Utilization of cloud point extraction for enhancement the efficiency of spectrophotometric estimation of milnacipran HCl as anti-depression drug in dosage forms and application to its tablets uniformity testing

Two spectrophotometric techniques that are straightforward, sensitive, accurate, and precise have been created and validated for the detection of a particular antidepressant drug, milnacipran HCl (MCN), in both pure and dosage forms. The method A was based on the reaction of MCN with p-dimethylaminobenzaldehyde (p-DMAB) in an acidic medium, to give a yellow colored Schiff’s base product that can be measured at a maximum wavelength of 400 nm. The concentration range of 60–300 μg/mL, with an excellent correlation coefficient (r2 = 0.9991) and with a relative standard deviation (RSD% = 2.0), complies with Beer’s law under ideal circumstances. The method B approach uses a cloud point extraction (CPE) methodology to measure the amount of the yellow color product utilizing Triton X-114 and cetyltrimethylammonium bromide (CTAB) as surfactants at a maximum wavelength of 410 nm. Beer’s law was obey in the concentration range of 0.2–4.0 μg mL−1, a correlation coefficient (r2 = 0.9995) and a relative standard deviation of (RSD% = 1.70). The best optimized reaction circumstances, including the type of solvent, reagent concentration, reaction time, and molar ratio. Calculations were made about the detection and quantification limits. No interference was seen with the frequently used excipients and additives. The proposed methods for determining MCN in its pharmaceutical formulations were effectively used, and the results for pure MCN and commercial tablets were in good agreement with those from the reported approach.

Green chemistry is the practise of reducing or eliminating the use or production of harmful compounds for the environment and human health [19]. For the following reasons, cloud point extraction (CPE) is a green method: (a) it uses inexpensive, diluted solutions of surfactants as the extractor media, economic reagents and producing few laboratory residues; and (b) unlike the organic solvents used in liquid-liquid extraction, surfactants are not toxic, volatile, or easily flammable.
Some adjusted conditions are needed in order to preconcentrate hydrophilic analytes employing CPE, such as MCN. Due to the greater surface activity of mixed micelles used in the CPE approach as well as their co-stabilizing and co-sensitizing capabilities, they demonstrate synergism when compared to a single surfactant [20]. In order to create both ideal hydrophobic and non-ideal electrostatic interactions within the same extraction system, mixed surfactants with various charges are utilised. Ideal hydrophobic and non-ideal electrostatic interactions may emerge from the addition of a modest amount of ionic surfactants to non-ionic surfactants in the same extraction solution. It has been demonstrated that using cationic (cetyltrimethylammonium bromide (CTAB)) and nonionic (Triton X-114) surfactants in combination can boost the extraction efficiency of both organic and inorganic substances [21][22][23][24]. To the best of our knowledge, preconcentration of MCN has never been documented for applications of CPE.
Spectrophotometric technique using Schiff's base reaction coupling p-dimethylaminobenzaldehyde, has been successfully used for spectrophotometric detection of many raw materials of drugs [25][26][27][28], followed by extraction and preconcentration using CPE.
The current study is aimed to combine and improve the CPE technique with a spectrophotometric technique to identify MCN as a highly sensitive process. This technique has been validated in accordance with ICH recommendations [29] and has also been used to test the studied tablet preparations' content uniformity in accordance with USP guidelines [30].

Materials and reagents
All reagents used were of analytical grade (El Nasr chemical co., Abu Zaabal, Cairo, Egypt).
Milnacipran hydrochloride (MCN, 99.8%) was kindly supplied by Averroes pharma for Pharmaceutical Industries, 6th industrial zone, Sadat City, Menoufia, Egypt.   An accurate weight equal to 100 mg of MCN was carefully and separately transferred into 100 mL volumetric flask. The powder was dissolved in 25 mL methanol and diluted to the final mark with the same solvent to obtain a stock solution of 1000 µg mL −1 . Further dilutions were made with methanol to obtain working standard solution in the range of calibration curves.

General procedure for method A (without CPE)
In 5 mL volumetric flasks, experimental standard MCN drug solutions equivalent to (300-1500 µg mL −1 ) were transferred and 1.0 mL of (0.5%, w/v) p-DMAB was added. The solutions were thoroughly mixed, then left to stand for 15 min at room temperature (25 ± 5 °C) before being diluted to the final mark with methanol. At 400 nm, the absorbance of the yellow product was measured in comparison to a reagent blank. The calibration curve was created, the regression equation was derived, and the absorbance was plotted versus the final concentration of MCN.

General procedure for method B (with CPE)
Schiff's base solutions formed at various MCN concentrations (0.2-4.0 µg mL −1 ) were transferred to a 15 mL centrifuge tube, and then 1.0 mL of Triton X-114 (10%, v/v), 1.0 mL of CTAB (0.01 mol L −1 ), and 1.0 mL of Na 2 SO 4 (5%, w/v) were added. The final product was equilibrated in a water bath at 40 °C for 15 min. Centrifugation was used to quicken the phase separation for 5.0 min. at 4000 rpm. A syringe pipette was used to extract the aqueous phase after the mixture was chilled in an ice bath to make the surfactant-rich phase more viscous. 0.5 mL of methanol was used to dilute the phase that was high in surfactants. The absorbance at 410 nm of the methanolic surfactantrich phase was then measured in a quartz cell against the appropriate reagent.

Application to dosage forms
Twenty of the aforementioned MCN-containing tablets were precisely weighed, pounded to a fine powder in a mortar, and thoroughly combined. A 100 mL volumetric flask was filled with an accurately weighed quantity of the ground tablets equal to 100 mg of MCN, along with around 80 mL of methanol. The flask was then subjected to 20 min of sonication [28], completed to the mark with the same solvent, and then filtered. The first batch of filtrate was thrown out. A series of 5-mL volumetric flasks were filled with aliquots of this solution and analysis was carried out as before.

Absorption spectrum
The chemical compound p-dimethylaminobenzaldehyde (p-DMAB) condenses with aromatic or aliphatic amino groups in an acidic medium to create a yellow color [28]. The development of accurate, reproducible, and sensitive enough spectrophotometric techniques, both with and without CPE, for recognizing MCN as an antipsychotic agent in pharmaceutical formulations and bulk powder are the focus of the current work. The method is based on the formation of a yellow Schiff base colored product after the removal of water and can be measured at 400 nm and 410 nm without CPE and with CPE, respectively (Fig. 2). This product is formed when the primary amino group of MCN reacts with the aldehyde moiety of p-DMAB in the presence of sulfuric acid (0.5 mol L −1 ).

Method A (without CPE)
The experimental conditions such as (volume of p-DMAB, type and concentration of acid, temperature and time of reaction and the effect of diluting solvent) were investigated. , the effect of the coupling reagent p-DMAB (0.5%, w/v) concentration was examined. According to Fig. 3, the volume of 1.0 mL of p-DMAB coupling reagent is ideal since it produced the highest absorbance, thus it is used in the following steps.

Effect of type of acids
With the use of hydrochloric acid, nitric acid, sulfuric acid, and o-phosphoric acid, the impact of various acids on the intensity of colored product absorption was investigated (0.5 mol L −1 ). Sulfuric acid was discovered to be the best acid since it had the highest absorption intensity (Fig. 4).

Effect of sulfuric acid concentration
For the general test process, different sulfuric acid concentrations ranging from (0.4-1.0 mol L −1 ) were utilised; it was discovered that the maximum absorbance intensity was obtained at the concentration of acid when it was 0.5 mol L −1 (Fig. 5).

Effect of reaction time, temperature and stability of the Schiff's base product
The condensation process between the MCN and p-DMAB and the production of the yellow-colored product have been optimised for time and temperature effects. The results are presented in Fig. 6. It was discovered that full colour generation occurs instantly, and that the maximum absorption intensity was reached after 15 min and remained constant for at least 40 min after dilution at ambient temperature (25 ± 5 °C).

Effect of surfactants
Because of its low cloud point temperature (CPT) and high density, the type of surfactant employed in CPE is an important consideration. One of the non-ionic surfactants that is frequently utilised in CPE is Triton X-114. This is because of its benefits, including commercial availability with high purity, a relatively low cloud point temperature, low toxicity, a low cost, and a high density of the phase rich in surfactants that makes phase separation by centrifugation possible. The impact of non-ionic surfactant concentration on CPE efficiency was tested within the Triton X-114 concentration range of (1.0-15%, v/v). The Triton X-114 concentration was raised to a maximum of (10%, v/v), in order to increase the absorbance of the Schiff's base coloured product. Increasing the surfactant concentrations above 10% (v/v) results in a significant drop in absorbance. This is explained by a rise in the micellar phase's volume and viscosity. Because there are not enough molecules of the surfactant to quantitatively entrap the Schiff's base product at concentrations below this threshold, the extraction efficiency of the Schiff's base product was low. Additionally, several doses of Triton X-114 (10%, v/v) were utilised between 0.2 and 2 mL to see how they affected CPE efficiency. The product's maximum absorbance was attained with 1.0 mL of Triton X-114 (10%, v/v). Therefore, 1.0 mL of Triton X-114 (10%, v/v) was used for the studies that followed ( Fig. 7a and b).
In the concentration ranges of 1.0 × 10 -4 to 1.0 × 10 -1 mol L −1 , the impact of ionic surfactant (CTAB) concentration on the CPE and determination of MCN was investigated. The absorbance was enhanced and maintained constant at greater concentrations by increasing CTAB concentration till 1.0 × 10 -2 mol L −1 .

Effects of incubation time and equilibration temperature
The effect of incubation time and equilibration temperature was examined between 5.0 and 30 min. The separation procedure only required a 15 min incubation period. By adjusting the temperature in the range of 30-70 °C, the impact of the equilibration temperature was examined. The outcomes showed that the temperature range of 40-45 °C maintained the extraction efficiency and maximum absorbance of the Schiff's base colored product (Fig. 8). Higher temperatures cause the Schiff's base colored product to break down and reduce extraction efficiency. The speed of phase separation, rather than micelle formation, is generally affected by centrifugation conditions. As a result, the ideal centrifugation time for complete separation and a successful CPE process was determined to be 5.0 min at 4000 rpm.

Effect of diluting solvents
Different solvents, including water, dimethyl formamide, dichloromethane, ethanol, and methanol, were investigated as diluent solvents to reduce the viscosity of the surfactant-rich phase formed following CPE and facilitate the absorbance measurements. It was discovered that surfactantrich phase was freely soluble in methanol. Methanol was chosen as the diluent solvent as a result. As a result, methanol was used to finish the surfactant-rich phase to a volume of 0.5 mL. So, employing the suggested CPE approach, a preconcentration factor of 30 was attained. In Fig. 9.

Investigation of the molar ratio
Using (3.3 × 10 -2 mol L −1 ) master equimolar solutions of both p-DMAB and MCN, the stoichiometric ratio between MCN and p-DMAB reagent was established using Job's method of continuous variation [31]. In Job's approach, different quantities of the MCN solution, ranging from (0.1-0.9 mL), and varied volumes of the reagent solution, ranging from (0.9-0.1 mL), are mixed in a series of 10 mL volumetric flasks before the suggested procedures are used. Figure 10s findings demonstrate that the molar ratio was determined to be 1:1 and was consistent with the reaction mechanism hypothesis. When the main amine group of MCN reacts with the aldehyde moiety of p-DMAB in an acidic environment and water is removed, a yellow-colored product results. The predicted reaction process is shown in (Fig. 11).

Validation of the proposed method
To demonstrate that the established process complies with the requirements of the given analytical performance, the recommended assay was confirmed and validated in accordance with International Conference on Harmonization (ICH) criteria [29]. Regarding linearity, specificity, accuracy, repeatability, and precision, the method's validity was examined.

Linearity
By examining a series of MCN concentrations and using the mean of three results for each concentration to reduce relative error, a standard calibration graph for MCN has been created under the optimal reaction circumstances. Without CPE, the relationship between MCN concentration and absorbance was largely linear in the concentration ranges of 60 to 300 μg mL −1 (Method A). The linear equation was A = 0.0012 + 0.0087C, where A is the absorbance and C is the MCN concentration (in μg mL −1 ), with a correlation coefficient of (r 2 = 0.9991). The equation A = 0.1119C + 0.0282 (r 2 = 0.9995) was discovered using CPE (0.2-4.0 μg mL −1 ) (Method B). The analytical traits of the suggested procedures without and with CPE are compiled in Table 1. The improvement factor, calculated as the difference between the calibration graph's slope after the preconcentration procedure with CPE and the calibration graph's slope before the procedure without CPE, was also close to 93.25. For six repeat measurements of 200 and 2.0 μg mL −1 of MCN, Methods A and B were found to have relative standard deviations (RSD) and relative errors of 2.0% and 1.70%, respectively.

Limits of detection and quantitation
The limits of detection were determined using the minimal concentration at which the analyte may be reliably recognised for MCN (LOD). The lowest concentration that can be measured with sufficient accuracy and precision served as the limit of quantification (LOQ). The following formulae were used to determine LOQ and LOD [29]: where s is the standard deviation of replicate determination values under the same conditions as for the sample analysis in the absence of the analyte and k is the sensitivity, namely the slope of the calibration graph. The outcomes are displayed in Table 1. The formula revealed that the LOD and LOQ were 13.1 and 40 μg mL −1 , respectively, without CPE and 0.06 and 0.2 μg mL −1 , respectively, with CPE. Table 1 contrasts the % recoveries of pure MCN achieved using the published approach and the new procedure [9]. By contrasting the results from the suggested approach with those from the stated method, statistical analysis [32] was utilised to evaluate the validity of the proposed method. When comparing the proposed and reported approaches using the estimated Student's t test and variance ratio F test, there is no discernible difference in terms of accuracy and precision (Table 1).

Accuracy and precision
To evaluate the accuracy and precision of the proposed techniques, three repeat investigations on pure MCN medication solution at three different concentration levels were conducted (within the working range). Relative error percentage (RE%) and relative standard deviation percentage (RSD%) were used to calculate the suggested method's accuracy and precision. The relative error percentage determined using the following equation: %R.E. = Found − Taken Taken × 100. Within − 2.20 to − 0.80% and 0.61-0.84%, respectively, are determined to be the intra-day findings for Method A and within − 2.20 to − 0.80% and 0.61-0.84%, respectively, for Method B. Inter-day findings for Method A are found to be between − 1.80 to − 1.50% and 0.61-0.81%, respectively, whereas those for Method B are within − 2.20 to − 0.80% and 0.61-0.84%, respectively ( Table 2). The findings showed that the devised approaches had good repeatability and reproducibility. The RSD% was less than 2.0% in every case, demonstrating the suggested approach's strong repeatability and reliability. The level of precision of the proposed methodology was adequate for MCN quality control analysis.

Robustness
The devised assay's robustness was examined for minor, fixed changes in the following variables: the volume of p-DMAB, the sulfuric acid concentration, and the reaction time. One parameter was changed during the test while the other parameters stayed the same, and the recovery Table 1 Analytical parameters for the analysis of MCN by the proposed methods A and B a A = a + bC, where C is the concentration in μg/ml, A is the absorbance units, a is the intercept, b is the slope b SD standard deviation; RSD% percentage relative standard deviation; RE% percentage relative error c LOD limit of detection; LOQ limit of quantification; ε molar absorptivity d 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)   (Table 3).

Specificity and effect of excipients
The specificity of the recommended method was assessed by looking for any interference from the excipients of the common pill. The traditional addition method was applied by adding known concentrations of pure MCN to a tablet solution that had already been evaluated. The recovery of the added MCN was calculated by comparing the concentration of the spiking mixtures with the previously established value ( Table 4). The proposed approach's strong recovery values demonstrated that the excipients did not interfere with it, suggesting that the method has a high selectivity.

Applications
The recommended assay was utilised to identify the MCN tablet formulation, and the findings were compared to those of the previously reported approach [9] using a 95% confidence level t test and variance ratio F test. There was no discernible difference between the suggested and reported methods' values. This demonstrates the assay's high level of precision and accuracy for the substance under investigation (Table 5).

Content uniformity test
Due to the highly accurate suggested assay and its ability to quickly ascertain the content of the drug in only one tablet obtained with sufficient precision, the developed method was applied to the content uniformity test, which is a time-consuming procedure when applying standard experiment techniques. The same procedure that was used to examine the MCN medication in tablets was used to the analysis of ten other tablets. The United States Pharmacopeia rules have been used to check the uniformity    [30]. When the acceptance value (AV) for dosage forms was calculated, it was found to be far lower than the maximum permitted acceptance value (L1). The outcomes of the commercial preparation's content uniformity are displayed in (Table 6).

Conclusions
In the study that is being reported, p-DMAB has been chosen as the coupling agent to create a yellow product with MCN. For the analysis of the antipsychotic medication (MCN) in its bulk and commercial tablets, the presented work has the advantages of being sensitive, easy, quick, trustworthy, and accurate. Additionally, it saves time because the samples don't need to be pre-treated or extracted. Additionally, the recommended assay is well suited for use in routine assays and quality control inspections of the MCN antidepressant drug as well as in tests to determine the uniformity of tablet content.
Funding This work was supported by the Deanship of Scientific Research at Umm Al-Qura University (Grant Code: 19-MED-1-02-0009).
Data availability This manuscript has associated data in a data repository. [Authors' comment: Data will be made available on reasonable request.]

Declarations
Conflict of interest There are no competing interests declared by the authors.
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