Background: Banlangen granules are broad-spectrum effective antiviral drugs, and have a
large clinical demand in China. Free amino acid is one of the main antiviral active ingredients of Banlangen
granules. The pre-processing of samples by the existing pre-column derivatization reversed-
HPLC method is complicated. Therefore, the determination of free amino acids (AAs) by underivatized
ion-pair HPLC-CAD is advantageous for simplifying the preparation process and improving sensitivity.
Objective: To better optimize AAs analysis methods, here a sensitive SPE-HPLC-CAD method with a
better resolution was established for the determination of underivatized AAs in Banlangen Granule for
the first time.
Methods: The analytes were separated by HPLC using a Hypercarb column with gradient elution of
solvent A (20 mM nonafluorovaleric acid in water) and solvent B (0.3% trifluoroacetic acid in acetonitrile-
0.3% trifluoroacetic acid in water (1:9, v/v)) at a flow rate of 0.15 mL/min. N2 gas pressure and
evaporation temperature of CAD were held at a constant 58.6 psi and 60 °C respectively.
Results: This method was linear over the respective concentration range of six amino acids. The precision,
accuracy, stability and recovery were satisfactory in all samples examined. And the method was
successfully applied to the determination of free amino acids in Banlangen granules and its fractions.
The total contents of six amino acids in 28 batches of Banlangen Granule were between 1.36 mg/g-
Conclusion: The proposed method could be a simple, accurate and sensitive alternative approach for
the determination of free AAs in Banlangen Granule.