Chromatography
Simultaneous Analysis of Combination Drugs by HPLC-DAD
Ashleigh Mellor, Applications Specialist, SCION Instruments
Both prescription drugs and over the counter medications contain a single active pharmaceutical ingredient (API); the biologically active substance that treats illnesses. Often, more than one API is used in combination to treat symptoms. The demand for accurate qualitative and quantitative methods of combination drugs is increasing, in a variety of industries. During manufacturing of pharmaceuticals, the level of active ingredient must be highly regulated. Long term users of combination drugs must also be monitored to ensure the individual is not at risk of overdose and/or long term side effects associated with chronic exposure to the API’s. Most importantly, the analysis of combination drugs is vital for any toxicology laboratory, especially when the target analytes are common in overdose cases.
SCION Instruments developed an HPLC-DAD method for the simultaneous determination of two common over the counter medications; Ibuprofen and Paracetamol. Both Ibuprofen and Paracetamol are commonly used to treat the common cold or minor aches and pains. The Diode Array Detector enables a single injection for the identifi cation of the two API’s by utilising specifi c wavelengths of the two target compounds as well as using the absorbance spectra for further confi rmation. Targeting the compounds at the specifi c wavelengths ensures maximum absorbance of the compound, at the set wavelength, enhancing identifi cation.
Experimental
A SCION HPLC with Diode Array Detector was used for the analysis of Ibuprofen and Paracetamol, as shown in Figure 1.
CompassCDS software was used for the whole analysis including method acquisition, data interpretation and reporting of results. Calibration standards containing both Ibuprofen and Paracetamol were prepared at the following concentrations: 10,50,100,200 and 400mg/L in methanol. Over the counter Ibuprofen and Paracetamol were purchased and prepared. One of each tablet was dissolved in 100mL of the mobile phase (phosphoric acid buffer/ methanol (30:70) before being sonicated and diluted with another 100mL of mobile phase. Samples were then centrifuged for 10 minutes at 3000rpm before being fi ltered through a 0.2µm fi lter. The sample was then transferred to an HPLC autosampler vial. If blood is the sample for analysis, liquid-liquid extraction must be performed. Analytical conditions for this analysis can be found in Table 1.
Conditions Table 1. Analytical conditions of HPLC-DAD. Column
Column Temperature Mobile Phase Gradient
Flow Rate
Detection Wavelength Injection Volume
Figure 1. SCION HPLC-DAD.
C18 5µm x 4.6mm x 150mm 25°C
A:Phosphoric Acid Buffer B: Methanol
0-6 minutes: A30:B70% 6.1-15 minutes: A10:B90% 1mL/min
220-400nm (Ibup 237nm, Para 254nm) 20µL
Figure 2. Calibration Curve of Ibuprofen at 237nm and Paracetamol at 254nm. HPLC-DAD.
INTERNATIONAL LABMATE - SEPTEMBER 2020
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