Method Development and Validation of Simultaneous Estimation of Oxycodone and Acetaminophen in Bulk and Pharmaceutical Dosage Forms by RP-HPLC Method

Percocet® is a combination analgesic containing oxycodone and acetaminophen. Under conditions of inflammation or hyperalgesia, opioid receptors in tissues can become upregulated and transported to nerve terminals. Acetaminophen (10-11) contributes to analgesia by inhibiting the COX-1 and COX-2 isoforms of cyclooxygenase, enzymes responsible for prostaglandin (PG) synthesis. Since prostaglandins are key mediators of pain sensation, reducing their production increases the pain threshold.

Oxycodone (8-9) and its active metabolites selectively bind to the μ-opioid receptor, and to a lesser extent the κ and δ receptors, in both the central nervous system and peripheral tissues. This interaction activates G-protein–coupled receptor signaling. Activation of μ-opioid receptors inhibits N-type voltage-operated calcium channels, reducing neurotransmitter release and thereby diminishing the perception of pain.

II. MATERIALS AND METHODS

Preparation of Stock solution:

100 mg of Oxycodone and 100 mg of Acetaminophen API standards were accurately weighed and are transferred into two separate 100 ml volumetric flasks, dissolved in mobile phase, then sonicated for 20 minutes to obtain 1000μg/ml.

Preparation of working standard solution:

From the above standard stock solution, 4 ml from each solution was transferred into 100ml volumetric flasks, made up to the volume with mobile phase to get 40μg/ml of Oxycodone and 40μg/ml of Acetaminophen.

III. RESULTS AND DISCUSSION

Method validation:

Validation parameters include specificity, linearity, range, accuracy, precision, limit of detection, limit of quantification, robustness and assay (1-7).

Specificity:

Specificity is the ability to assessing equivocally the analyte in the presence of components which may be expected to be present. Typically these components include impurities, degradants, matrix etc. Blank solution and standard solutions of Oxycodone(40μg/ml) and Acetaminophen(40μg/ ml) were injected into the HPLC system. The peak purity data of Oxycodone and Acetaminophen were compared. There should not be any interference at the retention time of the main peaks.

Linearity:

Linearity for the drugs Oxycodone and Acetaminophen (12-19) was determined by preparing the standard solutions at six concentrations levels in the range of 20-70μg/ml Oxycodone and 20-70μg/ml for Acetaminophen from stock solution. The linearity charts of Oxycodone and Acetaminophen was shown in the figure no 2&3. The correlation coefficient was found to be 0.9997 and 0.9993 for Oxycodone and Acetaminophen respectively. Linearity results were tabulated in table 2.

Accuracy:

Accuracy was performed by spiking known amounts of standard solution to sample solution at three different concentrations levels (50%, 100%, 150%) and there by analyzed for %RSD which should not be more than 2.0.The % recovery was calculated and the results was reported in table no. 3 & 4.

Precision:

The precision (7-12) of the analytical method was studied by injecting six replicates of standard containing 40μg/ml of Oxycodone and 40μg/ml of Acetaminophen which were injected into HPLC system. The % RSD was calculated and the results were reported in the table no.5 & 6. 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection was defined as the concentration which yields a signal - to – noise ratio 3:1 whereas the limit of quantification was calculated to be the lowest concentration that could be measured with signal - to – noise ratio10:1. LOD and LOQ were calculated from slope and standard deviation. The results were tabulated in table no. 7.

Robustness:

The smallest deliberate changes in method like change in flow rate are made but there were no predictable changes in the results and are in the range as per ICH guidelines. Conditions like decrease in flow rate (0.8 ml/min), increase in flow rate (1.2 ml/min) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. % RSD was found to be within the limits and results were tabulated in table no. 8.

Assay:

Assay was conducted on marketed formulation and mean % assay was found. The results were tabulated in table no. 9.

Table1: Optimised Chromatographic conditions

Figure 1: Optimized chromatogram

Table 2: Linearity data of Oxycodone and Acetaminophen

Figure 2: Calibration Curve of Oxycodone

Figure 3: Calibration Curve of Acetaminophen

Table 3: Accuracy data of Oxycodone

Table 4: Accuracy data of Acetaminophen

Table 5: System Precision data of Oxycodone and Acetaminophen

Table 6: Method Precision data of Oxycodone and Acetaminophen

Table 7: LOD and LOQ data of Oxycodone and Acetaminophen

Table 8: Robustness data of Oxycodone and Acetaminophen

Table 9: Assay data Oxycodone and Acetaminophen