RP-HPLC QUANTIFIABLE TECHNIQUE DEVELOPMENT FOR EVALUATING PREGABALIN AND ETORICOXIB COMBINATION IN TABLET AND BULK KINDS

Objective: This evaluation study aims to initiate a relatively sensitive RP-HPLC quantifiable technique for evaluating pregabalin (PRBN) and etoricoxib (ETRB) combination in tablet and bulk kinds.
Methods: PRBN and ETRB chromatographic evaluations were carried off using the “KNAUER C18 Eurospher II column (250 mm × 4.6 mm × 5 μ)”. The mobile phase (MBP) was driven into the KNAUER C18 Eurospher II column at a 1.0 ml/min run rate with an isocratic elution programme of 65% volume of 0.5 mmol sodium perchlorate 35% volume methanol, detected and evaluated the PRBN and ETRB content at 217 nm.
Results: The analysis of PRBN and ETRB is executed inside a run period of 15 min. The RP-HPLC quantifiable technique was developed to separate PRBN and ETRB and likely degradants formed from stress testing by isocratic elution. The RP-HPLC quantifiable technique developed was successfully validated to existing ICH limit guidelines and was confirmed as robust, specific, accurate, selective, precise, sensitive, and linear.
Conclusion: The RP-HPLC quantifiable technique developed here is more valuable and worthy for routine PRBN and ETRB analysis of tablets and bulk kinds.


INTRODUCTION
Etoricoxib (ETRB) assuages inflammation and aching at joints and muscles of patients aged 16 and up who are impaired from rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis [1,2]. In gout, ETRB can be administered for a brief length of period [3]. ETRB operates by modulating the cyclooxygenase-II enzyme, which contributes to manufacturing a substance recognized as prostaglandin [4]. Inflammation and aching are triggered via prostaglandins, which are secreted at regions of hurt or damage. Lesser prostaglandins are triggered as an outcome of inhibiting the operation of the cyclooxygenase-II enzyme; thus, ETRB assuages inflammation and aching.
Pregabalin (PRBN) is a first-line medicinal drug that significantly eliminates the complaints of many sorts of neuropathic aches (fibromyalgia, peripheral diabetic neuropathy, post-herpetic neuralgia, Chemotherapy-persuaded neuropathic aches in cancer sufferers) with a high extent of safety and success [5][6][7]. PRBN is a voltage-dammed Ca 2+ canal antagonist and assists as an antiepileptic and analgesic representative by interacting with the alpha-II-delta subunit [8].
Fixed-dose composition formulation denotes the products encompassing two or more medicinal drugs amalgamated in a single formulation dose [9]. To treat neuropathic persistent back pain, a recently authorized fixed-dose composition formulation of PRBN (75 mg) and ETRB (60 mg) was advised [10]. For PRBN and ETRB combination, no appropriate and consistent RP-HPLC quantitative technique has been mentioned. The wish of this evaluation study is to initiate a simple and relatively sensitive RP-HPLC quantifiable technique for determining PRBN and ETRB combinations in tablet and bulk types with significant accuracy and precision.

Drug materials reference
Cipla Limited (Hyderabad) provided PRBN and ETRB references for this research.

Chemicals
HPLC and Lab reagent grading chemicals-methanol, sodium perchlorate, hydrochloric acid, perchloric acid, peroxide and sodium hydroxide were picked up from Merck chemicals (Mumbai).

Instrument
Combined PRBN and ETRB evaluation was performed utilizing Agilent HPLC 1100 series system fitted with UV detector model G1314A and Agilent chem software.

Conditions of chromatography
Chromatographic partitions and evaluations of PRBN and ETRB were carried off by using the "KNAUER C18 Eurospher II column (250 mm × 4.6 mm ×5μ)". The mobile phase (MBP) was driven into the KNAUER C18 Eurospher II column at a 1.0 ml/min run rate with an isocratic elution programme consisting of 65% volume 0.5 mmol sodium perchlorate pH 5.0, tuned using 0.1% perchloric acid and 35% volume methanol. The temperature at the KNAUER C18, Eurospher II column, was sustained at 25 ᵒC value with an injection measure of 20 μl volume. The evaluations of PRBN and ETRB were carried off at 217 nm using a UV detector.

Tablet test solutions
Ten tablets, Etoshine NP, were concisely weighed, and the average weight was calculated. Etoshine NP was crushed, and an Etoshine NP powder containing PRBN (75 mg) and ETRB (60 mg) was concisely weighed and put to a 100 ml volume size volumetric flask. Methanol (60 ml) was included and homogenized over ten min with a sonicator. To produce the stock Etoshine NP solution of concentration 750 µg/ml PRBN and 600 µg/ml ETRB, the total volume size was adjusted up to 100 ml volume size mark using the same. The resultant stock Etoshine NP solution was sieved using a 0.45micron mesh membrane. Appropriate dilutions of stock Etoshine NP solution in MBP were produced to create workable Etoshine NP solutions of concentrations 75 µg/ml PRBN and 60 µg/ml ETRB.

Linearity curves
Linearity standard samples formulated in MBP at concentrations ranging from 18.75-150 µg/ml for PRBN and 15-120 µg/ml for ETRB were chromatographed employing RP-HPLC quantifiable technique developed. The peak responses of PRBN and ETRB were made out. The linearity curves of PRBN and ETRB were made out by applying their relative peak responses. Next, regression equations for PRBN and ETRB curves were built.

Assay of chosen drugs in etoshine tablets
20 μl of workable Etoshine NP solution was injected into the KNAUER C18 Eurospher II column. Chromatographed, the Etoshine NP solution, employing RP-HPLC quantifiable technique developed. Peak areas for PRBN and ETRB were worked off from PRBN and ETRB chromatograms. The amount of PRBN and ETRB in Etoshine NP solution was made out from PRBN and ETRB responses.

Stability of chosen two drugs
Stability analysis was performed on stock Etoshine NP solution of concentration 750 µg/ml PRBN and 600 µg/ml ETRB including photo, acidic, thermal, alkaline and oxidation degradation analysis [11,12].

Acid hydrolysis test
Ten ml of stock Etoshine NP solution (concentration-750 µg/ml PRBN and 600 µg/ml ETRB) was put to a 100 ml volume size volumetric flask. Ten ml HCl (strength-0.1N) was included and mixed over 30 min with a sonicator. This acid hydrolysis test was made out at room temperature. The complete volume size was adjusted up to 100 ml volume size mark using MBP.

Photo hydrolysis test
Etoshine NP powder containing PRBN (75 mg) and ETRB (60 mg) was concisely weighed and exposed for over 6 hr to sunlight. The sample was made out as detailed in the subsection "Tablet PRBN and ETRB solutions" after 6 h of exposure.

Alkaline hydrolysis test
Ten ml of stock Etoshine NP solution (concentration-750 µg/ml PRBN and 600 µg/ml ETRB) was put to a 100 ml volume size volumetric flask. Ten ml NaOH (strength-0.1N) was included and mixed over 30 min with a sonicator. This alkaline hydrolysis test was made out at room temperature. The complete volume size was adjusted up to 100 ml volume size mark using MBP.

Peroxide oxidation test
Ten ml of stock Etoshine NP solution (concentration-750 µg/ml PRBN and 600 µg/ml ETRB) was put to a 100 ml volume size volumetric flask. Ten ml peroxide (concentration-3%) was included and mixed over 30 min with a sonicator. This peroxide oxidation test was made out at room temperature. The complete volume size was adjusted up to 100 ml volume size mark using MBP.

Thermal hydrolysis test
Etoshine NP powder containing PRBN (75 mg) and ETRB (60 mg) was concisely weighed and exposed for over 6 h to 60 °C in the oven. After 6 h of exposure, the sample was made out as portrayed in the "Tablet PRBN and ETRB solutions" section.
Chromatographed the degraded Etoshine NP solutions employing RP-HPLC quantifiable technique developed. Peak areas for PRBN and ETRB were worked off from PRBN and ETRB chromatograms. The degradation values of PRBN and ETRB in degraded Etoshine NP solution were made out from PRBN and ETRB responses.

Limit of detection and limit of quantification
Our method's sensitivity was checked by evaluating the limits of detection for PRBN and ETRB and the limits of quantitation PRBN and ETRB. Our calculations are dependent on the relevant ICH-based equations [10]. The limit of detections was weighed as 1.206 µg/ml (PRBN) and 1.253 µg/ml (ETRB). The limit of quantifications was considered as 3.979 µg/ml (PRBN) and 4.136 µg/ml (ETRB).

Precision
To check out the precision, the workable PRBN and ETRB solutions of concentrations 75 µg/ml PRBN and 60 µg/ml ETRB was evaluated using RP-HPLC quantifiable technique developed on an identical day (intraday-precision) and two days (interday-precision). The RSD for the PRBN and ETRB peak responses were worked off (table 1).  (table 2).

Recovery and selectivity
The accuracy of the RP-HPLC quantifiable technique developed is calculated using the conventional addition procedure. The additions detections and their retention period times were displayed in chromatograms ( fig. 1) of acid hydrolysis test, alkaline hydrolysis test, photo hydrolysis test, peroxide hydrolysis test and thermal hydrolysis test.

Robustness
The robustness of our formed methodology was verified by altering some experimental variables such as MBP, pH, and wavelength while running the general analytical procedure. With each variable, the peak areas of PRBN and ETRB and relative percent change were assessed (table 4).

Applicability
The RP-HPLC quantifiable technique developed was exercised with Etoshine NP tablets. The content of PRBN and ETRB in Etoshine NP tablets was worked off (table 5).  [20,21]. This specificity results also proved high point stability-indicating an aspect of our RP-HPLC quantifiable technique [20,21].