INTRODUCTION:

Chemically speaking, PAR is N-acetyl-p-aminophenol; as shown in Fig. 1, it was discovered by accident that this first aniline derivative has both analgesic and antipyretic qualities^1,3. Flupirtine maleate (FLU) is an aminopyridine that functions as a central non-opioid analgesic. Its chemical name is ethyl {2-amino-6-[(4-fluorobenzyl) amino] pyridine-3-yl} carbamate (Fig. 1).

The PAR and FLU tablet dose form combined is used to treat migraine. A thorough review of the literature found that only a single UV method¹¹and a small number of HPLC methods were reported for the simultaneous quantification of PAR and FLU in tablet dosage form ^{12, 16}. Few UV, HPLC, and other analytical methods are available for the quantification of PAR^4,6 and FLU^7,10 alone or in combination with other drugs. n order to simultaneously quantify PAR and FLU in solid dosage form, the current study was conducted with the goal of creating a straightforward, precise, quick, and validated stability indicating RP-HPLC method.

Fig.1: Chemical structure of PAR (A) and FLU (B)

Experimental procedure:

Chemicals:

The medications PAR and FLU were received as a gift sample from the Hyderabad, India-based laboratory of Dr. Reddy. We bought commercial LUPIRTIN-P tablets with 325mg of PAR and 100mg of FLU from the local market and used them before they went bad. MERCK chemicals provided AR grade potassium dihydrogen phosphate, sodium hydroxide, and HPLC grade methanol. This experiment also made use of Milli-Q water and a 0.45μm-sized Millipore nylon filter.

Instruments:

An ACE C18 (250 x 4.6mm, 5µ) column with a PDA detector and an EMPOWER software connection was used for the chromatographic separation on an HPLC. Utilizing a Rheodyne injector with a 20µl loop installed, samples were injected. The Mettler Toledo MS2O5DU analytical balance, the POLMON LP139SA pH meter, and the Sonica Ultrasonic Cleaner model sonicator were also utilized. Furthermore, employed in this investigation were an electronic balance, a micropipette, and a micropore filtration assembly

Preparation of stock solution:

Precisely measured and transferred 325mg of PAR and 100mg of FLU into a 50mL volumetric flask. Diluents (methanol: water 20:30) were added to achieve the desired volume, mixed, and subjected to sonication for a duration of 20 minutes. Five milliliters of the solution were extracted from this and added to a hundred milliliters using diluent.

Method optimization:

A range of method conditions were explored during the development of the HPLC method in order to develop an analytical method that would be suitable for the simultaneous quantification of PAR and FLU in tablets. These conditions included methanol-water (50:50v/v), acetonitrile-water (50:50 v/v), and methanol-phosphate buffer (40:60v/v; pH 3.0-7.5 adjusted with sodium hydroxide and ortho phosphoric acid). Different columns, such as C8 and C18, with different injection volumes were investigated. Good resolution, suitability for stability studies, analysis time, and solvent cost were taken into consideration when optimizing the method conditions ¹⁷.

Procedure for assay of marketed tablets:

Twenty Lupirtin-P tablets, each containing 100mg of FLU and 325mg of PAR, were weighed and finely powdered. One tablet's worth of powder was precisely weighed, added to a 50mL volumetric flask along with 20mL of diluent, and sonicated for 20 minutes. The mixture was then brought to volume with diluent and passed through a 0.45-micron membrane filter. To quantify drugs in tablets using an optimized HPLC method, a 5mL filtered solution was transferred to a 100 mL volumetric flask and made up to volume with mobile phase. This solution was then further diluted with mobile phase to obtain the concentration of both drugs within the linearity range.

Method validation:

This optimized HPLC method was validated as per international guidelines^18-20.

Linearity:

2.5, 3.75, 5, 6.25, and 7.5mL were removed from the stock (PAR and FLU) solution, and the volume was then adjusted to 25mL using the mobile phase. These solutions underwent sonication and membrane filter filtration. The concentrations of the standard solutions are approximately 10–30µg mL-1 of FLU and 32.5–97.5 µg mL-1 of PAR. Using optimized method conditions, these solutions were injected six times into the HPLC system to obtain the chromatograms. Plotting the peak area against drug concentration allowed for the determination of the calibration line.

Accuracy (recovery studies):

Standard addition was used to calculate the accuracy. A pre-quantified sample was spiked in triplicate with three different levels of standards (i.e., 50%, 100%, and 150%). Three preparations of the drug were estimated at each concentration level, confirming the recovery. There was a calculation of the recovery percentage and RSD (%).

Precision:

Six replicates of a single standard solution of PAR and FLU at concentrations of 65µg mL^-1 and 20µg mL^-1 were analyzed intra-day (n=6) to assess the repeatability (intra-day precision) of the procedure. The study assessed intermediate precision by conducting an inter-day (n=6) analysis on six replicates of a single standard solution containing PAR and FLU at concentrations of 65µg mL^-1 and 20µg mL^-1. The percentage RSD was then computed.

Limit of detection (LOD) and limit of quantification (LOQ)

From the linearity plot the LOD and LOQ of PAR and FLU were calculated by following formula:

3.3 σ 10 σ

LOD = --------- LOQ= --------

S S

Where σ is the standard deviation and S is the slope of the calibration curve.

Robustness:

The effect of minute but intentional changes to the chromatographic conditions was evaluated in order to examine the robustness. The robustness of the method was examined by varying multiple variables such as the temperature by ±5°C, the mobile phase composition, and the flow rate by ±0.2mL/min.

System suitability:

A system suitability test was developed to ensure the validity of the analytical method. Using working standard solutions, parameters such as retention time (Rt), plate number (N), resolution (RS), and tailing factor of samples were examined for this purpose.

Procedure for forced degradation studies:

To figure out if the analytical method was stability indicating, forced degradation studies were conducted on the powdered active pharmaceutical ingredient (API) of PAR and FLU under various stress conditions. In order to assess the proposed method's capacity to extract PAR and FLU from its degradation products, deliberate degradation studies were conducted under various stress conditions, including acidic (0.1 N HCl), basic (0.1 N NaOH), neutral (water), peroxide degradation (5% H2O2), and thermal treatment (heated at 80 0C). In order to carry out forced degradation in acidic media, 325mg of PAR and 100mg of FLU drugs were added to a 50mL volumetric flask, dissolved in methanol, and then diluted to the appropriate level with 0.1 N HCl. The flask was set aside. For twenty-four hours, the flask at room temperature. After extracting 5mL of that solution, 5 mL of 0.1N NaOH was added to neutralize it. It was then injected into the HPLC system after being diluted in accordance with the test protocol. The alkali (NaOH), peroxide (H2O2), neutral (NaOH), and thermal (80°C) degradation tests were also conducted¹⁷.

RESULTS AND DISCUSSION:

Numerous chromatographic trials were looked into in the process of developing a new HPLC method with the aim of determining the best possible chromatographic conditions for the simultaneous quantification of FLU and PAR in tablets. Various parameters have been carefully evaluated, include optimal pH, ideal mobile phases with various ratios, columns, injection volumes, detector wavelengths, flow rates, and standard solution concentrations. Using a mobile phase of 0.1 N potassium di hydrogen phosphate buffer, pH 7.5: methanol at 60:40 v/v with a flow rate of 1ml min^-1, a detection wavelength of 250nm, an injection volume of 2µL at room temperature (30 0C), and a column (ACE C18, 250 x 4.6mm, 5µm) was finally used to achieve the chromatographic separation. The optimized method's chromatogram was shown in the table 1

Fig.2: Optimized chromatogram of PAR and FLU

Table 1: System suitability parameters

S. No	Parameter	PAR	FLU	Limits
1	Retention time	3.093	4.548	--------
2	Theoretical Plates	11633	8801	NLT 2000
3	Tailing factor	1.203	1.285	NMT 2
4	Resolution	9.161	9.161	NLT 2
5	LOD	0.0452	0.0094	----------
6	LOQ	0.1356	0.0282	---------

Linearity and range:

The linearity was evaluated by the least square regression method. The responses for PAR and FLU at 250nm were found to be linear in the concentration range of 32.5 - 97.5µg mL^-1 (PAR) and 10 - 30µg mL^-1 (FLU), with a correlation co-efficient (r²) value of 0.999. It was observed that with the increase in PAR and FLU concentration, the peak area at 250nm was increased. The calibration plots were shown in (Fig. 3).

Fig.3: Calibration plot of PAR (A) and FLU(B)

Accuracy (recovery studies):

The mean of percentage recoveries and %RSD values were calculated and reported in Table 2. The % recoveries of PAR and FLU were found to be in the range 99.52 - 99.59 and 98 - 99.1, respectively which are satisfactory. The %RSD value for recovery studies of PAR and FLU was less than 2.0, which indicates the developed simultaneous method was accurate.

Precision:

The % RSD of intra- day and inter-day analysis was less than 2.0 for both the drugs. These statistical data were indicative of good precision and reported in the Table 3.

LOD and LOQ:

The limit of detection and limit of quantification values were found to be 0.0452 and 0.1356 µg mL^-1for PAR and 0.0094 and 0.0282 µg mL^-1for FLU, reported in Table 1, which indicates the sensitivity of the method.

Robustness:

The result of robustness indicates the selected parameters remained unaffected by small variations in chromatographic conditions. The robustness results revealed that the developed method having ability to unaffected by small and deliberate variations in chromatographic conditions.

Forced degradation studies:

Table 4 displays the results of forced degradation studies in acid, alkali, peroxide, neutral, and thermal conditions. It shows that PAR and FLU degradation increase under oxidation and thermal conditions compared to other stress conditions. The resolution was found to be greater than two, and the drugs of interest peaks were clearly separated from the degradation product peaks. Because of this, the RP-HPLC method that was developed can measure PAR and FLU even when degradation products are present.

Analysis of commercial tablets (assay):

A commercially available tablet called LUPIRTIN-P, containing 325mg of PAR and 100mg of FLU, was used to assay the proposed method's accuracy. Table 5 presents the comparison between the reported amounts and the obtained results for PAR and FLU. The suggested approach yielded %assay values for commercial formulations of 99.2 for PAR and 98.3 for FLU. Since the formulation's percent RSD (LUPIRTIN-P) was less than 2, it can be concluded that the suggested method was effective at separating PAR and FLU for quantification simultaneously without interfering with each other or the excipients and matrix materials.

Table 2: Accuracy of the method (recovery studies)

Formulation	Recovery level (%)	Recovery of analyte	Theoretical content (mg)	Amount found (mg) A.M± SD (n=3)	Recovery (%)	% RSD
Lupirtin-P	50	PAR	487.5	485.2±0.450925	99.52	0.10372
	50	FLU	150	148.06±0.61101	98.71	0.5459
	100	PAR	650	647.2±0.416333	99.56	0.04635
	100	FLU	200	196.03±0.305505	98.0	0.38962
	150	PAR	812.5	808.733±0.208167	99.59	0.09444
	150	FLU	250	247.76±0.472582	99.1	0.46429

Table 3: Precision of the method

Concentration (µg/mL)	Intra-day precision		Inter-day precision
Concentration (µg/mL)	Concentration estimated (µg mL^-1) A.M± SD (n=6)	% RSD	Concentration estimated (µg mL^-1) A.M± SD (n=6)	% RSD
PAR
65	64.507±0.365276	0.56626	65.17667±0.4231	0.64933
FLU
20	19.574±0.136192	0.69576	19.983±0.186082	0.93119

CONCLUSION:

The method that was developed was quick, easy to use, accurate, precise, and economical. The results showed that all validation parameters—specificity, linearity and range, accuracy, precision, ruggedness, LOD, LOQ, and robustness—were all very acceptable. The suggested method was used to validate the stress degradation studies of the PAR and FLU, and the degradation peaks were clearly distinguished from the sample peak. The developed technique was effectively used determine PAR and FLU in tablets simultaneously and without interference

ACKNOWLEDGEMENT:

The authors are thankful to Dr. CVS Subrahmanyam, Principal and Management of Gokaraju Rangaraju College of Pharmacy for providing facilities for this research work.

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Received on 16.12.2023 Revised on 09.04.2024

Accepted on 25.07.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):251-255.

DOI: 10.52711/2231-5675.2024.00045

FLU				PAR
Degradation type	R_t	Peak area	% Drug degraded	Rt	Peak area	% Drug degraded
Acid	4.541	3640556	4.32	3.088	4538289	2.92
Alkali	4.541	3557134	6.52	3.090	4417549	5.5
Water	4.542	3632077	4.55	3.089	4412484	5.6
Peroxide	4.542	3582886	5.84	3.091	4271037	8.63
Thermal	4.541	3409279	10.41	3.091	4471084	4.35