Spectrofluorimetric protocol for antidepressant drugs in dosage forms and human plasma through derivatization with dansyl chloride

2.1 Apparatus

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  A Perkin Elmer LS 45 Luminescence spectrometer (United Kingdom) connected to an IBM PC computer loaded with the FL WINLAB™ software.

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  Spectronic™ Genesys™ 2PC. Ultraviolet/Visible spectrophotometer (Milton Roy Co, USA) with a matched 1 cm quartz cell connected to IBM computer loaded with winspec™ application software.

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  Milwakee SM 101 pH meter, Portugal.

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  Digital analytical balance (AG 29, Meltter Toledo, Glattbrugg, Switzerland).

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  Laboratory centrifuge 4000 c/s (Bremsen ECCO, Germany).

2.2 Materials and reagents

All the chemicals used throughout this work were of analytical reagent grade and their solutions were prepared with double distilled water. Samples of investigated drugs were generously supplied by their respective manufacturers and were used without further purification.

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  Sertraline hydrochloride (Pfizer Egypt, S.A.E., Cairo, Egypt), fluoxetine hydrochloride (EIPICO, El Asher Ramadan City, Cairo, Egypt), paroxetine hydrochloride (Pharaonia Pharmaceuticals Pharo Pharma, Alexandria, Egypt), amineptine hydrochloride (Servier Egypt Industries Limited, 6th October City, Giza, Egypt) and bupropion hydrochloride (Adwia Co., El Asher Ramadan City, Cairo, Egypt).

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  5-(Dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride), was purchased from Sigma (St. Louis, USA). A stock solution containing 3.71 × 10⁻³ M of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 3.71 × 10⁻⁵ M solution.

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  Sodium carbonate (El Nasr chemical co., Abu Zabbal, Egypt); 0.5 M aqueous solution (pH 10) was prepared by dissolving 13.3 g in 250.0 mL of double distilled water.

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  Acetone was purchased from El Nasr chemical co., Abu Zabbal, Egypt.

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  Plasma was kindly provided by Minia Hospital for psychiatric medicine and the study was performed and permitted according to institutional guidelines. Plasma samples were kept frozen until assay after gentle thawing.

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  Chloroform (Merck, Darmstadt, Germany).

2.3 Pharmaceutical formulations

The following available commercial preparations were analyzed;

Lustral® tablets (Pfizer Egypt, S.A.E., Cairo, Egypt) labeled to contain 50.0 mg sertraline per tablet. Flutin® capsules (EIPICO, El Asher Ramadan City, Cairo, Egypt) labeled to contain 20.0 mg fluoxetine per capsule. Paxetin® tablets (Pharaonia Pharmaceuticals Pharo Pharma, Alexandria, Egypt) labeled to contain 20.0 mg of paroxetine per tablet. Survector® tablets (Servier Egypt Industrial Limited, 6th October City, Giza, Egypt) labeled to contain 100.0 mg of amineptine hydrochloride per tablet. Abstain® tablets (Adwia Co., El Asher Ramadan City, Cairo, Egypt) labeled to contain 150.0 mg of bupropion per tablet.

2.4 Preparation of standard solution

An accurate weight 40.0 mg of hydrochloric salts of each investigated CNS drugs was transferred into a 100-mL separating funnel containing about 20 mL distilled water. The resultant solution was rendered distinctly alkaline by a drop wise addition of 33% w/v aqueous ammonia then the librated free base was extracted with three potions of 20 mL chloroform and the combined chloroformic extracts were filtered through anhydrous sodium sulfate supported on Whitman filter paper into a 100-mL volumetric flak. The filter paper was washed thoroughly with two portions of 5 mL chloroform. The combined extracts and washings were diluted to volume with chloroform to provide a standard solution containing 400.0 μg mL⁻¹. 10.0 mL of chloroform extract was further distilled under vacuum and the oily residue was dissolved in 20.0 mL of mixture of acetone −0.5 M sodium carbonate (3:2) and transferred into a 100 mL volumetric flask then completed to the volume with the same solvent mixture to obtain a standard solution containing 40.0 μg mL⁻¹. This solution was further diluted with the same solvent mixture to prepare working standard solutions containing 0.20–1.40 μg mL⁻¹ (200.0–1400.0 ng mL⁻¹). The standard solutions were stable for 7 days when kept in the refrigerator.

2.5 General procedure

Into a series of 10-mL volumetric flasks, 1.0 mL of working standard solution of drugs was transferred over the cited concentration range of 0.20–1.40 μg mL⁻¹ and 0.7 mL of 3.71 × 10⁻⁵ M of dansyl chloride reagent was added and mixed well. The reaction mixture was left for 25 min, and then completed to the volume with acetone. The fluorescence intensity of the reaction products was measured at 450 nm after excitation at 347 nm. Blank experiment was carried out simultaneously. The relative fluorescence intensity of each sample solution for each investigated drug was accurately measured and plotted against the final concentration of the drug (ng mL⁻¹) to get the calibration graphs.

3.1 Fluorescence spectrum

The fluorescence spectra of the sertraline as a representative example for investigated drugs in its reaction with dansyl chloride forming a highly intense yellow fluorescent derivative with emission at 450 nm after excitation at 347 nm are shown in Fig. 1. All other studied drugs exhibited similar spectra at the same excitation and emission wavelengths.

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Figure 1

Fluorescence spectra where A and B are excitation and emission spectra of blank, while C and D are excitation and emission spectra of sertraline, as a representative example, (140.0 ng mL⁻¹) with dansyl chloride.

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3.2 Optimization of variables

The spectrofluorimetric properties of the fluorescent products as well as the different experimental parameters affecting the development of the reaction product and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. The factors include pH, acetone and sodium carbonate ratio, concentration of the reagent, type of buffer, reaction time and dilution solvent

3.3 Stoichiometry and mechanism of the reaction

The stoichiometry of the reaction mechanism was studied adopting job’s method (Job, 1964) of continuous variation. The molar ratio of dansyl chloride to each of investigated drugs was 1:1. Based on the observed molar reactivity of the reaction, and depending on the presence of secondary amino group and by an analogy to similar reports dealing with the reaction of dansyl chloride with compounds containing secondary amino groups (Pesez and Bartos, 1974), the reaction pathway proposed is presented in Fig. 2.

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Figure 2

Proposed reaction pathway between dansyl chloride and investigated antidepressant drugs.

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3.4 Validation of the proposed method

3.5 Application to pharmaceutical dosage forms

The proposed method was applied for the determination of investigated CNS drugs in commercial pharmaceutical dosage forms. The results of proposed method were statistically compared with those of reported methods (Basavaiah and Sameer, 2010; Darwish, 2005; Mohamed et al., 2007), in respect to accuracy and precision. The obtained mean recovery values of the obtained amount were 99.49–100.67 ± 0.98–1.09%, as shown in Table 7. According to t- and F-tests, no significant difference was found between the calculated and theoretical values of both the proposed and the reported methods at 95% confidence level. This indicates a good level of precision and accuracy.

3.6 Application to spiked human plasma

The high sensitivity attained by the proposed method allowed the determination of the studied drugs in spiked human plasma. The concentrations of investigated CNS drugs were computed from its corresponding regression equations. The obtained mean recovery values of the obtained amount were 97.44–101.61 ± 0.425–1.78%, as shown in Table 8. Thus it can be seen that the proposed method is suitable for the analysis of the investigated CNS in human plasma.

3.7 Analysis of cited drugs in real human plasma

Fluoxetine is metabolized into its active metabolite norfluoxetine (Lemberger et al., 1985). Norfluoxetine concentrations are approximately equal to those of the parent drug during chronic therapy (Brunswick et al., 2002). After a fixed daily dose of fluoxetine (20.0 mg day⁻¹), the concentration of the drug and its active metabolite in the blood continues to grow through the first few weeks of treatment, and their steady concentration in the blood is achieved only after 4 weeks (Pérez et al., 2001). The paroxetine is completely absorbed after oral administration and metabolized in the liver forming three main metabolites: the two isomers (3S,4R)-4-(4-fluorophenyl)-3-[(4-hydroxy-3-methoxyphenoxy)methyl]-piperidine (M1) and (3S,4R)-4-(4-fluorophenyl)-3-[(3-hydroxy-4-methoxyphenoxy)methyl]-piperidine (M2) and (3S,4R)-3-hydroxymethyl-4-(4-fluorophenyl) piperidine (M3) (Hiemke and Hartter, 2000). Steady-state plasma paroxetine concentrations were achieved after approximately 10 days following a 40-mg once daily dose (Mandrioli et al., 2007). The bupropion is mainly metabolized into hydroxybupropion. Steady state plasma level was achieved within 5 days while its metabolites within 8 days in healthy volunteers following a 150-mg dose of the extended-release tablet every 12 h (Briggs et al., 1993). The sertraline is mainly metabolized into N-desmethylsertraline. Steady state plasma concentration level for sertraline and its metabolite was achieved after approximately one week of a 50-mg once-daily dosing (Mandrioli et al., 2006; Package Insert, 1992). Amineptine is mainly metabolized by beta-oxidation of the side chain, its principle metabolites have the same structure as the parent compound except that its side chain is reduced to five carbon atom (Lachatre et al., 1989). Steady state plasma level was achieved at 8th day following two 100.0 mg doses per day (Rop et al., 1990).

% recovery of investigated CNS drugs and their metabolites in plasma was calculated by using the following equation $$\%{\text{Recovery}}_{\text{in vivo}}=({\text{concentration}}_{\text{found}}/{\text{concentration}}_{\text{taken}})\times 100$$ where% recovery_{in vivo} is % recovery for drug in real human sample.Concentration_found is concentration of the drug found in the real human sample.Concentration_taken is concentration of the drug reported in the real human sample.

% Recoveries after application of the proposed method for the determination of the investigated CNS drugs in the real human plasma sample by intra and inter day assay are shown in Table 9.

3.8 Determination of stability constant

The stability constant of the formed product was calculated using the following equation (Sawyer et al., 1984): $$K_s=(A/A_{\mathit{ex}}{C}_x)/[(C_m-A/A_{\mathit{ex}}{C}_x){(C_L-\mathit{nA}/A_{\mathit{ex}}{C}_x)}^n$$ where K_s is the stability constant of the formed product; N = X/(1−X) where X is the mole fraction of the dansyl chloride at the maximum of the continuous variation curve; A/A_ex is the ratio of the observed relative fluorescence intensity to that indicated by the tangent for the same wavelength; C_m is the molar concentration of the dansyl chloride and C_L is the molar concentration of the investigated drugs. $$C_x=C_L/n$$ The calculated stability constants for the formed fluorescent product of the investigated drugs ranged from 33.19 × 10⁷ to 53.54 × 10⁷ as shown in Table 10 indicating good stability of the formed product. The high stability constants of the formed products may account for their high relative fluorescence intensity.