Development And Validation of RP HPLC Method for Estimation of Galantamine in Tablet Dosage Form

What is Dementia

Dementia is a general term for a group of conditions characterized by a decline in cognitive functioning severe enough to interfere with daily life and independence¹. It primarily affects memory, thinking, reasoning, and socialabilities. Alzheimer'sdisease isthe most commoncause of dementia, followed by vascular dementia and other types².

What are the Different types of dementia?

Types of dementia include:

• Alzheimer’s disease, the most common dementia diagnosis among older adults. It is caused by changes in the brain, including abnormal buildups of proteins known as amyloid plaques and tau tangles.³
• Frontotemporal dementia, a rare form of dementia that tends to occur in people younger than 60. It is associated with abnormal amounts or forms of the proteins tau and TDP-43.
• Lewy body dementia, a form of dementia caused by abnormal deposits of the protein alpha-synuclein, called Lewy bodies.⁴
• Vascular dementia, a form of dementia caused by conditions that damage blood vessels in the brain or interrupt the flow of blood and oxygen to the brain.

Mixed dementia, a combination of two or more types of dementia. For example, through autopsy studies involving older adults who had dementia, researchers have identified that many people had a combination of brain changes associated with different forms of dementia.

Basic Principles of HPLC

HPLC works on the principle of differential migration of analytes based on their affinity for the stationary and mobile phases. The process can be summarized as follows:

1. Injection of Sample: The liquid sample is introduced into the system.
2. Separation in Column: As the sample travels through the column, components interact with the stationary phase to different extents, resulting in separation.
3. Detection: A detector measures the separated analytes as they elute at different times (retention times).

Components of method validation: The following are typical analytical performance   characteristics which may be tested during methods validation:^[15-17]

1. Accuracy
2. Precision
3. Linearity
4. Detectionlimit
5. Quantitationlimit
6. Specificity
7. Range
8. Robustness
9. Quantitationlimit
10. Specificity
11. Range
12. Robustness

Accuracy

Accuracy is defined as the nearness of a measured value to the true or accepted value. Practically accuracy indicates the deviation between the mean value foundand the true value. It is determined by applying the method to samples to which known amounts of analyte have been added. These should be analysed against standard and blank solutions to ensure that no interference exists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay. It may often be expressed as the recovery by the assay of known, addedamounts of analyte.

Precision

It expresses closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision is a measure of the reproducibility of the whole analytical method. It consists of two components: repeatabilityand intermediate precision. Repeatabilityisthe variationexperienced by a single analyst on a single instrument. It does not distinguish between variation from the instrument or system alone and from the sample preparation process. During validation, repeatability is performed by analysing multiple replicates of an assaycomposite sample by using the analytical method. The recovery value is calculated. Intermediate precision is the variation within a laboratory such as different days, with different instruments, and by different analysts. Accuracyandprecision are not the same, as the diagram below indicates. A method can have good precision and yet not be accurate.

Linearity

Linearity is the ability of analytical procedure to obtain a response that is directlyproportional to the concentration (amount) of analyte in the sample. If the methodis linear, the test results are directly or by well-defined mathematicaltransformation proportional to concentration ofanalyte in samples within a givenrange. Linearity is usually expressed as the confidence limit around the slope of the regression line.

Limits of detection and quantitation:

The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. LOD is expressed as a concentration at a specified signal: noise ratio, usually 3:1. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyteinasamplethat can be determined with acceptable precision and accuracyunder the stated operational conditions of the method. For LOQ, ICH has recommended a signal: noise ratio 10:1. LOD and LOQ may also be calculated based on the standard deviation of the response (SD) and the slope ofthe calibrationcurve(s) at levels approximatingthe LOD according tothe givenbelow formulae.

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Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which maybe expected to be present. Typically, these might includeimpurities, degradants, matrix, etc. Lack of specificityof an individual analytical procedure may be compensated byother supporting analyticalprocedure(s). Thisdefinition has the following implications: Identification: to ensure the identityof an analyte. PurityTests: to ensure that all the analyticalprocedures performed allow an accurate statement of the content of impurities of an analyte, i.e. related substances test, heavy metals, residualsolventscontent,etc. Assay(content or potency): to provide anexact result which allows an accurate statement on the content or potency of the analyte in a sample.

Range

The range ofthe method is the interval between the upper and lower levels ofan analyte that have been determined with acceptable precision, accuracy and linearity. It is determined on either a linear ornonlinear response curve (iewheremorethanone range is involved, asshownbelow) and is normally expressed in the same units as the test results.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Validation parameter

AIM

The aim of the present work is to develop and validate a precise, accurate, and robust RP-HPLC method for the quantification of Galantamine in tablet dosage form.

OBJECTIVE

• To develop a simple and reproducible RP-HPLC method for estimating Galantamine.
• To validate the developed method based on parameters such as specificity, precision, accuracy, linearity, robustness, and detection limits.
• To ensure suitability of the method for routine quality control analysis in pharmaceutical formulations

MATERIALS AND METHODS

Table: Chemical and Reagents Used

Table: Instruments Used

Table: Preparation of Standard and Sample Solutions

Table: Chromatographic Conditions

Table: Method Validation Parameters

Fig: Structure of Galantamine

Chromatographic condition

The chromatographic separation of Galantamine was achieved on C-18 (id4.6x250mm, 5 µm) by using mobile phase composed of Methanol: Water (60:40 v/v %), at flow rate 1.0 ml/min with run time of 10 minutes. Detection of drug was carried out at 210 nm by using diluent as mobile phase.

Method of validation

As per ICH guideline (Q2R1), the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

A Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay(100% Conc.) and inject into the HPLC system in triplicate.

A Linearity and Range

Preparation of Solution for linearity studies: For the purpose of linearity, accurately weighed amount of Galantamine (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Galantamine. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 2, 4, 6, 8 and 10 µg/ml of Galantamine respectively.

Precision

Repeatabiliy

Prepared standard working solution of mixtures having concentration of Galantamine (4 μg/ml) was injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD. Intra-day and Inter-day Precision

• Method precision was determined by performing intraday and interday precision.
• Mixture that represents overall range (Galantamine =5,15 and 25µg/ml) was analyzed on same day at different time interval for intraday precision.
• Mixture that represents overall range (Galantamine =5,15and25 µg/ml) was analyzed on different days for inter-day precision.

System Suitability Parameters

Solution of Galantamine (15 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters for selected concentration were determined by C.V.

Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50,100 and 150 % of the target concentration

Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ=10σ/s and LOD=3.3σ/s

Where, σ = the standard deviation of the response.

S =the slope of the calibration curve

Robustness

Following parameters were alteredone byone for determinationofrobustness ofthe method and their effect was observed by comparing with the standard preparation. Mobile phase flowrate (± 0.1mL/min),optimizedflowratewas1.0mL/min.Mobilephasecomposition(±2mL),in optimized ratio2 determinations of Galantamine= 2 µg/mL for each alteration were carried outand RSD was measured

Result and Discussion

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Galantaminewas scannedbetween 200-400 nm against diluents. Absorbance maxima of Galantamine have detected at 210nm. Chromatogramwas taken at 219 nm, druggive good peak height and shape. So, 210 nm was selected for estimation of Galantaminein formulation.

Selection of Mobile phase

Trail  1

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Acetonitrile: Water 30:70 v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• RunTime:10minutes
• Observations: No peak detected.

Fig: Trial 1: Chromatogram of Galantamine (15µg,ml-1)

Trail 2

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Methanol: Water (50:50v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• RunTime:10minutes
• Observations: Peaks detected and separated, but broad peaks observe.

Fig:Trial 2:Chromatogram of Galantamine (15 µg,ml-1)

Trial 3

• Column: C-18 (id 4.6 x 250 mm, 5 µm)
• Mobile Phase: Methanol: Water (60:40v/v)
• Detection: 210 nm
• Flow rate:1ml/min
• RunTime: 10minutes
• Observations: Good peak with Adequate solution was observed.

Fig: Trial 3: Chromatogram of Galantamine (15 µg,ml-1)

Table 6.10 Mobile phase Trial for Galantamine

Chromatographic conditions for optimized mobile phase trial

• Stationary phase: C-18 (id4.6x250mm,5µm)
• Mobile Phase: Methanol: Water(60:40v/v)
• Detection: 210 nm
• Flow rate:1 ml/min
• Run Time: 10 minutes
• Detector: UV detector
• Injection volume:20 μl
• ColumnTemperature:40ºC
• Mode: Isocretic

Fig6.6: Optimized mobile phase trial for optimized chromatogram of Std. Galantamine: 4.315min

Fig : Chromatogram of blank Methanol: Water (60:40v/v)

Method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Galantamine(10 mg)was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml ofGalantamine. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 2, 4, 6, 8 and 10µg/ml of Galantamine

Table: Linearity data for Galantamine

Fig: Overlain Linearity Spectra of Galantamine

Fig : Calibration curve of Galantamine

Table: Linearity results for Galantamine

Precision

Repeatability

The data for repeatability for Galantamine is shown in table 6.13. The %R.S.D For Repeatability data was found to be 1.10 % for Galantamine.

Table: Repeatability data for Galantamine

Inter-day precision

The data for interday precision for Ranitidine and Ondansetron is shown in table 6.14. The % R.S.D for intraday precision was found to be 0.33-0.63% for Galantamine.

Table: Inter-day precision data for estimation of Ranitidine and Ondansetron

Intra -day precision

The data for intra-day precision for Galantamine is shown in table 6.15. The % R.S.D for intraday precision was found to be 0.35-0.57% for Galantamine.

Table: Intra-day precision data for estimation of Galantamine

Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Galantaminewas found to be 99.48- 99.78%. The results are shown in table.6.16.

Table: Recovery data for Galantamine

LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Table LOD and LOQ Limit for Galantamine

Selectivity

There is no interference in the mixture.

Robustness

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 6.19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table: Robustness data for Galantamine

Analysis of marketed product

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table: Analysis of marketed formulations

SUMMARY OF METHOD VALIDATION

Table: Summary of validation parameter of RP-HPLC method