The Estimation of Sapropterin Dihydrochloride in Tablet dosage form by RP-HPLC.

 

P. Janaki Pathi1*, N. Appala Raju2 and G.Parvathalu1

1Analytical Department, Vishnu Chemicals Limited, Hyderabad.

2Department of Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy Mount Pleasant, Road # 3, Banjara Hills, Hyderabad-500 034.

*Corresponding Author E-mail: pjp02002@yahoo.com

 

 

ABSTRACT:

A simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of Sapropterin Dihydrochloride in tablet dosage form. An XTerra(R) C18 analytical column (250x4.6 mm, 5 μm particle size) with mobile phase consisting of mixture of buffer 0.02M Ammonium Acetate in water and acetonitrile in the gradient program was used. The flow rate was 1.0 mL/min and the effluents were monitored at 238 nm. The retention time was 2.9 min. The detector response was linear in the concentration of 20-120 mcg/mL. The respective linear regression equation being y= 3234.6x-3233.6. The limit of detection and limit of quantification was 0.01mcg/mL and 0.03mcg/mL respectively. The percentage assay of Sapropterin Dihydrochloride was 99.4%. The method was validated by determining its accuracy, precision and system suitability.

 

The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Sapropterin Dihydrochloride in bulk drug and in its pharmaceutical dosage form.

 

KEYWORDS: Sapropterin Dihydrochloride, RP-HPLC and Tablets.

 


INTRODUCTION:

Sapropterin dihydrochloride, the active pharmaceutical ingredient in Kuvan (saproterin dihydrochloride tablets) Tablets, is a synthetic preparation of the dihydrochloride salt of naturally occurring tetrahydrobiopterin (BH4). Sapropterin dihydrochloride is an off-white to light yellow crystals or crystalline powder. Saproterin dihydrochloride tablets is indicated to reduce blood phenylalanine (Phe) levels in patients with hyperphenylalaninemia (HPA) due to tetrahydrobiopterin- (BH4-) responsive Phenylketonuria (PKU). Saproterin dihydrochloride tablets is to be used in conjunction with a Phe-restricted diet.

 

 

The chemical name of sapropterin dihydrochloride1 is (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro -4(1H)-pteridinone dihydrochloride and the molecular formula2 is C9H15N5O32HCl with a molecular weight of 314.17. Literature survey3-6 reveals no chromatographic methods for the estimation of Sapropterin Dihydrochloride from pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Sapropterin Dihydrochloride in pharmaceutical formulations.

 

 

Fig 1: Structure of Sapropterin Dihydrochloride


 

Fig 2: Typical Chromatogram of Sapropterin Dihydrochloride by HPLC

 

 

Fig 3: Calibration curve of the Sapropterin Dihydrochloride by RP-HPLC.

 


The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Sapropterin Dihydrochloride in bulk drug samples and in pharmaceutical dosage form.

 

MATERIALS AND METHODS:

Sapropterin Dihydrochloride was obtained as a gift sample from M/s. Vishnu Chemicals Ltd., Hyderabad. Acetonitrile, Ammonium acetate and water used were of HPLC grade (Qualigens). Commercially available Sapropterin Dihydrochloride tablets (Kuvan 100 tablets BioMarin Pharmaceutical Inc, USA) were procured from local market.

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Shimadzu LC 2010 dual λ detector equipped with automatic injector with injection volume 20 L. The HPLC system was equipped with LC solution Software.

 

HPLC Conditions

The contents of the mobile phase were mixture of buffer 0.02M Ammonium acetate in water and acetonitrile in the gradient program (shown in table-IV). They were filtered before use through a 0.45 μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 mL/min.

Table I: Linear Regression Data for Calibration curves:

Drug

Sapropterin Dihydrochloride

Concentration range (mcg/mL)

Slope (m)

Intercept (b)

Correlation coefficient

% RSD

20-120

3234.6

-3233.6

0.9999

0.83

 

Table II: Results of HPLC Assay and Recovery studies:

Sample

Amount claim

(mg/tablet)

% Found by the proposed method

% Recovery*

1.

2.

3.

100

100

100

99.49

99.36

99.37

99.36

99.52

99.56

*Average of three different concentration levels.

 

The run time was set at 30.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 238 nm.

 

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 10 mg of Sapropterin Dihydrochloride in 10 mL volumetric flask and dissolved in diluent (Acetonitrile and Water:50:50), sonicated for about 15 min and then made up to 10 mL with diluent get 1000 mcg/mL standard stock solution.

 

Working Standard solution:

1mL of the above stock solution was taken in 10 mL volumetric flask and thereafter made up to 10 mL with diluent (Acetonitrile and Water: 50:50) to get a concentration of 100 mcg/mL.

 

Preparation of Sample solution:

Twenty tablets (Kuvan 100 tablets BioMarin Pharmaceutical Inc, USA) were weighed, and then powdered. A sample of the powdered tablets, equivalent to 50mg of the active ingredient, was mixed with 30 mL of diluent in 50 mL volumetric flask. The mixture was allowed to stand for 15 min with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter, followed by adding diluent up 50 mL to obtain a stock solution of 1000mcg/mL. 5 mL of the above solution was taken and further diluted with diluent up to 50 mL to get working sample solution of 100 mcg / mL.

 

Linearity:

Aliquots of standard Sapropterin Dihydrochloride stock solution were taken in different 10 mL volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Sapropterin Dihydrochloride are in the range of 20-120 mcg/mL. Each of these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 238 nm and a Calibration graph was obtained by plotting peak area versus concentration of Sapropterin Dihydrochloride (Fig 3).

 

The plot of peak area of each sample against respective concentration of Sapropterin Dihydrochloride was found to be linear in the range of 20120 mcg/mL with correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being y= 3234.6x-3233.6. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in table I.

 

Assay:

20 L of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 2.9 minutes. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in table II.

 

Recovery Studies:

Accuracy was determined by recovery studies of Sapropterin Dihydrochloride, known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in table II. The study was done at three different concentration levels.

 

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Sapropterin Dihydrochloride. Parameters that were studied to evaluate the suitability of the system are given in table III.

 

Table III Validation Summary:

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor

Retention time in minutes

% Area

 

4528

1.20

2.9

99.96

LOD (mcg/mL)

LOQ (mcg/mL)

0.01

0.03

 

Table IV: Gradient Program in HPLC method:

Time in mins

Buffer

Acetonotrile

0.01

90

10

15

10

90

25

10

90

26

90

10

30

90

10

 

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) for Sapropterin Dihydrochloride were found to be 0.01mcg/mL and 0.03 mcg/mL respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. From the typical chromatogram of Sapropterin Dihydrochloride as shown in fig 2, it was found that the retention time was 2.9 min. A mixture of buffer 0.02M Ammonium acetate in water and acetonitrile in the gradient program (shown in table-IV) was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extractions were involved. A good linear relationship (r2=0.9999) was observed between the concentration range of 20-120 mcg/mL. Low values of standard deviation are indicative of the high precision of the method. The assay of Sapropterin Dihydrochloride tablets was found to be 99.4%. From the recovery studies it was found that about 99.5% of Sapropterin Dihydrochloride was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the tablets. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible.

 

Thus, the developed method can be easily used for the routine quality control of bulk and tablet dosage forms of Sapropterin Dihydrochloride within a short analysis time.

 

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Vishnu chemicals Limited, Hyderabad for the supply of as a gift sample Sapropterin Dihydrochloride and to the Management, Vishnu Chemicals Limited, Hyderabad, for providing the necessary facilities to carry out the research work.

 

REFERENCES:

1.       The Merck Index, 13, 1501, (2001)

2.       Martindale-The Complete Drug Reference, 36, 2383, (2009).

3.       Phenylketonuria (PKU): Screening and management. NIH Consens Statement. 2000;17:133.

4.       Donlon J, Levy H, Scriver C. Hyperphenylalaninemia: Phenylalanine hydroxylase deficiency. In: Scriver C, Beaudet A, Sly W, et al, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001:16671724.

5.       Friedman EG, Azen CG, Koch R, for the Writing Committee for the PKU Collaborative Study. Effects of dietary treatment on children with classical phenylketonuria (PKU): United States PKU Collaborative Study, 19671984. http://www.nichd.nih.gov/ publications/pubs/pku/sub15.cfm. Accessed January 22, 2010.

6.       Walter JH, White FJ, Hall SK, et al. How practical are recommendations for dietary control in phenylketon-uria. Lancet. 2002;360:5557.

7.       Rutherford P, Poustie VJ. Protein substitute for children and adults with phenylketonuria. Cochrane Data- base Syst Rev. 2005;CD004731.

8.       Kaufman S. The structure of the phenylalanine-hydroxylation cofactor. Proc Natl Acad Sci U S A. 1963;50: 10851093.

 

 

 

 

Received on 14.05.2012 Accepted on 16.10.2012

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Asian J. Pharm. Ana. 2(4): Oct. - Dec. 2012; Page 110-113