UPLC Separation and QTof–MS Identification of Major Alkaloids in Plumula Nelumbinis

Zongtao Lin; Ruinan Yang; Zheng Guan; Ailiang Chen; Wei Li

doi:10.1002/pca.2517

. Author manuscript; available in PMC: 2017 Mar 22.

Published in final edited form as: Phytochem Anal. 2014 Apr 14;25(6):485–494. doi: 10.1002/pca.2517

UPLC Separation and QTof–MS Identification of Major Alkaloids in Plumula Nelumbinis

Zongtao Lin ^a,¹, Ruinan Yang ^a,¹, Zheng Guan ^b, Ailiang Chen ^b, Wei Li ^a,^*

PMCID: PMC5361407 NIHMSID: NIHMS753557 PMID: 24733684

Abstract

Introduction

As an essential medicine and tea source in many countries, Plumula Nelumbinis potentially exerts its major biological activities through its alkaloids. However, its activities are not fully understood due to the lack of studies on its chemical components.

Objective

To establish an Ultra Performance Liquid Chromatography–Diode-Array Detector (UPLC–DAD) method, combined with an Electrospray Ionization–Quadrupole Time-of-flight Mass Spectrometry (ESI–QTof MS), for the separation and identification of Plumula Nelumbinis alkaloids.

Methods

The eluant from an UPLC separation of an ethanol extract of Plumula Nelumbinis was directly infused into an ESI–QTof MS system. Both positive and negative ion modes of ESI with low and high Collision Energy (CE) were used to obtain sufficient MS information.

Results

21 alkaloids were tentatively identified based on their chromatographic characteristics, UV spectra, exact mass, MS fragments, and literature reports. They consist of 6 bis-1-benzyltetrahydroisoquinoline, 11 benzyltetrahydroisoquinoline (containing 2 glycoalkaloids and 2 quaternary ammoniums), 2 aporphine, one proaporphine, and one indole alkaloids. Eleven were identified in Plumula Nelumbinis for the first time and 7 were firstly reported in Nelumbo nucifera Gaertn. Five compounds, namely norcoclaurine-4′-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4′-methyl-N-methylcoclaurine and N-norisoliensinine, were characterized and proposed as new compounds.

Conclusion

The established UPLC–DAD–ESI–QTof–MS method is efficient for systematic identification of the alkaloids in Plumula Nelumbinis extract.

Keywords: Alkaloids, Plumula Nelumbinis, Ultra Performance Liquid Chromatography, QTof–MS

Introduction

Plumula Nelumbinis (called “Lian Zi Xin” in Chinese), the dried embryo of the seed of Nelumbo nucifera Gaertn., is a widely used traditional Chinese medicine and green tea in most Asian countries. Previous studies on its activities suggested its usage for treatment of Alzheimer’s disease (Lin et al., 2013), hypertension (Hu et al., 1990; Yang et al., 1999), arrhythmia (Qian 2002), platelet aggregation (Yu and Hu 1996), and stomach cancer (Shi et al., 2003). Alkaloids (Chen et al., 2007; Lin et al., 2013) including liensinine, isoliensinine, and neferine, are demonstrated as main bioactive compounds responsible for these activities. These isolated alkaloids exhibit various activities such as butyrylcholinesterase (BChE) inhibition (Lin et al., 2013), anti-human immunodeficiency virus (anti-HIV) (Kashiwada et al., 2005), and anti-oxidation (Jia et al., 1994) activities.

Chen and co-authors (Chen et al., 2007) has previously reported pronuciferine, lotusine, nuciferine, liensinine, isoliensinine and neferine using high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC–ESI–Tof–MS). Similarly, (Lin et al., 2013) reported norisoliensinine, 6-hydroxy-norisoliensinine, liensinine, isoliensinine and neferine using HPLC–ESI–IT–Tof–MS (IT: Ion-trap) and liensinine, isoliensinine and neferine were quantified using HPLC–ESI–MS/MS (Li et al., 2008).

In the present study, an Ultra Performance Liquid Chromatography with Quadrupole Time-of-flight Mass Spectrometry (UPLC–QTof MS) was applied to systematically identify major alkaloids in Plumula Nelumbinis.

EXPERIMENTAL

Materials and Chemicals

A Plumula Nelumbinis sample was purchased from Beijing Tong-Ren-Tang Group Co., Ltd. (Beijing, China), and identified by Dr. Ailiang Chen (Institute of Quality Standards and Testing Technology for Agro-products, Chinese Academy of Agricultural Sciences, Beijing, China) as the embryo of the seed of Nelumbo nucifera Gaertn. Analytical grade ethanol, HPLC-grade acetonitrile and water were purchased from Thermo Fischer Scientific (Fremont, CA, USA); ammonium acetate, formic acid and other reagents were purchased from Sigma (St. Louis, MO, USA).

Sample preparation

The sample was extracted as previously described (Lin et al., 2013). Briefly, Plumula Nelumbinis was crushed into fine powder (approx. 2 g) which was weighed accurately and extracted with 90% ethanol (50 mL) in an ultrasonic water bath for 30 min. This extraction procedure was repeated three times. Solvent from the combined extracts was removed under reduced pressure and the powder residue dissolved in methanol (50 mL). The methanol solution was filtered through a 0.45 μm membrane prior to use and an aliquot (1 μL) was injected into the UPLC system.

UPLC conditions

The UPLC analyses were performed on a Waters ACQUITY I-Class UPLC system (Waters, Milford, USA) consisting of a binary pump, a column manager, an autosampler, a degasser, and a diode-array detector (DAD). An Agilent Zorbax Eclipse Plus C18 Column (2.1 × 50 mm, 1.8 μm) (Agilent Technology, Santa Clara, CA) maintained at 35 °C was used with a mobile phase consisting of a linear gradient of A (0.02 M acetic ammonium in water) and B (acetonitrile) under the following conditions: 0 → 1→ 3.5 → 7 → 7.5 → 8.5 → 9.5 → 10 min, 5% → 10% → 15% → 20% → 25% → 70% → 85% → 100%B. The flow rate was 0.3 mL/min and the DAD was operated in the range of 200–400 nm but 282 nm was used (Chen et al., 2007; Lin et al., 2013) for displaying chromatograms.

MS conditions

The UPLC was coupled to a Xevo™ G2-S QTof Mass Spectrometer (Waters, Milford, USA), a quadrupole hybrid with orthogonal acceleration time-of-flight tandem mass spectrometer. The scan range was set at 50 to 1200 Da in both positive and negative modes. The capillary and cone voltages were set at 3.0 kV and 30 V, respectively. The desolvation gas was maintained at 800 L/h at a temperature of 500 °C. The cone gas was set at 100 L/h with a source temperature of 150 °C. The data acquisition rate was set to 0.3 s, with a 20 s interval. All data were collected in centroid mode, the lockspray frequency was set at 20 s. Leucine–enkephalin solution (100 ng/mL) was utilized as the lock mass compound with a flow rate of 5 μL/min (m/z 556.2771 and 554.2615 for positive and negative modes, respectively). The MS data were collected with full scan mode for both positive and negative ion modes with low (6 V) and high (ramp from 20 V to 40 V) collision energy (CE) data channels to get the intact precursor ions (MS) and the fragment ions (MS/MS). All the data acquisition and analyses were controlled by Waters MassLynx v4.1 software.

RESULTS AND DISCUSSION

Using extracted ion chromatograms (EIC) displaying the protonated molecular ion ((M + H)⁺, positive ion mode) or the deprotonated molecular ion ((M – H)⁻, negative ion mode) for anticipated or potential alkaloids allowed tentative identification of 21 alkaloids (Fig. 1). Both the positive and negative ion modes were found to be efficient for the identification of alkaloids in Plumula Nelumbinis.

The UV Chromatogram was shown in Figure 2A and MS data produced by low CE were shown in Figure 2B and 2C. As an accurate tool, QTof MS can largely overcome the separation problem associated with certain overlapping peaks in UPLC. As shown in Figure 2D, all peaks can be distinguished well based on the extracted ion chromatogram (EIC) of the ions for 21 alkaloids. As shown in Figure 1, the chemical structures of these compounds are quite similar. Among them, lotusine, liensinine, isoliensinine and neferine are the major alkaloids in the present study, consistent with existing literature reports (Chen et al., 2007; Li et al., 2008). MS and MS/MS data in both positive and negative modes were listed in Table 1, each characteristic fragment was predicted to yield the molecular formula within 5 ppm. These 21 components were classified into five groups based on their structures: bis-1-benzyltetrahydroisoquinolines, benzyltetrahydroisoquinolines, aporphines, proaporphines and indole alkaloids. Among them, eleven alkaloids were identified in Plumula Nelumbinis for the first time, seven were first reported in the plant Nelumbo nucifera GAERTN., and five compounds (namely norcoclaurine-4′-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4′-methyl-N-methylcoclaurine and N-norisoliensinine) were proposed as new compounds according to their MS spectra.

The chromatogram at 282 nm (A), TIC in positive (B) and negative (C) ion modes at low CE, and EICs (D1–D12) of the components in Plumula Nelumbinis sample.

Table 1.

Characterization of constituents in P. nelumbinis by UPLC-QTof-MS and MS/MS analysis in positive and negative ion modes. All source spectra are shown in Supplementary Materials.

No.	Identification	t_R^d (min)	(+/−) experimental MS (error in ppm)	(+/−) predicted formula	(+) experimental MS^E (formula)	(−) experimental MS^E (formula)
1^a^,^b^,^c	Norcoclaurine-4′-O-glucoside	0.49	434.1814 (−0.2)/432.1660 (0.5)	C₂₂H₂₈NO₈/C₂₂H₂₆NO₈	272.1285(C₁₆H₁₈NO₃), 255.1021(C₁₆H₁₅O₃), 161.0602(C₁₀H₉O₂), 107.0496(C₇H₇O)	270.1131(C₁₆H₁₆NO₃), 239.0704(C₁₅H₁₁O₃), 162.0555(C9H8NO2)
2^a^,^b^,^c	Norcoclaurine-6-O-glucoside	1.34	434.1817 (0.5)/432.1663 (1.2)	C₂₂H₂₈NO₈/C₂₂H₂₆NO₈	272.1288(C₁₆H₁₈NO₃), 255.1023(C₁₆H₁₅O₃), 161.0602(C₁₀H₉O₂), 145.0650(C₁₀H₉O), 123.0447(C₇H₇O₂), 107.0497(C₇H₇O)	270.1136(C₁₆H₁₆NO₃),, 162.0555(C₉H₈NO₂), 137.0603(C₈H₉O₂)
3^a^,^b	2-methyl-3-formylindole	1.05	160.0763 (0.6)/−	C₁₀H₁₀NO/−	146.0606(C₉H₈NO), 130.0655(C₉H₈N), 115.0545(C₉H₇)	–
4^b	Norcoclaurine	2.02	272.1290(1.1)/270.1128(0.3)	C₁₆H₁₈NO₃/C₁₆H₁₆NO₃	255.1023(C₁₆H₁₅O₃), 161.0606(C₁₀H₉O₂), 143.0496(C₁₀H₇O), 123.0450(C₇H₇O₂), 115.0551(C₉H₇), 107.0500(C₇H₇O)	162.0556(C₉H₈NO₂) 135.0441(C₈H₇O₂)
5	Lotusine	2.26	314.1761 (1.6)/312.1602 (0.6)	C₁₉H₂₄NO₃/C₁₉H₂₂NO₃	269.1181(C₁₇H₁₇O₃), 237.0917(C₁₆H₁₃O₂), 209.0969(C₁₅H₁₃O), 175.0761(C₁₁H₁₁O₂), 143.0499(C₁₀H₇O), 121.0656(C₈H₉O), 107.0500(C₇H₇O)	239.0703(C₁₅H₁₁O₃), 221.0602(C₁₅H₉O₂), 209.0599(C₁₄H₉O₂), 193.0652(C₁₄H₉O), 162.0554(C₉H₈NO₂)
6^a^,^b^,^c	Isolotusine	2.64	314.1757 (0.3)/312.1599 (−0.3)	C₁₉H₂₄NO₃/C₁₉H₂₂NO₃	269.1179(C₁₇H₁₇O₃), 255.1021(C₁₆H₁₅O₃), 237.0924(C₁₆H₁₃O₂), 209.0969(C₁₅H₁₃O), 161.1605(C₁₀H₉O₂), 143.0500(C₁₀H₇O), 107.0501(C₇H₇O)	239.0706(C₁₅H₁₁O₃), 221.0603(C₁₅H₉O₂), 209.0600(C₁₄H₉O₂), 193.0652(C₁₄H₉O), 145.0289(C₉H₅O₂)
7	4′-methyl-N-methylcoclaurine	4.18	314.1759 (1.0)/312.1597 (−1.0)	C₁₉H₂₄NO₃/C₁₉H₂₂NO₃	283.1333(C₁₈H₁₉O₃), 269.1179(C₁₇H₁₇O₃), 237.0918(C₁₆H₁₃O₂), 209.0969(C₁₅H₁₃O), 190.0873(C₁₁H₁₂NO₂), 175.0760(C₁₁H₁₁O₂), 143.0499(C₁₀H₇O), 121.0655(C₈H₉O), 107.0501(C₇H₇O)	239.0710(C₁₅H₁₁O₃), 221.0599(C₁₅H₉O₂), 147.0452(C₉H₇O₂)
8	Armepavine	5.50	314.1758 (0.6)/312.1594 (−1.9)	C₁₉H₂₄NO₃/C₁₉H₂₂NO₃	283.1331(C₁₈H₁₉O₃), 237.0911(C₁₆H₁₃O₂), 206.1177(C₁₂H₁₆NO₂), 190.0880(C₁₁H₁₂NO₂), 151.0753(C₉H₁₁O₂), 145.0650(C₁₀H₉O), 121.0649(C₈H₉O), 107.0493(C₇H₇O)	–
9^a^,^b	Nornuciferidine	2.53	298.1447 (1.3)/296.1284 (−1.0)	C₁₈H₂₀NO₃/C₁₈H₁₈NO₃	283.1213(C₁₇H₁₇NO₃), 268.0977(C₁₆H₁₄NO₃), 254.1188(C₁₆H₁₆NO₂), 240.1027(C₁₅H₁₄NO₂), 226.1236(C₁₅H₁₆NO), 190.0869(C₁₁H₁₂NO₂), 176.0715(C₁₀H₁₀NO₂), 148.0766(C₉H₁₀NO), 107.0500(C₇H₇O)	239.0706(C₁₅H₁₁O₃), 221.0603(C₁₅H₉O₂), 209.0603(C₁₄H₉O₂), 193.0652(C₁₄H₉O), 145.0289(C₉H₅O₂)
10^b	N-methylisococlaurine	3.53	300.1603 (1.0)/298.1441(−0.7)	C₁₈H₂₂NO₃/C₁₈H₂₀NO₃	269.1180(C₁₇H₁₇O₃), 237.0919(C₁₆H₁₃O₂), 209.0971(C₁₅H₁₃O), 175.0757(C₁₁H₁₁O₂), 145.0653(C₁₀H₉O), 107.0497(C₇H₇O)	133.0289(C₈H₅O₂)
11^b	N-methylcoclaurine	3.84	300.1605 (1.7)/298.1441 (−0.7)	C₁₈H₂₂NO₃/C₁₈H₂₀NO₃	269.1177(C₁₇H₁₇O₃), 237.0912(C₁₆H₁₃O₂), 209.0964(C₁₅H₁₃O), 143.0496(C₁₀H₇O), 107.0496(C₇H₇O)	223.0762(C₁₅H₁₁O₂)
12^a^,^b^,^c	6-demethyl-4′-methyl-N-methylcoclaurine	5.96	300.1599 (−0.3)/298.1440 (−1.0)	C₁₈H₂₂NO₃/C₁₈H₂₀NO₃	269.1177(C₁₇H₁₇O₃), 237.0914(C₁₆H₁₃O₂), 209.0965(C₁₅H₁₃O), 192.1018(C₁₁H₁₄NO₂), 175.0756(C₁₁H₁₁O₂), 143.0494(C₁₀H₇O), 107.0496(C₇H₇O)	–
13^b	Coclaurine	5.62	286.1444(0.3)/284.1285 (−0.7)	C₁₇H₂₀NO₃/C₁₇H₁₈NO₃	269.1178(C₁₇H₁₇O₃), 237.0910(C₁₆H₁₃O₂), 209.0961(C₁₅H₁₃O), 143.0492(C₁₀H₇O), 115.0544(C₉H₇), 107.0496(C₇H₇O)	176.0708(C₁₀H₁₀NO₂)
14	Norisoliensinine	7.06	597.2966 (0.2)/595.2803 (−0.8)	C₃₆H₄₁N₂O₆/C₃₆H₃₉N₂O₆	566.2549(C₃₅H₃₆NO₆), 554.2543(C₃₄H₃₆NO₆), 489.2400(C₂₉H₃₃N₂O₅), 475.2241(C₂₈H₃₁N₂O₅), 446.1977(C₂₇H₂₈NO₅), 296.1298(C₁₈H₁₈NO₃), 206.1184(C₁₂H₁₆NO₂), 192.1030(C₁₁H₁₄NO₂), 175.0765(C₁₁H₁₁O₂), 107.0499(C₇H₇O)	282.1125(C₁₇H₁₆NO₃)
15	6-hydroxynorisoliensinine	8.08	597.2970 (0.8)/595.2813 (0.8)	C₃₆H₄₁N₂O₆/C₃₆H₃₉N₂O₆	566.2535(C₃₅H₃₆NO₆), 554.2540(C₃₄H₃₆NO₆), 489.2370(C₂₉H₃₃N₂O₅), 475.2228(C₂₈H₃₁N₂O₅), 432.1815(C₂₆H₂₆NO₅), 312.1243(C₁₈H₁₈NO₄), 296.1271(C₁₈H₁₈NO₃), 206.1177(C₁₂H₁₆NO₂), 192.1021(C₁₁H₁₄NO₂), 107.0499(C₇H₇O)	282.1127(C₁₇H₁₆NO₃), 268.0969(C₁₆H₁₄NO₃)
16^a^,^b^,^c	N-norisoliensinine	8.42	597.2969 (0.7)/595.2813 (0.8)	C₃₆H₄₁N₂O₆/C₃₆H₃₉N₂O₆	566.2534(C₃₅H₃₆NO₆), 554.2534(C₃₄H₃₆NO₆), 436.2114(C₂₆H₃₀NO₅), 418.2005(C₂₆H₂₈NO₄), 314.1381(C₁₈H₂₀NO₄), 206.1172(C₁₂H₁₆NO₂), 192.1017(C₁₁H₁₄NO₂), 176.1066(C₁₁H₁₄NO), 121.0646(C₈H₉O), 107.0499(C₇H₇O)	581.2669(C₃₅H₃₇N₂O₆), 548.2445(C₃₅H₃₄NO₅), 473.2079(C₂₈H₂₉N₂O₅), 190.0880(C₁₁H₁₂NO₂), 176.0715(C₁₀H₁₀NO₂)
17	Pronuciferine	8.28	312.1606 (1.9)/−	C₁₉H₂₂NO₃/−	283.1330(C₁₈H₁₉O₃), 269.1175(C₁₇H₁₇O₃), 177.0905(C₁₁H₁₃O₂)	–
18	Liensinine	8.47	611.3129 (1.3)/609.2966(0.2)	C₃₇H₄₃N₂O₆/C3₇H₄₁N₂O₆	580.2709(C₃₆H₃₈NO₆), 568.2702(C₃₅H₃₈NO₆), 503.2549(C₃₀H₃₅N₂O₅), 489.2389(C₂₉H₃₃N₂O₅), 446.1977(C₂₇H₂₈NO₅), 312.1235(C₁₈H₁₈NO₄), 206.1179(C₁₂H₁₆NO₂), 192.1018(C₁₁H₁₄NO₂), 174.0911(C₁₁H₁₂NO), 162.0912(C₁₀H₁₂NO), 107.0492(C₇H₇O)	595.2799(C₃₆H₃₉N₂O₆), 577.2704(C₃₆H₃₇N₂O₅), 487.2221(C₂₉H₃₁N₂O₅), 473.2070(C₂₈H₂₉N₂O₅), 386.1748(C₂₅H₂₄NO₃), 297.1366(C₁₉H₁₈N₂Na), 282.1129(C₁₇H₁₆NO₃)
19	Isoliensinine	8.60	611.3123 (0.3)/609.2970 (0.8)	C₃₇H₄₃N₂O₆/C₃₇H₄₁N₂O₆	580.2710(C₃₆H₃₈NO₆), 568.2699(C₃₅H₃₈NO₆), 503.2549(C₃₀H₃₅N₂O₅), 489.2383(C₂₉H₃₃N₂O₅), 475.2234(C₂₈H₃₁N₂O₅), 432.1811(C₂₆H₂₆NO₅), 206.1180(C₁₂H₁₆NO₂), 192.1025(C₁₁H₁₄NO₂), 176.1071(C₁₁H₁₄NO), 121.0650(C₈H₉O)	577.2701(C₃₆H₃₇N₂O₅), 534.2271(C₃₄H₃₂NO₅), 487.2234(C₂₉H₃₁N₂O₅), 473.2079(C₂₈H₂₉N₂O₅), 441.1808(C₂₇H₂₅N₂O₄), 386.1748(C₂₅H₂₄NO₃), 326.1392(C₁₉H₂₀NO₄), 312.1602(C₁₉H₂₂NO₃), 296.1284(C₁₈H₁₈NO₃), 190.0868(C₁₁H₁₂NO₂), 176.0711(C₁₀H₁₀NO₂), 121.0291(C₇H₅O₂)
20	Neferine	8.90	625.3280 (0.3)/623.3117(−0.6)	C₃₈H₄₅N₂O₆/C₃₈H₄₃N₂O₆	611.3129(C₃₇H₄₃N₂O₆), 594.2869(C₃₇H₄₀NO₆), 582.2859(C₃₆H₄₀NO₆), 503.2543(C₃₀H₃₅N₂O₅), 489.2391(C₂₉H₃₃N₂O₅), 446.1963(C₂₇H₂₈NO₅), 328.1549(C₁₉H₂₂NO₄), 206.1182(C₁₂H₁₆NO₂), 192.1023(C₁₁H₁₄NO₂), 176.1071(C₁₁H₁₄NO), 135.0806(C₉H₁₁O), 121.0651(C₈H₉O)	591.2861(C₃₇H₃₉N₂O₅), 548.2443(C₃₅H₃₄NO₅), 518.1972(C₃₃H₂₈NO₅), 487.2234(C₂₉H₃₁N₂O₅), 455.1969(C₂₈H₂₇N₂O₄), 440.1858(C₂₈H₂₆NO₄), 402.1708(C₂₅H₂₄NO₄), 386.1756(C₂₅H₂4NO₃), 312.1602(C₁₉H₂₂NO₃), 296.1287(C₁₈H₁₈NO₃), 264.1026(C₁₇H₁₄NO₂), 176.0712(C₁₀H₁₀NO₂), 121.0290(C₇H₅O₂)
21	Nuciferine	9.20	296.1654 (1.0)/−	C₁₉H₂₂NO₂/−	265.1231(C₁₈H₁₇O₂), 250.0996(C₁₇H₁₄O₂), 235.0765(C₁₆H₁₁O₂), 219.0807(C₁₆H₁₁O), 191.0860(C₁₅H₁₁), 179.0855(C₁₄H₁₁)	–

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firstly identified in Nelumbo nucifera Gaertn.

firstly identified in Plumula Nelumbinis;

proposed as a new compound;

retention time.

Identification of bisbenzyltetrahydroisoquinoline alkaloids for peaks 14–16 and 18–20

For peaks 18, 19 and 20 (Figure 2 D10–D11) in positive ion mode, the molecular formula of ions at m/z 611.3116 and 625.3278 were predicted as C₃₇H₄₃N₂O₆ ([M+H]⁺) and C₃₈H₄₅N₂O₆ ([M+H]⁺). The UV spectra of these peaks showed maximal absorption wavelengths at 229 and 281 nm, 227 and 283 nm, and 224 and 282 nm, respectively. In MS/MS, peak 18 showed ions at m/z 489.2389 (C₂₉H₃₃N₂O₅), 312.1235 (C₁₈H₁₈NO₄) and 206.1179 (C₁₂H₁₆NO₂). These data were consistent with 18 being liensinine (Chen et al., 2007; Li et al., 2008). As an isomer of liensinine, twoions at m/z 475.2234 (C₂₈H₃₁N₂O₅, [489–CH₂]⁺) and 192.1025 (C₁₁H₁₄NO₂, [206–CH₂]⁺) of compound 19 indicated a different substitution position, and was tentatively identified as isoliensinine (Chen et al., 2007; Li et al., 2008; Lin et al., 2013). 20 showed one more methyl group than 18 and 19; and its two major ions (m/z 489 and 206) are the same as that of liensinine. Thus 20 was tentatively identified as neferine based on its MS behaviors (Chen et al., 2007; Li et al., 2008; Lin et al., 2013). Data in Table 1 were also consistent with their identifications.

The EIC of m/z 597.2969 (C₃₆H₄₁N₂O₆, [M+H]⁺) in positive ion mode exhibited three peaks (peak 14, 15 and 16 shown in Figure 2 D8). This ion indicates one less methyl group than isoliensinine They exhibited a major ion (m/z 192.1024, C₁₁H₁₄NO₂), the same one of isoliensinine, in MS/MS. This indicated possible demethylation of R₃ (Figure 1). In addition, detailed fragments (Table 1) strongly suggested their similarity with isoliensinine. Compound 14 and 15 were thus tentatively identified as norisoliensinine and 6-hydroxynorisoliensinine (Lin et al., 2013), and 16 was tentatively proposed to have a secondary amine due to its latter retention time and was given the trial name N-norisoliensinine. The proposed fragmentation pathway of 16 was depicted in Figure 3.

The proposed fragmentation pathway of N-norisoliensinine (compound 16) in positive ion mode.

Identification of benzyltetrahydroisoquinoline alkaloids for peaks 4–8 and 10–13

The EIC of m/z 314.1751 (C₁₉H₂₄NO₃, [M+H]⁺)of 5, 6, 7 and 8 in Figure 2 D4 indicated they were substituted benzyltetrahydroisoquinoline alkaloids (Li et al., 2008; Sagrero-Nieves 1986). In positive MS/MS, both of 5 (Figure 4A) and 6 yielded same fragments m/z 269.1181 (C₁₇H₁₇O₃, cleavages of C₁-N₂ and N₂-C₃), 237.0917 (C₁₆H₁₃O₂, additional loss of CH₃O- at C₆), 209.0969 (C₁₅H₁₃O) and 175.0761 (C₁₁H₁₁O₂), while peaks 7 (Figure 4B) and 8 showed an extra ion (m/z 283.1333, C₁₈H₁₉O₃) indicating a neutral fragment loss of CH₃NH₂- from a tertiary amine,. This is the same behavior of 16 (from m/z 597 to 556 Figure 3). The loss of CH₃(CH₃)NH- from 314 for 5 and 6 indicated a quaternary ammonium group which was consistent with their greater tailing effect and shorter retention times. Therefore 5 and 6 were tentatively identified as lotusine (Li et al., 2008) and isolotusine, and 7 and 8 were tentatively identified as 4′-methyl-N-methylcoclaurine and armepavine as known constituents in Plumula Nelumbinis (Sagrero-Nieves 1986). The fragmentation pathways of lotusine (5) and 4′-methyl-N-methylcoclaurine (7) are shown in Figure 4A and Figure 4B.

The MS/MS spectra (high CE) and possible fragmentation schemes of lotusine (A, compound 5), 4′-methyl-N-methylcoclaurine (B, compound 7), 6-demethyl-4′-methyl-N-methylcoclaurine (C, compound 12), norcoclaurine-6-O-glucoside (D, compound 2), coclaurine (E, compound 13) and nuciferidine (B, compound 9) in positive ion mode.

Compounds 10, 11 and 12 in Figure 2 D6 ([M+H]⁺, C₁₈H₂₂NO₃) have one less methyl group than 7 (or 5, 6, 8). In (+)MS/MS, their spectra (Figure 4C of peak 12) are almost the same, very similar to 7 (Figure 4B) except absences of m/z 314 and 283. The loss of CH₃NH₂- from m/z 300 suggested they were all N-methyl tertiary amines. Since 10 and 11 are close to each other in UPLC, the second CH₃- was tentatively assigned at C₇ for 10 (N-methylisococlaurine) or C₆ for 11 (N-methylcoclaurine) (Kunitomo et al., 1973; Mukherjee et al., 2009). Methylation of C₁₃ OH group was possibly assigned for 12 and named 6-demethyl-4identifieN-methylcoclaurine as a new compound after comparing with 7.

The ion at m/z 286.1444 (C₁₇H₂₀NO₃, 13) in Figure 2 D7 indicated one less methyl group than 10 (11 or 12). The (+)MS/MS data of this compound (Table 1) was similar with other identified benzyltetrahydroisoquinoline compounds, suggesting their structure similarities. The loss of NH₃- (m/z 269) suggested 13 a secondary amine, and it was tentatively identified as coclaurine as a common component in Nelumbo nucifera (Kashiwada et al., 2005). The fragments of 13 were further explained in Figure 4 E. For peak 4 (EIC of m/z 272.1281, C₁₆H₁₈NO₃) in Figure 2 D3, it indicated one less methyl group than coclaurine (13), andwas tentatively identified as norcoclaurine by reported MS information (Kashiwada et al., 2005; Koshiyama et al., 1970).

Identification of glycoalkaloids for peaks 1 and 2

The EIC of m/z 434.1809 (C₂₂H₂₇NO₈) gave two polar compounds (Figure 2 D1). The ion at m/z 272.1288 (C₁₆H₁₈NO₃) in MS/MS (Figure 4 D) indicated a loss of glucose (C₆H₁₀O₅) moiety (Agnihotri et al., 2008; Kashiwada et al., 2005; Mukherjee et al., 2009). The ions at m/z 255.1021(C₁₆H₁₅O₃, [272–NH₃]⁺) and 161.0602(C₁₀H₉O₂, [272–NH₃–C₆H₅O]⁺) indicated the aglycone of the two glycosides was norcoclaurine. The glycosylated compounds were tentatively assigned as norcoclaurine-4′-O-glucoside (1) and norcoclaurine-6-O-glucoside (2), respectively.

Identification of aporphine alkaloids for peaks 9 and 21

Peak 21 (m/z 296.1645, C₁₉H₂₂NO₂) (Figure 2 D1) displayed m/z 265.1231 (C₁₈H₁₇O₂, [M+H–CH₃NH₂]⁺), 250.0996 (C₁₇H₁₄O₂), 235.0765 (C₁₆H₁₁O₂), 219.0807 (C₁₆H₁₁O), 191.0860 (C₁₅H₁₁), 179.0855 (C₁₄H₁₁) in their MS/MS, suggesting the presence of N-methyl group. It was tentatively identified as nuciferine (Agnihotri et al., 2008; Luo et al., 2005). Peak 9 (m/z 298.1438, C₁₈H₂₀NO₃) in Figure 2 D5 indicated one more OH group and one less methyl group than nuciferine, it also showed similar UV spectrum indicating its aporphine structure. MS/MS spectrum have shown successive losses of two methyl groups (m/z 283 and 268 in Table 1) and one H₂O (m/z 254), indicating two methoxy and one hydroxyl groups. This compound was tentatively identified as nornuciferidine reported as a natural alkaloid (Rasamizafy et al., 1987) shown in Figure 4 F.

Identification of proaporphine alkaloid for peaks 17 and indole alkaloid for peak 3

Peak 17 (m/z 312.1606, C₁₉H₂₂NO₃) in Figure 2 D9exhibited ions at m/z 283.1330 (C₁₈H₁₉O_3, [M+H–CH₃N]⁺), 269.1175 (C₁₇H₁₇O₃), 177.0905 (C₁₁H₁₃O₂) (Table 1) in (+)MS/MS. It was tentatively identified as pronuciferine after comparing its UV and MS spectra (Kunitomo et al., 1973; Li et al., 2008; Mukherjee et al., 2009). For peak 3 (Figure 2 D2), the precursor m/z 160.0763 (C₁₀H₁₀NO, [M+H]⁺) yielded m/z 146.0606 (C₉H₈NO), 130.0655 (C₉H₈N) and 115.0545 (C₉H₇), indicating the losses of one methoxy and one carbonyl groups. Based on its UV spectrum (UV_max at 220 and 273 nm), compound 3 and it was tentatively identified as a known compound 2-methyl-3-formylindole.

In summary, the acquired UPLC/DAD/QTof-MS data are powerful for rapid identification of various alkaloids in Plumula Nelumbinis. This study may provide the helpful chemical basis to explain the pharmacological and diary efficacies of Plumula Nelumbinis, and facilitate further drug development and quality control of this material.

Supplementary Material

NIHMS753557-supplement-1.docx^{(2MB, docx)}

Acknowledgments

This work was partially supported by an NIH instrument grant 1S10OD010678-01 to WL and funds from the Department of Pharmaceutical Sciences, College of Pharmacy, the University of Tennessee Health Science Center.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS753557-supplement-1.docx^{(2MB, docx)}

[R1] Agnihotri VK, ElSohly HN, Khan SI, Jacob MR, Joshi VC, Smillie T, Khan IA, Walker LA. Constituents of Nelumbo nucifera leaves and their antimalarial and antifungal activity. Phytochem Lett. 2008;1:89–93. doi: 10.1016/j.phytol.2008.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] Chen Y, Fan G, Wu H, Wu Y, Mitchell A. Separation, identification and rapid determination of liensine, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera Gaertn. by liquid chromatography coupled to diode array detector and tandem mass spectrometry. J Pharm Biomed Anal. 2007;43:99–104. doi: 10.1016/j.jpba.2006.06.016. [DOI] [PubMed] [Google Scholar]

[R3] Hu W, Guo L, Feng X, Jiang M. Hypotensive effects of neferine. Chin J Pharmacol Toxicol. 1990;4:107–110. [Google Scholar]

[R4] Jia J, Ao M, Hu B, Li Y, Guo M, Mo C. The Effects of Neferine on Lipid Peroxides and Active Oxygen Free Radical. Acta Universitatis Medictnae Tangji. 1994;23:62–64. [Google Scholar]

[R5] Kashiwada Y, Aoshima A, Ikeshiro Y, Chen YP, Furukawa H, Itoigawa M, Fujioka T, Mihashi K, Cosentino LM, Morris-Natschke SL, et al. Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids. Bioorg Med Chem. 2005;13:443–448. doi: 10.1016/j.bmc.2004.10.020. [DOI] [PubMed] [Google Scholar]

[R6] Koshiyama H, Ohkuma H, Kawaguchi H, Hsu H, Chen Y. Isolation of 1-(p-hydroxybenzyl)-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline (demethylcoclaurine), an active alkaloid from Nelumbo nucifera. Chem pharm bull. 1970;18:2564–2568. [Google Scholar]

[R7] Kunitomo J, Yoshikawa Y, Tanaka S, Imori Y, Isoi K, Masada Y, Hashimoto K, Inoue T. Alkaloids of Nelumbo nucifera. Phytochemistry. 1973;12:699–701. [Google Scholar]

[R8] Li W, Chen J, Yin Y, Wang X, Lee FS. Analysis of Alkaloids in Semen Nelumbinis by Accelerated Solvent Extraction-High Performance Liquid Chromatography-Diode Array Detection-Electrospray Ioninztion-Time of Flight-Mass Spectrometry. Chin J Anal Chem. 2008;36:79–82. [Google Scholar]

[R9] Lin Z, Wang H, Fu Q, An H, Liang Y, Zhang B, Hashi Y, Chen S. Simultaneous separation, identification and activity evaluation of three butyrylcholinesterase inhibitors from Plumula nelumbinis using on-line HPLC-UV coupled with ESI-IT-TOF-MS and BChE biochemical detection. Talanta. 2013;110:180–189. doi: 10.1016/j.talanta.2013.02.033. [DOI] [PubMed] [Google Scholar]

[R10] Luo X, Chen B, Liu J, Yao S. Simultaneous analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by high-performance liquid chromatography–photodiode array detection–electrospray mass spectrometry. Analytica Chimica Acta. 2005;538:129–133. [Google Scholar]

[R11] Mukherjee PK, Mukherjee D, Maji AK, Rai S, Heinrich M. The sacred lotus (Nelumbo nucifera) - phytochemical and therapeutic profile. J Pharm Pharmacol. 2009;61:407–422. doi: 10.1211/jpp/61.04.0001. [DOI] [PubMed] [Google Scholar]

[R12] Qian J. Cardiovascular pharmacological effects of bisbenzylisoquinoline alkaloid derivatives. Acta Pharmacol Sin. 2002;23:1086–1092. [PubMed] [Google Scholar]

[R13] Rasamizafy S, Hocquemiller R, Cavé A, Fournet A. Alcaloïdes des Annonacées, 78. Alcaloïdes des Écorces d’un Duguetia spixiana de Bolivie. J Nat Prod. 1987;50:674–679. [Google Scholar]

[R14] Sagrero-Nieves L. Isolation of lapachol from Diphysa robinoides. J Nat Prod. 1986;49:547. doi: 10.1021/np50045a035. [DOI] [PubMed] [Google Scholar]

[R15] Shi S, Zhuang Y, Cao J. Effects of neferine on gastric carcinoma apoptosis induced by vincristine. Chin Pharmacol Bull. 2003;19:928–930. [Google Scholar]

[R16] Yang J, He W, Zhang X. Study on the pharmacological effects of neferine. Academic J Guangdong College Pharm. 1999;15:183–185. [Google Scholar]

[R17] Yu J, Hu W. Effects of neferine on platelet aggregation and concentration of cytoplasmic free calcium. Chin J Pharmacol Toxicol. 1996;10:120–122. [Google Scholar]

PERMALINK

UPLC Separation and QTof–MS Identification of Major Alkaloids in Plumula Nelumbinis

Zongtao Lin

Ruinan Yang

Zheng Guan

Ailiang Chen

Wei Li