The German psychiatrist and neuropathologist Dr. Alois Alzheimer is credited with describing for the first time a dementing condition which later became known as AD. In his landmark 1906 conference lecture and a subsequent 1907 article, Alzheimer described the case of Auguste D, a 51-year-old woman with a ‘peculiar disease of the cerebral cortex,’ who had presented with progressive memory and language impairment, disorientation, behavioral symptoms (hallucinations, delusions, paranoia), and psychosocial impairment.13 Remarkably, many of the clinical observations and pathological findings that Alzheimer described more than a century ago continue to remain central to our understanding of AD today.There is no cure for AD, and drug therapy for the disease is still in its infancy. Approved medications for the treatment of probable AD help control the symptoms of AD but do not slow down the progression or reverse the course of the disease itself. At present, the mainstay of AD therapy are drugs that target neurotransmitter systems in the brain. AD primarily damages glutamate and acetylcholine-producing neurons and their associated synapses, and this damage correlates well with early cognitive symptoms of AD. Acetylcholinesterase inhibitors help improve memory function and attention in AD patients by interfering with the breakdown of acetylcholine, thereby increasing the levels of the neurotransmitter at the synapse. There are currently three FDA-approved cholinesterase inhibitors: rivastigmine and galantamine (for mild to moderate AD), and donepezil (for all stages of AD). Memantine is another FDA-approved medication for use in moderate to severe AD but belongs to a different class of drugs known as NMDA (glutamate) receptor antagonists. Both classes of medications are generally well-tolerated, with gastrointestinal upset, dizziness, and headache being the most common adverse effects observed. UPLC’s high sensitivity is beneficial for detecting trace levels of donepezil and memantine in biological samples, providing valuable insights into drug metabolism and distribution. It allows for the simultaneous detection of multiple compounds. This is particularly relevant when studying drug combinations, as it enables researchers to monitor the presence and concentrations of both donepezil and memantine is a single analysis. This is important in clinical research where timely results are essentials for treatment adjustments and decision-making. While the initial investment in UPLC technology may be higher, the operational costs can be lower due to reduced solvent consumption and shorter analysis times. UPLC systems typically require smaller sample volumes, which can be crucial when working with limited or precious samples. UPLC systems often exhibits higher peak capacity, allowing for the analysis of more compounds in a single run. Hence the current work serves to be first to elucidate the systematic implementation and to the development of simple, accurate and robust validated RP-UPLC method.

MATERIALS AND METHODS

Memantine and Donepezil pure drugs (API), Combination Donepezil and Memantine Capsules (Namzaric), Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dihydrogen 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 UPLC SYSTEM with Auto Injector and Acquity TUV detector. Software used is Empower 2, UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Memantine and Donepezil solutions.

Methods:

Diluent:

Based up on the solubility of the drugs, diluent was selected methanol and Water taken in the ratio of 60:40.

Preparation of buffer:

0.01N Kh2Po4 Buffer: Accurately weighed 1.38gm of Potassium phosphate 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 PH adjusted to 4.8 with dil. OPA.

Buffer: (0.1% Ortho phosphoric acid)

1ML of Ortho phosphoric acid solution in a 1000ml of volumetric flask add about 100ml of milli-Q water and final volume make up to 1000 ml with milli-Q water.

Preparation of Standard stock solutions:

Accurately weighed 14 mg of Memantine, 10mg of Donepezil and transferred to 50ml volumetric flask and 3/4 th of diluents was added to these flasks and sonicated for 10 minutes. Flask was made up with diluents and labeled as Standard stock solution. (280µg/ml of Memantine and 200µg/ml of Donepezil)

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. (28µg/ml Memantine of and 20µg/ml of Donepezil)

Preparation of Sample stock solutions:

10 Capsules were weighed and the average weight of each Capsule was calculated, then the weight equivalent to one Capsule(14mg/10mg) was transferred into a 100 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (140µg/ml of Memantine and 100µg/ml of Donepezil)

Preparation of Sample working solutions (100% solution):

2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (28µg/ml of Memantine and 20µg/ml of Donepezil)

Validation:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Memantine (28ppm) and Donepezil 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 Capsules were weighed and the average weight of each Capsule was calculated, then the weight equivalent to one Capsule(14mg/10mg) was transferred into a 100 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (140µg/ml of Memantine and 100µg/ml of Donepezil).

Preparation of Sample working solutions (100% solution):

2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (28µg/ml of Memantine and 20µg/ml of Donepezil).

The Precision were determined by preparing Sample solutions of Memantine (28ppm) and Donepezil 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 14 mg of Memantine, 10mg of Donepezil and transferred to 50ml volumetric flask and 3/4 th of diluents was added to these flasks and sonicated for 10 minutes. Flask was made up with diluents and labeled as Standard stock solution. (280µg/ml of Memantine and 200µg/ml of Donepezil)
• After the preparation of standard stock solution using stock solution preparation of 25% to 150% (0.25ml , 0.5ml,0.75ml,1ml,1.25ml,1.5ml.) standard solution was prepared.

Accuracy:

Preparation of Sample stock solutions:

10 Capsules were weighed and the average weight of each Capsule was calculated, then the weight equivalent to one Capsule(14mg/10mg) was transferred into a 100 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (140µg/ml of Memantine and 100µg/ml of Donepezil).

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. (28µg/ml Memantine of and 20µg/ml of Donepezil)

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. (14µg/ml Memantine of and 10µg/ml of Donepezil)

Preparation of 50% Spiked Solution:

 0.5ml 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 100% Spiked Solution:

1.0ml 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: 1.5ml 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.

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 (0.3ml/min), Flow plus (0.5ml/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 effected 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 Memantine, Donepezil 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 Memantine, Donepezil 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 Memantine and Donepezil, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For HPLC study, the resultant solution was diluted to obtain 28µg/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 Memantine and Donepezil, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600 C. The resultant solution was diluted to obtain 28µg/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 Memantine and Donepezil, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 28µg/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 HPLC study, the resultant solution was diluted to 28µg/ml & 20µg/ml solution and 10µl 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 280µg/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 HPLC study, the resultant solution was diluted to obtain 28µg/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 60º. For HPLC study, the resultant solution was diluted to 28µg/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 are within range and satisfactory as per ICH guidelines.

Table: 1 System suitability studies of Donepezil and Memantine method

Specificity-

       
           
       
 Fig : 1 Chromatogram of blank

       
           
       
    Fig:2 Typical chromatogram of Donepezil and Memantine.

Linearity:

6 Linear concentrations of Donepezil (5ppm-30ppm) and Memantine (7ppm to 42 ppm) are prepared and injected. Regression equation of the the Donepezil and Memantine are found to be, y = 8018.2x + 4057.2 and y = 8913.2x + 10674. and the regression co-efficient was 0.999.

Table: 2 Calibration data of Donepezil and Memantine method.

       
           
       
 Fig: 3 Calibration curve of Donepezil

       
           
       
    Fig: 4 Calibration curve of Memantine

Precision:

Intraday precision (Repeatability): Intraday Precision was performed and % RSD for Donepezil and Memantine were found to be 1.0% and 0.4% respectively.

Table: 3 Repeatability results for Donepezil and Memantine.

Intraday precision (Method Precision):Inter day precision was performed with 10 hrs time lag and the %RSD Obtained for Donepezil and Memantine were 0.4% and 0.5%

Table 4 Method precision results for Donepezil and Memantine.

Inter-day precision (Intermediate Precision):

Inter day precision was performed with 10 hrs time lag and the %RSD Obtained for Donepezil and Memantine were 0.6% and 0.4%.

Table 5 Intermediate precision results for Donepezil and Memantine.

Accuracy:

Three concentrations 50%, 100%, 150%, were injected in a triplicate manner and amount Recovered and % Recovery were displayed in Table 6.5 & 6.6.

Table: 6 Accuracy results of Memantine

Table: 7 Accuracy results of Donepezil

LOD:

LOD for Donepezil was found to be 0.09 and Memantine was 0.13 respectively.

2LOQ:

LOQ for Donepezil and Memantine were found to be 0.41 and 0.26 respective

Assay:

 Standard preparations are made from the API and Sample Preparations are from Formulation. Both sample and standards are injected six homogeneous samples. Drug in the formulation was estimated by taking the standard as the reference. The Average %Assay was calculated and found to be 99.29% and 99.99% for Donepezil and Memantine respectively.

Preparation of Sample stock solutions:

10 Capsules were weighed and the average weight of each Capsule was calculated, then the weight equivalent to one Capsule was transferred into a 100 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (140µg/ml of Memantine and 100µg/ml of Donepezil)

Preparation of Sample working solutions (100% solution):

2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (14µg/ml of Memantine and 10µg/ml of Donepezil)

Table 8 Assay Chromatogram Table:

       
           
       
    Fig No.4. Standard Assay Chromatogram Donepezil and Memantine

       
           
       
    Fig 5 Working Sample Assay Chromatogram Donepezil and Memantine

Degradation data

Degradation chromatograms

 Acid degradation chromatogram

           
       

Base degradation chromatogram

       
           
       
    Fig 7 base

Peroxide degradation chromatogram

       
           
       
    Fig. 8 peroxide

Thermal degradation chromatogram

       
           
       
    Fig. 8.Thermal

Uv degradation chromatogram

       
           
       
    Fig. 9 UV

Water degradation chromatogram

       
           
       
    Fig. 10 water

CONCLUSION: