HPLC/PDA determination of carminic acid and 4-aminocarminic acid using relative molar sensitivities with respect to caffeine

To accurately determine carminic acid (CA) and its derivative 4-aminocarminic acid (4-ACA), a novel, high-performance liquid chromatography with photodiode array detector (HPLC/PDA) method using relative molar sensitivity (RMS) was developed. The method requires no analytical standards of CA and 4-ACA; instead it uses the RMS values with respect to caffeine (CAF), which is used as an internal standard. An off-line combination of ¹H-quantitative nuclear magnetic resonance spectroscopy (¹H-qNMR) and HPLC/PDA was able to precisely determine the RMSs of CA^274nm/CAF^274nm and 4-ACA^274nm/CAF^274nm. To confirm the performance of the HPLC/PDA method using RMSs, the CA and 4-ACA contents in test samples were tested using four different HPLC-PDA instruments and one HPLC-UV. The relative standard deviations of the results obtained from five chromatographs and two columns were less than 2.7% for CA^274nm/CAF^274nm and 1.1% for 4-ACA^274nm/CAF^274nm. The ¹H-qNMR method was directly employed to analyse the CA and 4-ACA contents in test samples. The differences between the quantitative values obtained from both methods were less than 5% for CA and 3% for 4-ACA. These results demonstrate that the HPLC/PDA method using RMSs to CAF is a simple and reliable quantification method that does not require CA and 4-ACA certified reference materials.