Spectrofluorimetric determination of adrenaline and dopamine

A sensitive fluorometric method, with few steps and suitable for the daily routine, was made for examining adrenaline hydrochloride and dopamine hydrochloride. The reliance in this paper was on the nucleophilic substitution interaction of the mentioned drugs with 1,2-naphthoquinone sulfonate (NQS) in an aqueous pH 6 to give a fluorescent product with a maximum emission wave at ʎ em 471 nm after being excited at a maximum excitation wave at ʎ ex 300 nm. The plots have complied within the range of 0.01- 4.0, 0.01-2 µg/ml, and The detection limits (0.0062, 0.0027) and quantitation limits were (0.0207, 0.0091) µg/ml, for adrenaline and dopamine respectively. The accuracy (% recovery) was between (99.21% - 100.72%) and the relative standard deviation (RSD%) is better than 0.95%. It was also found that the formed product was in a ratio of 1:2 reagent to the drug. The estimation of adrenaline and dopamine has been successfully tested on the injection, and it is in good agreement with its approved value and with that of the British Pharmacopoeia method.


Introduction
Adrenaline hydrochloride, C9H14ClNO3, (R)-4-(1-hydroxy-2-(methylamino)ethyl)benzene-1,2diol hydrochloride (Figure 1). is classified as a hormone and is secreted by the adrenal gland (adrenaline gland). It was used as a medicine to treat some conditions, such as hypersensitivity caused by taking doses of medicines, especially penicillin, as a pupil dilatant, and to reduce intraocular pressure [ .  Figure 2)] is one of the catecholamine neurotransmitters in the brain. It is classified as a cardiac stimulant, and it is a hormone secreted from the adrenal gland and contributes to the fight-and-flight response processes, and it is called the happiness hormone. The right balance of dopamine in the human body is extremely important and vital, as it plays a role in controlling motor skills and emotional responses, making it essential for physical and mental health. The effect of dopamine lies in the vital areas of the brain, as it affects mood, sleep, study, focus, and learning, so its deficiency may lead to certain diseases, including; Parkinson's and depression [3][4]. Numerous analytical methods are used to determine adrenaline and dopamine, such as chromatographic methods [5][6][7][8], voltammetry [9][10][11][12][13][14], capillary electrophoresis [15], flow injection [16][17][18], ion-selective electrode [19][20][21][22][23], spectrophotometric [24][25][26][27][28][29][30] and spectrofluorimetric [31][32][33][34] methods, It is known that the fluorescence measurement methods are sensitive, so in this paper, an easy method was described to measure the fluorescence of adrenaline and dopamine and it does not require a lot of materials, that is, it is economically feasible in addition to being in an aqueous medium, while most of the methods used expensive organic solvents, which areharmful to the environment.

Experimental Apparatus
The fluorometric measurements were carried out using an RF-5301 PC-Spectrofluorophotometer xenon lamp with transparent quartz cells on all sides with a thickness of 1 cm. Heating processes were also carried out using a nüve NB 20 water bath, the acidity of the solutions was measured using a Philips PW 94 device connected to a CE 10-12 pH electrode, and the weighing processes were carried out using a sensitive balance of the type D0001.A&D Company Limited

Chemical Reagents
The reagents used are all of the analytic reagent grades supplied by BDH, Fluka, and Molekula companies. Standard adrenaline.HCl, dopamine.HCl solution was prepared at a concentration of 100 μg/ml by dissolving 0.0100 g in 2.0 ml of ethanol and then diluting it to the mark with distilled water separately in two volumetric flasks of 100 ml capacity. Then different volumes were taken from them as needed. 1,2-Naphthoquinone-4-Sulfonic Sodium (NQS) 0.5% (w/v), prepared daily by dissolving 0.5 g of it in 100 ml of distilled water. 0.1 M sodium bicarbonate was prepared by dissolving 5.3 g in distilled water and then adjusting the volume to 500 ml in a volumetric flask. Hydrochloric acid was prepared by diluting 1.75 milliliters of acid at a concentration of 11.44 molar in 200 milliliters of distilled water and then taken from it as needed. All surfactants were prepared by dissolving 0.1 g of each one in 100 ml volumetric flasks.

General procedure
Aliquots containing 2 ml of 0.1 M HCl followed by optimal values (0.001-0.4, 0.001-0.2 ml) of adrenaline and dopamine hydrochloride were transferred then 3 and 2 ml of 0.5% NQS, respectively, were added to two sets of 10ml volumetric flasks. The mixture was then diluted to the mark with separately distilled water and kept at 50 °C for 15 mins until fluorescence appeared. At 471 nm the fluorescence of the resulting solution was checked after excitation at 334 nm versus the blank solution.

Pharmaceutical preparation Adrenaline.HCl
A solution of 100 µg/ml was prepared after diluting the content of five epinephrine injections (each injection contains 1.0 mg/1.0 ml of epinephrine (Misr Company)) with distilled water then the volume was completed to 50 ml. Different volumes were taken from it to get concentrations covering the area of the standard plot of adrenaline in its pure form.

Dopamine.HCl
One injection (containing 200 mg/5 ml dopamine hydrochloride, Hospira, INC, LAKE FOREST, USA,) to make volume up to 200 ml was diluted with distilled water to get a solution at a concentration of 1000 μg/ml, from which solution was then prepared at a concentration of 100 µg/ml, different volumes of dopamine were taken within the standard curve range and treated according to the general procedure. The concentration of the medicinal compound in the syringe was found using the standard curve for the medicinal compound in its pure form.

Results and Discussion Preliminary test
The coupling of adrenaline and dopamine with the NQS reagent for the formation of fluorescent products were examined in the presence of each constant amount of hydrochloric acid and sodium hydroxide measured at 471 nm after excitation at 300 nm. It was also found that there is an increase in the intensity of fluorescence when heating the mixture at 50 ° C for a few minutes.

Optimization of conditions Effect of acid and base
To obtain a high fluorescence intensity, the effect of adding several acids and bases (0.1M) such as sodium hydroxide, sodium carbonate, sodium bicarbonate, hydrochloric acid, and acetic acid was examined. Later it was found that hydrochloric acid gave the highest intensity of fluorescence as shown in Table (1). The final pH of the final solution was measured. It was 6.

Effect of buffer solution
To study the effect of buffer solutions such as citrate, phthalates, and phosphate with pH 6, these buffer solutions were examined. A decrease in fluorescence intensity was observed so hydrochloric acid was used instead. The addition of increasing amounts of hydrochloric acid at a concentration of 0.1 M was also studied, and 2 ml was preferable ( Figure. 3), so it was used in subsequent studies.

Effect of NQS amount
In this study, the results of adding different concentrations of NQS to the solution containing a fixed amount of studied drugs were examined, and it is clear that the fluorescence intensity increases with the increase in the concentration of NQS, and this increase reaches a maximum when using 3.2 ml of 0.5% of adrenaline and dopamine reagent respectively ( Figure. 4). Therefore, these quantities were used in later studies.

Temperature and growing time
Fluorescence measurement of the solutions was tracked to determine the reaction time at room temperature and in a thermo-controlled water bath at various temperatures up to 70 °C. The fluorescence of the solutions was measured at 5-min intervals against the similarly treated blank reagent. The fluorescence was observed to peak after 15 min at 50 °C and remained constant for 80 mins, while a decrease in fluorescence intensity was observed with increasing time and temperature ( Figure. 5). Hence, 15 minutes at 50°C was the optimum temperature for this work.

Sequence of addition
To search for high sensitivity, the addition sequence of reagents under optimal conditions was tested. Table 2 shows that sequence III is optimal, so it was adopted in the next study.

Final spectra
The final spectrum for each adrenaline hydrochloride and dopamine hydrochloride with the reagent NQS was taken, which are nucleophilic substitutions. Figure 6 shows the fluorescence spectrum of the two drugs mentioned above.

Calibration plot and results
Calibration graphs were drawn using Optimum experimental conditions by plotting the fluorescence intensity (F) as a function of Adrenaline and dopamine hydrochloride ppm concentrations, Excellent linearity graphs are shown in the range of 0.01-4.0 and 0.01-2 μg/ml for the above drugs, respectively ( Figure 7). The specification of the curve is shown in Table 3.

A B
Concentration, μg/ml

Application of methods
To prove the efficiency of the proposed method and its success in estimating both adrenaline HCl and Dopamine HCl in their pharmaceutical preparations by injection, a comparison was made between the present analytical method and the standard method contained in British pharmacopeia using t and F tests at a confidence level of 95% with six degrees of freedom. By applying the statistical laws, the results of the experimental t-test and the F-test were less than the calculated value (t = 2.45, F = 6.39). This results in the proposed method were free of significant differences in comparison with the standard method. The results of the two methods are shown in Table 4. The nature of complex and the mechanism of reactions To predict the final products, a stoichiometric study of the interaction of adrenaline and dopamine with the NQS reagent was used by the mole ratio method using 1x10 -2 M solutions for each drug and NQS reagent. As shown in (Figures 8 A and B ), the results that 1:2 reagents to 2 of each drugs were formed using both of the above-mentioned methods. This indicates that a nucleophilic substitution product was formed in the weak acidic medium, as shown in the proposed mechanism [35] (Figure9).