Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets

doi:10.21203/rs.3.rs-4892587/v1

The present research aims to establish new stability indicating RP-UPLC method for concurrently estimating Pregabalin and Etoricoxib in blended powder and their combined tablet formulation. The effective separation of Pregabalin and Etoricoxib achieved with HSS column C18 (150x2.1mm,1.8µm), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, and isocratic elution at 228nm. The elution of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach. PRB and ETB have shown linear responses from 18.75 to 112.5µg/mL and 15 to 90µg/mL, respectively. The range of the % RSD for intraday and inter-day precision was 0.33 to 0.81. The LOD and LOQ of Pregabalin and Etoricoxib were computed to be 0.07 µg/mL, 0.21 µg/mL, and 0.01µg/mL, 0.04 µg/mL, respectively, by standard deviation method. The validation method was carried out using ICH standards. The stability- indicating the feature of the method was confirmed by the forced degradation studies where degradants generated by stress testing were clearly distinguished from the peaks of analytes. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of Pregabalin and Etoricoxib.

Pregabalin

Etoricoxib

RP-UPLC

isocratic elution

Stability indicating

C18 column

Pregabalin (PGB) is an anticonvulsant and analgesic agent [1]. It is well tolerated for managing neuropathic pain and chronic pain conditions [2]. It has a wide safety margin. PGB is a first-line or adjunctive therapy for many conditions, including generalized anxiety disorders, diabetic peripheral neuropathy, and partial seizures [3–6]. Etoricoxib (ETB) is an NSAID and COX-2 inhibitor [7]. This is primarily used to treat people with gout, osteoarthritis, rheumatoid arthritis, and spondylitis-related joint pain and swelling [8–10]. It is also employed to lessen edema and stiffness in the joints [11].

A Fixed-Dose Combination (FDC) is a product that combines two or more active ingredients in a fixed ratio and is used for treatment by integrating drugs, devices, or biological products. Defined by the U.S. FDA, FDCs enhance compliance, effectiveness, and cost-effectiveness while reducing pill burden and side effects [12]. They are widely prescribed globally for managing complex diseases such as HIV/AIDS, diabetes, hypertension, tuberculosis, neuropathic pain, asthma, and malaria. This approach streamlines treatment regimens, making them more efficient and patient-friendly. The chemistry of the PRB and ETB is illustrated in Fig. 1 [13, 14]. The combination of PRB and ETB provides good therapeutic benefits compared to a single regimen treatment in pinched nerves with muscle weakness [15].

Analytical methods are essential for accurately measuring drug amounts and purity, ensuring they are safe and effective. They check the quality of medicines, find impurities, and meet regulatory standards. These methods help develop new drugs by providing essential data. They also monitor drug levels in patients for proper dosing and fewer side effects. Overall, they ensure that medicines work correctly and safely.

As of now, numerous UV techniques, chromatographic methods, and hyphenated methods like LC-MS and LC-MS/MS have been reported for PGB as a single drug moiety or in combination with other drugs [16–23]. Similarly, ETB possesses different analytical methods for identification and estimation [24–30]. Few HPLC methods were reported in the literature for simultaneous estimation of PRB and ETB [31–34]. In the reported methods, only some possess longer ETB retention time [31, 32]. Few methods need stability-indicating properties [33, 34]. UPLC method is superior to HPLC in terms of lower RT, improved peak resolution, efficiency of peaks, and less solvent consumption due to less particle size of stationary phase and high-pressure pumps [35].

PRB and ETB in pure form were purchased from Spectrum Pharma in Hyderabad. All analytical and HPLC grade solvents were purchased from a nearby Merck India Limited vendor. Waters UPLC (TUV detector and Empower-2 software) was used to develop the method. Water (Milli-Q) and a 1 mg sensitive balance (SCALETEC-SAB224CL) were also utilized.

2.1 Method development

The method development was done using a trial-and-error concept. Different polar solvents with different rations were used to optimize the method by considering system suitability as per ICH. The effective separation of PRB and ETB was achieved with HSS column C18 (150x2.1mm,1.8µm), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30⁰C. Equal volumes of acetonitrile and water were used as a diluent to prepare the working standard, sample, and forced degradation solution.

2.1.1 Preparation of standard solution

Accurately transferred about 75mg of PRB and 60mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 µg/mL and 600 µg/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75 µg/mL and 60 µg/mL of PRB and ETB, respectively, which is referred to as 100% level concentration.

2.1.2 Preparation of sample solution

The tablet powder equivalent 75 mg of PRB and 60 mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 µg/mL and 600 µg/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75µg/mL and 60 µg/mL of PRB and ETB, respectively. The sample solution was filtered through the 0.45µ filter to exclude the particulate matter.

Method development

2.2 Method validation

The validation of the method has been performed with the provision of ICHQ2R1 guidelines [35–37].

2.2.1 System suitability

Six replicate injections of a standard solution consisting of PRB (75µg/ mL) and ETB (60 µg/mL) were analyzed by the UPLC system with optimized conditions. The ICH recommended system suitability parameters, including theoretical plates, tailing factor, %RSD, and resolution, were determined to ensure the method's suitability toward the UPLC system.

2.2.2 Linearity

The Linearity of the proposed method was established for PRB and ETB in the range of 18.75µg/mL to 112.5µg and 15µg/mL to 90µg/mL, respectively. A linear curve between concentration and peak area is used to get linear regression data to assess the slope, intercept, and regression coefficient(R²). The concentrations of about 18.75, 37.5,56.25,75,93.75 and 112.5µg/mL of PRB and 15,30,45,60,75 and 90µg/mL of ETB were analyzed in triplicate by the proposed approach to ensure the linearity range.

2.2.3 Accuracy

The proposed UPLC method’s accuracy was ascertained by the standard addition method, where different levels (50%, 100%, and 150%) of standard solutions were separately spiked to a known amount of sample solution. The average (n = 3) percentage recovery of various levels of standard solution from different spiked solutions was calculated from the recorded chromatograms.

2.2.4 Precision

Precision is referred to as the reliability of the observed responses of a homogeneous sample over numerous replications. In most cases, Precision should be done on the same day (intraday) and distinct days(inter-day) as per ICH recommendations. Six repetitive injections of standard solution in a day and three repetitions per day for three consecutive days were analyzed to perform both intraday and inter-day precision. The %RSD was calculated for the peak areas that were obtained.

2.2.5 Robustness

The ability of the approach to withstand altering the parameters of the method in the analysis of the intended analyte without any changes in the analytical results. In the present approach, deliberate changes have been made to the mobile phase (± 1mL), column temperature (± 5^oC), and flow rate (± 0.1mL/min). The system suitability parameters of chromatograms were obtained for standard solutions by injecting each altered parameter in triplicate.

2.2.6 Specificity

The method's capacity to recognize the selected drug in the presence of other substances is referred to as specificity. Individual injections of 1µL of prepared blank solution, working standard solution, sample solution, and forced degradation solution were injected to detect any interference with the peaks of PRB and ETB from other peaks in the acquired chromatograms.

2.2.7 Sensitivity

Sensitivity in terms of Limit of Detection (LOD) and Limit of quantification (LOQ) was assessed by the standard deviation (SD) method, where the SD of the intercept and average slope of the linear curve (n = 3) were considered.

LOD = 3.3 σ/S

LOQ = 10 σ/S

σ- SD of intercept of the linear plot, S- the average slope of the linear plot (n = 3)

2.3 Forced degradation (FD) studies

FD studies HPLC are intended to develop the stability-indicating method where stability and degradation of the intended analyte can be determined [38]. These FD studies help to assess the degradation behaviour and storage conditions to ensure drug safety and efficacy as per ICH requirements. These studies used the ICH Q1A(R2) and Q1B provisions [39–41].

The stability of PRB and ETB was examined under various FD conditions. In acid hydrolysis, 1mL of 2N HCl was mixed with 1mL of the standard stock solution, refluxed for 30 minutes at 60°C, and neutralized with the same strength NaOH. The resultant solution was diluted in such a way as to attain 75µg/mL and 60µg/mL of PRB and ETB, respectively. Base hydrolysis shadowed a similar process with 2N NaOH. Similarly, 20% H₂O₂ was used in oxidative degradation. All three samples were kept aside for 48 hr. while thermal degradation involved exposing the stock solution for 72 hr at 105°C/75% RH. Photostability was determined by exposing the standard stock solution to UV light in the dark control for 7 days. In neutral degradation, equal portions of HPLC-grade water and standard stock solution were mixed uniformly. In each case, further dilution was done to get a solution of 75µg/mL of PRB and 60µg/mL of ETB. Each solution was analyzed in triplicate to determine the percentage degradation of PRB and ETB.

2.4 Assay

The % purity of the commercial formulation can be assessed using an assay. It was carried out by injecting standard and sample solutions containing 75µg/mL of PRB and 60µg/mL of ETB, respectively.

3.1 Optimized method

The method development starts with initial conditions such as HSS column C18 (150x2.1mm,1.8µm), methanol: water in 50:50 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30⁰C. A broad peak with a low plate count was observed for ETB. A further modification was made by replacing water in the state mobile phase with KH2PO4, which produced broad peaks for both PRB and ETB. Finally, a mobile phase of 0.1% OPA and acetonitrile (65:35 v/v) provided sharp and efficient peaks with plate counts of more than 2000. The retention time of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach (Figure-2).

3.2 Method validation

3.2.1 System suitability

The six replicate injections of the standard solution (PRB − 75µg/mL and ETB − 60 µg/mL) resulted in an average USP plate count more fabulous than 2000 (i.e., 2980) and a tailing factor of 2 or less (i.e., 1.4). The % RSD of peak response for PRB and ETB is two or less(i.e.,0.30). The mean resolution between PRB and ETB in the chromatographic peaks is 6.2, more significant than two. All the system suitability parameters results obeyed the ICH recommended limits (Table 1).

Table 1

Results of system suitability of the developed RP-UPLC method.
Drug name	Statistical parameter	RT (Min)	Peak response	USP Plates	Tailing factor	Resolution
PRB	Mean (n = 6)	1.56	818272.67	2353.00	1.41	-
	SD	0.01	6765.03	109.95	0.04	-
	%RSD	0.50	0.83	4.67	2.61	-
ETB	Mean (n = 6)	2.04	785714.33	2814.67	1.24	6.2
	SD	0.02	2403.16	95.54	0.01	0.13
	%RSD	0.11	0.03	0.34	0.06	0.21

3.2.2 Linearity

The developed method has shown significant linearity with an R² value of 0.999 for the concentration ranges of PRB (18.75µg/mL to 112.5µg/mL) and ETB (15µg/mL to 90µg/mL)). These results reveal that the established method's linearity aligns with ICH recommendations. The obtained chromatogram showing this linearity is shown in Table 2 and Fig. 3.

Table 2

Results of linearity of PRB and ETB by developed method.
S. No	PRB		ETB
	Concentration (µg/mL)	Peak area	Concentration (µg/mL)	Peak area
1	18.75	203260	15	201015
2	37.5	400240	30	416912
3	56.25	610155	45	622526
4	75	820299	60	826289
5	93.75	1020461	75	1031996
6	112.5	1230021	90	1235425
(Correlation coefficient) R²		0.999		0.999

3.3.3 Accuracy

The average percentage recovery of PRB and ETB in 50%, 100%, and 150% levels of spiked solutions was calculated to be in the range of 99.0–99.845% (Table 3). These results were within 100% ± 2 limits as per ICH recommendations.

Table 3

Results of percentage recovery of PRB and ETB.
Drug name	% Level	Amount of standard solution		*% Recovery
Drug name	% Level	Added (µg/mL)	Recovered (µg/mL)	*% Recovery
PRB	50	37.5	37.2	99.2
	100	75	74.3	99.0
	150	112.5	112.1	99.64
ETB	50	30	29.72	99.0
	100	60	59.61	99.35
	150	90	89.87	99.85
*Mean of three determinations

3.3.4 Precision

Intraday and inter-day precision found that the computed % RSD values of the peak area responses standard solution of PRB and ETB in both intraday and inter-day precision were observed to be in the range of 0.27 to 0.55 (Table-4). The %RSD value less than or equal to two emphasizes the considerable precision of the established method.

Table 4

Intraday and inter-day results of PRB and ETB in standard solution.
Type of precision		Peak area
Type of precision	Parameter	PRB 75 (µg/mL)	ETB 60 (µg/mL)
Intraday	Mean ± SD (n = 6)	826041.5 ± 2274.95	784085.7 ± 2274.95
Intraday	%RSD	0.27	0.42
Inter-day	Mean ± SD (n = 9)	817685.8 ± 4549.40	785597.8 ± 3624.8
Inter-day	%RSD	0.55	0.46

3.3.5 Robustness

The slight and intentional alterations in the mobile phase, flow rate, and column temperature could not affect the method's performance. The results of the obtained system suitability parameters complied with ICH guidelines (Table 5), confirming the developed method's robustness.

Table 5

System suitability parameter data of PRB and ETB in the method's robustness.
Variation of Parameter		Pregabalin(n = 6)
Variation of Parameter		Peak Area (Mean ± SD)	%RSD	Plate count	Tailing Factor
Mobile phase ratio (± 1mL)	66:34	820830 ± 6509.8	0.8	2599	1.21
Mobile phase ratio (± 1mL)	64:36	815294 ± 4901.2	0.6	2562	1.28
Flow rate (± 0.1mL)	0.2	816438 ± 3517.0	0.4	2443	1.35
Flow rate (± 0.1mL)	0.4	816820 ± 3868.9	0.5	2578	1.29
Temperature (± 5ºC)	25	818876 ± 5841.5	0.7	2582	1.31
Temperature (± 5ºC)	35	816538 ± 6349.9	0.8	2544	1.28
		Etoricoxib (n = 6)
Mobile phase ratio (± 1mL)	66:34	819353 ± 4884.5	0.6	2855	1.19
Mobile phase ratio (± 1mL)	64:36	820997 ± 5994.3	0.7	2808	1.19
Flow rate (± 0.1mL)	0.2	820276 ± 5772.2	0.7	2964	1.21
Flow rate (± 0.1mL)	0.4	823831 ± 4652.9	0.6	2893	1.21
Temperature (± 5ºC)	25	821884 ± 8372.1	1.0	2806	1.20
Temperature (± 5ºC)	35	813344 ± 2297.9	0.3	2829	1.20

3.3.6 Sensitivity

The LOD and LOQ of PRB and ETB were computed to be 0.07 µg/mL, 0.21 µg/mL, and 0.01µg/mL, 0.04 µg/mL, respectively, by standard deviation method.

3.3.7 Specificity

No interference from the placebo, blank, and degradant solution was found at the Pregabalin and Etoricoxib retention times (Fig. 4). It shows that the method is specific for estimating PRB and ETB under the stated method conditions.

3.4 Forced degradation studies

The percentage degradation of PRB and ETB in degradation solutions was determined by comparing the peak responses of both analytes in freshly prepared standard solution with that of forced degradation solutions. The percentage degradation of PRB is higher in acidic conditions and very low in neutral degradation studies. Similarly, ETB has shown higher degradation in photolytic conditions and very low degradation in neural conditions. The above results significantly confirm the susceptibility of PRB and ETB toward acidic and photolytic environments, respectively (Fig. 5). The degradation of PRB and ETB in stated degradation conditions was identified by the developed approach regarding reduced peak response, and degradant peaks ensure the stability indicating feature of the established RP-UPLC method.

3.5 Assay

The percentage purity of PRB and ETB in the marketed tablet was determined to be 99.92 ± 0.52% and 99.86 ± 0.61%, respectively (Table 6).

Table 6

Percentage purity of PRB and ETB in commercial tablet.
Drug name		RT (Min)	Areas	Tailing factor	Plate count	Assay ± SD (%w/w) (n = 6)
PRB	Standard	1.55	817249	1.42	2365	99.92 ± 0.52
PRB	sample	1.54	818637	1.40	2355	99.92 ± 0.52
ETB	Standard	2.01	787484	1.23	2806	99.86 ± 0.61
ETB	sample	2.00	789479	1.20	2877	99.86 ± 0.61

The developed UPLC method exerts advantages over the reported methods. No single UPLC method is available in the literature for determining PRB and ETB in combined dosage forms. In the reported HPLC methods, longer RT is needed, and ETB is needed more than 5 minutes [31, 32]. The RT of PRB and ETB in the current method is 1.5 and 2.0 min in that order. The LOD and LOQ values of the current approach were highly superior to the existing methods [31–34]. Stability-indicating features for HPLC or UPLC is highly significant in current scenarios in analytical methods to adopt the methods in regular analysis. All the reported methods lacked stability, indicating property, compared to the current method [32–34]. The stated validation forced degradation studies results of the current method were aligned with the recommendations of ICH guidelines. The above results emphasize the developed approach's efficacy, compatibility, and economic aspects.

An economical, robust, sensitive, and specific reverse-phase liquid chromatography approach was established, offering improved sensitivity and sample analysis time. The developed method was validated and verified under varied stress conditions, ensuring stability-indicating property and specificity towards PRB and ETB. This approach magnificently distinguishes PRB and ETB, along with their potential degradants, with high resolution. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of PRB and ETB, confirming reliable results and effective separation.

FUTURE PROSPECTIVE RESEARCH

In the current research study, the UPLC stability-indicating method was established. This method is highly effective in routinely analyzing the mentioned drugs in manufacturing units' bulk and dosage forms. To know the nature of degradants and impurities, further investigation of pharmaceuticals should be conducted using advanced hyphenated techniques like LC-NMR and LC-MS/MS.

ETB- Pregabalin

PRB- Etoricoxib

FD- Forced Degradation

ICH- International Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use

RP- HPLC- Reverse Phase High-Performance Liquid Chromatography

LOD: Limit of Detection

LOQ: Limit of Quantification

RSD: Relative Standard Deviation

SD: Standard Deviation

RT: Retention Time

AUTHORS’ CONTRIBUTIONS

All the authors contributed equally to the design and frame of the work, acquisition, interpretation of data, and manuscript preparation; all authors have read the prepared manuscript and approved it for publication.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data and supportive information are available within the article.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS The authors are thankful to the GITAM School of Pharmacy, GITAM Deemed to be University, and the Malla Reddy Institute of Pharmaceutical Sciences.

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GRAPHICAL ABSTRACT

Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Method development

2.1.1 Preparation of standard solution

2.1.2 Preparation of sample solution

2.2 Method validation

2.2.1 System suitability

2.2.2 Linearity

2.2.3 Accuracy

2.2.4 Precision

2.2.5 Robustness

2.2.6 Specificity

2.2.7 Sensitivity

2.3 Forced degradation (FD) studies

2.4 Assay

3. RESULTS AND DISCUSSION

3.1 Optimized method

3.2 Method validation

3.2.1 System suitability

3.2.2 Linearity

3.3.3 Accuracy

3.3.4 Precision

3.3.5 Robustness

3.3.6 Sensitivity

3.3.7 Specificity

3.4 Forced degradation studies

3.5 Assay

4. CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1