Objective: The purpose of this research was to conduct an analysis with a high-performance liquid chromatography (HPLC) method for determining a simultaneously mixture of acetaminophen, propyphenazone and caffeine. Methods: This descriptive-experimental study was conducted by the HPLC method. This research was conducted by optimizing the ratio of the mobile phase of methanol: water (10:90), (20:80), (30:70), (40:60), (50:50), (60:40), (70: 30), (80:20), (90:10), flow rates 0.5-2.0 mL/min and column temperatures 30, 40, and 50°C. The analytical parameters in the validation test include accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ). Results: The results showed that the mobile phase of methanol: water (70:30) was a good optimization result. The average results of determining the levels of acetaminophen, propyphenazone and caffeine in the preparation of Bodrex Migraine® were 101.35 ± 0.16%, 99.24 ± 0.14%, 97.95 ± 0.16%, and Saridon® were 99.50 ± 0.07%, 100.25 ± 0.12%, 98.94 ± 0.06%. The results of the validation parameters performed on acetaminophen, propyphenazone and caffeine were 99.66%, 98.89% and 100.24% for the average recovery results, and 0.01738, 0.01751, 0.02557 for precision, and 1.00026, 0.69104, 0.38920 for the average LOD results and 3.33423, 2.30347, 1.29735 for LOQ average results. Conclusion: The mobile phase of methanol: water (70:30) and the flow rate of 1 mL/min with a column temperature of 30°C was a good optimization result for use in the simultaneous analysis of a mixture of acetaminophen, propyphenazone and caffeine
Keywords: HPLC; determination; acetaminophen; propyphenazone; caffeine
The combination of drugs in a preparation provides increased potential and onset of action of the drug so that it can relieve pain faster and with fewer side effects [1].
The combination of drugs in multicomponent preparations needs to be balanced with increased quality control, so that the drugs in body circulation can be guaranteed its safety and efficacy. According to Indonesian Health Law no. 36 of 2009 in article 105 paragraph 1, it is stated that "pharmaceutical preparations in the form of drugs and drug raw materials must meet the requirements of the Indonesian Pharmacopoeia V Edition or other standard books". There are many methods carried out still require extraction processes for further separation because they still contain several similar compounds that cannot be separated [2]. One of the methods used to determine levels of a combination of drugs such as acetaminophen, propyphenazone and caffeine was UV- Visible spectrophotometry method, but it need more efficient methods in determining levels of drug Table 1. Flow rate optimization data (0.5), (0.75), (1.0), (1.5) mL/min with mobile phase ratio (methanol: water) 70:30
combinations such as high performance liquid chromatography (HPLC).
The use of HPLC method for quality control purposes of preparations containing acetaminophen, propyphenazone and caffeine due to fast analysis time, good separation, sensitivity, selection of columns and eluents varies greatly, columns can be reused, can be used to analyze large and small molecules, easy samples recovered, the detector does not damage the components of the substance being analyzed, it can be used to count samples with very low levels [3].
The condition of the HPLC instrument used in determining the levels of the mixture of acetaminophen, propyphenazone and caffeine must be in the optimum state. The purpose of this optimization is to reduce analysis time, minimize the amount of solvents, and maintain the life of the equipment and columns.
This descriptive-experimental study was conducted using HPCL with column Zorbax ODS (4.5x150mm). This research was conducted by optimizing the ratio of the mobile phase of methanol: water (10:90), (20:80), (30:70), (40:60), (50:50), (60:40), (70: 30), (80:20), (90:10), flow rate 0.5, 0.75, 1.0, 1.5, 2.0 mL/min and column temperatures 30, 40, and 50°C. The results obtained from optimization are used in the determination of tablet dosage levels and validation test. Analysis parameters in the validation test include accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) [4].
The development of High Performance Liquid Chromatography Method which is carried out is the optimization of the comparison of the mobile phase, flow
0.5 5.769 603 6.137 2750 10.401 14336 0.75 3.879 5120 4.203 998 6.809 14078 1.0 3.093 18991 3.808 5337 5.845 31356 1.5 2.003 5009 2.306 6443 3.463 15109 rate and column temperature. Optimization of the ratio of the mobile phase (methanol: water) is the initial stage carried out in this study. In this study the comparison of mobile phase (methanol: water) which is optimized is (10:90), (20:80), (30:70), (40:50), (50:50), (60:40), (70:30), (80:20), (90:10). Optimal phase ratio results are used for optimization of the flow rate ratio (0.5), (0.75), (1.0), (1.5), (2.0) mL/minute. Optimal phase and flow rate comparisons are used for column temperature optimization of 30°C, 40°C, 50°C.
The selection of optimization results performed in this study is based on short retention time, theoretical plate, tailings factor ≤ 2 and resolution ≥ 1.5 [5]. Flow rate optimization data can be seen in table 1 and column temperature optimization data can be seen in table 2, and the chromatogram in seen in figure 1. The optimization results obtained are used for identification, determination of calibration curves, determination of levels and validation.
The results showed that the comparison of the mobile phase of methanol: water (70:30) and the flow rate of 1 mL/min with a column temperature of 30°C is a good optimization result for use in this study, because of the short retention time of acetaminophen 3.093 minutes, propyphenazone 5.845 minutes, caffeine 3.808 minutes, where as the value meets the requirements of the Tailing factor ≤ 2 and Resolution ≥ 1.5 [5, 6].
Identification of comparative standards of acetaminophen, propyphenazone and caffeine and samples carried out aims as a qualitative analysis. The results of the identification of comparative standards and samples can be seen in figure 2.
The results of identification carried out on the standard of acetaminophen, propyphenazone, caffeine and samples gave almost the same retention time, namely acetaminophen (2.984), propyphenazone (3.566) and caffeine (5.430). The data obtained above shows that the results obtained are true the test sample contains acetaminophen, propyphenazone and caffeine.
- - - 0.20 - - - 0.335 2.085 - - 1.857 - - - 1.030
Preparation of calibration curve is done by making a standard main solution comparing each acetaminophen, propyphenazone and caffeine with various concentrations. Calibration curves can be seen in figure 3, figure 4 and figure 5.
The results of the calculation and determination of the calibration curve linearity obtained by each regression line equation and the correlation coefficient for acetaminophen Y = 84902.95194X + 32179.955 with the correlation coefficient (r = 0.99996), propyphenazone Y = 46474.55631X + 6824.3351 with correlation coefficient (r = 0.99997) and caffeine Y = 30564.12183X-118.1324 with a correlation coefficient (0.99993).
The correlation coefficient (r) is used to determine the existence of a linear relationship between concentration and area. A perfect relationship if the price (r) approaches one. The correlation coefficient (r) obtained is still within the limits of acceptance of the correlation coefficient value that is r = 0.995[7].
In the trade of tablet preparations that contain acetaminophen, propyphenazone and caffeine are found with trade names that contain various compositions. The results of determining the concentration levels of a mixture of acetaminophen, propyphenazone and caffeine in tablet preparations can be seen in table 4 and table 5. Table 4 and 5 shows that the levels of a mixture of acetaminophen, propyphenazone and caffeine in tablet preparations obtained by several pharmaceutical industries meet the requirements for the general levels of tablet preparations that contain acetaminophen, Propyphenazone and caffeine not less than 90.0% and not more than 110.0% of the amount listed on the label [2].
The validation of an HPLC method is the procedure that shows information to determine whether the operating system is intended to use, precise, reliable and accurate.In this study, a validation test was done using a standard addition method to the Saridon® (Roche) tablet sample which included an accuracy test with a percent recovery parameter with a requirement of 98-102%, a precision test with an RSD parameter ( Relative Standard Deviation) with requirements ≤ 2%, linearity with correlation coefficient parameters with requirements r = 0.995, detection limit (LOD) and quantitation limit (LOQ) [8]. The results can be seen in table 6, table 7, and table 8.
From the above data, the percent recovery results meet the accuracy test requirements where the average range of recovery results is 98-102% [9]. With Acetaminophen Standard Deviation (SD) of 0.01732, propyphenazone of 0.01732 and caffeine in the amount of 0.02574. The precision test results with the Relative Standard Deviation (RSD) parameters are acetaminophen of 0.01738, propyphenazone of 0.01751 and caffeine of 0.02557 where the Relative Standard Deviation (RSD) results meet the precision requirements, where the RSD value is ≤ 2%. While the results of the detection limit (LOD) and the limit of quantitation (LOQ) obtained acetaminophen 1.00026 and 3.33423, propyphenazone 0.69104 and 2.0347, caffeine 0.38920 and 1.29735 [9, 10]
From the results of the study it was concluded that the determination of levels of a mixture of acetaminophen, propyphenazone and caffeine in tablet preparations with high performance liquid chromatography methods provided good accuracy and precision.