fast and sensitive hplc method for the determination of ... - wjpps

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Radha et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 3, 1315-1322.

Research Article

ISSN 2278 – 4357

FAST AND SENSITIVE HPLC METHOD FOR THE DETERMINATION OF ZOLPIDEM IN BULK AND FORMULATION S.V.V. Dhanu Radha*, K.M. Ch. Appa Rao and K. Rama Krishna Department of Chemistry, Gitam Institute of Sciences, Gitam University, Visakhapatnam, Andhra Pradesh. ABSTRACT

Article Received on 06 Jan 2016,

A simple, rapid, accurate, precise method has been developed for the

Revised on 26 Jan 2016, Accepted on 16 Feb 2016

estimation of Zolpidem in bulk and formulation using reverse phase high pressure liquid chromatography. The method was developed on C18, HYPERSIL250 mm length, 4.6 mm inner diameter column in an

*Correspondence for Author

isocratic mode using mobile phase containing a ternary mixture of

S.V.V. Dhanu Radha

ammonium acetate, methanol and acetonitrile. (30:45:25, v/v/v) The flow rate and detection wavelength were 1.0ml/min and 248nm

respectively. The retention time of the drug was found to be 3.05min and the method produced was linear in the concentration range of 30-90 µg/ml. KEYWORDS: Benzodiazepines, Zolpidem, HPLC, Ammonium acetate and Acetonitrile. INTRODUCTION Zolpidem is a new generation hypnotic, sedative and psychoactive structurally different from the Benzodiazepines (BZPs) but have the same pharmacological effect. They have a rapid onset of action and a short half-life, unlike BZPs; have weak myore- laxant and anticonvulsant effects used for the treatment of anxiety and sleep disorders.[1] It is selective for the gaba receptor due to its high affinity for this subtype. α 1-subtype is believed to be associated with sedation, i.e used as a positive control in a study of a novel sleep-promoting. Hence Zolpidem is a prescribed drug used for the short term treatment of insomnia, as well as some brain disorders. It is a short-acting non benzodiazepine hypnotic that potentiates gamma-amino butyric acid (GABA), an inhibitory neurotransmitter, by binding to gammaamino butyric acid (GABA) receptors at the same location as benzodiazepines.[2] Literature survey reveals that few analytical methods were reported for the determination of Zolpidem

and

www.wjpps.com

zolpidem

tartarate

in

bulk

and

Vol 5, Issue 3, 2016.

dosage

form

like,

HPTLC[3], 1315

Radha et al.


Spectrophotometric[4-7], HPLC[8-13], pulse voltammetry[14] capillary electrophoresis method for the determination of Zolpidem tartrate in tablet dosage.[15] The present study is to develop a rapid and sensitive HPLC method for Zolpidem in bulk and formulation. MATERIALS AND METHODS Pure sample of Zolpidem was purchased from SMS pharmaceutics, Hyderabad. Ammonium acetate, methanol and acetonitrile used are of HPLC grade purchased from MERCK and double distilled water for the reagent preparation. Zolpidem stock solution contains 100mg of Zolpidem dissolved in 100ml methanol. The stock solution was diluted to different concentrations accordingly for the experiment. Instrumentation The RP-HPLC method was performed using PEAK LC 7000 Series HPLC with LC 7000 software UV Detector. The column chosen was C18, HYPERSIL250 mm length, 4.6 mm inner diameter. METHOD DEVELOPMENT AND OPTIMIZATION OF CHROMATOGRAPHIC CONDITIONS For developing the method, a systematic study of the effect of various factors were undertaken by varying one parameter at a time and keeping all other conditions constant. Method development consists of selecting the appropriate wave length and choice of stationary and mobile phases. The following studies were conducted for this purpose. Detection wavelength The spectrum of 50ppm solution of the Zolpidem in methanol was recorded separately on UV spectrophotometer. The spectra observed its maximum absorbance at 248nm. Choice of stationary phase Preliminary development trials have performed with octadecyl columns with different types, configurations and from different manufacturers. Finally the expected separation and peak shapes were obtained on C18, HYPERSIL 250 mm length, 4.6 mm inner diameter. Selection of the mobile phase In order to get a sharp peak, a number of experiments were carried out by varying the composition of various solvents and flow rate. To have an ideal separation of the drug under

www.wjpps.com

Vol 5, Issue 3, 2016.

1316

Radha et al.


isocratic conditions, mixtures of solvents like ammonium acetate, methanol and acetonitrile in different combinations were tested as mobile phases on a C18 column. A ternary mixture of ammonium acetate, methanol and acetonitrile in the ratio of 30:45:25 v/v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was better defined and resolved and almost free from tailing. Flow rate The flow rate of the mobile phase was fixed as 1.0 mL/min for optimum separation. Table 1 Optimized chromatographic conditions for the estimation of Zolpidem using HPLC. S.No Parameters 1 Column

Chromatographic conditions C18, HYPERSIL250 mm length, 4.6 mm inner diameter

2

Mobile phase

CH3COONH4 : MEOH:ACN 30:45:25

3 4 5

Diluent Elution Wave length

Methanol Isocratic 248 nm

6 7 8 9 10 11 12 13 14

[A.P.I] pH Flow rate Runtime Retention time Area Theoretical plates Tailing factor Pump pressure

60 µg/ml 5.1 1ml/min 6 min 3.05 268027 7784 0.13 17 psi

www.wjpps.com

Vol 5, Issue 3, 2016.

1317

Radha et al.


Fig. 1 Standard chromatogram of Zolpidem. RESULTS AND DISCUSSION The developed method was validated as per standard guidelines.[16-17] The parameters studied for validation were linearity, accuracy, intra and inter precision, limit of detection, limit of quantification, robustness and formulation. Linearity The linearity of an analytical method is its ability to obtain results, which is directly proportional to the concentration of analyte in the sample. It was carried out by preparing the sample solution containing 30-90µg/ml. A calibration curve was drawn by plotting concentration on x-axis Vs area on y-axis.

[ZPM], µg/ml Fig. 2 Standard calibration graph of Zolpidem in linear range.

www.wjpps.com

Vol 5, Issue 3, 2016.

1318

Radha et al.


Table 2 Optical and regression characteristics of the proposed method. S. No 1 2 3 4 5 6 7 Regression equation Correlation coefficient Slope (b) Intercept (a) Average peak area

[ZPM],(µg/ml) Peak area 30 140654 40 182425 50 223445 60 267603 70 317115 80 353306 90 397295 Y = 4397.9x + 4341.8 0.9993 4397.9 4341.8 263883

Accuracy The accuracy of an analytical method was determined by analysis of standard additions at three levels, as recovery studies. Reference standard at three different concentrations (50,100 and 150%) was added to a fixed amount of pre- analyzed sample and the amount of drug was analyzed. Table 3 Accuracy in the assay determination of Zolpidem. S.No %[ZPM] 1 2 3

50 100 150

[ZPM], µg/ml 30 60 90

Recovery (µg/ml) 29.98 59.99 90.31

% of Recovery 99.86 99.98 100.35

Precision Precision is a measure of the random error associated with a series of repeated measurements of the same parameter within a sample. It describes the closeness with which multiple analyses of a given sample agree with each other, and is sometimes referred to as reproducibility. Precision is determined by the absolute standard deviation, relative standard deviation, variance, coefficient of variation, relative percent difference, or the absolute range of a series of measurements. The present method developed was observed the precision in the form of intra and inter precision was evaluated by analyzing the assay of six times and each time duplicate preparations of Zolpidem sample on two different days.

www.wjpps.com

Vol 5, Issue 3, 2016.

1319

Radha et al.


Table 4 Intra and Inter Precision of [ZPM], 60µg/ml. S.No 1 2

Area-1 270335 268444

Area-2 269205 267559

3

271512

271544

4 5 6

268941 268102 269166

269621 267828 270698

%RSD

INTRA=0.475 INTER=0.58

Limit of detection The Limit of Detection is the lowest concentration at which the analyte with certainty can be detected in the sample analyzed with the given method. The analyte could be detected if the concentration is the same or higher than the LOD. The Limit of detection is affected by sample content, volume of injection, solvent used and efficiency of the separation of the peaks. The extraction method may affect the LOD by the different parameters tested in the three- phase system e.g. pH of the donor- and acceptor phase and organic solvent. The presence of unwanted particles and other objects in the sample may also affect the LOD. LOD is determined by measuring the signal to noise ratio(S/N) in the sample with a given concentration where S is the height of the signal and N is the height of the noise. The LOD determined for the present method was 0.1µg/ml. Limit of quantification Limit of Quantification is defined as the lowest concentration analyte that could be determined with an acceptable precision. It is a compromise between the concentration and the precision. The analytical results usually differ more widely when the analyte concentration is low. LOQ is determined the same way as LOD, but the quantification limit is defined as S/N ≥ 10. The LOQ determined for the present method was found to be 0.3µg/ml. Robustness Robustness can be described as the ability to reproduce the analytical method in different laboratories or under different circumstances without the occurrence of unexpected differences in the obtained results. The robustness study was carried out by taking concentration of the sample and changing the mobile phase composition, wavelength and pH. It was observed that the method is unaffected by small changes in experimental conditions with resolution greater than 2.0 confirming robustness of the method.

www.wjpps.com

Vol 5, Issue 3, 2016.

1320

Radha et al.


Table 5 Robustness data. S. No

Parameter

1

Mobile phase

2 3

Wave length pH

Modification CH3COONH4 : MEOH:ACN: 30:35:35 246 nm 5.3

Peak Area

% of change

26.8652

0.39

26.7441 27.2016

0.06 1.65

Formulation The determination of the drug in its dosage form as tablet is assessed. 5mg of ZANLOP tablet was dissolved in 100ml methanol and filtered in 0.45micro membrane filter paper to remove exceptions. A solution of 50µg/ml was injected and estimated the drug concentration in terms of recovery. Table 6 Formulation data. S. No

Tablet

Dosage

[ZNP]µg/ml

1

ZANLOP

5 mg

50

[Estimated] µg/ml 49.92

% of drug estimated 99.84

CONCLUSION The proposed HPLC method for the determination of Zolpidem was found to be simple, sensitive and selective with rapid, reasonable precision and accuracy. All the validated parameters were found to be within the range and measured in terms of % RSD. Thus the method developed can be used as alternative method for the routine determination of the drug in quality control laboratories as it has taken very short run time of around 6min. ACKNOWLEDGEMENT The authors convey thanks to chemistry department, Gitam University for providing necessary facilities and their kind co-operation. REFERENCES 1. Mohler H, Fritschy JM, Rudolph U; A new Benzodiazepine pharmacology; J pharmacol Exp Ther, 2002; 300: 2-S8 2. Vermeeren A. Residual effects of hypnotics: epidemiology and clinical implications. CNS 3. Drugs, 2004; 18: 297-328. 4. Zeany, B.A.E., Moustafa, A.A. and Farid, N.F., J. Pharm. Biomed. Anal.; Determination of zolpidem hemitartrate by quantitative HPTLC and LC, 2003; 33: 393-401.

www.wjpps.com

Vol 5, Issue 3, 2016.

1321

Radha et al.


5. Mathrusri Annapurna M, Sai Pavan Kumar B, Venkatesh B and Raj Prakash J; International Journal of Research in Pharmaceutical and Biomedical Sciences; Spectrophotometric Quantification of Zolpidem Tartrate: Application to Quality Control and Quality Assurance Processes, 2012; 3(3): 1220-1227. 6. Rajiv Chomwal, Amit Kumar and Anju Goyal. Spectrophotometric methods for determination of Zolpidem tartrate in tablet formulation, J. Pharm. Bioall. Sci., 2010; 2(4): 365-368. 7. K.S. Patil, Y.V. Pore and S.B. Bhise; Spectrophotometric Estimation of Zolpidem in tablets; J Pharm sci res., 2010; 2(1): 1-4. 8. Mahajan, M. P; Sawant, S. D; International Journal of Chem Tech Research, 2012; 4(1): 403-408. 9. Saeid Yaripour, Saeed Nezami Rashid, Hesam Alibakhshi and Ali Mohammadi; Development and Validation of a Stability Indicating Reversed Phase HPLC Method for the Quality Control of Zolpidem in Bulk and Tablet Dosage Forms; Journal of Analytical Chemistry, 2015; 70(6): 738–743. 10. S. Ayesha Siddiqua, B. Raj kumar, B. Bharadwaja, S. Priyanka, B. Ramachandrudu, 11. M. Bhagyavathi, M. Aruna; International journal of pharma world research, 2012; 3(1). 12. Malesevic, M., Zivanovic, L., Protic, A. and Jovic, Z.; Chromatographia, 2011; 74: 197. 13. Souri, E., Shirvin, A., Ravari, N.S., Alvandifar, F. and Tehrani, M.B; The Open Conf. Proc. J., 2012; 3: 13. 14. Konoz, E., Mohsen Sarrafi, A.H., Abdolahnejad, R., Bahrami-Zonoz, M; Journal of chemistry, 2013; 21(7): 1-6. 15. Nanthakumar, R., Chitra, K., Vinodhini, C., Reddy, U.M.C.; Indian Drugs, 2010; 47(3): 57-61. 16. Radi, A., Bekhiet, G. and Wahdan, T; Chem. Pharm. Bull; Electrochemical study of zolpidem at glassy carbon electrode and its determination in a tablet dosage form by differential pulse voltammetry, 2004; 52(9): 1063. 17. Al Azzam, K.M., Yit, L.K., Saad, B. Shaibah, H.; Scientia Pharmaceutica, 2014; 82(2): 341-356. 18. ICH Draft Guidelines on Validation of Analytical Procedures (1995), Definitions and Terminology, Federal Register, vol 60, IFPMA, Switzerland, 11260. 19. Validation of compendial methods (2007) The United States, Pharmacopeia, 30th edn. USP30.

www.wjpps.com

Vol 5, Issue 3, 2016.

1322