Metabolite discovery of helicidum in rat urine with XCMS based on the data of ultra performance liquid chromatography coupled to time-of-flight mass spectrometry

The present study demonstrates the use of XCMS (various forms (X) of chromatography coupled to mass spectrometry), an open-source software tool primarily used in bioinformatics, on the data of ultra-performance liquid chromatography connected online with a mass spectrometer (UPLC/MS) for the discovery of the metabolites of helicidum in urine after oral single dosage to rats. Helicidum (formaldehydephenyl-O-β-D-pyranosyl alloside) is the major active component of the fruits of Helicid hilagirica Beed. In China, it is often used in the clinic to treat neurasthenic syndromes, vascular headache, and trigeminal neuralgia with high efficacy and low side effect and toxicity. The urine samples of five rats were collected during 0-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-32, 32-40, and 40-48 h, respectively, after oral administration of helicidum at a dosage of 25.0 mg/kg. A UPLC coupled to time-of-flight MS (UPLC/TOF MS) was used to analyze the samples. Concerning XCMS, the ".raw" format files were preliminarily converted to the open mzXML format using massWolf-4.3.1 (http://sourceforge.net/projects/sashimi/files/massWolf%20(MassLynx%20converter)/). For converting lots of files a time, we wrote a tool rawTomzXML which also uses massWolf-4.3.1. The data were processed using XCMS version l.26.0 (http://www.bioconductor.org/packages/2.8/bioc/html/xcms.html) running under R version 2.13 (http://http://www.r-project.org/) which provided the running platform for XCMS. The "centWave" method from XCMS was used for chromatographic peak detection. Based on the m/z data of the metabolites obtained by XCMS, MS was used to identify the molecular formula. Nine metabolites were finally found and identified. For six of them, the bio-transformation mechanisms of the parent compound was elucidated: glucuronide conjugation (C(19)H(24)O(14)), reduction (C(13)H(18)O(7)), oxidation (C(13)H(16)O(8)), methylation (C(14)H(18)O(7)), and the mixed transformation of reduction, methylation, and acetylation (C(16)H(22)O(8)). For the other three metabolites, C(11)H(19)N(3)O(9), C(11)H(21)N(3)O(9), and C(14)H(15)NO(7), the bio-transformation mechanisms remain unknown and need further investigation. Calculated as mass of helicidum, the cumulative urine excretion rate of the metabolites was 8.39%. The amount of oxidized helicidum was more than 50% among the metabolites while the parent compound helicidum was 13.28% and the reduced helicidum 11.72%, indicating that oxidation was the major bio-transformation that occurred in vivo.