

       
           
       
    Fig.9  peroxide

Thermal degradation chromatogram

       
           
       
    Fig.10  Thermal

Uv degradation chromatogram

       
           
       
    Fig.11. UV

Water degradation chromatogram

       
           
       
    Fig.12.water

CONCLUSION: