In the present work; an accurate and precise TLC densitometric method has been developed for simultaneous determination of PHZ and TMP in their bulk and dosage form. The proposed method based on determination of the UV-visualized bands after TLC separation of PHZ and TMP. The studied drugs were quantitatively separated on 60 F₂₅₄ silica gel plates using mobile phase consists of methanol: toluene: ethyl acetate (1:1:1, by volume)) with UV detection at 228nm over a concentration range of (0.5–6) and (1-10)μg/spot with mean percentage recoveries 99.66±0.984 and 99.72±1.232 for PHZ and TMP, respectively. The proposed method has been validated according to ICH guidelines. The validity of the proposed method was further evaluated by applying standard addition technique. The obtained results were statistically compared with the reported method, showing no significant difference with respect to accuracy and precision.

Keywords: phenazopyridine hydrochloride, trimethoprim, TLC, pharmaceutical analysis

Phenazopyridine hydrochloride (PHZ), [2, 6-diamino-3-(phenylazo) pyridine hydrochloride] Figure 1A exerts an analgesic effect on the mucosa of the urinary tract and provides symptomatic relief of pain in conditions such as cystitis and urethritis. It is given in conjunction with an antibacterial agent for the treatment of urinary tract infections.¹ Trimethoprim (TMP), [5-(3,4,5-Trimethoxybenzyl)pyrimidine-2,4-diamine] Figure 1B exerts antibacterial effect that is used for the treatment of infections due to sensitive organisms, including gastro-enteritis and respiratory tract infections, and in particular for the treatment and prophylaxis of urinary tract infections.¹

Figure 1A Chemical structure of phenazopyridine hydrochloride.

Figure 1B Chemical structure of trimethoprim.

Few spectrophotometric² and chromatographic³ methods have been reported for the determination of a mixture of PHZ and TMP while, multivariate spectrophotometric⁴ and electrochemical⁵ methods were used in combination with sulphamethoxazole in pharmaceutical dosage form. The aim of the present work was to develop simple, rapid, sensitive and selective TLC densitometric^6,7 method for the determination of binary mixture of PHZ and TMP in pharmaceutical formulation.

Instruments

1. Camag Linomat autosampler (Muttenzl, Switzerland), a Camag microsyringe (100μL) and a Camag 35/N/30319 TLC scanner with win CATS software; an ultraviolet (UV) lamp (Desaga, Wiesloch, Germany).
2. Aluminum TLC plates precoated with silica gel 60 GF₂₅₄ (20×20cm), (Merck, Darmstadt, Germany).
3. Chromatographic tank (25×25×9cm).

Pure standard

Standard PHZ (certified to contain 99.94%) and TMP (certified to contain 99.91%) were kindly supplied by kahira for pharmaceuticals, Cairo, Egypt.

Pharmaceutical preparation

Carmurit-T® tablets: (batch number 314467) each tablet is claimed to contain 25mg phenazopyridine hydrochloride and 100mg trimethoprim, manufactured by Memphis company for pharmaceuticals and chemical industries, purchased from local market.

Reagents and solvents
Methanol, acetonitrile, ethyl acetate and toluene, all of analytical grade, (Sigma-Aldrich, Germany).

Standard solutions

A standard solutions of (1mg/ml) was prepared by dissolving 0.1g of PHZ and TMP in 50ml methanol and complete to 100ml with the same solvent.

Construction of calibration curves

Into two separate series of 10 ml volumetric flasks, aliquots of standard solutions equivalent to (0.5-6mg) and (1-10mg) of PHZ and TMP respectively, were transferred separately and diluted to volume with methanol. So the series of flasks contain (50-600)µg/ml and (100-1000) µg/ml of each solution were applied to a TLC plate using Camag Linomat auto sampler with micro syringe (100μl). The plate was then developed by the ascending technique using methanol: toluene: ethyl acetate (1:1:1, by volume) as a mobile phase. The plate was then removed and air-dried. The chromatograms were scanned at 228nm. Calibration curves representing the relationship between integrated peak area and the corresponding concentrations of each drug were plotted.

Application to pharmaceutical formulation

Ten Carmurit-T® tablets were grinded to a fine powder with the help of mortar and pestle. An amount of the powder equivalent to 25mg PHZ and 100mg trimethoprim was transferred into 100-ml volumetric flask, dissolved in methanol, shaken for about 10 minutes and filtered through filter paper to obtain a solution labeled to contain (0.25mg/ml) of PHZ and (1mg/ml) of TMP. Transfer 4 ml into 10-ml volumetric flask then complete to volume with methanol to obtain a solution labeled to contain (100µg/ml) of PHZ and (400µg/ml) of TMP. The proposed method was applied for the determination of the pharmaceutical preparation solution using the procedure described above under the construction of the calibration curve. The cited drugs concentrations were calculated from the corresponding regression equations.

Reported method²

The reported method depends on direct spectrophotometric determination of PHZ at 429nm while TMP was determined by first derivative spectrophotometry and measuring the amplitude at 241nm.

This study is concerned with the simultaneous determination of PHZ and TMP using TLC densitometric method. Various systems were tried, where complete separation of PHZ and TMP was achieved using methanol: toluene: ethyl acetate (1:1:1, by volume) as the mobile phase. The R_f values were 0.68 for TMP and 1.49 for PHZ as shown in Figures 2 & 3.

Figure 2 Two-dimensional densitometric chromatogram of (1) TMP (6µg/spot) and (2) PHZ (4µg/spot) at 228nm.

Figure 3 Three-dimensional densitometric chromatogram of TMP (1–10µg/spot) and PHZ (0.5–6µg/spot) at 228nm.

Method validation

Method validation was performed according to the International Conference on Harmonization (ICH) guideline⁷ for the proposed method as follows:

Linearity and range

TLC-Densitometric technique permits selective determination of PHZ and TMP in the range of 0.5–6 and 1–10μg/spot, respectively. The linear regression equations were calculated and found to be:

y=4605.73 x- 66.22 (r=0.9996) for PHZ at 228nm.

y=3099.62 x-206.19 (r=0.9997) for TMP at 228nm.

Where y is the peak area at 228 nm, x is the drug concentration in µg/spot and r is the correlation coefficient.

Calibration graphs were constructed by plotting the area under peak versus drug concentrations.

Limits of detection and quantitation

The limits of detection (LOD) and the limits of quantitation (LOQ) were determined according to ICH guidelines from the following equations:

 LOD=3.3σ/S LOQ=10σ/S

Where σ is the residual standard deviation of the regression line and S is the slope of the calibration curve. LOD and LOQ values were mentioned in Table 1.

Accuracy

The accuracy of the proposed method was calculated by the average of three determinations for three concentrations for TMP or PHZ repeated three times as mentioned in Table 1.

Precision

The Precision was evaluated by calculating intraday (repeatability) and interday (Intermediate precision) after repeating measuring of the three different concentrations three times in the same day and in three successive days using the proposed method. The calculated RSD% values were listed as mentioned in Table 1 indicating acceptable precision of the proposed method.

Robustness

The robustness of the method was evaluated by slight changes in the chromatographic parameters such as working wavelengths (±2nm) and the mobile phase contents ratio (±2%). In each case only one parameter was changed while other parameters were kept constant. These minor changes did not have any significant effect on the peak area or separation of both drugs and % RSD of the responses were <2% confirming robustness of the procedure, as mentioned in Table 1. The validity of the proposed procedures was assessed by applying the standard addition technique showing no excipients interference as mentioned in Table 2. The results obtained by applying the proposed method were statistically compared with the reported method² and no significant difference was found regarding accuracy and precision as mentioned in Table 3.

In this study; sensitive and selective TLC-densitometric procedure for the simultaneous determination of PHZ and TMP in their pure form and in their pharmaceutical preparation has been developed and validated. The developed method is time saving where many bands can be run at the same time. This method is also economic since a small quantity of mobile phase as a developing system was used unlike HPLC procedures. Finally we can conclude that the described TLC-densitometric procedure can be used in routine analysis of PHZ and TMP in their pure forms and pharmaceutical dosage form without previous separation.

Authors are deeply thankful to ALLAH, the Almighty, by the grace of whom this work was realized. Deep gratitude and appreciation to pharmaceutical analytical chemistry department, faculty of pharmacy Al Azhar University, for providing facilities and continuous support.

The author declares that there is no conflict of interest.

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