UV Spectrophotometric Method Development and Validation for Estimation of Doxycycline Hydrochloride in Bulk and Capsule Dosage Form

 

Singh Kumar Rakesh¹*, Patel Singh Pankaj¹, Sharma V. K.¹, Roy R. K.²

¹KNGD Modi Institute of Pharmaceutical Education and Research, Modinagar, U. P.-201204

²Dr. K. N. Modi Institute of Pharmaceutical Education and Research, Modinagar, U. P.-201204

*Corresponding Author E-mail: rakeshbluebalance@gmail.com

A novel, simple, sensitive and rapid UV Spectrophotometric method was developed for the estimation of Doxycycline hydrochloride in bulk and its pharmaceutical capsule dosage forms. Doxycycline hydrochloride exhibited maximum absorbance at 271 nm in distilled water and obeyed linearity in the concentration range of 5-35 ppm. The proposed method has been applied successfully for the analysis of Doxycycline hydrochloride either in bulk or pharmaceutical capsule dosage forms with good accuracy and precision. The method herein described can be employed for quality control and routine analysis of Doxycycline hydrochloride in pharmaceutical capsule dosage forms.

 

 

INTRODUCTION:

Doxycycline hydrochloride is, chemically, (4S,4aR,5S,5aR,6R,12aS)-4- dimethylamino-1,4,4a,5,5a,6, 11,12a-octahydro-3,5,10,12, 12a-pentahydroxy-6-methyl-1,11-dioxonaphthacene-2-carboxamide hydrochloride hemiethanolate hemihydrate. The molecular formula is C₂₂H₂₄N₂O₈.HCl, (fig. 1) representing a molecular weight of 480.9 ^[1]. Doxycycline is a member of the tetracycline antibiotics group, and is commonly used to treat a variety of infections. In addition to the general indications for all members of the tetracycline antibiotics group, doxycycline is frequently used to treat Lyme disease, chronic prostatitis, sinusitis, pelvic inflammatory disease, acne, rosacea and rickettsial infections ^[2]. The drug is official in Indian Pharmacopoeia ^[1], British Pharmacopoeia ^[3] and United States Pharmacopoeia ^[4].

 

Fig. 1 Structure of Doxycycline hydrochloride

 

Doxycycline hydrochloride is a pale yellow, crystalline powder, hygroscopic that is freely soluble in water and in methanol; sparingly soluble in ethanol (95%); practically insoluble in chloroform and in ether ^[1].

 

Literature survey revealed that several methods have been proposed for the analysis of doxycycline both in pharmaceutical preparations and biological samples, such as spectrophotometry ^[5-7], fluorimetry ^[8-9], phosphorimetry ^[10], thin-layer chromatography ^[11-13], LC ^[14-16], flow injection analysis ^[17-18], and capillary electrophoresis ^[19]. Hence, an attempt has been made to develop new, simple and sensitive method based on UV spectrophotometry for its estimation either in bulk or pharmaceutical capsule dosage form with good accuracy, simplicity, precision and economy.

 

EXPERIMENTAL:

Instrumentation: Spectral and absorbance measurements were made on UV spectrophotometer. Digital balance (1 mg sensitivity) Sansui was used for weighing the samples. Commercially available capsules of Doxycycline hydrochloride were procured from the local market and estimated. 

 

Chemicals and reagents: -

1	Distilled Water	In-house
2	Doxycycline hydrochloride (98%)	Shreya Life Sciences Pvt. Ltd., Roorkee

 

 

Fig. 2 Spectrum of Standard Doxycycline hydrochloride (20 ppm)

 

Fig. 3 Linearity curve of Doxycycline hydrochloride

 

Apparatus / Instruments: -

Name	Model	Manufacturer
UV spectrophotometer	UV-1800	Shimadzu
Micropipettes	----	Impulse
Tube and micro-pipette tips	----	Eppendorf

 

OPTIMIZATION

Scanning and determination of maximum wavelength (l_max): In order to ascertain the wavelength of maximum absorption (λ _max) of the drug (20 ppm) in distilled water was scanned using UV spectrophotometer within the wavelength region of 200 – 300 nm against distilled water as blank. The resulting spectrum is shown in fig. (2) And the absorption curve showed characteristic absorption maxima at 271 nm for Doxycycline hydrochloride.

 

METHODS

Preparation of standard stock solutions: Standard stock solution was prepared by dissolving 100 mg of Doxycycline hydrochloride in distilled water and volume was made up to 100 ml with distilled water to get concentration of 1mg/ml (1000 ppm) solution.

 

Table 1: Linearity table of Doxycycline hydrochloride in working standard

S. No.	Concentration (ppm)	Absorbance
1.	5	0.147
2.	10	0.288
3.	15	0.427
4.	20	0.536
5.	25	0.666
6.	30	0.817
7.	35	0.956

 

 

Fig. 4 Spectrum of Doxycycline hydrochloride sample (20 ppm)

 

Table (2) Linearity curve data

Beer’s Law limit (ppm)	5-35
Correlation coefficient (R2)	0.99828
Regression equation (y*)	y= 0.02656x + 0.01617
Slope (m)	0.02656
Y-Intercept (c)	0.01617
Chi square	0.00152
Standard error of estimate	0.01306
RSD	0.01192

* y=mx+c where ‘x’ is the concentration of Doxycycline hydrochloride in ppm and y is the absorbance

 

 

Preparation of working standard solutions and construction of standard graph: The prepared stock solution was further diluted with distilled water to get working standard solutions of 5, 10, 15, 20, 25, 30 and 35 ppm of Doxycycline hydrochloride to construct Beer’s law plot for pure drug. The absorbance was measured at λ _max 271 nm, against distill water as blank. The results are shown in table (1). The standard graph was plotted by taking concentration of drug on X-axis and absorbance on Y-axis and is shown in fig (3). The drug has obeyed Beer’s law in the concentration range of 5-35 ppm. The linearity curve data is shown in table (2).

 

 

Table 3: Recovery from the formulation

Formulation	Labeled amount (Doxycycline HCl)  (mg)	UV Spectrophotometry method*
		Mean ± s. d  (amount mg recovered)	% Labeled amount	% RSD
Doxylab (capsules)	108.2	108.67± 0.115	100.46± 0.1039	0.106

* Each value is average of three determinations ± standard deviation.

 

Table 4: Accuracy Readings

Sample ID	Concentration (ppm)		Absorbance	%Recovery of Pure drug	Statistical Analysis
	Pure drug	Formulation
S1 : 80 %	4	5	0.267	104.93	Mean=104.305   SD=0.575   %RSD=0.551
S2 : 80 %	4	5	0.264	103.68
S3 : 80 %	4	5	0.267	104.93
S4 : 80 %	4	5	0.268	105.35
S5 : 80 %	4	5	0.267	104.93
S6 : 80 %	4	5	0.266	104.51
S7 : 100 %	5	5	0.292	103.85	Mean=104.603   SD=0.859   %RSD=0.821
S8 : 100 %	5	5	0.294	104.60
S9 : 100 %	5	5	0.292	103.85
S10 : 100 %	5	5	0.293	104.23
S11 : 100 %	5	5	0.298	106.11
S12 : 100 %	5	5	0.295	104.98
S13 : 120 %	6	5	0.323	105.02	Mean=105.533   SD=0.7099   %RSD=0.673
S14 : 120 %	6	5	0.324	105.36
S15 : 120 %	6	5	0.323	105.02
S16 : 120 %	6	5	0.326	106.05
S17 : 120 %	6	5	0.323	105.02
S18 : 120 %	6	5	0.328	106.73

% Recovery= amount recovered / amount introduced X 100

Preparation of sample stock solutions and working sample solutions:

Twenty capsules were accurately weighed and average was calculated. The capsules were then crushed to obtain fine powder. An accurately weighed quantity of capsule powder equivalent to about 100 mg of Doxycycline hydrochloride was transferred to 100 ml volumetric flask, added 50 ml of distilled water and the contents of the flask were shaken for 15 min. The volume was made up to the mark with distilled water and filtered through Whatman filter paper. The required dilutions were made from sample stock solution. The spectrum of sample is shown in fig. 4 and recovery study from formulation is shown in table (3).

 

VALIDATION

Accuracy:

To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of bulk samples of Doxycycline hydrochloride within the linearity range and added to the preanalyzed formulation of concentration 5 ppm. From that percentage recovery, values were calculated. The results are shown in table (4)..

 

Precision:

The precision of the proposed method was ascertained by actual determination of six replicates of fixed concentration of the drug within the Beer’s range and finding out the absorbance by the proposed method. From this absorbance, mean, standard deviation and % RSD was calculated. The system precision and method precision readings are shown in table 5 (a) and 5 (b) respectively.

 

Table 5 (a): System precision readings

Concentration (ppm)	Absorbance	Statistical analysis
20	0.520	Mean = 0.5195   SD = 0.0005477   %RSD = 0.105
20	0.520
20	0.520
20	0.519
20	0.519
20	0.519

 

Table 5 (b): Method precision readings

Concentration (ppm)	Absorbance	Statistical analysis
20	0.508	Mean = 0.5085   SD = 0.00451   %RSD = 0.8869
20	0.507
20	0.507
20	0.515
20	0.512
20	0.502

 

Limit of detection (LOD): LOD calculation is based on the standard deviation of the response (σ) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula:

LOD = 3.3(σ /S)

LOD=1.481 ppm

 

Limit of Quantitation (LOQ): LOQ calculation is based on the standard deviation of the response (σ) and the slope of the calibration curve (S) at levels approximating the LOQ according to the formula:

LOQ = 10(σ /S)

LOQ = 4.488 ppm

RESULTS AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that Doxycycline hydrochloride obeyed linearity within the concentration range of 5-35 ppm. From the results shown in accuracy table (4), it was found that the percentage recovery values of pure drug from the preanalyzed solution of formulation were in between 103.68 – 106.73, which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method. From the results shown in table 5 (a) and 5 (b) it was found that the % RSD is less than 2, which indicates that the system as well as method has good reproducibility. LOD and LOQ were found to be 1.481 and 4.488 ppm respectively.

 

CONCLUSION:

The proposed method was simple, sensitive and reliable with good accuracy and precision. The proposed method is specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the routine determination of Doxycycline hydrochloride in bulk and pharmaceutical capsule dosage form.

 

ACKNOWLEDGEMENTS:

The authors thank Shreya Life Sciences Pvt.  Ltd., Roorkee for providing the gift sample of Doxycycline hydrochloride. Authors are also thankful to Dr. K. N. Modi foundation, Modinagar, Uttar Pradesh for providing necessary facilities for the work.

 

REFERENCES:

1.       Indian Pharmacopoeia (1996)

2.       Tripathi K. D., “Essential of Medical Pharmacology”, 6^th Edition, Jaypee Brothers Medical Publishers (P) Ltd., pp- 710, 712-715.

3.       The British Pharmacopoeia (2009) Vol. 2 (Her Majesty’s Stationery Office, London) pp- 2139-2142.

4.       The United States Pharmacopoeia 30- NF 25 (2007) pp- 1996.

5.       U. Saha, A.K. Sen, T.K. Das, S.K. Bhowal, Talanta 37 (1990) 1193.

6.       J. L. Lopez Paz, J. Martinez Calatayud, J. Pharm. Biomed. Anal. 11 (1993) 1093.

7.       A. Espinosa-Mansilla, F. Salinas, I. De Orbe Paya, Anal. Chim. Acta 313 (1995) 103.

8.       F. Salinas, A. Munoz de la Pena, I. Duran Meras, Anal. Lett. 23 (1990) 863.

9.       N. Hu, D.-Y. Wang, Y. B. Zhao, J. G. Xu, Fenxi Shiyan Shi 15 (1996) 46.

10.     H. Z. Xie, C. A. Dong, W. J. Jin, Y. S. Wei, C. S. Liu, S. S. Zhang, B. L. Zhou, Anal. Chim. Acta 319 (1996) 239.

11.     W. Naidong, S. Greelen, E. Roets, J. Hoogmartens, J. Pharm. Biomed. Anal. 8 (1990) 891.

12.     W. Naidong, S. Hua, E. Roets, J. Hoogmartens, J. Planar Chromatogr. Mod. TLC 7 (1994) 297.

13.     H.Z. Xie, C.A. Dong, Y.L. Fen, C.S. Liu, Anal. Lett. 30 (1997) 79–90.

14.     P. D. Bryan, J. T. Stewart, J. Pharm. Biomed. Anal. 12 (1994) 675.

15.     S. Croubeks, H. Vermeersch, P. De Backeer, M.D.F. Ssantos, J.P. Remon, C. van Peteyhem, J. Chromatogr. B 708 (1998) 145.

16.     D. Farin, G. Piva, I. Gozlan, R. Kitzes, Chromatographia 47 (1998) 547.

17.     P. Izquierdo, A. Gomez Hens, D. Perez Bendito, Anal. Lett. 27 (1994) 2303.

18.     W. Oungpipat, P. Southwell-Keely, P.W. Alexander, Analyst 120 (1995) 1959.

19.     A. Van Schepdael, R. Kibaya, E. Roets, J. Hoogmartens, Chromatographia 41 (1995) 367.

 

 

Received on 08.04.2013       Modified on 19.04.2013

Accepted on 25.04.2013      © AJRC All right reserved