Qualitative and quantitative analysis of glucosinolates and nucleosides in Radix Isatidis by HPLC and liquid chromatography tandem mass spectrometry

Abstract

Multi-component fingerprinting and quantitation of the glucosinolates and nucleosides in samples of Radix Isatidis have been carried out using high-performance liquid chromatography with diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/MS). Five nucleosides together with one glucosinolate were identified by comparing retention times, ultraviolet spectra, mass spectra and/or empirical molecular formulae of reference compounds. Quantitation of these six compounds was carried out simultaneously by HPLC on a Phenomenex Luna C18 column using gradient elution with methanol and water and detection at 254 nm. All calibration curves were linear (r>0.9994) within test ranges. Limits of detection and quantitation were 0.33 ng and 2.50 ng on column, respectively. Intra- and inter-day precision (as relative standard deviation) for all analytes was <2.19% with recoveries in the range 99.6%-101.8% at three concentration levels. The validated method was successfully applied to fingerprinting and assay of 25 batches of Radix Isatidis sourced from different geographical regions of China. The method is simple and reliable and has potential value in the quality control of Radix Isatidis.

Keywords: Glucosinolate; HPLC–DAD–ESI/MS; Nucleoside; Quality control; R,S-Goitrin; Radix Isatidis.

Graphical abstract

Figure 1

Typical chromatograms of (A) a mixed standard solution and (B) an aqueous extract of Radix Isatidis showing peaks from six bioactive compounds. 1, cytidine; 2, uridine; 3, adenine; 4, guanosine; 5, R,S-goitrin; 6, adenosine.

Figure 2

HPLC fingerprints of 25 samples of Radix Isatidis and the reference standard fingerprint generated by similarity evaluation software based on identification and quantitation of six compounds. 1, cytidine; 2, uridine; 3, adenine; 4, guanosine; 5, R,S-goitrin; 6, adenosine.

Figure 3

Similarity evaluation of the HPLC fingerprints of 25 Radix Isatidis samples classified into three groups (A, B and C) based on the three pharmaceutical companies from which they were sourced.

Figure 4

Discrimination analysis based on a PLS-DA plot of the fingerprints of 25 samples of Radix Isatidis generated by SIMCA-P (version 12.0, Demo). Samples are classified into three groups (A, B and C) based on the three pharmaceutical companies from which they were sourced.

Figure 5

Comparison of the average content (mg/g) of the six marker compounds in 25 samples of Radix Isatidis classified into three groups (A, B and C) based on the three pharmaceutical companies from which they were sourced.