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Nonionic surfactants like polysorbates (Tweens) and co-surfactant like propylene glycol are used in pharmaceutical dosage forms, like microemulsion of diazepam. These additives interfere significantly with the estimation of diazepam by UV spectrophotomery method. The aim of this work was to develop a first-order derivative UV-spectrophotometry method that can estimate diazepam in presence of Tween-20 and propylene glycol. The experimental results clearly suggested that, in comparison with the UV-spectrophotometry method, the first-order derivative UV-spectrophotometry is a simple method to estimate diazepam with sufficient accuracy, specificity, and precision even in the presence of 282-times Tween-20 and 2,072-times propylene glycol.
Diazepam, a benzodiazepine derivative (7-chloro-1,3-dihydro-1-methyl-5-phenyl 2H-1,4-benzodiazpein) [CAS:(439-14-5)], is used for managing convulsion/epilepsy, pre-operative medication, and sedation of short time duration and to relieve generalized anxiety and sleep disorder (1). Diazepam is generally available as conventional tablets and intravenous injection. Official compendia suggest liquid chromatographic method for estimation of diazepam in tablets and injectables. Besides the official methods, numerous analytical techniques like gas chromatography (2), HPLC (3,4), capillary electrophoresis (5), TLC–densitometry (6), electrochemical method (7), and flow injection fluorometry (8) have been reported for analysis of the drug. Spectrophotometry methods (9) are also available for assay of diazepam in conventional pharmaceutical dosage forms.
During the last few decades, a great interest has been seen in the development of various novel drug delivery systems. In many of these formulations, surfactants like polysorbates (Tweens) and co-surfactants like propylene glycol and polyethylene glycol (PEG) are used. For instance, a patented method disclosed the use of Tweens, propylene glycol, and PEG in intranasal microemulsion dosage form of diazepam (10). However, as those additives exhibit considerable absorbance at the wavelength of maximum absorbance (λmax) of diazepam UV-spectrometry method cannot be used to estimate diazepam accurately in their presence.
This paper presents first-order derivative UV-spectrophotometry as an accurate, precise, and simple method in comparison to UV-spectrophotometry method for the estimation of diazepam in presence of Tween-20 and propylene glycol.
Diazepam (IP 2007) was obtained as gift sample from Torrent Pharmaceutical Ltd., India. It contains not less than 98.5% and not more than 101.0% diazepam, calculated with reference to the dried substance. Tween-20, propylene glycol (SD FINE-CHEM Limited, Mumbai, India), methanol AR, potassium–dihydrogen–phosphate GR (Merck Ltd., Mumbai, India), and all other reagents were obtained commercially and used as received. Bi-distilled water was used to prepare phosphate buffer solution (pH 7.4; 11), standard solution of drug, and other reagents.
Spectrophotometric measurements were performed on a Varian Cary-50, Bio Spectrophotometer (Varian, Inc.) under the following optimized working settings—scan range, 200 to 400 nm; slit width, 1.5 nm; average time interval, 0.5 s; data interval, 2 nm; scan rate, 240 nm/min.
Derivative spectra were obtained mathematically from the absorbance spectrum with FindGraph, Version 1.924.
Accurately weighed amount of diazepam was dissolved in 5 ml methanol and diluted to 100 ml with phosphate buffer solution. This drug solution was diluted five times to get a stock solution having concentration of 25 μg/ml. Aliquots from this stock solution were further diluted to 25 ml with buffer solution to get standard solutions having concentrations from 1 to 9 μg/ml. Each of these solutions was scanned from 200 to 400 nm against phosphate buffer as blank. Standard solutions were prepared three times a day from the same stock solution (25 μg/ml) and subsequently scanned to get the absorbance value (A) at 2-nm interval. The entire experiment was replicated for three consecutive days.
The absorbance values (A) at various wavelengths (λ; ranging from 200 to 400 nm) of each standard solution was used to calculate the slope of the tangent at each data point and plot of against λ generated the corresponding first-order derivative spectra.
Calibration curves were constructed by linear regression analysis, using the method of least square. The absorbance values (A) at 230 nm and values at 260 nm of five standard solutions were used to construct the calibration curves for UV-spectrophotometry and first-order derivative UV-spectrophotometry methods, respectively.
The standard solutions of diazepam in phosphate buffer prepared for the calibration curves were analyzed by both methods at 0, 2, 4, 6, 24, 48, and 72 h after preparation. The behavior of diazepam remained unchanged during the whole period of study.
Graded volumes (1.5 to 4 ml) of a 20-μg/ml stock solution of diazepam in phosphate buffer, 0.1 ml 100 mM Tween-20 solution, and 0.08ml propylene glycol were taken in 10-ml volumetric flasks, and the volumes were made up to the mark with buffer solution. Each solution was scanned in the range of 200 to 400 nm. The absorbance values (A) at 230 nm and values at 260 nm were used to calculate the concentration of diazepam in presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol from the respective calibration curves. The recovery analysis was triplicated each day for three consecutive days.
Limit of detection (LOD) and limit of quantification (LOQ) of diazepam were determined in presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol. 0.1 ml Tween-20 and 0.08 ml propylene glycol were diluted to 10 ml with phosphate buffer. The absorbance (A) and value of this solution at 230 nm and 260 nm, respectively, were determined against phosphate buffer as blank. These measurements were replicated for 12 times with freshly prepared sample, and the standard deviation (SD) of the determined parameters (A and ) were calculated. Then, the respective calibration curves were used to estimate the LOD and LOQ from the following equations.
The wavelength of maximum absorbance (λmax) of diazepam in phosphate buffer solution (pH 7.4) was found to be 230 nm (Fig. 1). Presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol in standard solution of drug having 3 μg/ml concentration caused λmax to shift from 230 to 228 nm and increased the absorbance value by 38.40%. Such interference was observed throughout the total range of drug concentration studied, although the level of interference decreased with increase in drug concentration (Fig. 2).
The first-order derivative spectra of diazepam consisted of one positive peak at 224.1 nm and two major negative peaks at 238 and 260 nm (Fig. 3). Though the first-order derivative spectrum of diazepam in the presence and absence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol were not superimposed; the change in value at 260 nm was much smaller than the corresponding change in A value at 230 nm. For example, the value at 260 nm of 3 μg/ml diazepam solution was increased by about 10% due to the presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol (Fig. 4). This deviation (10%) was much less than that of absorbance value at 230 nm (38.4%). Moreover, as the concentration of diazepam was increased, the effect of Tween-20 and propylene glycol on value at 260 nm reduced more sharply than absorbance (A) value at 230 nm.
According to ICH guidelines, the essential estimates to establish linearity are (1) correlation coefficient, (2) Y-intercept, (3) slope of regression line, and (4) residual sum of squares. The statistical parameters for the calibration curves obtained by the two methods have been shown in Table I. From the comparative data, it was obvious that the first-order derivative UV-spectrophotometry method provided a calibration curve with r2 value closer to unity, Y-intercept value closer to 0, and residual sum of square about 30,000 times smaller than that obtained from UV-spectrophotometry method. Thus, the concentration of diazepam in phosphate buffer solution was much better correlated linearly with value at 260 nm than the absorbance value at 230 nm.
Since the 95% confidence interval of intercept (Table I) did not include the null point, the calibration curve for first-order derivative spectroscopy method did not pass through the origin. This may be due to “systemic error”.
The recovery analysis of diazepam at four concentration levels in presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol have been shown in Table II. It was evident that, while the range of recovery varied from 98% to 108.5% with a bias of −2% to 8.5% by the first-order derivative UV-spectrophotometry method, the same varied from 110% to 137% with a bias of 10% to 37% by the UV-spectrophotometry method. Thus, the first-order derivative UV-spectrophotometry method was more accurate than the UV-spectrophotometry method.
The first-order derivative UV-spectrophotometry method was found to quantify diazepam at 4 μg/ml concentration in the presence of as much as 282 times Tween-20 and 2,072 times propylene glycol with approximately 104% recovery (i.e., 4% bias) and 0.45% relative standard deviation (RSD). On the other hand, although percent RSD was 0.1359, the bias was approximately 24.25% when quantification was done with UV-spectrophotometry method. High percentage of bias suggests that, in comparison to UV-spectrophotometry method, first-order derivative UV-spectrophotometry method is specific enough to quantify diazepam with acceptable limit of accuracy.
Furthermore, the intermediate precision (ruggedness) of first-order derivative UV-spectrophotometry method was higher as the inter-day percent RSD values (≤1) were less than that of UV-spectrophotometry method (percent RSD≤3).
The intra-day relative standard deviation (percent RSD), representing the repeatability of the analytical methods, are shown in Table III. A small value represents better repeatability. Hence, in comparison to UV-spectrophotometry method, the first-order derivative UV-spectrophotometry method had better repeatability.
The LOD and LOQ for the UV-spectrophotometry method were found to be 0.153 μg/ml and 0.389 μg/ml, respectively. The values of the same parameters for the first-order derivative UV-spectrophotometry method were 0.084 μg/ml and 0.168 μg/ml. Thus, in presence of 1 mM Tween-20 and 0.8% (v/v) propylene glycol, the first-order derivative UV-spectrophotometry method was found to detect and quantify diazepam at much lower concentration than the UV-spectrophotometry method.
UV-spectrophotometry method appeared to be unsuitable for the estimation of diazepam in presence of Tween-20 and propylene glycol used as additives in pharmaceutical formulations. However, the first-order derivative UV-spectrophotometry method can be used to measure diazepam in the presence of those additives with sufficient accuracy and precision. The proposed method is simple, rapid, and economical for routine analysis of diazepam in pharmaceutical laboratories.
We want to express our thanks to University Grants Commission (UGC), New Delhi, India, for financial support. We also gratefully acknowledge Torrent Pharmaceutical Ltd., India for providing gift sample of diazepam.