Amlodipine (AML), is a dihydropyridine compound with antihypertensive and antianginal effects with a nomenclature of 3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate, (Fig. 1-a). Various analytical techniques have been reported for determination of AML, either alone or in combination with other drugs, including, spectrophotometric techniques (Attimarad et al. 2019; Erden et al. 2016; Hegazy et al. 2015; Karadurmuş et al. 2018; Khachornsakkul and Dungchai 2020; Mahant et al. 2016; Saad et al. 2016; Siridevi et al. 2019), spectrofluorimetric techniques (Attala et al. 2020; Dinç et al. 2017; El-Kosasy et al. 2015; Ibrahim et al. 2015; Mabrouk et al. 2018; Mohamed et al. 2016; Mohammed 2015; Nasr and Shalan 2020; Sh Shalan et al. 2015; Shereen Shalan and Nasr 2019), electrochemical techniques (Erden et al. 2016; Wei et al. 2016) and chromatographic techniques such as TLC-densitometry (Patel et al. 2020a, b), LC–MS/MS (Rezk and Badr 2020) and HPLC (Alaama et al. 2015; Attimarad et al. 2020; Desai et al. 2019; Duraisamy and Jaganathan 2017; Ebeid et al. 2015; Karadurmuş et al. 2018; Mahant et al. 2016; Nagamani et al. 2020; Naik et al. 2020; Patel et al. Patel et al. 2020a, b; Saleh et al. 2020; Saputri et al. 2018). Perindopril (PER), is an antihypertensive non-sulfhydryl angiotensin converting enzyme (ACE) inhibitor used to treat hypertension and stable coronary artery disease with nomenclature of (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-ethoxy-1-oxopentan-2-yl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (Fig. 1-b). For determination of PER alone or in combination, a variety of analytical techniques have been reported including spectrophotometric techniques (El-Bagary et al. 2016; Hegazy et al. 2015; Karadurmuş et al. 2018; Magdy et al. 2018; Rahman et al. 2017; Saad et al. 2016; Siridevi et al. 2019), spectrofluorimetric techniques (Al-Haj Sakur et al. 2015; Fael and Sakur 2015a, b; Sakur et al. 2015) and chromatographic techniques including TLC-densitometry (Patel et al. 2020a, b), LC–MS/MS (Rezk and Badr 2020) and HPLC (Duraisamy and Jaganathan 2017; Hassan et al. 2020; Hemdan et al. 2018; Karadurmuş et al. 2018; Naik et al. 2020; Patel et al. 2020a, b; Saleh et al. 2020).

Fig. 1
figure 1

Chemical structures of Amlodipine besylate (a), perindopril (b) and Eosin Y(c)

Eosin Y is a sodium salt where the carboxyl and phenolic hydroxyl groups lost their protons and the resultant charge is neutralized by two sodium cations, with nomenclature of (2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen- 9-yl) benzoate) (Fig. 1-c). It has been frequently utilized as histological dye and was applied for estimation the concentration of certain amino compounds by forming ion pair complexes.

The spectrofluorimetric methods are simple, more sensitive than some other analytical methods compared for example with spectrophotometric methods, introduce a chance for estimation of different drugs in biological fluids in very small concentrations and more economic compared for example with chromatographic methods. On the other hand, the spectrofluorimetric methods are limited for certain types of drugs.

Until now, there is no spectrofluorimetric method for determination of the cited drugs via Eosin Y has been published, so in this work a simple, sensitive, validated and cheap spectrofluorimetric method was developed primarily in order to quantify AML and PER in their powders, pharmaceutical formulations and biological fluids.



A Perkin Elmer LS-45 Luminescence spectrometer (UK) was used to get the required emission spectra. It has a xenon flash lamp and a quartz cell of 1 cm, and connected to an IBM PC. A Biochrom Ultrospec 700/700 PC, dual beam, ultraviolet (UV)–visible spectrometer (Cambridge, UK), with 1 cm quartz cells were accustomed. A centrifuge (Bramsen ECCO, Germany) and pH meter were used.


Chemicals and reagents

Without additional purification, all compounds and solvents are of analytical grade. Eosin Y was acquired from Sigma-Aldrich Chemie GmbH, (Steinheim, Germany) and was prepared in distilled water (5 × 10−4 M). Methanol, sodium acetate and acetic acid were obtained from El-Nasr Pharmaceutical Chemicals Co., (Abu-Zabaal, Cairo, Egypt). The acetate buffer is obtained by mixing 0.1 M acetic acid and 0.1 M sodium acetate then completed with distilled water to get pH range of 3.0 to 5.5. Amlodipine of 98.76% purity and Perindopril of 99.45% purity regarding to the reported methods (Basavaiah et al. 2006) and (Suresh 2012), respectively, were kindly provided by Eva Pharma Pharmaceutical Industries, (Giza, Egypt).

Pharmaceutical formulation

Alkapress® oral tablets (batch No: 302, manufactured by Hikma Pharma, Cairo, Egypt) which contain 5 mg of AML per one tablet. Protectopril® oral tablets (batch No: 200282, manufactured by Al Andalous for Pharmaceutical Industries, 6th October City, Egypt) which contain 8 mg of PER per one tablet.

Standard solutions

Ten mg of either AML or PER was accurately weighed, transferred to a volumetric flask of 100-mL and dissolved in 50 mL methanol by shaking. The volume was completed to the line with same solvent to get 0.1 mg/mL stock solution of cited drugs. To get a working solution of 10.0 μg/mL, 10 mL of AML and PER stock solutions (0.1 mg/mL) was accurately transferred into a volumetric flask of 100-mL, and volume was filled up with distilled water.


General analytical procedure

AML and PER solutions were taken from their working solutions (10.0 μg/mL) to a set of arranged volumetric flasks of 10 mL at different concentrations levels from 0.3 to 3.0 μg /mL for AML and 0.2 to 2.0 μg /mL for PER. After that, 1.2 mL of acetate buffer solution (pH 4.4) and 1.0 mL of Eosin Y solution (5 × 10−4 M) were added. Then, the volume was completed with distilled water. After excitation at 425 nm, the fluorescence of the produced solutions was recorded at 544 nm. In their calibration curves, the decrease in fluorescence intensity for the produced solutions were recorded and plotted against both AML and PER concentrations.

Procedure for pharmaceutical formulations

Ten tablets of each Alkapress® tablets (AML) and Protectopril® tablets (PER) were finely ground and thoroughly combined. Amounts of accurately weighed powdered tablets, containing (10 mg) of both AML and PER were transferred to a volumetric flask of 100 mL. A volume of 50 mL methanol was added and sonication for 20 min was performed before cooling and completing the volume with same solvent. After that, filtration was performed to have a stock solution of 0.1 mg/mL of both AML and PER. Then, dilution with methanol was done to get 10.0 μg/mL for both AML and PER solutions. The steps described in the general analytical procedure were followed to estimate AML concentration in Alkapress® tablets and PER concentration in Protectopril® tablets using their corresponding regression equation.

Procedure for spiked human plasma samples

A volume of 5-mL drug free blood sample was drained from the arm of a healthy male volunteer of 35 years into a previously heparinized tube. The separation of needed plasma is done through centrifugation of the obtained blood sample for 20 min at 4000 rpm. Aliquot of 1.0 mL of the separated plasma was taken and 1.0 mL of either standard drug solution having concentration of (50, 100, 150 μg/mL) for AML and (50, 80, 100 μg/mL) for PER, were added, mixed well, stand for 10 min and 2.0 ml acetonitrile was added for deproteinization, then completed to the 10 mL with distilled water. The mixed components were centrifuged at 4000 rpm for 30 min. One mL of the clear supernatant was taken from these solutions and analyzed using the previously described spectrofluorimetric method.

Result and discussion

Eosin is a dye with acidic characters which is commonly used as an ion-pairing tool for the spectrofluorimetric determination of amino compounds via the production of binary complexes. The electrostatic interaction between the cited drugs cationic functional groups and the eosin anion formed at (pH 4.4) resulted in the complex formation. After excitation at 425 nm, the intensity of Eosin Y's fluorescence at 544 nm decreased upon complex formation (Fig. 2).

Fig. 2
figure 2

Excitation and emission spectra at 425 & 544 nm) a & a) of (5 × 10−4 M (eosin Y solution and (b & b) of 5 × 10−4 M Eosin with 1.0 µg/mL AML and (c & c) of (5 × 10−4 M) eosin with 1.0 µg/mL PER after excitation at 425 nm at pH 4.4 using acetate buffer

Experimental parameters optimization

All various parameters that influence the production and stability of the generated complex between the cited drugs and Eosin Y such as: buffer volume, buffer pH, and Eosin volume were studied and optimized.

Buffer pH and volume optimization

The medium had to be acidic to induce ion pair reaction between cited drugs and Eosin Y completely and hence, pH was regarded as an important variable. The pH of acetate buffer solutions of the range of 3.0 to 5.5, were examined but the pH of 4.4 was chosen since it produced the highest ΔRFI readings, (Fig. 3). The effect of various volumes of the acetate buffer (pH 4.4) were investigated (0.5 to 2.0 mL). The best result that showed the greatest ΔRFI values was attained when 1.2 mL of acetate buffer solution was used, (Fig. 4).

Fig. 3
figure 3

Effect of acetate buffer pH on ΔRFI of a 1.5 µg/mL of AML and b 1.0 µg/mL PER

Fig. 4
figure 4

Effect of acetate buffer volume (pH4.4) on ΔRFI of a 2.0 µg/mL of AML and 1.0 µg/mL b PER

Eosin volume optimization

To achieve the highest sensitivity of the proposed method, several volumes of Eosin Y (5 × 10−4 M), (0.5–2.0 mL) were tested. The ΔRFI values showing the highest intensity when1.0 mL of Eosin Y solution was used, (Fig. 5).

Fig. 5
figure 5

Effect of (5 × 10−4 M (eosin Y solution volume on ΔRFI of 2.0 µg/mL of a AML and b PER

Method validation

The described method was validated in accordance with three commendations of International Conference on Harmonization (ICH) (2005).

Linearity of calibration curve

Calibration curves of both AML and PER were built by plotting ΔRFI value versus their corresponding concentrations. Linearity of the described method was recorded in concentration area of 0.3–3.0 µg /mL and 0.2–2.0 µg/mL with 0.9993 and 0.9995 as correlation coefficient for AML and PER, respectively. The parameters of the regression equation that were obtained are shown in Table 1.

Table 1 Regression parameters for analysis of AML and PER by the proposed spectrofluorometric approach

Limits of quantitation and detection

Values of 0.224 and 0.074 µg/mL were calculated as quantitation and detection limits for AML while the values for PER were 0.121 and 0.0399 µg/mL. The lowest values of these limits provide the developed method's highest sensitivity.


The developed method's precision was tested at inter-day and intra-day precision levels by estimating standard solution at three concentrations (0.5, 1.0, and 1.5 µg/mL) for AML and (0.5, 0.8, and 1.0 µg/mL) for PER. Each concentration was measured three times applying the general procedure, through three consecutive days for intra-day or in three different times within the day, for inter-day precision. The acceptable percentage recoveries, SD and RSD values were obtained and displayed in Table 2.

Table 2 The intra-day and inter-day precision for determination of AML and PER by the proposed spectrofluorometric approach


Small variations in method characteristics have no effect on the created method's effectiveness. Buffer solution volume, pH, and Eosin volume were examined. Table 3 shows that the developed method was robust as minor variations in any of these factors had no discernible impact on the method's effectiveness.

Table 3 Robustness study of the proposed spectrofluorometric approach for determination of AML and PER


The suggested method's accuracy was tested by evaluating three concentration levels within the chosen calibration ranges (0.5, 1.0, 1.5 µg/mL) for AML and (0.5, 0.8, 1.0 µg/mL) for PER. For each single concentration, the percentage recovery, SD and RSD were estimated and shown in Table 4. The proposed method was then used to estimate AML and PER in pharmaceutical formulations applying standard addition technique, that involved spiking each of Alkapress® oral tablet and Protectopril® oral tablet solutions with suitable amounts of AML and PER standard solutions and analyzing the solutions as previously mentioned. The concentrations of AML and PER were then calculated using their corresponding regression equations, and percentage recoveries, SD and RSD were calculated. The accuracy of the developed method was proved by the satisfactory results of the standard addition technique, also that results prove that there is no interference of the occurred additives with the drugs concentrations as shown in Table 5.

Table 4 Accuracy of the proposed spectrofluorometric approach at three concentration levels of AML and PER within the specified range
Table 5 Standard addition technique for the determination of AML and PER in their pharmaceutical formulations by the proposed spectrofluorimetric approach

Applications to pharmaceutical formulations

Oral tablets of Alkapress® and Protectopril® were evaluated as described in the general procedure. Accebtable percentage recoveries and standard deviations of 98.96 ± 0.91 and 99.04 ± 0.23 were obtained, which statistically comparable to the values obtained using the reported methods (Basavaiah et al. 2006) and (Suresh 2012) for AML and PER, respectively, using the tabulated t-test and F- test at 95% confidence limit. Table 6 illustrates the acquired t-test and F- test values, which were detected to be lower than the tabulated ones, indicating that the developed method has no discernible change in precision and accuracy between the proposed and the reported methods.

Table 6 statistical comparison of the obtained results using the reported approaches for AML (Basavaiah et al. 2006) and PER (Suresh 2012)

Applications to spiked human plasma

As the proposed method is sensitive, so it was expected to give acceptable recovery in spiked human plasma. The developed method was applied for the analysis of the studied drugs in spiked human plasma by mixing certain volumes of plasma and standard drugs solutions with different concentration levels within the recommended range of AML and PER. The investigated concentration levels were (0.5–1.0–1.5 µg/mL) and (0.5–0.8–1.0 µg/mL) for AML and PER, respectively. Percentage recoveries, SD and RSD of each concentration level show suitability of this method for analysis of AML and PER in spiked human plasma as shown in Table 7.

Table 7 Application of the developed approach for the determination of AMD and PER in spiked human plasma


The aim of that work is to introduce a new spectrofluorimetric method for quantitation of AML and PER in raw material, pharmaceutical formulations and spiking human plasma that is simple, precise, sensitive and time efficient. The developed method is a good technique for quality control of the cited drugs because of its sensitivity and simplicity. The developed method is a more sensitive spectrofluorimetric method than some other existing spectrofluorimetric methods that shown in Table 8.

Table 8 A comparison between the reported method and the proposed method for AML (Sunitha et al. 2016) and PER [Patel et al. 2020a, b)