Modernizing the USP Monograph for Acetaminophen
In the USP method for acetaminophen (1), the related compound p-aminophenol is determined using spectrophotometry while another related compound, p-chloroacetanilide, is determined using TLC.
In the USP method for acetaminophen (1), the related compound p-aminophenol is determined using spectrophotometry while another related compound, p-chloroacetanilide, is determined using TLC. We developed a new HPLC method to analyze both compounds in acetaminophen simultaneously using 5-µm columns, including ZORBAX Eclipse XDB-C8 and ZORBAX Eclipse Plus C18. The methods were then transferred to 2.7-µm superficially porous columns, which have efficiencies similar to those of sub-2-µm totally porous particles. This is attributed primarily to a shorter mass-transfer distance and a narrower particle size distribution. Furthermore, the larger particle size results in lower back pressure, allowing these columns to be used on virtually any LC system. The benefits of transferring from larger particle columns include very significant time and cost savings, because superficially porous particles are optimally run at faster flow rates and achieve similar resolution with a much shorter column length. The method was run on several Poroshell 120 phases of different selectivity. The results easily met the requirements of the USP monograph for acetaminophen.
Figure 1: System suitability and test solution for USP acetaminophen analysis using Agilent Eclipse XDB-C8. Standards, corresponding to 50 ppm (A), test solution (B).
Materials and Methods
The test solution was 20 mg/mL acetaminophen in the mobile phase. The standard solution was 1 µg/mL p-aminophenol, acetaminophen, and p-chloroacetanilide in the mobile phase, corresponding to 0.005% in 20 mg/mL acetaminophen.
Figure 2: System suitability and test solution for USP acetaminophen analysis using Agilent ZORBAX Eclipse Plus C18. Standards, corresponding to 50 ppm (A), test solution (B).
The HPLC analysis was performed with an Agilent 1260 Infinity Binary LC including a G1312B Binary Pump SL, G1376C Automatic Liquid Sampler SL, G1316B Thermostatted Column Compartment SL, and G1316C Diode Array Detector SL.
Figure 3: System suitability and test solution for USP acetaminophen analysis using Agilent Poroshell 120 EC-C8. Standards, corresponding to 50 ppm (A), test solution (B).
Results and Discussion
The European Pharmacopoeia (EP) 5.0 includes an HPLC method for determination of related substances in acetaminophen (paracetamol) (2). An isocratic method with an EP-recommended C8 column is used for separation of impurities in acetaminophen. The isocratic method allows a very long analysis time with a broadening peak of p-chloroacetanilide, which leads to low sensitivity. A gradient method using phosphate buffer and acetonitrile was developed on 4.6 × 250 mm, 5-µm columns for separating p-aminophenol, acetaminophen, and p-chloroacetanilide at 1 µg/mL. The three compounds were well separated. The resolution between p-aminophenol and acetaminophen was sufficient for limit detection of p-aminophenol in a high concentration of acetaminophen. The signal-to-noise of p-chloroacetanilide at 1 µg/mL was also sufficient for limit detection. Both columns, the ZORBAX Eclipse XDB-C8 and Eclipse Plus C18, were appropriate for determination of the two related compounds, but the ZORBAX Eclipse XDB-C8 column resolved more impurities around 10 min, as shown in Figure 1 and Figure 2.
Figure 4: System suitability and test solution for USP acetaminophen analysis using Agilent Poroshell 120 EC-C18. Standards, corresponding to 50 ppm (A), test solution (B).
The method was then transferred to 4.6 × 75 mm, 2.7-µm Poroshell 120 EC-C8, Poroshell 120 EC-C18, and Poroshell 120 SB-C18 columns, shown in Figure 3, Figure 4, and Figure 5. The total analysis time was reduced from 30 min to only 8 min, while still meeting the requirements of the method. Of the three phases, the Poroshell 120 EC-C18 column provided the best peak shapes and resolutions between additional impurities that eluted around 3 min.
Figure 5: System suitability and test solution for USP acetaminophen analysis using Agilent Poroshell 120 SB-C18. Standards, corresponding to 50 ppm (A), test solution (B).
In addition to these Poroshell 120 phases, the original method was also transferred to other phases, including Poroshell 120 Bonus-RP, a polar embedded amine column that gave unique selectivity, and Poroshell 120 Phenyl-Hexyl, a phenyl-hexyl bonded column that has improved selectivity for aromatic compounds. Results are shown in Figure 6 and Figure 7. Bonus-RP provided good resolution for all the impurities, while the Phenyl-Hexyl column showed several coeluted peaks. Therefore, the Bonus-RP column could be an alternative to a C18 phase due to its unique selectivity.
Figure 6: System suitability and test solution for USP acetaminophen analysis using Agilent Poroshell 120 Bonus-RP. Standards, corresponding to 50 ppm (A), test solution (B)
According to the EP method for related-substance analysis, the resolution between p-aminophenol and acetaminophen, and signal-to-noise ratio for the peak p-chloroacetanilide are required for system suitability. Table I lists the resolution and S/N with the new gradient method we developed, which all meet the EP method requirements.
Figure 7: System suitability and test solution for USP acetaminophen analysis using Agilent Poroshell 120 Phenyl-Hexyl. Standards, corresponding to 50 ppm (A), test solution (B).
Conclusions
HPLC methods can easily determine p-aminophenol and p-chloroacetanilide in acetaminophen, replacing current spectrophotometer and TLC methods. Both traditional 5-µm columns and Poroshell 120 columns can be used for the analysis of related compounds. The ZORBAX Eclipse XDB-C8 phase is good for the separation of additional impurities. Poroshell 120 EC-C18 provides the best peak shapes and separation for this analysis, while Poroshell 120 Bonus-RP provides unique selectivity for these impurities. For the full application note with larger images, search for pub number 5991-3679EN at www.agilent.com/chem/library.
Table I: Chromatographic system requirements and measured values for related compounds of acetaminophen
References
(1) "Acetaminophen," in United States Pharmacopeia 30–National Formulary 25 (United States Pharmacopeial Convention, Rockville, MD, USA, 2006).
(2) "Paracetamol" in European Pharmacopoeia 5.0 (European Directorate for the Quality of Medicines & Healthcare, Council of Europe, Strasbourg, France, 2004).
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