Simultaneous ultra-sensitive analysis of tamsulosin hydrochloride and tolterodine tartrate binary mixture in their dosage form via high-performance thin-layer chromatography with fluorimetric detection

A simple, sensitive, selective, and stability-indicating high-performance thin-layer chromatography method with fluorescence detection was developed for the analysis of a mixture of tamsulosin hydrochloride and tolterodine tartrate, used in the management of benign prostatic hyperplasia in their laboratory-prepared binary mixture and dosage form. The separation was employed on thin-layer chromatography silica gel 60 aluminum sheets. As developing system, ethyl acetate–n-hexane–diethylamine (9:3:1, V/V) was used. Tamsulosin and tolterodine had retention factors (RF) of 0.40 ± 0.03 and 0.85 ± 0.03, respectively. A 225 nm excitation wavelength was used for the fluorescence detection. Linearity was in the range of 10.0–200.0 and 100.0–900.0 ng/band for tamsulosin and tolterodine, respectively. The method was validated successfully according to the International Council for Harmonisation guidelines. Our suggested method for determining tamsulosin and tolterodine in their dosage forms and in the presence of their degradation products could be conveniently applied in quality-control laboratories.


Introduction
Benign prostatic hyperplasia (BPH) is a prevalent disease in males over 50 years that causes bladder outlet obstruction and lower urinary tract symptoms (LUTS), which can be managed according to the symptoms only [1]. Combining alpha-blockers with muscarinic receptor blockers improves LUTS in men with BPH [2].
The combination of TAM and TOL is given orally in modified-release formulations in a usual dose of 0.4 and 4.0 mg once daily for TAM and TOL, respectively [6].
For the analysis of TAM and TOL in combination, spectrophotometry [25], spectrofluorimetry [9], HPLC [26][27][28], UPLC [29], and only two HPTLC with ultraviolet (UV) detection [30,31] methods were reported. No chromatographic method coupled with fluorescence detection has been reported for the determination of this mixture. This encouraged us to develop and validate a stability-indicating HPTLC method with fluorescence detection for the determination of TAM and TOL in their laboratory-prepared mixture and in the presence of their degradation products. The known advantages of HPTLC analysis with fluorescence detection are the high sensitivity, simplicity, selectivity, speed of analysis, and low sample and solvent consumption compared to HPLC.

Instruments
For densitometric scanning, a TLC Scanner 4 densitometer, model S/N 230826 (CAMAG, Muttenz, Switzerland); CAMAG Linomat 5 auto-sampler with CAMAG microliter syringe (100 µL); 20 cm × 10 cm twin-trough glass chamber (CAMAG Automatic Developing Chamber 2) were used. Optical filter K 320 was used to measure the intensity of the emitted light after excitation at 280 nm by a mercury (Hg) lamp. CAMAG HPTLC software visionCATS Version 3 was used to analyze the peaks. Neutralization steps after stability studies and buffer preparations were done using a digital pH meter (Hanna HI 2211, Sigma Aldrich, St. Louis, MO, USA) equipped with a glass-calomel electrode combination. The dosage form solutions were sonicated using Powersonic 410 micro-process controlled bench-top ultrasonic cleaner (Hwashin, Yeongcheon, South Korea).

Standard stock solutions
Standard stock solutions of TAM (100.0 µg/mL) and TOL (1.0 mg/mL) were prepared separately by transferring 10.0 mg of TAM and 100.0 mg of TOL into 100-mL volumetric flasks, then dissolving and completing the volume to the mark with methanol. The volumetric flasks were protected from light by wrapping them with aluminum foil and stored in a refrigerator.

Standard working solution
By transferring 1 mL from each of the stock solutions into a 10-mL volumetric flask and filling to the mark with methanol, then wrapping with aluminum foil, a working standard solution of the binary mixture of TAM (10.0 µg/mL) and TOL (100.0 µg/mL) was prepared.

Construction of the calibration curve
Using a CAMAG microliter syringe (100 μL), aliquots from the working stock solution were prepared as per the procedure described in Sect. 2.3.2, and spotted in triplicate as bands on TLC silica gel plates to yield a concentration range of 10.0-200.0 ng/band for TAM and 100-900 ng/band for TOL. Table 1 lists the optimal chromatographic conditions for developing and scanning the plates. To obtain the calibration curve and compute the regression equation, the average peak areas were plotted against the final concentrations in ng/band.

Procedure for capsule analysis
The contents of ten Roliflo-OD® caps were weighed and grinded to fine powder. Amounts equivalent to 2.5 mg and 25.0 mg of TAM and TOL, respectively, were transferred to a 25-mL volumetric flask, dissolved in methanol, and sonicated for 15 min, and diluted to the mark with methanol. The prepared solution (0.1/1.0 mg/mL) from TAM and TOL, respectively, was filtered using a disposable syringe filter (0.45 μm), then diluted with methanol to obtain a working stock solution of (10.0/100.0 µg/mL) from TAM and TOL, respectively. Aliquots from this solution were spotted in triplicate as bands on the silica gel plates to give concentration ranges of 30.0, 70.0, 90.0 ng/band of TAM and 300.0, 700.0, 900.0 ng/band of TOL. The plates were developed and scanned under the optimized chromatographic conditions ( Table 1). The content of the capsules was then determined from the previously plotted calibration curve.

Procedures for stability-indicating assay
Forced degradation was performed on the Roliflo-OD® caps. A solution of 0.1/1.0 mg/mL from TAM and TOL, respectively, was prepared as per the procedure described in section Sect. 2.4.2. For each of these tests, 1 mL of this solution was transferred into a series of 10-mL volumetric flasks.  Table 1. The calibration curve was used to determine the nominal contents of the aliquots.

Oxidative degradation 1 mL of 10% (V/V) aqueous
solution of H 2 O 2 was added to the prepared solution mixture in two volumetric flasks, wrapped with aluminum foil, and left at room temperature for 2 h. After the required time, the solution was evaporated in front of a fan to get rid of H 2 O 2 , and the volume was then completed to the mark with methanol and filtered using a syringe filter (0.45 µm). The rest was proceeded as in Sect. 2.4.3.1.

Photolytic degradation
One volumetric flask was exposed to sunlight for one day before being completed to the mark with methanol and filtered using a syringe filter (0.45 µm). The rest of the procedure was performed as described in Sect. 2.4.3.1.

Results and discussion
An ultra-sensitive and stability-indicating analysis of TAM and TOL via HPTLC with fluorimetric detection method was developed and validated with various experimental parameters accurately tested and optimized as shown in Table 1. Fluorescence detection is a sensitive method for determining fluorescing compounds like TAM and TOL in planar chromatography. When compared to UV-visible absorption, fluorescence emission gives better selectivity and sensitivity.

Method development and optimization
Several chromatographic conditions were tried to get the highest sensitivity, greatest difference between the R F values of TAM and TOL, and the best resolution of the peaks over the reported HPTLC methods [30,31].

Mobile phase system
Several solvent systems were tried to ensure sharp symmetric peaks, including different ratios of mixtures of methanol, ethyl acetate, and n-hexane. The medium was rendered alkaline by using ammonia, triethylamine, or diethylamine. The results provided that the best resolution, separation of the binary mixture, was provided by ethyl acetate-n-hexane-diethylamine system with a ratio of 9:3:1 (V/V).

Optimum wavelength selection
Several excitation wavelengths were tried, such as 200, 220, 225, and 280 nm, and it was found that 225 nm gave the highest fluorescence intensity, peak area, and sensitivity of the two drugs.

Validation of the method
The following parameters were evaluated for validation of the proposed method: linearity and range, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, specificity, and robustness, according to the International Council for Harmonisation (ICH) guidelines Q2 (R1) [32].

Linearity and range
The linearity of the proposed method was estimated via constructing the calibration curve by plotting the peak areas against TAM and TOL concentrations in ng/band as shown in Fig. 2. The regression equations were computed,  Table 2. The linearity of the calibration curves was proved by the high value of correlation coefficients and the small value of residual standard deviations as shown in Table 2.

Limit of detection and limit of quantification
LOD and LOQ of TAM and TOL were calculated according to the ICH guidelines, as shown in Table 2, based on the equations: where σ is the residual standard deviation of the response and S is the slope of the curve. LOD = 3.3 ∕S and LOQ = 10 ∕S,

Accuracy
As shown in Table 2, accuracy was calculated as percent relative error. Using the previously published enhanced spectrofluorimetric determination method of TAM and TOL [9], we were able to prove the accuracy of our proposed method. As shown in Table 3, a statistical comparison of the results obtained by our proposed method and those obtained by the reported method using mean recoveries, Student's t test, and variance ratio F test revealed no significant difference between our proposed method and the reported one.

Precision
The precision of the method was determined in terms of intra-and inter-day precision by the replicate analysis of three different concentrations of the pure drugs (30.0, 70.0, 90.0 ng/band for TAM and 300.0, 700.0, 900.0 ng/band for TOL). The aliquots from the working stock solution were prepared as per the procedure described in Sect. 2.3.2 and spotted in triplicate. Each concentration was measured three successive times within one day to prove the intraday precision and on three consecutive days to prove the inter-day precision. The same procedure was done for the capsules but aliquots were prepared as per the procedure described in Sect. 2.4.2. The results are summarized in Tables 4 and 5.

Selectivity
The specificity of the method was evaluated by peak purity of TAM and TOL spectrum in the calibration curve and forced degradation studies using the TLC scanner as shown in Fig. 3. The 3D purity of the peak spectrum was assessed at three levels peak start, peak apex, and peak end. The correlation coefficient was 0.9996 for both drugs. The method was able to determine both drugs in their pure form, pharmaceutical preparations, and in the presence of their degradation products without interference from excipients or degradants.

Robustness
The robustness of the proposed method was assessed upon making minor deliberate changes in the method parameters, including room temperature ± 5 and changing the amounts of solvents in the mobile phase by ± 1 mL for ethyl acetate and ± 0.05 mL for other solvents, scanning wavelength ± 1 nm and saturation time ± 5 min. It was found that there was no significant difference regarding the response.

Application
Our proposed method was able to successfully determine the content of TAM and TOL in the prepared laboratory mixture from their dosage forms, as shown in Table 3. A standard addition technique was used to determine the matrix effect of the excipients, as shown in Tables 6 and 7, and it was found that there was no significant effect of the matrix.

Results of the stability-indicating assay
TAM and TOL were found to be liable to all tested degradation conditions, as shown in Table 8. The degradation of both drugs increased over time. There were no additional peaks for the degradation products as shown in Fig. 4C. It could be concluded that the degradation products have no fluorescence.

Conclusion
A simple, sensitive, selective, and stability-indicating HPTLC method with fluorescence detection could be easily used in quality-control laboratories for the determination of TAM and TOL in their dosage forms and in the presence of their impurities or degradation products without the need of large amounts of sample or sample pre-treatment. The results also show the validity of our proposed method.

Conflict of interest
The authors state that they have no known competing financial interests or personal relationships that would have influenced the work presented in this research.
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