Determination of Sapropterin Dihydrochloride in Solid Dosage form by Visible Spectrophotometry

 

U. Viplava Prasad¹, M. Syam Bab¹*, B. Kalyana Ramu²*

¹Department of Organic Chemistry and Analysis of Foods Drugs and Water Laboratories, School of Chemistry, Andhra University, Visakhapatnam-530003 Andhra Pradesh (India)

²Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

*Corresponding Author E-mail: kalyanaramu23566@gmail.com, msyambab@gmail.com

ABSTRACT:

Purpose: The aim of the investigation was to see the simple and sensitive visible spectrophotometric method for the determination of the sapropterin dihydrochloride either in bulk or tablet dosage forms. Method: A simple, sensitive indirect visible spectrophotometric method has been developed for the estimation of sapropterin dihydrochloride in bulk and dosage forms. The method is based on the oxidation of drug with excess N-Bromo succinimide and determining the unreacted NBS with an oxidisable dye Celestein blue by spectrophotometrically at 522nm Results: Beer’s law obeyed in the concentration range of 4-12 µg/ml. No interference was observed from the usually existing additives in pharmaceutical formulations and the applicability of the method was examined by analyzing kuvan tablets containing SAP. Conclusion: The reported methods for its assay involve sophisticated equipment, which are very costly and pose problems of maintenance. To overcome these problems, the use of visible spectrophotometric technique is justifiable. The statistical data indicates the accuracy, reproducibility and the precision of the proposed method.

 

 

 

INTRODUCTION:

Sapropterin (SAP) (Figure 1) is an enzyme cofactor and oral form of a synthetic preparation of the dihydrochloride salt of naturally occurring tetrahydrobiopterin(BH₄)^[1-3]. It is chemically designated as (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-,6,7,8-tetrahydro-4(1H)-pteridinone dihydrochloride. Its empirical formula is C₉H₁₅N₅O₃·2HCl representing molecular weight of 314.17  It is an off white to slightly yellow crystalline powder that is soluble in water and methanol.

 

BH₄ works with phenylalanine hydroxylase to metabolize phenylalanine (Phe). Saproterin dihydrochloride tablets are indicated to reduce blood phenylalanine (Phe) levels in patients with hyperphenylalaninemia (HPA) due to tetrahydrobiopterin- (BH₄-) responsive Phenylketonuria (PKU) and to be used in conjunction with a Phe-restricted diet.

 

Figure 1: Chemical structure of sapropterin dihydrochloride

 

Literature survey reveals that no spectrophotometric method was reported. Only analytical methods such as HPLC ^[4-6], have been reported for the determination of SAP in biological fluids and formulations. For routine analysis, simple, rapid and cost effective visible spectrophotometric methods are required and preferred. The availability of the UV-Visible spectrophotometric methods with high sensitivity and selectivity will be very useful for quality control analysis and small scale pharmaceutical industries. The functional groups present in the drug have not been exploited properly in developing visible spectrophotometric methods. Nevertheless, there is a need for development of sensitive accurate and flexible visible spectrophotometric methods for the determination of SAP in pharmaceutical preparations. So the authors have made some attempts in this direction and succeeded in developing this method based on the redox reaction of drug with NBS-CB as a reagent ^[7] under specified experimental conditions. The proposed method for SAP determination has many advantages over other analytical methods due to its rapidity, normal cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and not costly. All the analytical reagents are inexpensive and available in any analytical laboratory. The method can be extended for the routine quality control analysis of pharmaceutical products containing SAP.

 

MATERIALS AND METHODS:

Apparatus and Chemicals:

A Shimadzu double UV/Visible spectrophotometer model-1800 with 10mm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade. NBS solution: (Loba, 0.01%, 5.618x10^-4M) Prepared by dissolving 10 mg NBS in100ml distilled water and standardized iodometricallly.  CB solution: (Chroma, 0.02%, 5.49x10^-4M) Prepared by dissolving 20 mg of Celestine blue in 100ml distilled water. Hydrochloric acid :( Merck, 5M) Prepared by diluting 217.5 ml of Con. Hydrochloric acid to 500 ml with distilled water and standardized.

 

Preparation of Standard drug stock solution:

The standard stock solution (1mg/ml) of SAP was prepared by dissolving 100mg of SAP in 100 ml distilled water. This solution was further diluted stepwise with the same solvent to obtain working standard solution concentration of 100μg/ml. The prepared stock solution was stored at 4⁰ C protected from light. From this stock solution, a series of standards were freshly prepared during the analysis day.

 

Preparation of Sample Solution:

About 20 tablets were weighed to get average tablet weight and pulverized. The powder equivalent to 100mg of SAP was weighed and extracted with CHCl₃ (2x15ml), and filtered through whatman filter paper no.41. The filtrate was evaporated and the residue was used for the preparation of working sample solutions in the same way as under working standard solution.

 

Determination of wavelength maximum (λ _max):

The 3.0 ml of working standard solution of SAP (100µg/ml) was taken in 25 ml standard flask. To this, 1.3 ml of 5.0M HCl, 3.0ml of (5.618x10^-4 M) NBS solution were added and the volume was made up to 20 ml with distilled water. After 10 min, 5 ml of (5.49x10^-4M) CB was added and mixed thoroughly. In order to investigate the wavelength maximum, the above solution was scanned in the range of 350-750nm by UV-Visible spectrophotometer. From the spectra (Figure 2), it was concluded that 522nm is the most appropriate wavelength for analyzing SAP with suitable sensitivity.

 

Figure 2: Absorption spectra of SAP-NBS-CB system

 

Preparation of calibration curve:

Aliquots of the standard SAP solution ((1.0-3.0ml, 100µg/ml) were transferred into a series of 25ml calibrated tubes, 1.3 ml of 5.0M HCl, 3.0ml of (5.618x10^-4 M)NBS solution were added and the volume was made up to 20 ml with distilled water. After 10 min, 5 ml of (5.49x10^-4M) CB was added and mixed thoroughly. The absorbances were measured after 5 min at 522 nm against distilled water. The blank (omitting drug) and dye (omitting drug and oxidant) solutions were prepared in a similar manner and their absorbances were measured against distilled water. The decrease in absorbance corresponding to the consumed oxidant NBS, which reflects the drug concentration, was obtained by subtracting the decrease in absorbance of the test solution (dye-test) from that of the blank solution (dye-blank). Calibration graph was prepared by plotting the decrease in the absorbance of dye against the amount of drug. The amount of drug in a sample was calculated from its calibration curve (Figure 3).

 

Figure 3: Beer’s Law plot of SAP-NBS-CB system

 

RESULTS AND DISCUSSION:

In developing this method, a systematic study of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed. The effect of various parameters such as time, volume, temperature and strength of reagents and order of addition of reagents, stability of colored species and solvent for final dilution of the colored species were studied and the optimum conditions were established. The optical characteristics such as Beer’s  law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits ) were calculated  and the results are summarized in Table-1. Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e) and % range of error (0.05 and 0.01 confidence limits) were calculated and are shown in Table 1.

 

Kuvan tablets containing SAP were successfully analyzed by the proposed method. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table 2. The interference studies in the determination of SAP in pharmaceutical formulation revealed that the normally existing excipients and additives like starch, talc, stearic acid, boric acid, gelatin, magnesium carbonate and sodium lauryl sulphate were found not to interfere even when present in excess (1-100 folds). However, preliminary clean up procedure with CHCl₃ is necessary prior to the estimation of SAP in formulations if lactose is present.

 

 

Table 1: Optical Characteristics, Precision and Accuracy of Proposed Method.

Parameter	Values
גmax  (nm)	522
Beer’s law limit(µg/ml)	4-12
Sandell’s sensitivity  (µg/cm2/0.001 abs. unit	0.001568627
Molar absorptivity (Litre/mole/cm) Correlation Coefficient	200283.375 0.997
Regression equation  (Y)*
Intercept (a)	-0.046
Slope(b)	0.030
%RSD**	1.93
%  Range of errors (95% Confidence  limits)
0.05 significance level	2.02
0.01            significance level	3.17

*Y = a +b x, where Y is the absorbance and x is the concentration of SAP in µg/ml

**calculated from six determinations

 

 

TABLE 2: ANALYSIS OF SAP IN PHARMACEUTICAL FORMULATIONS

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method  ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
SAP-NBS-CB	Tablet-1	100	98.4±1.01	2.1	1.13	98.04±1.07	98.4±1.01

* Tablet- 1: KUVAN tablets of Bio Marin Pharmaceuticals Inc USA

**Average ± Standard deviation of six determinations, the t- and f-values refer to comparison of the proposed method with UV reference method. Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre analyzed sample (average of three determinations).

Reference method (UV method) using methanol developed in our laboratory (λ _max=224nm).

 

Figure 4: probable sequence reactions of the proposed method

 

 

Chemistry of Colored Species:

This method involves two steps. First step is the oxidation of drug with NBS. The second step is quantitative decolorization of Celestine blue (CB) by unreacted NBS. The probable sequence of reactions in two steps (drug-NBS, NBS-CB) based on analogy are presented in Figure 4.

 

CONCLUSION:

The reagents utilized in the proposed method are normal cost, readily available and the procedure does not involve any critical reaction conditions or tedious sample preparation. The proposed method possesses reasonable precision, accuracy and is simple, sensitive and can be used as alternative method to the reported ones for the routine determination of SAP depending on the need and situation.

 

ACKNOWLEDGEMENTS:

The authors (BK Ramu and MS Bab) are thanks to the University Grants Commission, New Delhi for providing financial assistance under Teacher fellowship and also thanks to University authorities for providing facilities in this work.

 

REFERENCES:

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

2.       Martindale The complete drug reference, 36, 2383(2009).

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6.       A. Khan, M .Bhojani, M. Rajput, P. Revathi, S. Biswas, Arifkhan, International Journal of Pharmaceutical and Chemical Sciences 2(2),695-704(2013).

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Received on 01.02.2017       Accepted on 21.04.2017     

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