High Speed Analysis of Paracetamol and its Process Impurities

December 2, 2010

The Application Notebook

The Application Notebook, The Application Notebook-12-02-2010, Volume 0, Issue 0

KNAUER Application Note

Anke Knöfel and Silvia Marten, KNAUER, Berlin, Germany.

Paracetamol is a major ingredient in numerous medications due to its analgesic and antipyretic properties. During its synthesis (Figure 1), a total of ten process-related impurities are observed. Several HPLC applications have been developed for the monitoring of these impurities,1,2 including the European Pharmacopoeia which has adopted an isocratic HPLC method using a silica-based C8 column with 5 μm particle size, requiring a run time of 45 min.3 By using a gradient method and standard HPLC instrumentation, the analysis can be reduced to 7 min.4

Figure 1: Synthesis of paracetamol (N-(4-hydroxyphenyl)acetamide).

To remain competitive however, pharmaceutical laboratories require even faster methods with higher efficiency and rapid resolution. These priorities have led to the development of stable sub-2 μm columns which can meet these demands. Compared to 5 μm columns, sub-2 μm columns offer shorter analysis times, improvements in resolution, sensitivity and peak capacity. This study describes a gradient method using a sub-2 μm column for the simultaneous determination of nine impurities and one degradation product.


The analysis was performed on a KNAUER high pressure gradient PLATINblue UHPLC system, equipped with two P-1 Pumps, M-1 Degasser, AS-1 Autosampler, T-1 Column Temperature Manager and UV-1 Detector.

Column: BlueOrchid 120 C18, 100 × 2 mm, 1.8 μm

Mobile phase: A: ACN / B: Buffer (pH 3.7)


Time (min)……% A……% B





Flow rate: 0.8 mL/min

Detection: UV at 245 nm (80 Hz, 0.005 s)

Temperature: 50 °C

Injection volume: 1 μL


Using a KNAUER PLATINblue UHPLC system and a BlueOrchid C18 1.8 μm column, paracetamol and ten impurities were separated in under 2 min (Figure 2), more than 3× faster than the conventional HPLC gradient method.4 Moreover, the UHPLC method required only one-fifth of the sample volume and eluent consumption was reduced by 18%.

Figure 2: High speed separation of paracetamol mixture on BlueOrchid 120 C18, 1.8 μm, 100 × 2 mm (blue: standard; red: pharmaceutical formulation). 1. 4-aminophenol; 2. paracetamol (N-(4-hydroxyphenyl) acetamide); 3. N-(4-hydroxyphenyl) propanamide; 4. N-(2-hydroxyphenyl) acetamide; 5. N-(3-chloro-4-hydroxyphenyl) acetamide; 6. 1-(4-hydroxyphenyl) ethanone oxime; 7. N-phenylacetamide; 8. 4-(acetylamino) phenyl acetate; 9. 4-nitrophenol; 10. chloroacetanilide; 11. 1-(2-hydroxyphenyl) ethanone.

The limit of detection (LOD) for all compounds was in the range of 0.01–0.08 μg/mL. Retention time reproducibility of the UHPLC method was in the range of 0.1–0.7% RSD (n = 5).


The high speed analysis of paracetamol and its related process impurities in a pharmaceutical formulation illustrates how analytical labs can benefit from sub-2 μm columns like BlueOrchid in combination with a UHPLC system like PLATINblue, in terms of faster separations, higher resolution, higher sensitivity and reduced mobile phase consumption.


1. R.R. Nageswara and A. Narasaraju, Anal. Sci., 22, 287–292 (2006).

2. B.S. Nageralli et al., J. Chromatogr. B, 798(1), 49-54 (2003)

3. European Pharmacopoeia Monograph, Paracetamol 01/2005:0049/2.2.29

4. Knauer, Applications Journal, V7801, 07/2008, 79, www.knauer.net

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