Stability Indicating Validated HPLC Method for the Determination of Aceclofenac and Misoprostol in Bulk and Pharmaceutical Formulation

For the simultaneous evaluation of Aceclofenac and Misoprostol using RP-HPLC, an accurate, rapid, economical, and straightforward, reliable assay technique was developed and demonstrated. The proposed method achieved effective chromatographic separation by using an inertsil ODS column (150mmx4.6mm, 3.5), acetonitrile, and 0.1 percent orthophosphoric acid (OPA) (50:50 v/v) as a mobile phase with a (cid:976)low rate of 1 ml/min and a wave-length of 227 nm. The retention time (Rt) of Aceclofenac was 3.189 minutes and Misoprostol was 6.966 minutes. Chromatography was performed isocrat-ically at room temperature with a run time of around 10 minutes. The suitability parameters of the system were investigated by multiplying the quality six times, and the results were well within acceptable limits. The linearity analysis was conducted at 10 percent to 150 percent stages, with a regression coef(cid:976)icient of 0.999. Aceclofenac and Misoprostol had LOD and LOQ values of 0.063 ug/ml, 0.063 g/ml, and 0.208 ug/ml and 0.208 g/ml, respectively. The drug was recovered at a rate of 98-102 percent, which means that the recovery is within reasonable limits. The validation results were satisfactory, and the approach was found to be suitable for bulk and formulation analysis. As a result, it was obvious that the proposed approach was ideal for routine pharmaceutical preparation review and quality control. Validation results were very close to the appropriate maximum. RSD values of less than 2.0 percent indicate that this approach is accurate and precise. The above method was used to perform a retail formulation assay, which showed that 100.24 percent of the formulation was present. Degradation stress conditions in acidic, alkaline, peroxide, and thermal media were investigated. Under ideal conditions, the established method provided ef(cid:976)icient, precise, and accurate results. According to ICH guidelines, the approach was justi(cid:976)ied.


Aceclofenac, Misoprostol, Validation, HPLC, Stress studies
A For the simultaneous evaluation of Aceclofenac and Misoprostol using RP-HPLC, an accurate, rapid, economical, and straightforward, reliable assay technique was developed and demonstrated. The proposed method achieved effective chromatographic separation by using an inertsil ODS column (150mmx4.6mm, 3.5), acetonitrile, and 0.1 percent orthophosphoric acid (OPA) (50:50 v/v) as a mobile phase with a low rate of 1 ml/min and a wavelength of 227 nm. The retention time (Rt) of Aceclofenac was 3.189 minutes and Misoprostol was 6.966 minutes. Chromatography was performed isocratically at room temperature with a run time of around 10 minutes. The suitability parameters of the system were investigated by multiplying the quality six times, and the results were well within acceptable limits. The linearity analysis was conducted at 10 percent to 150 percent stages, with a regression coef icient of 0.999. Aceclofenac and Misoprostol had LOD and LOQ values of 0.063 ug/ml, 0.063 g/ml, and 0.208 ug/ml and 0.208 g/ml, respectively. The drug was recovered at a rate of 98-102 percent, which means that the recovery is within reasonable limits. The validation results were satisfactory, and the approach was found to be suitable for bulk and formulation analysis. As a result, it was obvious that the proposed approach was ideal for routine pharmaceutical preparation review and quality control. Validation results were very close to the appropriate maximum. RSD values of less than 2.0 percent indicate that this approach is accurate and precise. The above method was used to perform a retail formulation assay, which showed that 100.24 percent of the formulation was present. Degradation stress conditions in acidic, alkaline, peroxide, and thermal media were investigated. Under ideal conditions, the established method provided ef icient, precise, and accurate results. According to ICH guidelines, the approach was justi ied.

INTRODUCTION
Aceclofenac is a nonsteroidal anti-in lammatory drug (Machado et al., 2017) (NSAID) analog of diclofenac. It is used for the relief of pain and in lammation (Mantovani et al., 2008) in rheumatoid arthritis (Smolen et al., 2016), osteoarthritis (Glyn-Jones et al., 2015) and ankylosing spondylitis (Smith, 2015). Aceclofenac is not approved for infants or people with porphyria (Stein et al., 2017;Cherem et al., 2005). It is also not recommended for mothers who are breastfeeding (Kramer and Kakuma, 2012). It should be avoided in a pregnant woman near term due to the possibility of premature ductus arteriosus closure (Bouayad et al., 2001), which can lead to foetal hydrops (Isaacs, 2008).
Misoprostol is a synthetic prostaglandin (Ricciotti and FitzGerald, 2011) drug that is used to prevent and treat stomach ulcers, induce labour, induce abortion, and treat postpartum bleeding caused by uterine incompetence. When used to avoid gastric ulcers in people taking NSAIDs, misoprostol is taken by mouth. It may be used alone or in combination with mifepristone or methotrexate to induce abortions (Kulier et al., 2004). Abortion ef icacy varies between 66 and 90 percent on its own (Bryant et al., 2014;Raymond et al., 2019). It is taken by mouth, absorbed in the mouth, or injected into the vaginal canal for labour induction or abortion (Marret et al., 2015). It can also be used rectally for postpartum bleeding (Blum et al., 2007). Diarrhea and abdominal pain are two common side effects. It's a pregnancy category X drug, which means it's known to cause harm to the foetus if taken during pregnancy. Uterine rupture (Murphy, 2006) can happen in rare cases. It's a prostaglandin analogue (Winkler and Fautsch, 2014), more precisely a synthetic prostaglandin E1 (PGE1). The following Figure 1 shows the chemical structures of Aceclofenac and Misoprostol.

Equipment
Chromatographic system of e-2695 with a quaternary pump, and a PDA detector of 2996 was used. The chromatographic data was analyzed with Empower software of version 2.0.

Chromatographic Conditions
By using the Chromatographic conditions separation was administered in isocratic mode at temperature employing a inertsil ODS (150mmx4.6mm, 3.5µ) column. The combination of 0.1 % orthophosphoric acid and acetonitrile 50:50 v/v with a low 1ml/min was used as a mobile phase. The volume of injection was 10 µl and eluent was observed at 227 nm, so this was selected. The PDA spectrum of Aceclofenac and Misoprostol was shown in Figure 2.

Preparation of standard solution
Weigh 50 mg of Aceclofenac and 50 mg of Misoprostol working standards and transferred into lask volume of 100 ml and diluted to volume with diluents. Further dilute 5 ml of the prepared solution to 50 ml with diluents.

Preparation of sample solution
Transfer 69.5 mg of Aceclofenac and Misoprostol sample into a lask of 100 ml and add 70 ml of diluents, sonicate to dissolve it and make up to the mark. Further dilute 5 ml of the prepared sample solution to 50 ml with diluents.

Method validation
In this method validation parameter (system precision, linearity, precision, accuracy, LOD, LOQ, robustness, forced degradation and stability) studies were validated for the chosen drugs of Aceclofenac and Misoprostol. Standard and sample

System suitability
The HPLC system was stabilized for 60 min to urge a stable bottom line. Six replicate injections of the quality solution containing 50 µg/ml of Aceclofenac and 50 µg/ml of Misoprostol were assessed to see the system suitability. The amount of the theoretical plate count of Aceclofenac and Misoprostol were 3265 and 6487, tailing factor was 1.04 and 1.25, resolution was 5.47 respectively. These parameters were found to be within the acceptable limit.

Linearity
To assess the tactic's linearity, a standard solution containing 50 g/ml of Aceclofenac and 50 g/ml of Misoprostol was prepared (100 percent of the targeted level of the assay concentration). Sequential dilutions of the given solutions were performed at 10 percent, 25 percent, 50 percent, 100 percent, 125 percent, and 150 percent of the target concentrations. The peak areas are used to map calibration curves since they were pumped. These analytes had a correlation coef icient of 0.999. Table 1 summarized the conclusions and the Figures 5 and 6 shows the calibration plots of aceclofenac and misoprostol.

Limit of detection and quanti ication
Limit of detection and quanti ication is the minimum concentration level at which the analyte are often reliably detected, quanti ied by using the quality formulas (3.3 σ/s and 10 σ/s for LOD and LOQ respectively). LOD value of Aceclofenac and Misoprostol were 0.06 µg/ml, 0.06 µg/ml and s/n values

Precision
The method precision of the process was investigated by examining six samples of an identical batch that were prepared separately. From these six separate samples, solution was injected and therefore the peak responses obtained wont to calculate mean and percentage RSD values. This technique was observed to be precise and RSD was 2.0% and also the share assay values were on the brink of 100%. The results were represented in Table 2.

Accuracy
Accuracy decided by recovery studies which were administered in 3 individual concentration levels (50%, 100% and 150%). APIs with concentration 25, 50 and 75 µg/ml were prepared. As per the test method the test solution was injected to 3 preparations at each spike level and therefore the assay was performed. The percentage recovery values were observed in between 99.5-100.6 of Aceclofenac and 99.4-100.4 of Misoprostol. The share recovery values were observed as 2%. The results were given in Table 3.

Ruggedness
The tactic's sturdiness was investigated, and it was discovered that the chromatographic patterns did not change dramatically by using a different HPLC method, observer, or column. The fact that the RSD percentage was less than 2% demonstrates the robustness of the established process.

Robustness
Robustness of the tactic was found to attract RSD should be but 2%. Slightly variations were wiped out the optimized method parameters like low (±0.2 ml/min), organic content in mobile phase (±10%). The results were shown in Table 4.

Stability
By observing the stability techniques, stability of ordinary and sample solutions were studied from initial to 24 hrs stored at RT. They were injected at different time intervals and difference between initial to 24 hrs percentage of assay wasn't quite 2%. There is no effect in storage conditions for Aceclofenac and Misoprostol drug. The results are shown below in Table 5.

Forced degradation
Forced degradation conditions containing acidic, basic, peroxide, hydrolysis, reduction and thermal stress were studied in 1 N concentration level. Forced degradation results were shown in Table 6.

CONCLUSION
This method describes the quanti ication of Aceclofenac and Misoprostol in bulk and pharmaceutical formulation according to ICH guidelines. The evolved technique was observed to be accurate, precise, linear and reliable. The bene it comes from the ease with which the sample was prepared, as well as the use of less expensive reagents. The proposed HPLC conditions ensure adequate resolution and, as a result, accurate compound quanti ication. The precision and reproducibility data are satisfactory, according to the testing results. The developed chromatographic technique was widely used in drug testing for routine study.