A Validated RP-HPLC Method for Estimation of Paracetamol and Zaltoproen in Combined Pharmaceutical Preparations

PARACETAMOL

Paracetamol (Fever reducer and pain reliever) Non opioid Analgesic and antipyretic agent. It is a common over-the-counter medication. This is however compared to non-steroidal anti-inflammatory drugs (NSAIDs) which are safer to take short term than paracetamol. Paracetamol is used for lowering fever. The name of the paracetamol (often called paracetamol, paracetamol IUPAC name N-(4-hydroxyphenyl) ethanamide.

Figure 1: STRUCTURE OF PARACETAMOL

Frequent use of alcohol may increase the risk of overdose. Untreated, a paracetamol overdose can result in a slow and painful illness. In the early stage, no clear symptoms, or vague ones. Early symptoms of overdose usually do not develop until hours after ingestion and include nausea, vomiting, sweating, and pain, as acute liver failure starts to take hold.                                 

Pharmacokinetics:

Absorption: After taking paracetamol, your body quickly absorbs it almost entirely from the gut. You can expect to see the highest levels in the blood about 30 minutes to 2 hours later.

Distribution: This medication spreads widely in the body and can pass through the blood-brain barrier. It also shows up in small amounts in breast milk.

Metabolism: Most of the paracetamol is broken down in the liver. About 90% turns into safe substances, but around 5-10% changes into a harmful form (NAPQI), which can lead to liver damage if it builds up.

Excretion: The body get rid of these breakdown products through urine, mainly as glucuronide and sulphate forms.

Figure 2:  STRUCTURE OF ZALTOPROFEN

IUPAC name of zaltoprofen: 2-(6-Oxo-5H-benzo[b][1] benzothiepin-3-yl) propanoic acid. 

Molecular Formula of zaltoprofen: C₁₇H₁₄O₃S.

Molecular Weight: 238.27 g/mol.

Zaltoprofen is available in tablet and injectable forms. Common side effects include gastrointestinal discomfort, nausea, dizziness, and headache. Serious side effects may include Common side effects include gastrointestinal discomfort, nausea, dizziness, and headache. Serious side effects may include gastrointestinal bleeding, kidney damage, and cardiovascular risks.

Zaltoprofen represents a category of medications classified as non-steroidal anti-inflammatory drugs (NSAIDs), primarily indicated for the management of pain and inflammatory responses associated with a variety of musculoskeletal disorders, including arthritis and other inflammatory diseases. The pharmacological mechanism of Zaltoprofen involves the inhibition of cyclooxygenase (COX), an essential enzyme that catalyses the biosynthesis of prostaglandins, which serve as mediators in the processes of pain, fever, and inflammation.

Clinical applications of Zaltoprofen encompass conditions such as osteoarthritis and rheumatoid arthritis, wherein inflammation and pain constitute predominant clinical manifestations. It is noteworthy that Zaltoprofen is recognized for its comparatively reduced incidence of gastrointestinal adverse effects when juxtaposed with other NSAIDs available on the market.

While Zaltoprofen generally exhibits a favourable tolerance profile among patients, it is imperative to acknowledge that, like all pharmaceuticals, it may provoke certain side effects. These may include, but are not limited to, dizziness, headache and gastrointestinal disturbances. The administration of Zaltoprofen typically occurs via oral tablets, with dosages adjusted based on the severity of the underlying conditions being treated.

PREPARATION OF STOCK SOLUTION

Accurately weighed 10 mg each of paracetamol and zaltoprofen were transferred into separate 10 mL volumetric flasks, dissolved in a small quantity of Methanol and 0.1%    Ammonia, and diluted to volume with the same solvent to prepare their respective stock solutions. Subsequently, a series of dilutions was prepared, and the solutions were scanned in the UV range of 200–400 nm using methanol as a blank for baseline correction. The maximum absorbance for paracetamol was observed at 256 nm, and for zaltoprofen, it was at 244 nm, which were selected as the analytical wavelengths. An equimolar mixture of 1 mL each of paracetamol and zaltoprofen was prepared, and the absorbance spectrum was recorded to determine the Isosbestic point, which was found to be at 236 nm.

Figure 3:  Combined UV spectra of paracetamol and zaltoprofen

METHOD VALIDATION

Subsequently, method validation was performed, which included the assessment of linearity, accuracy, precision, and sensitivity. Linearity was evaluated by constructing calibration curves over a specific concentration range. Accuracy was determined by spiking known concentrations of paracetamol and zaltoprofen into the sample matrix, while precision was assessed through repeatability and intermediate precision studies. The sensitivity of the method was evaluated by calculating the Limit of Detection (LOD) and Limit of Quantification (LOQ), which were determined.

LINEARITY

Linearity is a key parameter in method validation, ensuring that the response (e.g., absorbance) is directly proportional to the concentration of the analyte within a specified range. To assess linearity, a series of standard solutions with varying concentrations of paracetamol and zaltoprofen were prepared. These solutions were then analysed under the same conditions, and the corresponding absorbance values were recorded.

The linearity of the method is determined by plotting the concentration of the analyte (x-axis) against the absorbance (y-axis) and evaluating the curve. Ideally, the relationship should be linear, and the data points should fit well to a straight line. The linearity is quantified by calculating the correlation coefficient (R²), which should be ≥ 0.999 for the method to be considered linear over the tested concentration range. The linearity range should cover the expected concentration range of the analyte in the sample for accurate quantification. A calibration curve represents the relationship between the instrument's response and the known analyte concentration. Linearity was determined by analysing five concentrations ranging from 10–18 µg/ml and 40–72 µg/ml, respectively, and plotting the peak area ratio against the corresponding concentrations.

Figure 4: LINEARITY CHROMATOGRAM

ACCURACY

Accuracy was evaluated by performing recovery studies at three concentration levels:

50%, 100%, and 150% of the standard concentration. A known quantity of paracetamol and zaltoprofen standard solutions was spiked into pre-analysed samples, and the mixtures were analysed using the developed method. The percentage recovery was calculated using the formula:

Percentage Recovery = (Observed Concentration) / (nominal concentration) ×100

The results, expressed as mean percentage recovery, were within the acceptable range of 98–102%, confirming the method's accuracy.

The accuracy of an analytical method refers to how closely the test results match the true value. It was evaluated using the Standard Addition Method. Accuracy was represented by the mean, while precision was assessed using the relative standard deviation.

Figure 5: Accuracy- 50% Chromatogram

Figure 6: Accuracy – 100% Chromatogram

Figure 7:Accuracy -150% Chromatogram

PRECISION

Precision was evaluated through repeatability (intra-day precision) and intermediate precision (inter-day precision). Repeatability: Six replicates of paracetamol and zaltoprofen at a specified concentration were analysed on the same day under identical conditions. The results were expressed as the percentage relative standard deviation (%RSD).

• Intermediate Precision: The same procedure was repeated on different days and by different analysts to assess variability between runs.

The %RSD values for both intra-day and inter-day precision were found to be within the acceptable limit of less than 2%, demonstrating the precision of the method.

The precision of an analytical procedure reflects the closeness of agreement (or degree of scatter) among a series of measurements taken from multiple samplings of the same homogeneous sample under specified conditions. Both inter-day (Day-I and Day-II) and intra-day (Morning and Evening) precision were evaluated.

Figure 8:PRECISION CHROMATOGRAM

Evaluation of Method Robustness:

The robustness of the analytical method was evaluated by implementing minor modifications to the chromatographic parameters. One specific alteration involved the adjustment of the mobile phase flow rate to 1 ml/min, in alignment with the guidelines established by the International Council for Harmonisation (ICH)

Figure 9:  ROBUSTNESS

Assessment of Sensitivity:

The determination of sensitivity, specifically the limit of detection (LOD) and the limit of quantitation (LOQ), was based on the lowest concentration standard included in the calibration curve. At this concentration, the peak corresponding to the analyte was still discernible, thereby affirming its capacity for quantitation.

MATERIALS AND INSTRUMENTATION

The following   listed were used in carrying   out the research    work represented in tables

Table2: Chemicals/solvents used

Table3: Instruments used

Drugs used were Paracetamol and Zaltoprofen

Marketed formulation:

• Drug : Paracetamol and Zaltoprofen
• Brand name : Zott P
• Label claim : zaltoprofen 80mg and paracetamol 325mg
• Manufactured by : J.B.  Chemicals & pharmaceuticals Ltd
• Batch No : ZP 250123
• Mfg. Dt : 02/2025
• Exp. Dt : 01/2027

RESULTS AND DISCUSSION

Determination of wavelength by UV-Spectrum: Soluble study

Table: Solubility study of Paracetamol and Zaltoprofen

Figure 10:  COMBINED UV SPECTRUM OF PARACETAMOL AND ZALTOPROFEN

Results: The analysis indicated that the detection wavelength was established at 236 nm through the overlaying of individual spectra.

Discussion: The absorbance was assessed by permitting the instrument to scan across a range of wavelengths, leading to the identification of the isosbestic point for Paracetamol  and Zaltoprofen, which was determined to be at 236 nm.

Conclusion: Consequently, subsequent investigations were conducted at the wavelength of 236 nm.

OPTIMIZED HPLC METHOD:

Optimized chromatographic conditions are as follows:                                                         

Chromatographic Conditions:                                                                                                           

Column: Inertsil ODS (250 x 4.6 mm, 5 µm)                                                                                         

Mobile Phase: Phosphate buffer : TEA  in  water : Methanol (40:10;50v/v/v)                                                                                                                                                                        

Flow Rate: 0.7 mL/min                                                                                                                    

Runtime: 10min                                                                                                                             

Wavelength: 236 nm                                                                                                                     

Injection Volume: 10 μL

Figure 11:Blank Chromatogram

Figure 12: Optimized standard chromatogram

Figure 13: Optimized sample chromatogram

Result: The analytical procedure yielded two distinct peaks characterized by well-defined baselines and satisfactory resolution. Consequently, this methodology has been deemed optimized. Notably, the chromatogram displayed no interference from analytes in the blank sample.

Description: In the observed data, sharp peaks were identified corresponding to retention times of 3.384 and 4.832 minutes. For the analysed sample, retention times were recorded at 3.388 and 4.842 minutes

Conclusion: So, this method was considered as optimized chromatographic method and further it will be validated according to the ICH Q2 (R1) guidelines.

SYSTEM SUITABILITY: 

Figure14: System suitability chromatogram

Results: The determined tailing factors for Paracetamol and Zaltoprofen were 3.559 and 4.583, respectively. The corresponding theoretical plate counts were recorded as 3132 and 7031, while the relative standard deviations (% RSD) were found to be 0.092 and 0.057. All parameters pertaining to system suitability were successfully met and remained within established limits.

Discussion: The evaluation of system suitability demonstrated that all measured parameters conformed to the required specifications. Consequently, it can be inferred that the instrument, reagents, and column utilized in this study are appropriate for both assay execution and validation processes.

Acceptance criteria:   Criteria for acceptance dictate that the chromatogram derived from the blank must not exhibit any peaks corresponding to the retention time associated with the analyte. Furthermore, it is essential that the retention times recorded in both the standard and sample chromatograms are identical.

Table10: Linearity results of paracetamol and zaltoprofen

Figure 15: Calibration curve of Paracetamol

Results: The determined tailing factors for Paracetamol and Zaltoprofen  were 3.559 and 4.583, respectively. The corresponding theoretical plate counts were recorded as 3132 and 7031, while the relative standard deviations (% RSD) were found to be 0.092 and 0.057. All parameters pertaining to system suitability were successfully met and remained within established limits.

Discussion: The evaluation of system suitability demonstrated that all measured parameters conformed to the required specifications. Consequently, it can be inferred that the instrument, reagents, and column utilized in this study are appropriate for both assay execution and validation processes.

Acceptance criteria:   Criteria for acceptance dictate that the chromatogram derived from the blank must not exhibit any peaks corresponding to the retention time associated with the analyte. Furthermore, it is essential that the retention times recorded in both the standard and sample chromatograms are identical.

METHOD PRECISION

Figure 16: METHOD PRECISION CHROMATOGRAM