Analysis of catalpol derivatives by characteristic neutral losses using liquid chromatography combined with electrospray ionization multistage and time-of-flight mass spectrometry.

Iridoid glycosides are found in some families such as Scrophulariaceae, Labiatae, Rubiaceae and Gentianaceae. They have been of particular interest due to their important biochemical and physiological effects, including anti-inflammatory properties, anti-microbial or anti-viral effects, detoxification, diuretic functions, and resolving phlegm effects, etc. Veronica linariifolia Pall. ex Link ssp. dilatata (Nakai et Kitagawa) Hong is a member of the family Scrophulariaceae. Phytochemical studies and our previous studies show that iridoid glycosides are its main components, with catalpol being the predominant derivative. However, a systematic study of the mass spectrometry of catalpol derivatives and the fragmentation pathways under electrospray ionization (ESI) conditions has not been conducted yet, due to the complexity of the compounds, the easy degradation of iridoid glucosides and lack of authentic standards. Liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) has become a powerful technique, because of the increased power of resolution, full-scan capabilities, and accurate mass measurements, for the sensitive analysis of the multiple active components found in herbal medicines. It provides the elemental compositions of unknown constituents with high accuracy (routinely within 5 ppm) in complex matrices. In comparison, liquid chromatography/electrospray ionization multistage tandem mass spectrometry (LC/ESI-MS) has been widely used to provide highly sensitive and abundant information on the molecular mass and structural features on-line. The structural elucidation of the unknown constituents with the same parent nucleus using the LC/ ESI-MS platform is based on their structural similarities. Using the product ion scan mass spectrum of the authentic standards as a substructural template, detailed structural information of the compounds with similar structures can be rapidly characterized by extracting protonated or deprotonated molecules, subsequent or simultaneous product ions and neutral losses. Utilizing the combination of LC/TOF-MS and LC/ESIMS to gather accurate mass measurements and complementary structural information, we apply the powerful approach for the identification and structural elucidation of the iridoid glycosides, especially the catalpol derivatives in V. linariifolia. Characteristic neutral losses and product ions of the catalpol derivatives were concluded, and appropriate fragmentation pathways were readily proposed based on definite composition of the product ions. Entire plants of V. linariifolia were collected in Wanrong City, Shan Xi Province in China and identified by Prof. Minjian Qin. High-performance liquid chromatography (HPLC)-grade methanol was purchased from Merk (Darmstadt, Germany). Standards were isolated and purified from V. linariifolia in our laboratory, including verproside, catalpol, geniposidic acid and aucubin. Their structures (Fig. 1) were identified by spectroscopic methods (UV, IR, MS, H-NMR and C-NMR). The TOF-MS was performed using an Agilent orthogonal TOF mass spectrometer equipped with an ESI source (Agilent Corp., CA, USA), and was performed using full scan mode. The mass range was set at m/z 50–1000 in negative mode. ESI-MS analysis was performed on a diode-array detector (DAD) and a LC/ MSD trap SL mass spectrometer (Agilent Technologies, USA). The wavelength range was set as 210–600 nm and monitored at 255 nm. The ESI source conditions were as follows: drying gas (N2) flow rate, 9.0 L min ; drying gas temperature, 3508C; nebulizer, 35 psi; capillary, 3800 V; scan range, 50–1000 u in positive and negative ion mode; The capillary exit voltage was set at 150 V for positive ion mode and 150 V for negative ion mode. All the operations, acquisition and analysis of data were controlled by Chemstation software (Agilent Technologies, USA). ESI mass spectra in both negative and positive modes were examined in this study. The iridoid glycosides were not very stable in positive mode; they are prone to yield fragment ions rather than protonated molecules, and the MS and MS fragment ions produced in negative ion mode gave better results in our study. Hence, the MS data were mainly obtained in negative mode in the following experiments. The base peak chromatogram and typical HPLC chromatograms of the extracts obtained by HPLC/ESI-MS are presented in Fig. 2. More than 25 peaks were separated and detected according to the spectrum of each peak in the base peak chromatogram. Among them, 21 major constituents including 12 iridoids, 4 flavonoids, 4 phenolic acids and 1 lignan were identified (see Supporting Information). Structures of iridoids are shown in Fig. 1. The accurate mass measurements (<5 ppm), retention times (tR), formulas, experimental and theoretical masses and errors (in ppm) of iridoid compounds 1–12 (marked as C1–C12) are listed in Table 1. The UV and ESI-MS data of C1–C12 are listed in Table 2. Compound 1–4, 6–7 and 10–12 were characterized in this plant for the first time. Most of the iridoids occur in V. linariifolia in the glycoside forms, whose 7 and 8 positions are linked through an aglycone oxygen bridge with different benzene substituent groups at the 6 position (Fig. 3). The most common sugar residues are hexoses (glucose) linked to aglycone through glycosidic bonds at the O-1 position. Additionally, the fragmentation patterns indicate that the glycosyl residues of iridoids in Veronica are mostly glucose, which was employed to elucidate other iridoids in V. linariifolia according to the standards isolated. There are a few reports I: 10.1002/rcm.4676