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 RF 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, and the analytical data of the calibration curves are listed in 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.

Fig. 2

A 3D densitogram of tamsulosin hydrochloride and tolterodine tartrate using fluorescence detection mode. B Calibration curve of tamsulosin hydrochloride (10.0–200.0 ng/band). C Calibration curve of tolterodine tartrate (100.0–900.0 ng/band)

Table 2 Regression parameters obtained from the calibration curves of tamsulosin hydrochloride and tolterodine tartrateLimit 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:

$$} = .\;} \right. \kern-\nulldelimiterspace} S}\;}\;} = 0\;} \right. \kern-\nulldelimiterspace} S},$$

where σ is the residual standard deviation of the response and S is the slope of the curve.

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.

Table 3 Statistical analysis of the results of our proposed method of tamsulosin hydrochloride and tolterodine tartrate in pure form, compared with the reported spectrofluorimetric methodPrecision

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 intra-day 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.

Table 4 Repeatability and reproducibility of the proposed HPTLC method for the determination of tamsulosin hydrochloride in pure and dosage formTable 5 Repeatability and reproducibility of the proposed HPTLC method for the determination of tolterodine tartrate in pure and dosage formSelectivity

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.

Fig. 3

A Tamsulosin hydrochloride and B tolterodine tartrate overlaid UV spectra, recorded using the HPTLC scanner

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.

Table 6 Results of the proposed HPTLC method for the determination of tamsulosin hydrochloride in its dosage form and results of standard addition techniqueTable 7 Results of the proposed HPTLC method for the determination of tolterodine tartrate in its dosage form and results of standard addition techniqueResults 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.

Table 8 Summary of the results of stability studiesFig. 4

HPTLC densitograms of A tamsulosin hydrochloride and tolterodine tartrate reference standard; B tamsulosin hydrochloride and tolterodine tartrate dosage form, C alkaline degradation