Development and Validation of a Reversed-Phase HPLCMethod for the Estimation of Zolpidem in Bulk Drug and Tablets

In the present study an isocratic reversed-phase high-performance liquid chromatographymethodwas developed for the estimation of zolpidem in bulk drug and pharmaceutical dosage forms. e quanti�cation was carried out on C18 columns. A mixture of acetonitrile-ammonium acetate (pH = 8.0, 0.02M) (60 : 40 v/v) was used as the mobile phase, at �ow rate of 1.0mL/min and the determination wavelength at 245 nm. e retention time of zolpidem was found to be 3–5min. e validation of the proposed method was carried out for speci�city, linearity, accuracy, precision, limit of detection, limit of quanti�cation, and robustness. e linear dynamic range was from 2.5 to 30 μμgmL. Regression equation was found to be yy = 0.1y1yyy y 0.018y with correlation coefficient rr = 0.rrry. e percentage recovery obtained for zolpidem was greater than 96.5%. Limit of quanti�cation and limit of detection were found to be 2.5 μμgmL and 0.83 μμgmL, respectively. e developed method can be used for routine quality control analysis of zolpidem in tablet formulations.

e free base has a molecular weight of 307.4,with the salt form (zolpidem hemitartrate) having a formula weight of 764.9.e salt has an appearance of a white to off-white powder, with a melting point of 193-197 ∘ C. e tartrate salt is slightly soluble in water (23 g/L at 20 ∘ C), sparingly soluble in methanol, and practically insoluble in methylene chloride.e Ultraviolet (UV) spectrum of zolpidem in 0.1 N HCL exhibits the  maximum of 294 nm [1][2][3][4][5][6].
Zolpidem is not a difficult drug to extract, detect over the therapeutic range, or detect at overdose levels.It has been measured in various biological specimens using a variety of different analytical techniques.Several analytical methods have been reported for quanti�cation of zolpidem in human body �uids and organ samples including capillary electrophoresis (CE) [7], GC [8][9][10], HPLC [1,3,[11][12][13][14][15][16][17], GC/MS [9,18,19], LC/MS [20], LC/MS/MS [21][22][23], and radioimmunoassay [24].In the HPLC methods, the sensitivity of zolpidem is higher with �uorimetric detection than with UV detection [25].Among these technologies, LC/MS and GC/MS are very expensive and are not oen affordable for a common laboratory nowadays, and the derivative procedure made GC difficult to be a robust tool for monitoring zolpidem in many samples.Hence, it is a great selection to develop sufficiently selective and sensitive analytical method in terms of oen used instruments such as HPLC-UV.
e main objective of the present work was to develop simple, fast, inexpensive, sensitive, and accurate method which could be applied to analyses of zolpidem in pure form and in pharmaceutical dosage form.

Experimental
2.1.Equipment.e HPLC apparatus was a Waters chromatographic system 1515 isocratic HPLC pump equipped with an injection valve 20 L and Waters 2487 UV-Visible detector.A reversed phase C 18 column (Waters Xterra, 4.6 × 250 mm, particle size 5 m) was used.e column oven temperature was set at 30 ± 2 ∘ C. Peak area integration was performed using Breez soware version 3.30.
Samples were injected using 25 L Hamilton analytical syringe.

Chemicals and
Reagents.HPLC grade acetonitrile, ammonium acetate (proanalysis), and orthophosphoric acid and ammonium solution 25% (pro analysis) were purchased from Merck (Germany).Triple distilled water from the Milipore equipment was used to prepare all solutions.Authentic samples of zolpidem were obtained from Hexal pharmaceuticals (Germany).
e buffer solution was prepared by dissolving 1.54 g ammonium acetate in 1000 mL of triple distilled water and the pH was adjusted to 8 by addition of ammonium solution 25%.

Preparation of Standard Solutions and
Samples.An accurate weighted zolpidem reference standard was transferred to a 50 mL volumetric �ask and dissolved in mobile phase to make solution of 20 g mL −1 .is solution contains about 0.02 mg of zolpidem RS per mL.
Twenty tablets were �nely powdered and weighed; the average tablet weight was determined and weighed into 25 mL volumetric �ask and dissolved in mobile phase.To dissolve it, the solution was sonicated into an ultrasonic bath for 5 min, and transfer 2 mL of this solution to a 20 mL volumetric �ask, dilute with mobile phase to volume, and mix [12].en this solution that contains 0.02 mg/mL of zolpidem was �ltered through a 0.45 m PTF� syringe �lter (Sartorius) and was used as sample solution.
For process control, 25 L of sample were pipetted accurately into 50 mL volumetric �asks and subsequently diluted to the desired volume with mobile phase.e solutions were sonicated into an ultrasonic bath for 1 min and �ltered through a 0.45 m PTF� syringe �lter (Sartorius) and then 20 L were injected into the HPLC apparatus.to 30 g mL −1 ; solutions were prepared in triplicate.e linearity was evaluated by linear regression analysis, which was calculated by the least square regression method.Before injection of solution, the column was equilibrated for 60 min with mobile phase �owing through the system.ree determinations were carried out for each solution.Peak area was recorded for all the solutions.e correlation graph was constructed by plotting the peak area obtained at the optimum wavelength of detection versus the injected amounts.
2.5.Chromatographic Conditions.e mobile phase was a mixture of acetonitrile/ammonium acetate buffer 0.02 M, pH � 8, (60 : 40 v/v).e �ow rate was 1 mL min −1 .e UV detector wavelength was set at 245 nm and the column oven temperature was set at 30 ± 2 ∘ C.

Results and Discussion
3.1.System Suitability.e chromatographic separation, as explained above, was carried out with HPLC to evaluate the chromatographic parameters including retention factor (), asymmetry of the peak, and tailing factor.Figure 2 showed that no interference peaks were observed in the representative chromatogram.e retention factor () of the zolpidem peak, the tailing factor parameter, and USP Plate Count were 4.4, 1.1 and >7500, respectively.It was concluded that the developed method is the optimum according to the studied parameters.e resulted retention factor was within the range of accepted value (>2).e tailing factor to be controlled was within the limits established by these guidelines.
It can be seen in Figure 2, which shows the chromatogram obtained according to these optimized conditions, good peak asymmetry in a reasonable run time, that is, 4 min.Alternatively, different gradient elution programs using acetonitrile as organic modi�er were tested and used photodiode array detector was published [6].e HPLC analyses were performed using an isocratic mobile phase consisting methanol-acetonitrileand 26 mM sodium acetate buffer (pH 2.0) containing 0.26 mM tetrabutyl ammonium phosphate (13 : 10 : 77 v�v�v) and �ow rate was set at 0.3 mL min −1 in another published paper [12].
erefore, the main objective of the present work can be applied properly to its intended purpose, and compared with past publishedpapers HPLC methods are very easy, simple, fast, and inexpensive.

Stability of the Solution.
Results obtained in the study of stocks solutions of zolpidem in mobile phase were found to be stable for 5 days (120 h), when kept in refrigerator.

Method Validation
4.�.Speci�city.Speci�city described as the ability of a method to discriminate the analyte from all potential interfering substances was evaluated by preparing the analytical placebo and it was con�rmed that the signal measured was caused only by the analytes.A solution of an analytical placebo (containing all the ingredients of the formulation except the analyte) was prepared according to the sample preparation procedure and injected to HPLC.A mixture of the inactive ingredients (placebo) before (Figure 3(a)) and aer being spiked with standards (Figure 3(b)), standard solutions (Figure 3(c)), and the commercial pharmaceutical preparations including zolpidem (Figure 3(d)) were analyzed by the proposed method, to identify the interference by these recipients.e obtained results show that the peak of analytes was pure and recipients in the formulation did not interfere with the analyte.

Accuracy and Recovery Studies.
Accuracy was studied using three different solutions, containing 10, 20, and 30 g mL −1 of zolpidem.Recovery data were reported in Table 1.e obtained values were within the range of 96.45 and 104.3%.

Precision. e precision of an analytical procedure
expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogenous sample under the prescribed conditions.e system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing six replicate analyses of the same working solution.e relative standard deviation (RSD) obtained for zolpidem was 0.46 (Table 1).
e intra-and interday variability or precision of data is summarized in Table 2. e intraday precision of the developed LC method was determined by preparing the tablet samples of the same batch in ten determinations with three concentrations, expressed as a parentage of the label claim, which were used to evaluate the method precision.e intraday precision was also determined by assaying the tablets in triplicate per day for consecutive 3 days.e results indicated the good precision of the developed method (Table 2).
4.4.Linearity.e linearity of the proposed method was estimated by regression analysis at six concentration levels in the range of 2.5-30 g mL −1 .e slope and intercept of calibration curves are shown in Table 4. e calibration curve was obtained by applying linear regression model based on the least square method.

4.�. Limit of �etection (LO�) and Limit of Quanti�cation (LOQ)
. LOD and LOQ of zolpidem were determined by calibration curve method [26].Solutions of zolpidem were prepared in the range of 2.5-30 g mL −1 and injected in triplicate.Average peak area of three analyses were plotted against concentration.e LOD and LOQ were calculated using the following equations: where   is residual variance due to regression;  is slope.e obtained LOD and LOQ for zolpidem were 0.83 and 2.5 g mL −1 , respectively (Table 3).
4.6.Robustness.Robustness relates to the capacity of the method to remain unaffected by small but deliberate variations introduced into the method parameters.Several experimental parameters as changes of pH of the mobile phase, �ow rate, percentage of acetonitrile in the mobile phase, column oven temperature, and detection wavelength were varied around the value set in the method to re�ect changes in different test environments.Analysis was carried out in triplicate and only one parameter was changed in the experiments at a time.e degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 4).

Analysis of Pharmaceutical Preparations.
Developed and validated method was applied to determination of zolpidem in ten different pharmaceutical preparations.ese preparations have the same amount of zolpidem (5 mg).Each pharmaceutical preparation was analyzed by performing eight independent determinations.Satisfactory results were T 2: Intra-and interday assay precision data (  ).obtained for each compound and were found to be in agreement with label claims (Table 5).e proposed method was compared to the UV spectrophotometric method, to verify the results obtained from HPLC.A calibration equation was obtained in the concentration range 2-10 g mL −1 at a wavelength of 245 nm by using methanol as blank.e relation between absorbance () and concentration of zolpidem () was [  11 − ,   ].e tablet dosage from analysis results was found to be 57 ± 7 (mean ± SD;   ) by UV spectrophotometric method.High reproducibility and insig-ni�cant di�erence between the two methods were obtained at the 0.05 probability level according to -and -tests.

Conclusions
A simple, speci�c, linear, precise, and accurate RP-HPLC method has been developed and validated for quantitative determination of zolpidem in tablet formation.e method is very simple and speci�c for zolpidem (chromatograms show no other peaks) and excipient peaks with total run time of 5 min, which make it especially suitable for routine quality control analysis work.Nominal concentration of each company was 20 g mL −1 .a Nominal mg per tab of these companies (Germany) was 10 mg.b Nominal mg per tab of these companies was 5 mg.

2. 4 .F 2 :
Calibration Procedure.e calibration curve was obtained with six concentrations of standard solution 2.5 Chromatograms of a solution containing zolpidem at a concentration of 20 g mL −1 at the described chromatographic conditions.

F 3 :
e chromatograms of (a) tablet excipients (placebo), (b) tablet excipients aer being spiked with standard, (c) standard solutions,and (d) commercial pharmaceutical preparation at the described chromatographic conditions.
a Mean of six determinations.b RSD: relative standard deviation.
Nominal concentration of sample solution was 20 g mL − and tablets were 5 mg.

T 3 :
Results of the data analysis for the quantitative determination of zolpidem by the proposed method.

T 5 :
Data from the analysis of commercial tablets by HPLC and UV method.