Sensitive Visible Spectrophotometric methods Development for Estimation of Sapropterin Dihydrochloride in tablet dosage form

 

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 & Autonomous), Vizianagaram-535002 (AP) India.

 

 

ABSTRACT:

The drug Sapropterin (SAP) dihydrochloride is an enzyme cofactor and oral form of a synthetic preparation of the dihydrochloride salt of naturally occurring tetrahydrobiopterin(BH₄) and  used to reduce blood phenylalanine (Phe) levels in patients with hyperphenylalaninemia (HPA).  Purpose: The aim of the present investigation was to see the simple and sensitive visible spectrophotometric methods for the determination of the sapropterin dihydrochloride in bulk and tablet dosage forms. Methods: Two simple, sensitive and cost effective visible spectrophotometric methods (M₁-M₂) were developed for the estimation of sapropterin dihydrochloride in bulk and dosage forms. The first method (M₁) is based on the formation of blue reduced product by treating drug with Folin Ciocalteu (FC) reagent in the presence of sodium carbonate solution with an absorption maximum of 770nm. The second method (M₂) is based on the reaction of drug with ferric chloride and potassium ferricyanide to form a dark green colored species having absorption maxima at 704nm. Results: Beer’s law obeyed in the concentration range of 2-6µg/ml and 2.5-12.5 µg/ml for methodM₁ and M₂ respectively. No interference was observed from the usually existing additives in pharmaceutical formulations and the applicability of the methods was examined by analyzing kuvan tablets containing SAP. Conclusion: The reported methods HPLC 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 proved the accuracy, reproducibility and the precision of the proposed methods.

 

 

 

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 a 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

 

No available UV-Visible spectrophotometric methods for its determination in literature, 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 not exploited. 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 two methods based on the reaction between the drug and FC reagent^[7] (M₁) or Fe(III)-Potassium ferricyanide^[8] (M₂) under specified experimental conditions.

 

The proposed methods for SAP determination have many advantages over other analytical methods due to its rapidity, normal cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. These methods 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. Commercial available FC reagent (Loba, 2N), 10%Na₂CO₃ (BDH, 9.43x10^-1M) solution was used for method M₁.  Fe (III) solution (Wilson labs, 0.9%, 1.10x10^-2M prepared by dissolving 900mg anhydrous ferric chloride in 100ml of distilled water), Potassium ferricyanide solution (0.1%, 3.02x10^-3M prepared by dissolving 100mg Potassium ferricyanide in 100ml of distilled water with worming) and 1N HCl.

 

Preparation of Standard stock solution:

The standard stock solution (1mg/ml) of SAP was prepared by dissolving 100mg of SAP in 100 ml distilled water. The working standard solutions of SAP were obtained by appropriately diluting the standard stock solution with the same solvent (M₁ and M₂- 50 µg/ml). The prepared stock solution was stored at 4°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 the average tablet weight and pulverized. The powder equivalent to 100mg of SAP was weighed, dispersed in 25ml of Isopropyl alcohol, sonicated for 15 minutes and filtered through Whatman filter paper No 41.The filtrate was evaporated to dryness and the residue was dissolved as under standard solution preparation.

 

Determination of wavelength maximum (λ _max): 

Method M₁:

3.0ml of Standard SAP solution was transferred into 25ml calibrated tube. To this 2.5ml of FC (2N) reagent was added. After 3 minutes 7ml of 10% Na₂CO₃ was added. The solutions were mixed and kept at room temperature for 30 minutes for complete color development and diluted to the mark with distilled water.  In order to investigate the wavelength maximum, the above colored solution was scanned in the range of 450-1100 nm UV-Visible spectrophotometers against a reagent blank. From the absorption spectra (Figure 2), it was concluded that 770nm is the most appropriate wavelength for analyzing SAP with suitable sensitivity.

 

Figure 2: Absorption spectra of SAP-FC system

 

Method M₂:

2.5ml of Standard SAP solution was transferred into 10ml calibrated tube. Then 0.5ml of FeCl₃ 1.10x10^-3M solution was added and shaken well for 5 minutes. Then 1.0ml of potassium ferricyanide (3.02x10^-3M) solution were added successively and kept aside for 10 minutes. To this 1.0ml of 1N HCl was added .After 10 minutes dark green color solution was obtained and total volume in tube was made to 10.0ml with distilled water. In order to investigate the wavelength maximum, the above colored solution was scanned in the range of 400-800 nm UV-Visible spectrophotometers against a reagent blank. From the absorption spectra (Figure 3), it was concluded that 704nm is the most appropriate wavelength for analyzing SAP with suitable sensitivity.

 

Figure 3: Absorption spectra of SAP-Fe(III)-K₃[Fe(CN)₆]- System

 

Preparation of calibration curve: 

Method M₁:

Aliquots of Standard SAP solution (1.0-3.0ml, 50µg/ml) were transferred into a series of 25ml calibrated tubes. To each tube 2.5ml of FC (2N) reagent was added. After 3 minutes 7.0ml of 9.43x10^-1M Na₂CO₃ was added. The solutions were mixed and kept at room temperature for 30minutes for complete color development and then diluted to the mark with distilled water.  The absorbance was measured at 770nm against a reagent blank prepared simultaneously. The amount of drug was computed from its calibration graph (Figure 4).

Figure 4: Beer’s Law Plot of SAP-FC system

Method M₂:

Aliquots of Standard SAP solution (0.5-2.5ml, 50µg/ml) were transferred into a series of 10ml calibrated tubes. Then 0.5ml of FeCl₃ 1.10x10^-3M solution was added and shaken well for 5 minutes. Then 1.0ml of potassium ferricyanide (3.02x10^-3M) solution were added successively and kept aside for 10 minutes. To this 1.0ml of 1N HCl was added and total volume in each tube was made up to 10.0ml with distilled water. The absorbances of the colored complex solution were measured after 10min. at 704 nm against the reagent blank prepared similarly. The content of the drug computed from the appropriate calibration graph (Figure5).

 

Figure 5: Beer’s Law Plot of SAP-Fe III- K₃[Fe(CN)₆] system

 

RESULTS AND DISCUSSION: 

Optimum operating conditions used in the procedure were established adopting variation of one variable at a time (OVAT) method.  The effect of various parameters such as time, volume and strength of reagents, the order of addition of reagents and solvent for final dilution of the colored species were studied. In method M₁, Na₂CO₃ preferred among other bases like NaOH or Pyridine as they were found to be inferior. Distilled water was found to be best solvent for final dilution. Other water miscible solvents like methanol, ethanol, propan-2-ol and acetonitrile have no additional advantage in increasing the intensity of the color in both methods.  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), 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 the results are summarized in TABLE 1.

 

Table 1: Optical characteristics, precision and accuracy of the proposed methods

Parameters	Method M1	Method M2
λ max(nm)	770	704
Beer’s law limit (µg/ml)	2-6	2.5-12.5
Sandell’s sensitivity (µg/cm2/0.001 abs. unit)	0.000509554	0.002628351
Molar absorptivity (Litre/mole/cm)	616558.625	119531.2127
Regression equation        (Y) *= a +b x
Intercept (a)	-0.106	-0.022
Slope(b)	0.104	0.039
%RSD	2.04	1.22
% Range of errors (95% Confidence limits) 0.05 significance level 0.01 significance level	2.15	1.28
	3.37	2.00

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

 

 

Commercial formulations containing SAP were successfully analyzed by the proposed methods. 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. MS Excel Software-2007 used for calculations.  These results are summarized in Table 2.

 

 

 

TABLE 2: Analysis of SAP in pharmaceutical formulations

*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) developed in our laboratory using methanol solvent

(λ _max=224nm).

 

 

 

Chemistry of Color species:

Method M₁:

The color formation by Folin- Ciocalteu reagent with SAP may be explained basing on the analogy with the reports of earlier workers. The mixed acids in the FC preparation are the final chromogen and involve the following chemical species.

 

3H₂O.P₂O₅.13WO₃.5MoO₃.10H₂O

                                                                  And

3H₂O.P₂O₅.14WO₃.4MoO₃.10H₂O

 

SAP probably effects a reduction of the 1,2 or 3 oxygen atoms from tungstate and /or molybdate in FC preparation (phosphomolybdo tungstate), thereby producing one or more several reduced species which have characteristic intense blue color.

 

Method M₂:

SAP drug exhibits reducing property due to the presence of functional moieties vulnerable to oxidation selectively with oxidizing agents such as Fe (III) under controlled experimental conditions. When treated with known excess of oxidant, SAP undergoes oxidation, giving products of oxidation (inclusive of reduced form of oxidant, Fe II from Fe III) besides unreacted oxidant. It is possible to estimate the drug content colorimetrically, which is equivalent to either reacted oxidant or reduced form of oxidant formed. The reduced form of Fe III (i.e. Fe II) has a tendency to give a colored complex on treatment with hexacyanate ferrate (III).

 

Step-1 :

SAP drug analyte + Fe(III) → oxidation products

+  Fe(II) + Fe(III) (unreacted)

 

Step-2:

3Fe2+ + 2[Fe(CN)6]3- → Fe3[Fe(CN)6]2  (colored species)

 

Scheme for Method M₂

 

 

CONCLUSION:

The proposed methods applicable for the assay of drug are the advantage of wider range under Beer’s law limits; at higher absorption maxima the involvement of excipients in formulation is very less. These methods are validated as per ICH guide lines and possess reasonable precision, accuracy and simple, sensitive. These methods can be extended for the routine assay of SAP formulations.  

ACKNOWLEDGEMENTS:

The authors (MS Bab and BKR) are thanks to the University Grants Commission, New Delhi for providing financial assistance under teacher fellow ship and also thanks to University authorities for providing necessary facilities in this work.

 

 

REFERENCES:

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

3.       http://www.drugbank.ca/drugs/DB00360.

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5.       V.Bhavani, T. Siva Rao, B. Jamelunnisa, Indo American Journal of Pharmaceutical Research 4(7), 3077-3092 (2014).

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

7.       O Folin and D Ciocaleau J. Biol. Chim., 73, 627(1927).

8.       K. Imai, s. Yamada, Kubota, H and Sakami, J. Eur. Pal. Appl., 17 (1964). 

 

 

 

 

Received on 09.02.2017       Modified on 15.02.2017

Accepted on 22.02.2017     ©A&V Publications All right reserved