Spectrophotometric and Reverse Flow Injection Method Determination of Nitrazepam in Pharmaceuticals Using O-Coumaric Acid as a New Chromogenic Reagent

A spectrophotometricreverse flow injection analysis (rFIA) method has been proposed for the determination of Nitrazepam (NIT) in pure and pharmaceutical preparations. The method is based upon the coupling reaction of NIT with a new reagent O-Coumaric acid (OCA) in the presence of sodium periodate in an aqueous solution. The blue color product was measured at 632 nm. The variation (chemical and physical parameters) related with reverse flow system were estimated. The linearity was over the range 15 450 μg/mL of NIT with detection limits and limit of quantification of 3.425 and 11.417 μg mL -1 NIT,respectively. The sample throughput of 28 samples per hour was achieved. Suggested method was successfully applied for the determination of NIT in its dosage forms.

Also various spectrophotometric methods have been reported for determination of NIT, after reduction by Zn/HCl, such as coupling with different reagents namely, N-(1-napthyl)-ethylene diamine dihydrochloride (14), m-aminophenol (15), acetyl acetone(16), vanillin (17), Metol (18), and phloroglucinol (19). Among the different techniques, the spectrophotometric and flow injection analysis methods are considered the most popular and simple methods existing for quick and trace analysis of different pharmaceutical forms (20)(21)(22). One of the flow injection analysis modes is reverse flow injection analysis (rFIA), in which the reagent is injected into the sample stream which is reversed to the usual FIA when the sample is injected. The main advantages of rFIA mode are the minimization of reagent consumption and waste disposal, improving the analytical sensitivity by enhancing mixing efficiency, and the possibility of making several different determination on the same sample solution stream by injection of different reagents (23,24). The present investigation method offers an improvement of the simple spectrophotometric-rFI technique for the estimation of NIT in both pure and pharmaceutical forms as tablet depending on the oxidative coupling reaction between NIT and O-Coumaric acid as a new chromogenic agent in the presence of sodium periodate in an aqueous medium to produce a blue-colored product.

Materials and Methods: Tools
•A Shimadzu UV-VIS 260 double beam spectrophotometer (Shimadzu, Kyoto-Japan) had been used for absorbance measurements in FIA procedure. The absorbance measurements were carried out using 1 cm path length of quartz flow matched cells (Cecil, 50 µL internal volume).
• A peristaltic pump of six channels (SHENCHEN, CHINA) was used for impelling the reagents and sample solutions using flexible polyvinyl chloride tubes of (0.8 mm i.d.).
• The injection valve of 6 -ways (KNAUER, Germany) with various sample loops was used to injected sample solutions. • Flexible Teflon tubes of (0.5 mm i.d.) were used for reaction coils with different lengths. And Tlinks were used to mix streams of reagents. For rFIA a two-channel manifold was used (Fig. 2), first channel used to transported the solution of (NIT). And the second channel was used to flow sodium periodate solution, while the reagent O-Coumaric acid (OCA) was injected through the injected valve then it was mixed with the stream of drug and oxidant and the mixing completed in the reaction coil. The blue colored was measured at 632 nm.

Chemicals and Reagents
All chemicals used were analytical grade reagents, and distilled water was used for the work. NIT Pharmaceutical grade was supplied from SDI, Samara-Iraq.

NIT Solution (500 µg mL -1 )
The reduction solution of NIT was prepared by dissolving 0.0500 g of NIT in 50 mL of ethanol. Then it was transferred into a beaker and 20 mL of distilled water, 20 mL of 11.65 N hydrochloric acid, and 3.0 g of zinc powder were added. The mixture was allowed to stand for about 15 min at room temperature (25°C). The solution was filtered into a 100 mL volumetric flask, and the residues were washed and diluted to the mark with D.W. Simple dilution was used daily to prepare further diluted solutions.

OCA Solutions (0.05 M)
The solution was prepared daily by diluting 0.8220g of OCA with 100 mL of D.W and then transferred into brown bottle for keeping it out from light. The diluting solutions were prepared with distilled water.

Sodium Periodate NaIO 4 (0.03 M)
This solution was prepared by diluting 0.6416 g of NaIO 4 with100 mL D.W. Working solutions was prepared by suitable dilution by using the same solvent.

Pharmaceutical Solutions
10 tablets were accurately weighed and powdered. Then, an amount of powder equivalent to 50 mg of NIT was dissolved in approximately 30 mL of ethanol. This solution was filtered into a 50 mL volumetric flask and then washed and diluted to the mark with ethanol to obtain 500 μg mL -1 of NIT. The resultant solution was transferred into 125 mL beaker and was reduced as described above. The appropriately diluted solutions of the medicinal formulations were made with distilled water.

Results and Discussion: Preliminary studies
The absorption spectra of the product and the blank were obtained. In a 10-mL volumetric flask a 25 µg mL -1 concentration of reduced NIT, 1 mL of 0.05 M of OCA, and 2 mL of 0.03M NaIO 4 were mixed and the blue -colored product was formed immediately. The flasks were made up to the mark with distilled water. The absorption spectra were recorded between 380 and 1000 nm. The maximum value of absorption was dignified at 632 nm versus the reagent blank (Fig. 3). After studying the reaction stoichiometry of reaction in a batch method using an equimolar concentration of NIT and OCA (0.00177M for both) and applying the mole ratio method and jobs method, it was found that a 1:1 NIT: OCA ratio was obtained. The mechanism of the reaction was summarized by reduction the nitro group of NIT compound in first step (using zinc powder with concentrated HCl) followed by oxidative coupling between reduced drug and OCA in the presence of NaIO 4 as oxidizing agent (25) as shown in Scheme1.The conditional stability constant of the blue colored product was 2914.092M -1 . The Gibbs free energy (ΔG)(26) of the reaction was also determinate to indicate the spontaneously of the reaction ΔG was calculated using the equation (ΔG= -2.303RT log K), where T is the absolute temperature (273+25°C), R is the universal gas constant (19.767 J/mol k). The negative value of ΔG refers to the spontaneously of the current reaction.

Optimization of rFIA System (Chemical and Physical Parameters)
In order to enhance the conditions of the method, preliminary studies were directed toward optimization of the experimental conditions, so as to select the most favorable parameters for the reaction, the absorbance was measured at a wavelength of 632 nm against reagent blank. The preliminary chemical and physical parameters are listed in Table1; the reagent was injected three times for each experiment.

Effect of Chemical Parameters
Different concentrations of the reagent (OCA) was injected (from 0.0005 to 0.15M) to choose the best concentration of the reagent. The results indicated that the concentration of 0.005 M of OCA offered the highest absorbance (Fig. 4). Different concentration of sodium periodate was also studied and optimized using the concentration range between (0.005 to 0.06 M), the concentration of 0.03 M showed the higher absorbance and therefore, it was used in all subsequent experiments (Fig. 5).

Effect of Physical Parameters
The reaction coil lengths effect was examined using variable reaction coils lengths ranged from 0(i.e. without reaction coil) to 250 cm and as shown in Fig. 6, the maximum absorbance and good repeatability were obtained when reaction coil length of 150 cm used. The higher length of reaction coils caused an increase in the dispersion.
The flow rate between 0.4 and 4.1 mL/min was also studied. As the flow rate increased, the absorbance also increased up to 1.8 mL/min (Fig.  7). Increasing the flow rate leads to the absorbance decrease, and this can be attributed to the dispersion. As a result, 1.8 mL/min was selected as the optimum rate. The injected reagent volume was examined by varying the sample loop in the range from 75 -250 µL. Above 100 μL, the absorbance decreased due to the high reagent to sample volume ratio which leads to insufficient ratio and the sample loop of 100 µL was the optimum loop in this work and it was used in further experiments (Fig. 8).
The sampling rate was calculated by recording the period from the injected instant to the appearance of maximum absorbance. The sampling frequency under the optimum conditions (Table 2)

Calibration Curve, Limit of Detection , Accuracy and Precision
A linear calibration curve was achieved over the range 15-450 µg mL -1 of NIT with a limit of detection (LOD) of 3.425µg mL -1 and limit of quantification (LOQ) of 11.417µg mL -1 under the optimal conditions. Table 3 summarizes the statistical analytical treatments for the curve and analytical values, the value of correlation coefficient indicated the high linearity of the suggested method.
To establish the accuracy and precision of the suggested rFIA technique, three different concentrations (five replicates) of NIT were analyzed. The small values of RSD% and Erel% indicated that the method provided good accuracy and repeatability (Table 4).  To assess the efficiency of the method and avoid the restrictions from interference by tablet additives, the determinate of NIT was accomplished by using the standard addition method at two different concentration and the results in Table 5 indicated the high accuracy and precision of the method.

Analytical Applications
The rFIA method was effectively applied for the determination of NIT in commercial pharmaceuticals by the analysis of two different concentrations directly, and the results are given in Table 6. The recoveries of NIT were between 100.693to101.137% which revealed that the current method is accurate and the relative standard deviation was between 0.636to 1.504% which revealed that the current method is repeatable for the determination of NIT in commercial pharmaceuticals. The results obtained from the suggested method were in comparison with those obtained by applying the standard method (UV   *theoretical value, t has degree of freedom = n 1 + n 2 -2= 2, F has degree of freedom = n 1 -1=1.

Conclusion:
The proposed rFIA method found to be sensitive, simple, low cost, and offers the advantages of the rapidity with wide linearity range and high sampling rate. OCA is an excellent choice as reagent due to its readily available and sensitivity In addition, the proposed method is applicable for determinate NIT in pharmaceutical formulations in microgram concentration level. Moreover, the method did not need extraction or heating steps and FIA instruments are available in most laboratories.