Spectrophotometric analysis of empagliflozin tablets as SGLT2 inhibitors in pharmaceutical samples

Azo dyes account for 70% of dye chemistry, and their importance may grow in the future. Empagliflozin is a sodium-glucose co-transporter-2 (SGLT2) inhibitor. SGLT2 transporters are primarily responsible for glucose reabsorption in the kidney. In 2014, empagliflozin was approved for medical use in the United States and the European Union. With over 4 million prescriptions in 2019, it was the 146th most commonly prescribed medication in the United States in 2019. The spectrophotometric determination of empagliflozin is described using coupling agents such as 3-chloro-4-nitroaniline or sulfanilamide. These methods are straightforward and are based on the reaction of empagliflozin with diazotized products of 3-chloro-4-nitroaniline or sulfanilamide to produce colored azo dyes with absorption maxima at 470 and 480 nm. Empagliflozin was linear from 1.2 to 26.6 µgml −1 or 0.8 to 20.4 µgml −1 when combined with diazotized 3-chloro-4-nitroaniline or sulfanilamide, respectively. Empagliflozin’s molar absorptivity and Sandell’s sensitivity to 3-chloro-4-nitroaniline or sulfanilamide azo dyes were 3.179 × 10 4 l mol −1 cm −1 or 4.367 × 10 4 l mol −1 cm −1 and 1.149 × 10 −2 µgcm −2 or 8.368 × 10 −3 µgcm −2 , respectively. The formed colored azo dyes are stable for more than 12 hours. The optimal reaction conditions and other analytical parameters are assessed. Foreign organic compound interference has been studied. The method has been successfully used to determine empagliflozin in pharmaceutical samples.

Empagliflozin (Fig. 1) is a competitive inhibitor of sodium-glucose co-transporter-2 that is orally active and has an antihyperglycemic effect (Hailat et al., 2022). It is approved for treating adults with type 2 diabetes in the EU, USA, and Japan, among other parts of the world (Frampton, 2018). This mechanism is independent of β-cell function; thus, these agents effectively treat type 2 diabetes mellitus at any disease stage (Levine, 2016;Mula-Abed and Aughsteen, 2005). Many methods have been adopted to determine empagliflozin (Ahmad et al., 2021). The liquid chromatography-mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4 and metformin D6 as an internal standard (Wattamwar et al., 2020). An Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) method was developed to determine empagliflozin and metformin using a bridged ethylene hybrid C18 column (Ayoub and Mowaka, 2017). Another univariate spectrophotometric method and multivariate chemometric approach were developed and compared to determine empagliflozin simultaneously and metformin manipulating their zero-order absorption spectra with application to their pharmaceutical preparation (Mabrouk et al., 2019). 4-Nitroaniline forms molecular adducts with 4-aminobenzoic acid. It reacts with nitrite ion in a hydrochloric acid medium to form 4-nitrophenyldiazonium chloride, which couples with naphth-1-ol in an alkaline medium to give a purple azo dye. (Figure 2) Photocatalytic degradation of 4-nitroaniline in the presence of TiO 2 suspensions in a batch and continuous annular reactor has been studied (Abed-Elmageed et al., 2020;Ayoub et al., 2021;Baveja et al., 1981;Marchewka et al., 2011;Wu et al., 2012). Sulfanilamide is an organic sulfur compound similar to p-aminobenzoic acid (PABA) with antibacterial properties. Sulfanilamide competes with PABA for the bacterial enzyme dihydropteroate synthase, thereby preventing the incorporation of PABA into dihydrofolic acid, the immediate precursor of folic acid (Dionisio et al., 2018;United States Pharmacopeial Convention, 2007).

Effect of acid, base concentration, and temperature used
The effect of acid and base on the diazotization reaction of empagliflozin (2 µgml −1 ) was studied by adding different acidic solutions (1 M) such as HCl, HNO 3 , H 2 SO 4 , and CH 3 COOH and basic solutions (1 M) such as KOH, NaOH, Na 2 CO 3 , and NH 4 OH. It was observed that CH 3 COOH gave low absorbance with low color stability. In contrast, HCl gave high absorbance with the highest color stability, whereas 1.0 ml of NaOH gave the maximum absorbance for the reaction of empagliflozin coupled with diazotized 3-chloro-4-nitroaniline or sulfanilamide. Therefore, 0.5 ml of 0.5 M HCl (Table 1) and 1.0 ml of 1 M NaOH solutions were preferred for the diazotization reaction of empagliflozin.
The effect of various acids such as HCl, HNO 3 , H 2 SO 4 , and CH 3 COOH (0.5 M) on the diazotization reaction was studied under the maximum absorbance by varying the volume of different acids between 0.25 and 1.0 ml while fixing all other parameters. It was found that 0.5 ml of HCl (0.5 M) gave the highest absorbance and was preferred for the diazotization reaction of empagliflozin (Table 2).
Room temperature (25°C ± 5°C) is recommended for these diazotization reactions because losses in color intensity and stability were observed at low or high temperature.

Effect of nitrite concentration and coupling reagents
The color is at maximum intensity when using 1 ml of a 0.1 M sodium nitrite solution using the current procedure with 2 µgml −1 of empagliflozin and adding 1 ml of 0.02-0.16 M solutions of the nitrite in hydrochloric acid (0.5 M) to a series of nitrite solutions. A higher concentration did not build up the absorbance further, and at a lower concentration, no good results were obtained (Table 3).
The detection limit (D L = 3.3 σ/S) and quantitation limit (Q L = 10 σ/S) of empagliflozin coupled with diazotized 3-chloro-4-nitroaniline or sulfanilamide were found to be 0.363 and 1.100 µgml −1 or 0.270 and 0.820 µgml −1 [where σ is standard deviation (n = 5) and S is slope of the curve] and the correlation coefficient of empagliflozin with 3-chloro-4-nitroaniline or empagliflozin with sulfanilamide was 0.999 or 0.998. The better optical characteristics and statistical data were obtained under optimum conditions (Table 5).

Applications
This simple and uncomplicated method is beneficial for determining empagliflozin in different pharmaceutical samples. The results of the offered method are in good agreement with the acknowledged content. The relative standard deviation and percentage recoveries for all five samples ranged from 0.81% to 2.27% and 98.00% to 100.40% at 95% confidence. The additional ingredients present in pharmaceutical sample appearances did form, not hinder. The results (Table 6) are compared with the endorsed spectrophotometric method (Ayoub, 2016;Patil et al., 2017). These confirm no significant differences between    Table 4. Effect of 3-chloro-4-nitroaniline or sulfanilamide solution on absorbance. the offered and endorsed methods. The precision and accuracy were evaluated by replicate analysis of three different samples containing empagliflozin at different concentrations.

CONCLUSION
Sulfanilamide and 3-chloro-4-nitroaniline, the first spectrophotometric coupling agents used to determine empagliflozin, are inexpensive and equitably selective. Compared to other methods, this one is simple, quick, sensitive, and reproducible, has good precision and accuracy, and has high dye stability (12 hours).
As low relative standard deviation and percentage recovery values highlighted good accuracy and precision of the proposed methods, no tedious separation or solvent extraction procedures were required. There is no interference from excipients in results obtained using the proposed methods. The proposed method examined empagliflozin levels in pharmaceutical samples, which can be applied to more complex samples. For example, a blood sample to determine the blood level of empagliflozin helps in various pharmacokinetic and toxicological studies.