Liquid Chromatographic Method for the Determination of Content Uniformity of Tetrabenazine


Kathirvel S.1*, Raju R.1, Seethadevi B.2, Suneetha A.3 and Jyothisree K.3

1Department of Pharmaceutical Analysis, National College of Pharmacy, Manassery, Kozhikode, Kerala, 673602, India.

2Department of Pharmacognosy, National College of Pharmacy, Manassery, Kozhikode, Kerala, 673602, India.

3Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi road, Guntur, A.P, 522002, India.

*Corresponding Author E-mail:



The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyse the chemical purity and assay of tetrabenazine in bulk drug and tablet formulation and also to apply the proposed method to determine the content uniformity according to USP specification. During computer assisted literature search only a few publications were found about tetrabenazine, so the ultimate objective of the present investigation was aimed to develop stability – indicating LC method and the development of the newer analytical method was achieved under optimised chromatographic conditions by using mobile phase composed of potassium phosphate buffer: acetonitrile in the ratio (40:60v /v). The seperation was achieved by using isocratic elution mode with a flow rate of 1.0 mL/min. The effluent was monitored on a UV detector at 284 nm. The retention time of tetrabenazine was found to be at 5.05min and the standard calibration curve was linear over a concentration range of 6.25-37.5 µg/mL with r value of 0.999.  The LOD and LOQ were found to be 0.562 and 1.704 µg/mL respectively. The recovery studies were performed and the percentage recoveries were found to be in the range of 98.64-98.88 %. The drug was also subjected to acidic, basic, oxidative and photolytic degradation as per the ICH guidelines. The developed method allows to separate all degradation from tetrabenazine and to quantitate the tetrabenazine amount. As there is no official monograph in the pharmacopeias about tetrabenazine, the performed full validation procedure makes the method ready to use in routine analysis.


KEYWORDS: Content uniformity, ICH Guidelines, Tetrabenazine and USP specification.




Tetrabenazine, (Fig. 1) used clinically for the management of movement disorders, functions to deplete brain mono- amine levels by inhibition of the vesicular monoamine transporter type 2 (VMAT2)1. In rodents and humans, tetrabenazine is rapidly and extensively metabolized by reduction of the 2-keto group, producing a- and b-dihydro- tetrabenazine2. These alcohols also have high in vitro affinity for the VMAT2, and are likely the pharmacologically active agents in the mammalian brain3.


Fig.1: Chemical structure of tetrabenazine


A-Dihydrotetrabenazine and related benzo [a] quinolizines have recently been labelled with tritium and carbon-11 radio iso- topes and used for in vitro and in vivo studies of the VMAT2 in animal and human brain4,5.  An extensive literature survey reveals that few analytical methods have been reported for the estimation of tetrabenazine6-10. However, there is no reported stability – indicating analytical method for the determination of tetrabenazine in the presence of degradation products. Hence, there is a need for the development of newer, simple, sensitive, rapid, accurate, and reproducible HPLC method for the estimation of tetrabenazine and to validate the method according to ICH Q2(R1) Guidelines.


Content uniformity or the Uniformity of Dosage unit is defined as the degree of uniformity in the amount of active substance among dosage units. The risk assessment strategy underlying content uniformity testing is the assumption that some pre-specified limits exist where safety and efficacy outcomes may change if content uniformity fails 11 moreover drug content and content uniformity depends on a number of processes associated with its manufacture, hence it is obviously unrealistic to presume every unit to be contained exactly the same amount of the active ingredient as of the label claim, and also due to increased awareness of physiological availability, pharmacopieal standards and specifications have been established to provide limits for allowable variations for the active ingredients in single dosage units 12 of all coated and uncoated tablets intended for oral administration where the range of size of the dosage form available include 25 mg or smaller sizes. So ultimately the present research work was aimed to determine content uniformity for estimation of tetrabenazine in its tablet dosage form and also to prove its application by comparing it with 10 different marketed samples. The optimization of the method separation, validation parameters and quantification of tetrabenazine in bulk and its formulation were reported in the following sections.




Pharmaceutical grade of tetrabenazine was kindly supplied as a gift sample by Finecure Pharma Ltd., Gujarat, India, used without further purification and certified to contain 99.72% (w/w) on dried basis. All chemicals and reagents used were of LC grade and were purchased from Merck Chemicals, India.


Instrumentation and chromatographioc conditions

The LC system consisted of a pump (model Jasco PU 1580, intelligent LC pump) with auto injecting facility (AS-1555 sampler) programmed at 20 µl capacity per injection was used. The detector consisted of a UV-VIS (Jasco UV 1575) model operated at a wavelength of 284 nm. The Software used was jascoborwin version 1.5, LC-Net II/ADC system. The column used was Kromasil C-18 (250 mm x 4.6mm, 5.0 µm) Flexit Jour Laboratories Pvt. Ltd., Pune, India. Diffčrent mobile phases were tested in order to find the best conditions for seperation of tetrabenazine in presence of its degradation products. The optimal concentration of mobile phase was determined to be0.01N Pottassium phosphate buffer : acetonitrile (40 :60V/V) and the pH was adjusted to 6.5. The flow rate was set to 1.0mL min -1withtotal run time of 10 minutes. The mobile phase and samples were filtered using 0.45µm membrane filter. Mobile phase was degassed by ultrasonic vibrations prior to use. All determinations were performed at ambient temperature.


Standard solutions and calibration graphs

Stock standard solution was prepared by accurately weighing 25 mg of tetrabenazine standard drug and transferring into a 100 mL volumetric flask, 50mL of diluent (mobile phase) was added and sonicated for 10min and the volume was made up to the mark using the diluent (250 µg/mL). The standard solutions were prepared by dilution of the stock solution with methanol to reach a concentration range 6.25- 37.5µg/mL (6.25µg/mL, 12.5µg/mL, 18.75µg/mL, 25µg/mL, 31.25 µg/mL and 37.5µg/mL) for tetrabenazine. Injection of 20 µl by triplicate were made six times for each concentration and chromatographed under the conditions described above. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.


Sample preparation

To determine the content of tetrabenazine in conventional tablets (Revocon-label claim: 25 mg of tetrabenazine per tablet), the 20 tablets were weighed, their mean weight determined and they were finely powdered and powder equivalent to 25 mg tetrabenazine was weighed and transferred into a 100mL volumetric flask and 50 mL of diluent was added. The mixture was subjected to sonication for 30min and filtered using 0.45-µm filter (Millipore, Milford, MA).Then 10mL of the filtratewas diluted to 100ml with mobile phase to get 25µg/mL concentration of sample and 20-µl volume of sample solution was injected into LC, six times. The peak was measured at 284 nm and concentrations in the samples were determined using multilevel calibration developed on the same LC system under the same conditions using linear regression equation.


Method Validation

After the method development, the method is validated13 in terms of parameters like assay, linearity, precision, accuracy, robustness, ruggedness, LOD and LOQ.



From the stock solution (250µg/mL) of tetrabenazine 2.5 ml, 5 ml, 7.5 ml, 10 ml, 12.5 ml, and 15 ml is taken in six different 100 ml volumetric flasks and diluted with the mobile phase to the give the following concentrations (6.25µg/mL, 12.5µg/mL, 18.75µg/mL, 25 µg/mL, 31.25 µg/mL and 37.5 µg/mL). These solutions were injected into the chromatographic system and the response was recorded. The calibration graph was plotted with peak area in the Y axis and concentration of standard solution in the X axis. The degree of linearity was estimated by calculating the correlation coefficient. Y- Intercept, slope of the regression line.



Precision of an analytical procedure referred to degree of scatterness between a series of observations obtained from multiple sampling of same homogenous sample in given conditions.


a) The system precision of test method was performed by injecting five 20 µL portions from a standard solution on to the analytical column and the peak area data obtained then %RSD was calculated.  

b) The method precision of test method was done by performing assay on five replicate determination of sample preparation at test concentration level (as per method of analysis) and the relative standard deviation of assay results was obtained.



Accuracy is the closeness of the test results to that of true value which can be determined in terms of percent recovery. Accuracy of the method confirmed by studying recovery at 3 different concentrations of 50, 100, and 150%, in accordance with ICH guidelines, by replicate analysis (n=3). Standard drug solution was added to a pre analysed sample solution and percentage drug content, % recovery and % RSD were measured.



For demonstrating the robustness of the developed method, experimental conditions were purposely altered and evaluated. The method must be robust enough to withstand such slight changes and allow routine analysis of the sample. Robustness of method was carried out with variation of flow rate (0.1 ml/min of set value i.e. 0.9 ml/min and 1.1 ml/min) and variation of Temperature (+/- 2 0C).


Limit of Detection and Quantification

LOD is the minimum concentration of the analyte that gives a measurable response, whereas LOQ is the minimum concentration which can be quantified accurately and precisely.



Specificity is the ability of the analytical method to measure the analyte free from interference due to other components. Specificity was determined by comparing test results obtained from analyses of sample solution with that of test results. Chromatograms for placebo, working and reference standards were recorded and it shows that there is no interference with the results.


Forced degradation studies

Forced degradation studies are performed as a part of specificity studies. Stability of drug product or a drug substance is a critical parameter which may affect purity, potency and safety. Changes in drug stability can risk patient safety by formation of a toxic degradation product(s) or deliver a lower dose than expected. Therefore it is essential to know the purity profile and behaviour of a drug substance under various environmental conditions.


Acid stressed degradation

Acid degradation was performed by taking 25mg into 100 mL volumetric flask followed by the addition of 1mL of 1N HCl and 80 mL methanol and the mixture was heated under reflux for 1 hour, and the volume was made up to mark with diluents and filtered. Further dilutions were made to obtain 25µg/mL solution. From the above solution 10µL were injected in to the system and the chromatograms were recorded.


Alkali stressed degradation

For alkali stressed degradation the 25 mg of the drug was taken and transferred in to 100 mL volumetric flask followed by the addition of 1mL of 1N NaOH and 80 mL methanol and the mixture was heated under reflux for 1 hour, and the volume was made upto mark with diluent and filtered. Further dilutions were made to obtain 25µg/mL solution. From the above solution 10µL were injected in to the system and the chromatograms were recorded.


Oxidative degradation.

The solution for the oxidative degradation was performed by taking 25mg into 100 mL volumetric flask then 1mL of 20 % H2O2and 80 mL of diluent was added and the mixture was heated under reflux for 1 hour, and the volume was made up to mark with diluent. Further dilutions were made to obtain 25µg/mL solution. From the above solution 10µL were injected in to the system and the chromatograms were recorded.


Photo stability degradation

The drug solution (1 mg/mL) for photostability testing was exposed to UV light for (365 nm) 1 h in UV light chamber and analysed. Appropriate aliquot was taken from the above solution and diluted to obtain a final concentration of 25 µg/mL. The chromatogram was recorded to assess the stability of sample.



Fig.2: Chromatogram of standard tetrabenazine ( 25 μg ml-1); tR: 5.05 ± 0.05) measured at 284 nm


Fig.3: Chromatogram of sample tetrabenazine (25 μg ml-1); tR: 5.05 ± 0.05) measured at 284 nm



The chromatographic conditions were optimized for the tetrabenazine within a short analysis time (10 min) using simple mobile phase and an acceptable peak tailing (<1.5). In order to achieve these goals, the chromatographic mobile phase and column were chosen first. Initially the mobile phase chosen with pH 8.0 where precipitation observed for standard preparation, hence the mobile phase pH was reduced and optimized as 6.5 with phosphate buffer. The 25 ppm of tetrabenazine solution was scanned between 200 – 400 nm in a UV spectrophotometer. The maximum absorbance (λmax) was obtained at 284 nm and hence the wavelength 284 nm was selected in further development. The run time was set at 10 min and the retention time was found to be 5.05 min. Each sample was injected 6 times and the retention time was recorded. The chromatogram of standard and sample tetrabenazine was shown in Fig. 2 and 3 respectively. When the concentrations of tetrabenazine and its respective peak areas were subjected to regression analysis by least squares method, a good linear relationship (r = 0.999) was observed between the concentration of tetrabenazine and the respective peak areas in the range of 6.25 – 37.5 μg/mL. The regression of tetrabenazine was found to be Y = 91933x - 2385. Where Y is the peak area and x is the concentration of tetrabenazine.


Table I. System precision of Tetrabenazine

S. No.











Std dev


System Precision-1

System Precision-2

System Precision-3

System Precision-4

System Precision-5

System Precision-6




















Table II.  Method precision of Tetrabenazine

S. No.











Std dev


Method Precision-1

Method Precision-2

Method Precision-3

Method Precision-4

Method Precision-5

Method Precision-6




















The proposed HPLC method was also validated for precision studies (system precision and method precision) and results were reported in Table I and II, respectively. The recovery studies were performed for the proposed method and the percentage recovery was found to be in the range of 98.64-98.88 %, respectively. The results were represented in Table III. Keeping the wavelength (284 nm) constant, the chromatograms of drug solution were recorded at different flow rates such as 0.9 mL and 1.1 mL /min. At the flow rate of 1.0 mL/min, the peaks were sharp with good resolution. Apart from above said flow rate, rest of the flow rates were found to be not satisfactory, So 1.0 mL/min flow rate was kept constant throughout the analysis. The LOD and LOQ were calculated and found to be 0.562 and 1.704 µg/mL respectively.


Table III. Recovery data (n=3)



Amount of drug added (μg/mL)*

Amount found (μg/mL)*

% Recovery

















Fig.4:  Chromatogram for acid (0.1N HCl) stressed degradation of tetrabenazine


Fig.5: Chromatogram for alkali (0.1N NaOH) stressed degradation of tetrabenazine


Chromatograms for placebo, working and reference standards were recorded and it shows that there is no interference with the results. So the method can be declared as specific.


Forced degradation studies were performed to show the inherent stability of the drug towards the different stressed conditions. Acid stressed degradation was performed using 0.1 N HCl. The % degradation of tetrabenazine was found to be 3.03% and the results are incorporated in the Table-IV and Fig.4. Alkali stressed degradation was performed using 0.1N NaOH. The %degradation of tetrabenazine was found to be 6.93% and the results are depicted in the Table-IV and Fig.5, respectively. Oxidative degradation was performed using 3% H2O2and the percentage degradation of tetrabenazine was found to be 3.53% and the results are shown in the Table-IV and Figure 6, respectively. Photolytic degradation was performed and the percentage degradation was found to be 2.28% and results are incorporated in Table IV and shown in Fig.7, respectively.


Table IV. Degradation data of tetrabenazine

Stress condition

Retention time

% purity

% Degradation

Acid degradation

Alkali degradation

Oxidation degradation

Photolytic degradation














Fig.6: Chromatogram for oxidative (3% H2O2) stressed degradation of tetrabenazine



Fig.7: Chromatogram for photolytic stressed degradation of tetrabenazine


The HPLC method development in the present study has been used to quantify tetrabenazine in tablet formulations. Tetrabenazine tablets (each containing 25 mg of drug) were analyzed as per the procedure described above. No interfering peaks were found in the chromatogram indicating these excipients usually present in the pharmaceuical dosage forms did not interfere with the estimation of drug by the proposed HPLC method. The amount of tetrabenazine found by using the proposed method was found to be 99.99%. The results are depicted in the Table V.


TableV.  Summary of Assay results (n=6)


Labeled amount

Amount found

% Assay

Tetrabenazine (Revocon)

25 mg




The developed analytical method has also been applied to investigate the samples of tetrabenazine in10 different marketed brands. For comparison, the samples were analysed by this validated method and the results were compared as per the USP, EP, JP pharmacopoeial guidelines for uniformity of dosage forms determination. All the samples comply with the acceptance value and the specifications limit according to the pharmacopeias. The sample acceptance value for content Uniformity tests to pass was that it should be less than 15.0 for 10 units, the % RSD should be less than 6.0 and the Mean value should be between 85% to 115% of average content, with the condition of only one dose unit is outside 85 to 115 % and between 75 – 125% of average content (Table VI)


Table VI. Percentage content in bulk and ten individual tablets of marketed formulation of tetrabenazine by HPLC method


Label claim/Taken (mg)

Percentage purity*

% C.V












































* Average of six determinations



The proposed LC method provide simple, accurate, reproducible and stability indicating method for quantitative analysis of tetrabenazine in pharmaceutical tablets, without any interference from the excipients and in the presence of its acidic, alkaline, oxidative and photolytic degradation products. This study is a typical example of development of a stability- indicating assay, established following the recommendations of ICH guidelines. The developed method was used for the content uniformity test of 10 different marketed tablet samples of tetrabenazine. The results were comparable and comply with different pharmacopeial limits. The method has significantly reduced runtime with better peak shape. Using the same method, assay was performed for individual samples and found that values are between good agreements. Hence this will be an excellent method for the assay determination and content uniformity of tetrabenazine in oral solid dosage form. The developed method may be extended for quantitative estimation of said drug in plasma and other biological fluids.



The authors are thankful to Bio Leo Analytical Labs for providing the gift sample of tetrabenazine.



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Received on 28.02.2015          Accepted on 21.03.2015        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 5(2): April-June 2015; Page 86-92

DOI: 10.5958/2231-5675.2015.00014.9