Multi-Detection Microplate Reader (BioTek Synergy 2, BioTek Instruments Inc., USA) was used for all measurements in 96-microwell plates. A Shimadzu model UV-1601 PC (Kyoto, Japan) UV-VIS double beam spectrophotometer with matched 1-cm quartz cells was used for recording the absorption spectrum. Polypropylene microplates, 96 well with U bottom were obtained from Santa Cruz Biotechnology, Inc. (USA). Finn pipette adjustable 8 channel-pipettes were purchased from Sigma Chemical Co., USA.

Pentoxifylline hydrochloride (POX), (Servier Egypt Industries Limited, Egypt). 1,2-Naphthoquinone-4-sulpho-nic acid (NQS) sodium salt (Aldrich Chemical Co., Milwaukee, USA) was 5 × 10⁻² M aqueous solution. Sodium hydroxide solution was 0.25 M aqueous solution. Trental^® (Servier Egypt Industries Ltd., Egypt), Vasotal^® (T3A, Egypt), and Pexal^® (Mepha, Egypt) tablets are labeled to contain 400 mg of pentoxifylline hydrochloride per tablet. All solvents and other chemicals used throughout this study were of analytical grade.

An accurately weighed amount (60 mg) of POX was quantitatively transferred into a 50-ml calibrated flask, dissolved in 30 ml distilled water, completed to volume with distilled water to produce a stock solution of 1.2 mg/ml. This stock solution was then diluted with distilled water to obtain working standard solutions of 100 - 1200 μg/ml.

Twenty tablets of each formulation were weighed and finely powdered. A quantity of the mixed powder equivalent to 40 mg of POX was transferred into a 50-ml calibrated flask and dissolved in 25 ml distilled water. The contents of the flask were swirled, sonicated for 5 min, and then completed to the mark with distilled water. The contents were mixed well, filtered, and the first portion of the filtrate was rejected. The filtrate solution yield working solutions of 800 μg/ml for analysis.

The Job’s method of continuous variation [25] was employed for determination of the stoichiometry of the reaction between POX and NQS. Master equimolar (3.6 × 10⁻⁴ M) aqueous solutions of POX and NQS were prepared. Series of 5ml portions of the master solutions of POX and NQS were made up comprising different complementary proportions (0:10, 1:9… 9:1, 10:0, inclusive) in 10.0 ml-calibrated flasks. Two milliliters of 0.25 M NaOH was added to each flask, and the solutions were diluted to volume with distilled water and mixed well. The absorbance of the solutions was measured after 10 min at 472 nm against reagent blank treated similarly.

The kinetic data recorded for the designed methods was transformed to the Microsoft Office Excel software, version 2010, for curve fitting, regression analysis, and statistical calculations. In the initial rate method of analysis, the initial rate (V) of the reaction was obtained from the slope of the absorbance-time curve at definite concentration. The calibration curve was constructed by plotting (log V) at different concentrations versus (log C) of POX. Alternatively, the fixed time method was used for constructing the calibration curve by plotting the absorbance measured after fixed time of 10 min vs. the corresponding POX concentrations. The least squares method was used for getting the regression equation.

The following validation parameters were assessed and evaluated according to ICH guidelines [26] . The standard curve for determination of POX was constructed applying the general assay procedure using either the initial rate or fixed time method. After getting the regression equation, the limit of detection (LOD) and limit of quantitation (LOQ) were determined [26] using the formula: LOD or LOQ = k∙SD_a/b, where k = 3.3 for LOD and 10 for LOQ, SD_a is the standard deviation of the intercept, and b is the slope (sensitivity) of the proposed method. Intra- and inter-day accuracy and precision of the proposed methods were assessed by recovery studies at three (low, medium and high) concentration levels. Recovery was determined by the standard addition method. Known amounts of POX were added to the pre-determined drug-containing dosage forms, and then determined by the proposed methods. The mean recovery was determined by dividing the measured concentrations to the concentrations taken for analysis, expressed in percentages. The intra-day precision and accuracy were determined by carrying out the assay of six replicate samples of each concentration as one batch in a single run, while the inter-day precision and accuracy were determined by repeating the assays for the same samples at three different days. The selectivity of the proposed method was studied by testing the interferences liability from the excipients of the dosage forms. Common excipients such as starch, glucose, lactose, acacia, and magnesium stearate were used in this study. The recovery was calculated each time. Ruggedness was also tested by two different analysts at two different elapsed times. Moreover, robustness was evaluated at small variation in the reagents’ concentrations, reaction temperature and reaction time

1,2-Naphthoquinone-4-sulphonate (Folin’s reagent) is frequently used as an analytical reagent for the determination of nucleophilic analytes in weak alkaline medium at normal or slightly elevated temperature. It has been used for the determination of primary amines [21] -[23] , secondary amines [24] , thiols [27] and active methylene containing compounds [28] [29] by spectrophotometry [21] , [23] [24] and spectrofluorometry [22] , [27] after the reduction of the condensation product to the fluorescent 1,2-dihydroxynaphthalene-4-derivative. In the present work, POX was found to react with NQS producing an orange-colored product measured at l_max 472 nm. Figure 1 shows the absorption spectrum of the reaction product of POX with alkaline NQS. The preliminary investigation of the reaction showed that the absorbance of the colored product increased with time to the maximum, then rapidly decreased without a stable period for the measurements. This finding attracted our notice to develop an automated kinetic spectrophotometric assay for the determination of POX to overcome this disadvantage. This can be done by carrying out the reaction in 96-microwell plate as a reaction vessel and measuring the signal with a microplate reader depending on our previous experience [19] [20] . In order to come to this conclusion, the reaction was investigated under various conditions of reagent concentration and alkalinity as well as reaction time and temperature. The following sections describe the conditions under which the reaction of POX with alkaline NQS fulfills the requirements necessary for its spectrophotometric analysis.

In order to investigate the effect of NQS concentration on the reaction product, different concentrations (1.0 - 10 × 10⁻² M) of NQS working solution were tested. The results revealed that the absorbance and the initial reaction rate increased with increasing the concentration of NQS and maximum values were attained when the NQS concentration reached 5 × 10⁻³ M in the final solution (5 × 10⁻² M working solution) as shown in Figure 2. Also, different volumes (0.5 - 4.0 ml) from NQS working solution (5 × 10⁻² M) were tested. The results indicated that 2 ml of 5 × 10⁻² M NQS working solution yielded the highest values. Therefore further testing was performed

using 2 ml of 5 × 10⁻² M solution of NQS using the conventional spectrophotometry. Using the microwell plate format, the results indicated that 50 μl of 5 × 10⁻² M NQS solution in total volume of 200 μl (plate well volume) resulted in the highest absorbance values.

To investigate the effect of sodium hydroxide concentration on the initial reaction rate, different concentrations (0.01 - 1.0 M) of NaOH solution were tested. The results showed that maximum absorbance was obtained when NaOH concentration of 0.025M in the final solution was used (0.25 M working solution) (Figure 2). Furthermore, different volumes (1.0 - 5.0 ml) from 0.25 M working solution were investigated and indicated that 2 ml of 0.25 M NaOH solution in total volume of 10 ml was the ideal concentration. Using the microwell plate format, the results indicated that 50 μl of 0.25 M NaOH solution in total volume of 200 μl gave the highest absorbance values.

Because the formation of the colored product increases with time, we had to optimize the time of the reaction between POX and NQS by monitoring the reaction at different times (0.2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30 min). Figure 3 showed that the highest absorbance values were obtained after 10 min followed by gradual decrease in the absorbance after 12 min. This is a strong disadvantage if we use the conventional spectrophotometry. However, we can overcome this disadvantage by using either the microwell plate format or the kinetic initial rate method for the analysis.

In spite of heating the reaction mixture in the range of 25˚C - 50˚C had slight positive effect on the reaction, however to simplify the procedure, further testing was performed at room temperature (25˚C ± 2˚C). The solvents used for NQS reagent as well as for dilution of the reaction mixture were carefully studied using different solvents: water, methanol, ethanol, isopropanol, and acetonitrile. Water was found to be the optimum solvent as the highest absorbance values were obtained. Table 1 shows the optimum conditions for the reaction between POX and NQS using both the conventional and 96-microwell spectrophotometric methods.

The stoichiometry of the reaction of POX with NQS was determined by the Job’s method of continuous variation [25] . The POX: NQS ratio was found to be 1:1. The POX molecule contains four tertiary amino groups and only one active methylene group. The tertiary amino groups cannot react with NQS, however, the active methylene group can react with alkaline NQS. Therefore, the mechanism of POX-NQS reaction was suggested to proceed according to the pathway given in Figure 4.

Using the general assay procedures (Table 1), the following validation parameters were assessed and evaluated according to ICH guidelines [26] .

Depending on the obtained validation results, the designed procedures (initial rate or fixed time) were found to

^aThe label claim % for Trental, Pexal and Vasotal tablets determined by the reported method [5] were 98.81 ± 1.51, 99.46 ± 1.79, and 98.1 ± 1.77, respectively. Values are mean ± RSD of six determinations. ^bThe tabulated values of t and F at 95% confidence limit are 1.81 and 5.05, respectively.

be suitable for the routine quality control analysis of POX. The concentration of POX was calculated from its corresponding regression equations. The proposed and the reference methods [5] have been tested on commercial tablets. The results obtained by the present methods were statistically compared with those obtained by the reference method with respect to the accuracy and precision. The recovery of the labeled amount was 97.91% - 101.92% ± 0.67% - 3.24% (Table 5). The results of t- and F-tests showed that there are no significant differences between the present and reference methods at 95% confidence level. This proved the applicability of the designed methods for analysis of POX in its pharmaceutical tablets with comparable analytical performance. However, the comparative evaluation and/or recommendations of these methods might be based on the high- throughput value, automation and the precision level of these methods. According to the above consideration, the designed microwell assay might be highly recommended for routine analysis of POX in quality control laboratories.