INTRODUCTION:
Bromocriptine mesylate (BMC) is a dopamine receptor agonist that selectively activates D2 dopamine receptors while concurrently functioning as a partial antagonist of D1 dopamine receptors. Dopamine agonism exhibits various effects contingent upon the target tissue. Bromocriptine mesylate (BMC) is frequently utilized for the inhibition of prolactin and the suppression of puerperal breastfeeding.
Despite bromocriptine's frequent hypotensive effects, instances of severe hypertension leading to stroke have been documented during the postpartum period. Individuals with pregnancy-induced hypertension have an elevated chance of developing hypertension. The use of bromocriptine for the suppression of lactation is no longer approved by the Food and Drug Administration1. BMC is chemically identified as Ergotaman-3',6',18-trione, 2-bromo-12'-hydroxy-2'-(1-methylethyl)-5'-(2-methylpropyl), (5'α)-monomethanesulfonate (salt) (Figure 1). To our knowledge, there is an official monograph for BMC in the USP pharmacopoeia2.
Figure 1: The chemical structure of bromocriptine mesylate (BMC)
The literature review revealed numerous analytical techniques employed for the detection of BMC in pharmaceuticals and biological fluids, including spectrophotometry3,4, spectrofluorimetry5 chromatography6-10, and electrochemistry11. The approaches are complex, requiring thorough and laborious preparation of samples and careful clean-up procedures prior to analysis. Spectrophotometry is esteemed as the most efficient analytical method in quality control laboratories, hospitals, and pharmaceutical industries owing to its simplicity, low cost, sensitivity, selectivity, accuracy, and precision.
In contrast, visible spectrophotometry is considered as the most convenient analytical technique in most quality control and clinical laboratories, hospitals and pharmaceutical industries for the assay of different classes of drugs in pure, pharmaceutical formulations and biological samples due to its simplicity, reproducibility, speed, less analysis time and reasonable sensitivity with significant economic advantages. An ion-pairs are formed through the electrostatic attraction between positive protonated drug and negative reagent 12-26.
The present investigation aims to develop simple, sensitive and cost-effective approaches for the estimation of BMC in pure form and pharmaceutical formulations using spectrophotometric technique. The methods based on the formation of ion–associate complexes between BMC and the dyes bromocresol purple (BCP), methyl orange (MO), and alizarin red S (ARS) in acidic buffer which is extractable into methylene chloride. The proposed approaches have been statistically validated for accuracy, precision, sensitivity, selectivity, robustness, and ruggedness according to ICH recommendations27.
MATERIALS AND METHODS:
Apparatus:
The absorption spectra were obtained using a Shimadzu UV-1601 UV/visible double beam spectrophotometer from Sweden, which was equipped with a 10mm quartz cell for measuring absorbance. The spectrophotometer offers a wavelength accuracy of ±0.2nm, a scanning speed of 200nm/min, and a bandwidth of 2.0nm within the wavelength range of 200–900nm. The pH values of different buffer solutions were measured using a Hanna pH-meter instrument (pH 211) (Romania) equipped with a combined glass-calomel electrode.
Chemical and reagents:
All chemicals, solvents, and reagents utilized in this study were of analytical reagent or pharmaceutical quality, and all solutions were freshly produced on a regular basis. Pure sample of BMC was kindly supplied by Amoun Pharmaceuticals (El Obour city, Cairo, Egypt), with a purity of 99.90±0.90%. Bromocriptine tablets contain 2.5mg of BMC per tablet and are produced by October Pharma S.A.E., 6th October city, Egypt.
Bromocresol purple (BCP), methyl orange (MO), and alizarine red S (ARS) (BDH Chemicals LTD, Poole, England) and used without further purification. Chloroform, methylene chloride, and carbon tetrachloride were obtained from (BDH Chemicals Ltd., Poole, England) and anhydrous sodium sulfate was obtained from (Prolabo).
Preparation of standard solutions:
Stock standard solutions (100μg mL-1) and (1.0 × 10−3 mol L-1) of BMC were prepared by dissolving 10 and 75.0mg of pure BMC, respectively in the least amount of methanol and further diluted with bidistilled water to the mark in a 100mL volumetric flask. The standard solutions were stable for at least 7.0 days when kept in the refrigerator. Serial dilution with the same solvent was performed to obtain the appropriate concentration range.
Stock solutions of BCP, MO and ARS reagents (0.1%, w/v) and (1.0×10−3mol L-1) were generated by dissolving 0.1g and (0.054, 0.033 and 0.034g of pure solid reagent in 10mL 96% ethanol and diluted to 100 mL with bidistilled water in 100ml a volumetric flask. The solutions remained stable for a minimum of one week when stored in the refrigerator.
Series of buffer solutions of KCl–HCl (pH=1.5-4.2), NaOAc–HCl (pH=1.99-4.92), NaOAc–AcOH (pH=3.0-5.6) and potassium hydrogen phthalate–HCl (pH=2.0-7.0) were prepared by following the standard methods28.
General procedures:
Precisely measured aliquots (0.1–2.4mL) of standard BMC solution (100μg mL-1) were put into 10mL volumetric flasks. 3.0 ml of NaOAc–AcOH buffer at optimal pH levels of 3.5, 4.0 and 3.0 were administered using BCP, MO, and ARS, respectively. Subsequently, 2.0mL of each reagent (0.1%, w/v) was included, and the total volume was adjusted to 10mL using bidistilled water. The ion association complexes were extracted using 10mL of methylene chloride. The solution was agitated for 2.0minutes, then permitted to stand for the distinct separation of the two phases, after which the methylene chloride layer was filtered through anhydrous sodium sulfate. The absorbance of the yellow ion-pair complexes was quantified at 409, 424, and 428nm utilizing BCP, MO, and ARS, respectively, against appropriately produced reagent blanks. All measurements were conducted at ambient temperature. In the three proposed methodologies, a standard curve was established by graphing absorbance values against concentrations of BMC to determine the quantity of medication in unknown analyte samples.
Applications to pharmaceutical formulations:
Twenty tablets containing BMC were meticulously ground and weighed. A measured amount of powdered tablets corresponding to 10mg of BMC was placed into a 100mL volumetric flask, about 20mL of ethanol was added, and the flask was subjected to sonication for 30 minutes. The volume was adjusted to the mark using bidistilled water, well mixed, and filtered through Whatman No.1 filter paper into a 100mL volumetric flask, discarding the initial 10mL. Subsequently, the conical flask was rinsed with bidistilled water. The wash was included into the same volumetric flask, which was subsequently filled to the designated volume with bidistilled water. Aliquots with BMC at the specified final concentration ranges were examined according to the "General recommended procedure." The concentration of BMC was ascertained either from the calibration curve or by employing the relevant regression equation. The conventional addition method was employed for the precise quantification of BMC content.
Stoichiometric relationship:
The stoichiometric ratios of the ion-associates produced between the investigated medications and the reagents were established using the continuous variation29 and molar ratio30 methods at the wavelengths of maximum absorbance. In the continuous variation approach, equimolar solutions were utilized: a 1.0 x 10-3 mol L-1 standard solution of BMC and a 1.0 x 10-3 mol L-1 solution of dye were employed. A series of solutions was produced in which the total volume of the examined medications and the dye was maintained at 2.0mL. The drug and reagent were combined in diverse complementary ratios (0:2, 0.2:1.8, 0.4:1.6, …, 2:0, inclusive) and brought to volume in a 10mL calibrated flask with the suitable solvent for extraction, adhering to the aforementioned protocol. In the molar ratio approach, the concentration of BMC is maintained at 1.0 mL of (1.0 x 10-3 mol L-1), while the concentration of dye (1.0 x 10-3 mol L-1) is systematically varied between 0.2 and 2.4mL. The absorbance of the produced solutions was assessed at the optimal wavelength for each complex.
RESULTS AND DISCUSSIONS:
Absorption spectra:
The suggested approaches rely on the reactivity of the secondary amine group of BMC with acid dyes (BCP, MO, and ARS). BMC generates an ion-association complex with acid dyes that may be extracted into dichloromethane from the aqueous phase. The protonated nitrogen of BMC is anticipated to attract the negatively charged components of sulphonpthalein anionic dyes in an acidic buffer solution at pH ≥ 3.0, functioning as a cohesive unit due to electrostatic attraction within the pH range of 2.5-5.0. This interaction results in the formation of a yellow ion-pair complex that can be extracted with an organic solvent. The absorbance spectra of the ion-pair complexes produced between BMC and reagents were recorded in the region of 350–550nm relative to the blank solution. The ion-pair complexes of BMC with BCP, MO, and ARS exhibit peak absorbances at 409, 424 and 428nm, respectively (Figure 2).
Figure 2: Absorption spectra of ion-pair complexes of 16, 24 and 20 µg mL-1 BMC using (0.1 %, w/v) BCP, MO and ARS reagents, respectively against reagent blank.
Optimum reaction conditions for complex formation:
The procedures were meticulously optimized to attain complete reaction production, optimal sensitivity, and maximal absorbance. The reaction conditions for the ion-pair complex were determined through preliminary studies that examined factors such as buffer pH, organic solvent type, dye quantities, reaction duration, and temperature for the extraction of ion-pair complexes.
Effects of pH on ion-pair formation:
The successful extraction of the complex is contingent upon the type of buffer employed and its pH value. The influence of pH was examined by isolating the colored complexes using several buffers, including NaOAc–HCl (pH=2.0-4.5), NaOAc–AcOH (pH=2.8-5.5), and potassium hydrogen phthalate–HCl (pH=3.0-6.0). The highest color intensity and absorbance were observed in the NaOAc-AcOH buffer. The maximal absorbances of the ion pair complexes between BMC and the reagents were observed at pH 3.5, 4.0 and 3.0 utilizing BCP, MO, and ARS techniques, respectively (Figure 3(a)). The buffer volume was ascertained by conducting the same experiment while systematically varying the volume from 0.5 to 5.0mL. A greater absorbance value and consistent results were achieved using 3.0mL of acetate buffer solutions.
Figure 3: Effect of (a) pH of buffer solution; (b) extraction solvent; (c) volume of (0.1%, w/v) reagent; (d) time on the ion pair complex formation between (16, 10 and 20μg mL-1) BMC and (0.1%, w/v) BCP, MO and ARS reagents, respectively, (n=3.0).
Effect of extracting solvents:
The impact of various organic solvents, including chloroform, carbon tetrachloride, methanol, ethanol, acetonitrile, n-butanol, benzene, acetone, ethyl acetate, diethyl ether, toluene, dichloromethane, and chlorobenzene, was examined for the efficient extraction of colorful species from the aqueous phase. Dichloromethane was identified as the best appropriate solvent for the quantitative extraction of colored ion pair complexes utilizing the examined reagents (Figure 3(b)). Experimental results demonstrated that twofold extraction with a total volume of 10 mL provided the optimal solvent, giving highest absorbance intensity, steady absorbance for BMC, significantly lower extraction capability for the reagent blank, and the quickest time to achieve equilibrium between both phases.
Effect of reagent concentration:
The impact of the reagents was analyzed by quantifying the absorbances of solutions with a constant concentration of BMC and varying volumes of the corresponding reagents (0.1%, w/v) within the range of 0.5 to 4.0mL. The results indicated that the absorbance of the extracted ion-pair rose with the reagent amount up to 2.0mL. The optimal color intensity of the complex was attained with 2.0mL of each reagent solution at a concentration of 0.1% (w/v). A greater amount of the reagent did not significantly affect the absorbance of the resulting ion-pair complexes (Figure 3(c)).
Effect of shaking time and temperature:
The optimal reaction time was examined from 0.5 to 5.0 minutes by monitoring the color development at ambient temperature (25±2°C). The maximum and consistent absorbance values were achieved after 2.0 minutes of shaking for all combinations (Figure 3(d)). Consequently, a shaking duration of 2.0 minutes was upheld for the entirety of the experiment. The influence of temperature on colored complexes was examined by assessing the absorbance values within the temperature range of 20-35°C. The absorbance of the colored ion pair complex remained consistent up to 30°C. At elevated temperatures, the concentration of the medication was shown to rise owing to the volatile characteristics of dichloromethane. Consequently, room temperature (25±2°C) was selected as the optimal temperature for the analysis of the investigated medicines in bulk and pharmaceutical formulations. The absorbance of the complexes remains constant for a minimum of 10hours at ambient temperature.
Stoichiometric ratio:
The molar ratio of the reagents (BMC: dye) in the ion-pair complexes was determined by the continuous variations (Job’s method) (Figure 4(a)) and the molar ratio method (Figure 4(b)). The results indicate that 1:1 for (BMC: dye) ion-pairs are formed through the electrostatic attraction between positive protonated BMC+ and negative BCP−, MO−, or ARS−. The extraction equilibrium can be represented as follows:
Where BMC + and D− represent the protonated BMC and the anion of the dye, respectively, and the subcript (aq) and (org) refer to the aqueous and organic phases, respectively.
Figure 4: (a) Job’s method of continuous variation graph (b) Mole ratio plots for the reaction of BMC with BCP, MO and ARS, [drug] = [dye] = (1.0×10−4 mol L-1).
Method Validation:
Linearity and sensitivity:
The absorbance-concentration relationship for BMC medicine demonstrated linearity across the concentration ranges of 1.0–16, 1.0–24, and 1.0–20μg mL-1 when employing BCP, MO, and ARS, respectively, under the specified experimental circumstances. The calibration graph is expressed by the equation (A = a + b C), where A represents absorbance, a designates the intercept, b identifies the slope, and C specifies concentration in μg mL-1. This equation is derived using the least squares approach. Table 1 displays the correlation coefficient, intercept, and slope for the calibration dataset. The molar absorptivity of the colored ion-pair complexes and the relative standard deviation were computed and displayed in Table 1.
The ICH guideline17 delineates multiple approaches for determining the limits of detection (LOD) and quantitation (LOQ). The factors include visual inspection, signal-to-noise ratio, and the application of standard deviation of the response along with the gradient of the calibration curve. The LOD and LOQ of the approach were determined by injecting progressively smaller quantities of reference solutions with the developed technique. The LOD and LOQ) for the suggested approaches were determined using a specific equation 27, 31:
LOD = 3s / k and LOQ = 10 s / k
where s is the standard deviation of ten replicate determinations values of the reagent blank or the standard deviation of intercepts of regression lines and k is the sensitivity, namely the slope of the calibration graph. In accordance with the formula, LODs were found to be 0.30, 0.28 and 0.30 μg mL-1 and LOQ were found to be 1.0, 0.93 and 1.0 μg mL-1 using BCP, MO and ARS, respectively.
Table 1. Statistical analysis and analytical data in the determination of BMC using the proposed methods.
|
Parameters |
BCP |
MO |
ARS |
|
Wavelengths λ max (nm) |
409 |
424 |
428 |
|
Beer’s law limits (μg mL-1) |
1.0-16 |
1.0-24 |
1.0-20 |
|
Molar absorptivity ε, (l mol-1 cm-1) x 104 |
2.7988 |
1.9907 |
1.6679 |
|
Sandell,s sensitivity (ng cm-2) |
26.82 |
37.71 |
45.01 |
|
Regression equation a |
|
|
|
|
Intercept (a) |
0.0078 |
0.0121 |
0.0026 |
|
Standard deviation of intercept (Sa) |
0.055 |
0.083 |
0.029 |
|
Slope (b) |
0.0349 |
0.0283 |
0.022 |
|
Standard deviation of slope (Sb) |
0.042 |
0.064 |
0.061 |
|
Correlation coefficient (r) |
0.9996 |
0.9995 |
0.9995 |
|
LOD (μg mL-1) b |
0.30 |
0.28 |
0.30 |
|
LOQ (μg mL-1) b |
1.0 |
0.93 |
1.0 |
|
Mean ± SD |
99.50 ± 1.20 |
99.40 ± 0.85 |
100.30± 1.30 |
|
Relative standard deviation; RSD% b |
1.20 |
0.85 |
1.30 |
|
Relative error, RE% b |
1.26 |
0.89 |
1.37 |
|
Variance |
1.44 |
0.72 |
1.69 |
|
t-test c |
0.60 |
0.9 |
0.57 |
|
F- test c |
1.78 |
1.12 |
2.09 |
aA = a + bC, where C is the concentration in μg mL-1, A is the absorbance units, a is the intercept, b is the slope.
bSD, standard deviation; RSD%, percentage relative standard deviation; RE%, percentage relative error.
c The theoretical values of t and F at P= 0.05 are 2.571and 5.05, respectively, at confidence limit at 95% confidence level and five degrees of freedom (p= 0.05).
Accuracy and Precision:
The accuracy of the proposed approach was evaluated by examining three different concentrations of BMC within the linearity range across six replicates. Accuracy was measured using recovery percentage and percent relative error (RE%) for BMC, whilst precision was evaluated by relative standard deviation (RSD%). Intra-day precision was assessed on a single day, whereas inter-day precision was evaluated on three distinct days (n=6 for each level). The results of this investigation are presented in Table 2. The minimal values of the relative standard deviation (RSD%) and percentage relative error (RE%) indicate a high degree of precision and accuracy in the proposed methodologies.