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Introduction - Radix Gentianae Macrophyllae, a traditional Chinese medicine, has been frequently used to dispel rheumatism and ease pain. There are four species of <i>Gentiana </i>(<i>G</i>. <i>macrophylla</i>, <i>G</i>. <i>straminea</i>, <i>G</i>. <i>dahurica</i> and <i>G</i>. <i>crassicaulis</i>) recorded as herbal drugs in the <i>Chinese Pharmacopoeia </i>and two other <i>Gentiana</i> species (<i>G</i>. <i>officinalis</i> and <i>G</i>. <i>siphonantha</i>) are often used as substitutes. Currently, the LC fingerprint comparison among different species and evidence for the equivalent application of these herbs are lacking.<br>Objective - To develop an HPLC method for the simultaneous determination of four iridoid and secoiridoid glycosides and a comparative study of six species of <i>Gentiana</i>.<br>Methodology - HPLC analysis was performed on a C<sub>18</sub> column (Phenomenex, 150 × 4.6 mm, 5 µm particle size) with gradient elution using 0.4% aqueous phosphoric acid and methanol at 242 nm.<br>Results - The proposed method was precise, accurate and sensitive enough for simultaneous quantitative evaluation of four iridoid and secoiridoid glycosides (loganic acid, swertiamarin, gentiopicroside and sweroside) in the six species of <i>Gentiana</i>. Contents of the four marker compounds varied from each other even among the samples from the same species and the LC chromatograms of the six species of <i>Gentiana</i> showed high similarities.<br>Conslusion - The close similarity of LC chromatograms and chemical composition of the four genuine <i>Gentiana</i> species explain their popular usage as Radix Gentianae Macrophyllae in Chinese medicine. By comparing the four genuine <i>Gentiana</i> species, it is suggested that the two substitutes could be used as Radix Gentianae Macrophyllae to relieve the scarcity of resources. Copyright © 2010 John Wiley & Sons, Ltd.<br>An HPLC method has been developed for simultaneous determination of four iridoid and secoiridoid glycosides and the fingerprints of the six <i>Gentiana</i> species was analyzed and compared. The close similarity of LC chromatograms and chemical composition of the four <i>Gentiana</i> species (<i>G. macrophylla</i>, <i>G. straminea</i>, <i>G. dahurica</i> and <i>G. crassicaulis</i>) explain their popular usage as Radix Gentianae Macrophyllae in Chinese medicine. By comparing the four genuine <i>Gentiana</i> species, it is suggested that <i>G. officinalis</i> and <i>G. siphonantha</i> could be used as Radix Gentianae Macrophyllae to relieve the scarcity of resources.