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Journal Pre-proofs Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS/MS) Hasanjan Ahad, Hongli Jin, Yanfang Liu, Jixia Wang, Guangying Sun, Xinmiao Liang, Haji Akber Aisa PII: DOI: Reference:

S1570-0232(19)31096-7 https://doi.org/10.1016/j.jchromb.2019.121923 CHROMB 121923

To appear in:

Journal of Chromatography B

Received Date: Revised Date: Accepted Date:

18 July 2019 20 November 2019 1 December 2019

Please cite this article as: H. Ahad, H. Jin, Y. Liu, J. Wang, G. Sun, X. Liang, H. Akber Aisa, Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/ MS), Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121923

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© 2019 Published by Elsevier B.V.

Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

Hasanjan Ahad 1, a, b, c, Hongli Jin 1, b, Yanfang Liu b, Jixia Wang b, Guangying Sun a, Xinmiao Liang b, *, Haji Akber Aisa a, *

1: these authors contributed equally to this work a

Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of

Physics and Chemistry, Chinese Academy of Sciences, Urumqi, Xinjiang 830011, People`s Republic of China b

Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics,

Chinese Academy of Sciences, Dalian 116023, China c

University of Chinese Academy of Sciences, Beijing 100039, China

Corresponding author: Prof. Dr. Haji Akber Aisa Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011 (P. R. China) Email: [email protected] Fax: +86-0991-3838957 Additional corresponding author: Professor Xinmiao Liang E-mail: [email protected]

Running title: Chemical profiling of spermidines in Goji Berry by strong cation exchange

solid-phase

extraction

(SCX-SPE)

combined

with

ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

1

Abstract Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance

liquid

chromatography-quadrupole

time-of-flight

mass

spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.

Keywords: Spermidines / Fragmentation Rules / Goji Berry / Selective Enrichment / UPLC-DAD/QTOF-MS/MS

2

1 Introduction Spermidines are a group of acylated hydroxycinnnamic acid amides, which mainly distributed in the family of Solanaceae, and responsible for the maintenance of general cell homeostasis [1,2]. These components are commonly represented as N, N′-bis- and N,N′,N′′-tris-hydroxycinnamoyl spermidines, where N1, N2, and N3 positions can be substituted with p-coumaroyl, caffeoyl, feruloyl, 5- hydroxyferuloyl, and sinapoyl residues, as well as the corresponding 4-O-hexosides [3]. Recently, this type of alkaloids has received growing attention, because of their outstanding antioxidant activity and low toxicity [4-6]. Mounting evidence has indicated that spermidine correlates with considerable health benefits in lifespan extension and neuroprotection [7-12]. In addition, natural spermidines can reduce cardiovascular pathologies and its co-morbidities such as obesity, diabetes and renal abnormalities. These reports demonstrated that natural spermidines have great potential to be a promising source for new drug discovery from natural products [13]. However, to date, only about 30 naturally occurring spermidines have been characterized [14-15]. It is imperative to carry out an intensive study on the separation and identification of spermidines from natural products, which would be of significance to the complement of structural pattern diversity and the applications in the medical field. Lycium barbarum is a deciduous shrub belonging to the family of Solanaceae. Its fruit, also known as goji berry, is a valued TCM. In the Chinese medicinal monographs “shennongbencaojing”, “ben cao gang mu” and “ben cao hui yan”, L. barbarum fruits were recorded as nourishing liver and kidney, enhancing eyesight [16], enriching blood, invigorating sex, reducing rheumatismā and so on [17-18]. In recent years, its fruit has been praised for the uses related to healthy aging, and human well-being effects with its immunity improvement [19,20], anti-oxidation, anti-radiation, anticancer, enhancing hemopoiesis properties [21-24]. Among the chemical compounds, polysaccharides and carotenoids are well documented as abundant bioactive ingredients of goji berry [25-27]. Nevertheless, few studies are reported the compositions of spermidines. To our best knowledge, only one report

3

illustrated that fifteen dicaffeoylspermidines were isolated and analyzed structurally by nuclear magnetic resonance (NMR) [15]. The chemical constituents of spermidine derivatives in this plant remain unexploited yet. Ultrahigh-performance liquid chromatography (UPLC) coupled to quadrupole Time-of-flight mass spectrometry (Q-TOF-MS) was expected to separate and identify spermidines in goji crude extracts due to its advantages of excellent peak capacity and determined masses of molecular ions [28]. This technique also permits MS/MS and thus provides corresponding fragment ions with accurate masses [29, 30]. The combination of UPLC and Q-TOF/MS was able to realize on-line and high sensitive detection prior to purification [31-34]. However, detailed studies on fragmentation behaviors of different types/groups of spermidines were hampered, due to the lack of available calibration standards [35]. Solid-phase extraction (SPE) is an effective pretreatment technique to simplify the crude extracts and improve qualitative sensitivity [36-38]. Various sorbents provide different selectivity to the targets, which plays a significant role in the wide applications of SPE [39]. Silica-based strong cation exchange (SCX) packing is a preferred material for selective enrichment of basic components, according to their charge property [40]. The SCX-SPE method has attracted considerable attention for separation and enrichment of alkaloids from natural products [41]. The aim of this study was to establish a UPLC-Q-TOF/MS/MS method for chemical profiling of spermidines from goji berry. The fragmentation behaviors of four different types/groups of spermidines standards (Fig. 1) were elucidated based on the MS/MS fragment ions in a positive mode. An SPE method was developed and optimized for the selective enrichment of spermidines. With the established online SPE coupled with UPLC-Q-TOF/MS/MS method, spermidine compositions from goji berry were fully investigated.

2 Materials and methods 2.1 Apparatus and reagents

4

HPLC-grade acetonitrile and methanol were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). HPCL-grade formic acid (FA) was purchased from J&K chemical (Hebei, China). Water for UPLC mobile phase was reverse osmosis Milli-Q water (18.2 MU) (Millipore, USA). N1, N3-bis-dihydrocaffeoyl-spermidine (S1), N3-dihydrocaffeoyl-N1-caffeoyl-spermidine

(S2),

N1-caffeoyl-N3-dihydrocaffeoyl-spermidine (S3), and N1, N3-dicaffeoyl spermidine (S4) were isolated from L. ruthenicum in our lab, and identified by MS and NMR. The exact structures were shown in Fig.1. The goji berry was purchased from Zhong Ning County of Ningxia Hui Autonomous Region, and authenticated by senior engineer Xiaoping Yang, Xi Yuan Hospital of China Academy of Traditional Chinese Medicine. 2.2 Sample preparation Dried goji berry fruit samples were ground and passed through a 60 mesh filter. The powdered sample (5 g) was placed in a flask and mixed with 50 mL of 90% methanol by ultrasound-assisted extraction for 3 h at room temperature. Subsequently, the filtrate was concentrated by rotary evaporation at 50ć and 0.1 MP conditions. The extraction yield was 16.67%. The SCX-SPE method, with an SCX packing column (1 g´ 6 ml, 60 μm, dia.) was used for the enrichment of spermidines from the goji berry extraction. The loading amount of the sample was 0.05 g (3 mL). The elution process included two steps: the sample was eluted with 90 % methanol to remove the non-alkaloid compositions, and 0.1 mol/L NaH2PO4˄V water: V acetonitrile: V phosphoric acid = 30: 70: 2˅ was used as an eluent for spermidine components. The target components were concentrated by centrifuge, and desalinated with a C18YE column (1 g´ 6 mL, 60 μm, dia.). Finally, the target components were filtered with a 0.22 mm filter membrane prior to UPLC-DAD/ESI-MS/MS analysis.

2.3 Chromatographic conditions The analysis experiments were performed on an Agilent 1290 Infinity LC instrument (Agilent, USA) coupled to an Agilent 6540 series QTOF-MS (Agilent, 5

USA) equipped with an ESI source, a diode-array detector (DAD), an automatic sample injector, a degasser and a column thermostat. S1-S4 samples were directly injected for MS/MS analysis. The scan range was from m/z 100 to 1500. The mass spectrometer was operated in the ESI+ mode. Other conditions were optimized as follows: drying gas flow rate, 8 L min-1; gas temperature, 350 Ԩ; nebulizer gas pressure, 35 psi; capillary and fragmentor potentials, 3500 and 175 V, respectively. The collision energies, from 10 to 50 eV, were examined for the characterization of dicaffeoylspermidines. To characterize the spermidines in goji berry, separation was conducted on a XCharge C18 column (purchased from Acchrom Co. Dalian, China). The flow rate was 0.2 mL min-1. The UV/Vis spectral data were recorded in range of 210–400 nm, and chromatograms were obtained at 280 nm. The column temperature was maintained at 30 Ԩ. The mobile phases A and B were 0.1% FA (v/v) in water and

0.1% FA (v/v) in acetonitrile, respectively. The gradient elution conditions were as follows: 0–5 min, 0% B; 5–20 min, 0–2% B; 20–40 min, 2–4% B; 40–80 min, 4–8% B; 80–95 min, 8–20% B. The UPLC separation was split by proportional valves and then delivered into the mass spectrometer. Conditions for the mass spectrometer were the same as above.

3 Result and discussion 3.1 Fragmentation rules of representative spermidines Spermidines were analyzed by MS using both negative and positive ion modes. The result showed that the positive ion mode offered more reliable identification, due to the predictable fragment ions. This result was consistent with previous reports [14, 42]. Therefore, in this study, all the mass spectra were collected by ESI in positive ion mode. The MS conditions were optimized to realize optimal sensitivity. Different collision energies from 10 to 50 eV were tested for S2. Abundant fragmentation ions were generated in the MS/MS spectra when the collision energy was raised to 30 eV. However, no other obvious fragment ions were detected when collision energy

6

increased from 30 to 50 eV (data not shown). Thus, all samples were analyzed under the collision energy of 30 eV. The detailed fragmentation behaviors of spermidines were carefully discussed below. S1 featured an [M+H]+ ion at m/z 474.2597, and fragment ions at m/z 310.2112, 293.1855, 222.1149, 165.0544, and 123.0441 were obtained (Fig. 2 (A)). The fragmentation behavior of S1 was inferred based on these fragment ions. The base peak at m/z 474 gave a fragment ion at m/z 310 [M+H-165]+ and the most abundant peak at m/z 165, which represented the dihydrocaffeoyl (dhc) group (165 Da) and the amide moiety, respectively. The fragment ion at m/z 310 generated the peak at m/z 293, and this ion was formed by a displacement rearrangement after the loss of ammonia (17 Da). The ion at m/z 293 produced the peak at m/z 222 through the loss of a cyclic (CH2)4-NH2 (72 Da) unit. The one at m/z 123 was observed by the loss of a unit at m/z at 100 from the ion at m/z 222. The displacement rearrangement was a type of common intramolecular rearrangements for long chain primary amines, and can form corresponding cyclic amine fragment ions in MSE analysis [43]. Fig. 3 showed the detailed fragmentation pathway of S1. Fig. 2 (B) represented the MS/MS analysis of S2. This compound exhibited an [M+H]+ ion at m/z 472.2423, and produced five major product ions at m/z 310.2120, 293.1848, 220.0970, 163.0387 and 123.0435. The daughter ion at m/z 310 ([M+H-163]+) was obtained by the loss of the caffeoyl molecule at m/z 163. Whereafter, the ion at m/z 293 ([M+H-163-17]+) was produced by ammonia leaving from the ion at m/z 310. The ion at m/z 222.1114 ([M+H-163-17-72]+) and the rearrangement ion at m/z 72 were produced after the breakage of a C−N2 bond from the ion at m/z 293. With the splitting of the C−N3 bond from the ion at m/z 222, the comparatively stable fragment peak at m/z 123.0441 [M+H-165-17-72-100]+ and the displacement rearrangement ion at m/z 100 were generated. The fragmentation pathway for S2 was proposed (shown in Fig. 4). S3 yielded an [M+H]+ ion at m/z 472.2423, and daughter ions at m/z 310.2119, 293.1852, 222.1114, 163.0381 and 123.0434, respectively (Fig. 2 (C)). The ion at m/z 310 [M+H-163]+ resulted from the loss of a caffeoyl group from the parent ion by the 7

break of C-N1 bond. The fragment ion at m/z 293.1855 [M+H-165-17]+ indicated the loss of ammonia from the ion at m/z 310. The product ion at m/z 222 [M+H-163-72]+ represented the elimination of rearrangement ion at m/z 72 from the ion at m/z 293. The fragment ion at m/z 123.0441 [M+H-165-17-72-100]+ was yielded by the loss of the substitution rearrangement production ion at m/z 100 from the ion at m/z 222 (Fig. 5). Noticeably, another series of fragment ions at m/z 253.3221, 236.0236, and 220.0970 were detected in the MS/MS spectra of S3. It was assumed that with the cleavage of the C−N2 bond, the parent ion produced two fragment ions at m/z 220.0970 and 253.3221. The fragment ion at m/z 253 produced another fragment ion at m/z 236 with an ammonia loss. The product ion at m/z 165 represented the elimination of rearrangement ion at m/z 72 from the ion at m/z 236. A simplified fragmentation pathway for S3 was proposed and shown in Fig.5. S2 and S3 were N, N′-caffeoyl-dhydrocaffeoyl-spermidine isomers. Because of the similar fragment ions, it was difficult to differentiate this type of spermidine isomers by MS analysis. However, the results of this study proposed that S2 and S3 had certain differences on the fragmentation behaviors. With the support of references and the two different series of fragment ions [14], it was assumed that because of the dissymmetrical structure, the linkage location of chemical groups had possibly made some particular differences on MS/MS fragmentation patterns. In the structure of S3, the C=C (in the caffeoyl unit) bond was located at the C-N3 side, and might increase the molecular conjugate effect of this side. In the light of the mass spectrometric cleavage stability of different ions and radicals, the enhancement of molecular conjugated effect lead to the C−N2 bond cleaved easily [43]. The appearance of another series of fragment ions, especially the co-existence of the fragment ions at m/z 220 and 222 was the specificity of S3, and they could be important characteristic fragment ions to distinguish spermidine isomers S2 and S3. S4 showed its molecular ion [M+H]+ at m/z 470.2225, and yielded product ions at 308.1955, 291.1980, 220.0962, 163.0387 and 123.0434, respectively. One fragment ion at m/z 308.1955 [M+H-163]+ corresponded to the caffeoyl ion lose. The ion at m/z 308 lost ammonia and produced fragment ion at m/z 291.1680 [M+H-163-17]+. 8

The fragment ion at m/z 220 [M+H-163-17-72]+ arose after the loss of ion m/z 72. The fragment ion at m/z 220 [M+H-163-17-72]+ resulted from the loss of substitution rearrangement production ion at m/z 100, and produced the relatively stable fragment ion at m/z 123. Proposed fragmentation pathway for S4 was revealed in Fig. 6. Spermidines generated aglycone ions and several characteristic product ions after the cleavage of different C-N bonds. S1-S4 exhibited characteristic fragmentation behaviors that were effectively used to confirm their structures by MS/MS. The fragmentation rule analysis of S1- S4 could offer more reliable information for rapid characterization and chemical profiling of spermidines from natural sources.

3.2 Selective enrichment of spermidines Goji berry extraction is a complex mixture contains variety of chemical compositions [16], and spermidines in goji berry were considered as relatively strong polar alkaloids. For comprehensive analysis of spermidines, pretreatment method was necessary to minimize the non-alkaloid components of the extracts before MS detection. In present work, a strong cation exchange SPE (SCX-SPE) combined with a polar modified RP-SPE method was developed to selective enrichment of spermidines in goji berry [40.44]. Two eluted fractions (named non-alkaloid fraction and alkaloid fraction) were obtained from the SPE processing of goji berry extraction. The extraction sample and the SPE fractions were analyzed by UPLC-DAD-MS (Fig. 7). In the goji berry extraction (Fig. 7, A), the alkaloids and non-alkaloids were co-eluted in the chromatographic separation. In the non-alkaloid fraction (Fig. 7, B), a variety of complex components were detected. Some main peaks, such as peaks 1 to 9, were identified and revealed that the non-alkaloid fraction contained phenols, flavonoids and some phenolic acids. For example, peak 6 was tentatively identified as acid red 73, and the peak 9 was determined as rutin. In the alkaloid fraction (Fig. 7, C), characterization of peak 10 to 21 showed that spermidine alkaloids were mainly enriched in this fraction (MS data shown in supporting information). This method 9

effectively solved the co-elution issue in chromatographic separation, and realized the selective enrichment of spermidines. Furthermore, several trace spermidinesˈwhich were ignored in the conventional chromatographic analysis, were detected by the optimized SPE procedure. This result indicated that developed SPE processing can be a feasible technique for the enrichment of spermidines. 3.3 Characterization of Spermidines from goji berry Chromatographic conditions were systematically optimized, including analytical column, mobile phase, and gradient elution conditions, to obtain optimal peak capacity and the most acceptable resolution for the target analytes between their adjacent peaks in the UPLC chromatogram. Several RP columns were tested, such as Unitary C18 column (150 × 2.1 mm I.D., 5 um), XCharge C18 column (150 × 2.1 mm I.D., 5 um), X Aqua C18 column (150 × 2.1 mm I.D., 5 um). Ultimately, the XCharge C18 column was selected for generating the symmetric peak shape, and well retention for spermidines. The mobile phase was investigated between acetonitrile-water and methanol-water, and the latter combination exhibited greater separation potency for the analytes. Afterward, gradient elution was carefully optimized to achieve better separation. The experiments were performed on UPLC-DAD-Q-TOF/MS and MS/MS modes to obtain the structural data rapidly. Spermidines from goji berry corresponded to the peaks (280 nm) in the UPLC-DAD chromatogram (Fig. 8). Each peak was characterized based on characteristic fragment ions that were obtained from MS and MS/MS analysis. Based on fragmentation behavior analyses of the standard samples (S1-S4) and typical cleavage of each peak, a total of 41 spermidines were tentatively identified. Table 2 summarized the characterization results of spermidines from goji berry. According to the fragment ions and comparing the fragmentation behaviors with standard samples, the peaks had similar fragment ions with S1, S2, S3 and S4 were classified as group 1, 2, 3 and 4, respectively. Other peaks were analyzed by their characteristic fragment ions and with the support of references. 10

Group 1: Peak 23 had the [M+H]+ at m/z 474, and yielded daughter ions at m/z 310.2171, 293.1909, 222.1175, 165.0582, and 123.0466. Possible fragmentation pattern of peak 23 was inferred. The m/z 310 [M+H-165]+ ion was yielded by the leaving of dihydrocaffeoyl group from the molecular ion. The ion at m/z 293 [M+H-165-17]+ was generated by losing ammonia from the ion at m/z 310 [M+H-165]+. The one at m/z 222.1149 [M+H-165-17-72]+ resulted from the loss of the

rearrangement

ion

m/z

72.

The

daughter

ion

at

m/z

123.0441

[M+H-165-17-72-100]+ was observed in the spectrum via the loss of another substitution rearrangement product ion m/z 100 from the ion m/z 222. All these fragmentation ions indicated the presence of characteristic fragment ions, such as caffeoyl group and amide moiety, in the structure. Compared to the fragment ions and fragmentation behaviors with standards, peak 23 was deduced to possess similar structure with S1, and initially identified as N1, N3-bis-dhc-spermidine. Some glycosidic products of S1 were detected and analyzed according to the identical fragment ions and fragmentation behaviors. Peak 24 appeared the precursor and produced the fragment ions at m/z 636, 474, 310, 222, 163, ion [M+H]+ at m/z 798ˈ and 123. The ion at m/z 636 [M+H-hexose]+ represented the leaving of a hexose unit (162 Da.). The one at m/z 474 [M+H-di-hexose]+ indicated the aglycon ion by the loss of another hexose moiety from the ion at m/z 636. The ions at m/z 310, 293, 222 and 123 were basically identical with S1. Therefore, peak 24 was tentatively identified as N1, N3-bis-dhc-spermidine-di-hexoside. With similar analysis, peaks 26 and 28 had identical daughter ions and cleavage rules with S1 after loss of three hexose units. Therefore,

peak

26

and

peak

28

were

preliminary

assigned

as

N1 ,

N3-bis-dhc-spermidine-tri-hexoside isomers. It is worth mentioning that the linkage position of hexose units was difficult to confirm based on the UPLC-DAD-MS analysis, and this will be one of the key directions in our future study. Group 2: Peak 40 had molecular ion [M+H]+ at m/z 472.2541, and eight fragment ions at m/z 310.2200, 293.1937, 236.1351, 222.1194, 163.0441, 123.0474, 100.0782, and 72.0831. The ions at m/z 310 and 163 were obtained after the loss of a caffeoyl unit from the protonated parent ion. The one at m/z 293 appeared after the loss of 11

ammonia unit from the ion at m/z 310. The ion at m/z 222 was detected with the loss of a rearrangement ion at m/z 72 from the ion at m/z 293. The daughter ion at m/z 123 was formed by the loss of the substitution rearrangement production ion at m/z 100 from the ion at m/z 222. The results showed that the fragmentation behavior of peak 40 corresponded with the spectrometric rules of S2. Based on the MS data and the fragmentation

pathway

analysis,

peak

40

was

characterized

as

N1-dhc–N3-caffeoyl-spermidine. According to the fragment ions and fragmentation pathways analysis, some peaks were identified as the glycosidic products of S2. Peaks 34 and 38 had similar protonated molecules at m/z 634, and shared the same fragment pattern with S2 after the loss of a hexose moiety. Hence, these peaks were tentatively identified as N1-dhc-N3-caffeoyl-spermidine hexoside isomers. Peak 44 displayed the protonated ion [M+H]+ at m/z 796, and the ions at 634.2981, 472.2468, 310.2170, 293.1912, 222.1167, 163.0421, 72.0820, respectively. According to the fragmentation pattern, this peak was tentatively characterized as N1-dhc-N3-caffeoyl-spermidine-di-hexoside. Group 3: Peak 34 had molecular ion [M+H]+ at m/z 472.2760. This peak produced fragment ions at m/z 310.2163, 293.1906, 236.1334, 222.1170, 220.1007, 163.0420, 123.0453 and 72.0819, respectively. The ions at m/z 310 and 163 were obtained after the cleavage of C-N1 bond from the parent ion. The one at m/z 293 appeared after the loss of ammonia unit from the ion at m/z 310. The ion at m/z 222 was detected with the loss of a rearrangement ion at m/z 72 from the ion at m/z 293. The daughter ion at m/z 123 was formed by the loss of the substitution rearrangement production ion at m/z 100 from the ion at m/z 222. The ions at m/z 220 and 253 were generated from another fragmentation pathway, after the division of C-N2 bond. The one at 253 generated a 236 with the loss of ammonia unit. Based on the MS data and the fragmentation pathway analysis and compared with the patterns of standards, peak 34 was characterized as N1-caffeoyl-N3-dhc-spermidine. Peaks 28, 32, 33 and 36 displayed similar protonated ion [M+H]+ at m/z 634. These peaks yielded the same fragment ions and exhibited identical fragmentation behaviors

with

S3,

and

thus, 12

initially

identified

as

N1-caffeoyl-N3-dhc-spermidine-hexoside isomers. Peaks 27, 29, 31, 36, and 39 produced the parent ion at 796, and displayed unanimous fragmentation pattern with S3 after two hexose parts loss. Peaks 25, 30, 35 and 38 had the same [M+H]+ at m/z 958, and they also shared the same fragment behaviors with S3 after loss of three hexose

moieties.

Thus,

these

peaks

were

N1-caffeoyl-N3-dhc-spermidine-di-hexoside

preliminary

identified

isomers

as and

N1-caffeoyl-N3-dhc-spermidine-tri-hexoside isomers, respectively. Group 4: Peak 47 had a [M+H]+ ion at m/z 470, and produced four fragment ions at m/z 308.1986, 291.1735, 220.1001, 163.0407, which were the characteristic fragment ions of S4. The one at m/z 308 was caused by the loss of m/z 163 from the molecular ion. Subsequently, the ion at m/z 291 arose after the loss of ammonia. The one at m/z 220 appeared when the ion at m/z 72 lost from the ion at m/z 291. According to the MS/MS fragment ions and fragmentation behavior, peak 47 was identified as N1, N3-bis-caffeoyl-spermidine. Some glycoside derivatives of S4 were also detected. Peaks 13 and 43 produced [M+H]+ ion at m/z 632, and displayed aglycon ion at m/z 470 [M+H-162]+ by the loss of a hexose unit. These peaks had presented the fragment ions at m/z 308, 220, 163. The fragment ions and fragmentation behaviors were basically similar with S4. Thus, peaks

13

and

43

were

tentatively

identified

as

N1 ,

N3-bis-caffeoyl-spermidine-hexoside isomers. Peaks 32, 40, 45, 46, 48 and 49 yielded an aglycon ion at m/z 470 after the loss of two hexose moieties. According to the MS/MS fragment ions and fragmentation rules, these peaks were initially characterized as N1, N3-bis-caffeoyl-spermidine-di-hexoside. Peaks 37 and 42 had the same fragment patterns with S4 after loss of three hexose moieties. Consequently, these peaks were initially confirmed as N1, N3-bis-caffeoyl-spermidine-tri-hexoside isomers. Moreover, several hydroxyl substituted spermidines were detected. According to the published reports and the MS/MS fragmentation pattern [3], peaks 14, 18, 20, 41, 46, 50 and 51 were preliminary predicted as the hydroxyl substituted spermidines. Peak 14 produced an aglycon ion at m/z 472 after loss of a hexose moiety and a 13

hydroxyl unit. Peaks 18, 41 and 46 appeared the same pseudo molecular ion [M+H]+ at m/z 648, and generated the fragment ion [M+H-17]+at m/z 632 , [M+H-162]+ at m/z 486, and aglycon ion [M+H-162-17]+ at m/z 470. Peaks 20 and 42 produced the aglycon ion at m/z 472, while peaks 50 and 51 yielded the same aglycon ion at m/z 470.

Thus,

peak

14

was

tentatively

confirmed

as

hydroxy-N1-dihydrocaffeoyl-N3-caffeoyl-spermidine-hexoside isomer, and peak 18 was

initially

identified

as

hydroxy-N1-dihydrocaffeoyl-N3-caffeoyl-spermidine-di-hexoside isomer. Peak 41 and 46

were

preliminary

characterized

as

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-hexoside isomers. The peaks 20, 42, and were initially characterized as hydroxy-N1-dhc-N3-caffeoyl-spermidine-hexoside isomers,

and

the

peaks

50

and

51

were

tentatively

confirmed

as

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-di-hexoside isomers. It was hard to confirm the linkage position of substituents through the UPLC-DAD-MS analysis, and follow up studies will be conducted in view of this topic. Some peaks were predicted as the cyclic products of spermidines based on the fragment ions. These peaks had similar parent ions with straight chain spermidines. However, the fragment ions were different. Peak 12 had the same parent ion [M+H]+ with S4 at m/z 470. The fragment ions at m/z 453ˈ383ˈ283ˈ256ˈ155 and 136 were obtained from the MS/MS analysis. Peaks 17, 19 and 21 exhibited similar fragmentation ions with peak 12. Early publications revealed some cyclic products of spermidines [15]. It was conjectured that these components may be the cyclic-spermidine compounds. Cyclic products were a new structural type of spermidine components, and owned a variety of differences compared to the straightchain spermidines. Investigation will be needed in further structural studies for these components. Due to the deficiency of references and structural information, some peaks were not detected out in this work. For instance, the peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15 and 16 were initially assumed as compounds belonging to spermidines. However, these peaks displayed totally different fragment patterns compared to the standards. 14

Further studies will be needed for precise identifications. In a similar manner, based on the MS and MS/MS analysis, 41 out of 58 spermidines in goji berry were initially characterized. As far as we known and compared with references, at least 26 spermidines were reported for the first time from this plant extraction.

4 Conclusions In summary, the chemical profiling of spermidines in goji berry was carried out by applying an SPE coupled with UPLC-DAD-Q-TOF/MS/MS method. The separated spermidines were characterized based on the fragmentation rules that were obtained from the straight-chain spermidine standards. Different structure types of spermidines could be distinguished by the unique fragmentation rules and characteristic fragment ions. More importantly, the co-existence of fragment ions at m/z 220 and 222 was the key clue for the differentiation for spermidine isomers. The developed SPE method exhibited a good prospect of high selective enrichment for spermidines, and several trace spermidines in goji berry were detected. According to the summarized fragmentation mechanisms and established method, a total of 41 spermidines were tentatively characterized. Compared with references and to our best knowledge, 26 of them were reported from goji berry for the first time. This result demonstrated that the SPE coupled with UPLC-DAD-Q-TOF/MS/MS method was a potential technique for characterizing the trace spermidines without purification. It also illustrated that goji berry was rich in bioactive natural spermidines, and could be a promising source of spermidines for medical application and new drug discovery.

15

Acknowledgement This work was supported by the State Key Program of National Natural Science of China(Grant No.U1508221 ).This work was supported by “Project of National Science Foundation of China (31670374 and 81803706)”. The innovation platform for the Supported by Central Asian Drug Discovery and Development Center of Chinese Academy of Sciences˄No. CAM201805 )

The authors have declared no conflict of interest.

16

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20

Fig 1. Chemical structures of spermidines

Fig. 2. MS/MS spectra of different groups of spermidines

21

Fig. 3 Proposed fragmentation pathway of S1

Fig. 4 Proposed fragmentation pathway of S2

Fig. 5 Proposed fragmentation pathway of S3

22

Fig. 6 Proposed fragmentation pathway of S4

Fig. 7 The SCX-SPE enrichment of dicaffeoyl spermidines from goji berry

23

Fig. 8 UPLC-DAD-Q-TOF-MS/MS analysis of spermidines from goji berry

24

Table 1 Dicaffeoylspermidines tentatively identified in goji berry extraction using UPLC-DAD-Q-TOF-MS/MS 1

tR

LE,m/z

Formula

Error/ppm

6.752

488.2728

470.2353,399.1613,370.1723,172.1496,155.1225,129.1419,112.1146,72.0848

HE,[M+1]/m/z

C25H33N3O7

3.57

Tentative structural assignment unknown

2

7.887

650.2967

632.2808,470.2315,453.2029,400.1511,172.1467,115.1183,129.1376,112.1119,72.0848

C31H43N3O12

5.84

unknown

3

10.151

650.2922

632.2815,470.1222,453.2056,400.1572,172.1478,115.1192,129.1389,112.1111,72.0868

C31H43N3O12

2.47

unknown

4

13.003

384.1291

320.1971,236.1318,165.0575,123.1456,114.0563,72.1821

C18H28N3O6

- 4.94

unknown

5

15.956

369.1828

285.1623,190.1243,162.1103,150.0928,70.0659

C18H28N3O5

10.57

unknown

6

17.182

546.0293

528.2540,470.1247,398.1818,236.1313,163.0431,84.0971

C28H39N3O8

0.2

unknown

7

18.259

486.1611

468.6878,400.1786,370.9505,172.1496,155.1311,112.1152

C25H31N3O7

4.25

unknown

8

23.536

413.1221

234.1611,198.1159,177.0582,163.0423,145.0320

C22H24N2O6

-5.04

unknown

9

24.002

413.1226

234.1628,198.1179,177.0561,163.0431,145.0328

C22H24N2O6

2.31

unknown

10

25.187

544.2701

488.1777,470.1380,455.2282,399.0769,236.9971,172.2560,163.6589,123.6453,112.0867,70.6657

C28H37N3O8

5.03

unknown

11

28.670

531.2716

293.1890,222.1160,163.0570,123.0450

C28H42N4O6

6.64

unknown

12

29.875

470.2308

453.2080,399.1566,382.1293,355.1432,324.0961,222.1194,172.1513,163.0428,155.1223,129.140

C25H31N3O6

2.64

unknown

6,112.1141,84.0820,72.0817 13 14

31.156 32.668

632.2897

470.2342,455.2435,398.1821,310.1189,293.1920,220.1026,163.6442,112.1154,84.0826,70.662

C25H31N3O6

5.05

dhc-caffeoyl-cyclic-spermidine-hex

490.3129

455.2112,310.2189,293.1925,236.1336,220.1024,172.1494,163.0427,155.1218,123.0471

C25H35N3O7

3.75

hydroxy-N,N-bis-dhc-cyclic-spermidine

652.3008

634.3015,472.2820,382.1570,293.1946,236.1309,220.1037172.1502,163.0437,155.1230,123.049

C31H39N3O11

4.01

hydroxy-N1-caffeoyl-N3-caffeoyl-cyclic-spermidine-hex

4,112.11153,70.0671 15

34.361

428.0104

384.1699,293.1915,222.1182,162.0585,123.0465,112.1145,84.0305

C23H29N3O5

-3.81

unknown

16

35.109

512.2377

474.2644,384.1696,293.1918,222.1168,165.0575,123.0459

C25H37N3O9

3.75

unknown

17

37.115

630.2619

468.2061,453.2227,308.1921,291.1683,220.0999,172.1476,163.0419,155.1216,112.1128,98.0983

C31H39N3O11

1.84

unknown

18

38.230

814.3529

796.3407,634.3931,472.2402,455.2205,310.2124,220.0993,,163.0415

C37H55N3O17

4.25

hydroxy-N1-dhc-N3-dhc-cyclic-spermidine-di-hex

19

39.116

468.2063

308.1940,293.2103,220.0916,163.0401

C25H31N3O6

2.01

unknown

20

40.296

812.3352

794.3252,632.2751,470.2456,453.2175,382.1446,310.2144,220.0987,163.0385,172.1450

C37H53N3O17

-0.92

hydroxy-N1-caffeoyl-N3-dhc-cyclic-spermidine-di-hex

21

41.627

486.1867

468.2500,453.2407,399.6974,308.2019,291.1749,220.0995,163.0417,155.1366

C25H31N3O7

-4.28

unknown

22

47.239

796.3562

634.3019,472.2499,382.1544,310.2183,220.1024,163.0432

C37H53N3O16

-5.24

N1-dhc -N3-caffeoyl-spermidine-di-hex

23

48.411

474.2641

310.2171,293.1909,236.1340,222.1175,165.0582,123.0466,72.0826

C25H35N3O6

3.12

N1,N3-bis-dhc-spermidine

24

49.218

798.3677

636.3148,474.2625,384.1689,222.1169,165.0576

C37H55N3O16

5.92

N1,N3-bis-dhc-spermidine-di-hex

25

50.070

958.4051

796.3563,634.2896,472.2436,382.1535,293.1912,220.1231,163.0609,123.0450,84.0830

C43H63N3O21

2.61

N1-dhc -N3-caffeoyl-spermidine-tri-hex

26

50.847

960.4134

798.3599,636.3099,474.2584,384.1665,222.1147

C43H65N3O21

-2.4

N1,N3-bis-dhc-spermidine-tri-hex

27

52.505

796.3412

634.2895,472.2412,384.1637,310.2127,293.1865,236.1298,222.1139,220.0982,163.0403

C37H53N3O16

4.41

N1-caffeoyl-N3-dhc-spermidine-di-hex

28

53.499

960.4159

798.3601,636.3059,474.2612,384.1632,222.1143,165.0544

C43H65N3O21

2.55

N1,N3-bis-dhc-spermidine-tri-hex

634.2961

472.2444,382.1515,310.2153,220.1014,163.0420

C31H43N3O11

3.66

N1-dhc -N3-caffeoyl-spermidine-hex

25

29

54.322

796.3532

634.3009,472.2481,382.1543,310.2174,220.1019,163.0427

C37H53N3O16

5.63

N1-dhc -N3-caffeoyl-spermidine-di-hex

30

55.801

958.4098

796.3582,634.3007,472.2448,382.1534,220.1015,163.6438

C43H63N3O21

1.42

N1-dhc -N3-caffeoyl-spermidine-tri-hex

31

57.415

796.3602

634.3055,472.2699,382.1511,310.2198,220.1035,163.0440

C37H53N3O16

1.73

N1-dhc -N3-caffeoyl-spermidine-di-hex

32

58.583

794.3420

632.2877,470.2561,382.1556,310.2200,220.1027,163.0437

C37H51N3O16

3.31

N1,N3-bis-caffeoyl-spermidine-di-hex

634.2961

472.2444,382.1515,310.2153,220.1014,163.0420

C31H43N3O11

6.11

N1-dhc -N3-caffeoyl-spermidine-hex

33

59.135

634.3034

472.2680,382.1566,310.2198,220.1039,163.0440

C31H43N3O11

4.67

N1-dhc -N3-caffeoyl-spermidine-hex

34

59.406

472.2760

310.2163,293.1906,236.1334,222.1170,220.1007,163.0420,123.0453,72.0819

C25H33N3O6

0.41

N1-dhc -N3-caffeoyl-spermidine

634.2952

472.2451,310.2153,293.1892,222.1158,163.0414

C31H43N3O11

5.23

N1-dhc -N3-caffeoyl-spermidine-hex

35

60.640

958.4095

796.3561,634.3016,472.2466,382.1553,220.1023,163.0426

C43H63N3O21

3.07

N1-dhc -N3-caffeoyl-spermidine-tri-hex

36

61.405

634.3001

472.2483,310.2174,220.1018,163.0425

C31H43N3O11

5.4

N1-dhc -N3-caffeoyl-spermidine-hex

796.3514

634.2998,472.2479,384.1689,310.2172,293.1890,222.1165,220.1012,163.0424,70.0681

C37H53N3O16

0.65

N1-dhc -N3-caffeoyl-spermidine-di-hex

37

63.349

956.3862

794.3280,632.2796,470.2629,380.1518,310.2136,220.0992,163.0416

C43H61N3O21

4.34

N1,N3-bis-caffeoyl-spermidine-tri-hex

38

64.575

634.3011

472.2487,310.2181,293.1912,222.1179,163.0429,72.0825

C31H43N3O11

6.21

N1-caffeoyl-N3-dhc-spermidine-hex isomer

958.4003

796.3465,634.2940,472.2609,382.1507,310.2137,220.0993,163.0411

C43H63N3O21

2.72

N1-dhc -N3-caffeoyl-spermidine-tri-hex

39

66.512

796.3413

634.3109,472.2409,310.2126,220.0982,163.0402

C37H53N3O16

0.25

N1-dhc -N3-caffeoyl-spermidine-di-hex

40

67.063

794.3335

632.2831,470.2517,3823.1525,308.2010,291.1167,234.1169,220.1009,163.0421

C37H51N3O16

8.23

N1,N3-bis-caffeoyl-spermidine-di-hex

472.2541

310.2200,293.1937,236.1351,222.1194,163.0441,123.0474,100.0782,72.0831

C25H33N3O6

5.72

N1-caffeoyl-N3-dhc-spermidine hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-hex

41

68.412

648.3134

632.2784,486.2631,470.2522,382.1533,308.2018,291.1742,234.1162,220.1012,163.0426,72.6827

C31H43N3O12

5.6

42

69.542

956.4047

794.3405,632.2993,470.2557,382.1547,308.2041,220.1018,163.0432

C43H61N3O21

-0.09

N1,N3-bis-caffeoyl-spermidine-tri-hex

812.3844

650.3257,488.2800,472.2513,310.2151,293.1995,238.3444,222.1170,220.1046,163.1344

C37H53N3O17

2.23

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-di-hex

43

70.494

632.2884

470.2562,308.2033,281.1771,220.1033,163.0441

C31H41N3O11

5.14

N1,N3-bis-caffeoyl-spermidine-hex

44

71.097

796.3506

634.2981,472.2468,310.2170,293.1912,222.1167,163.0421,72.0820

C37H53N3O16

1.28

N1-caffeoyl-N3-dhc-spermidine-di-hex

956.9988

794.3445,632.3056,470.2517,220.1041,163.0435

C43H61N3O21

3.31

N1,N3-bis-caffeoyl-spermidine-tri-hex

45

72.410

794.3411

632.2875,470.2558,382.1557,308.2035,291.1779,220.1028,163.0435

C37H51N3O16

3.88

N1,N3-bis-caffeoyl-spermidine-di-hex

46

74.739

648.3147

632.2843,486.2624,470.2451,382.1536,308.2010,291.1762,234.1168,220.1013,163.0423

C31H41N3O12

4.47

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-hex

794.3355

632.3042,470.2301,308.2027,291.1761,220.1008,163.0436

C37H51N3O16

3.37

N1,N3-bis-caffeoyl-spermidine-di-hex

47

77.125

470.2552

308.1986,291.1735,234.1171,220.1001,163.0407

C31H43N3O11

4.38

N1,N3-bis-caffeoyl-spermidine

48

78.015

794.3242

632.2999,470.2248,308.1963,291.1552,220.0982,163.0405

C37H51N3O16

4.27

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-di-hex

49

79.073

794.3284

632.2993,470.2276,382.1506,308.1985,220.0994,163.0412

C37H51N3O16

2.12

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-di-hex

50

81.686

810.3530

634.2945,486.2625,472.2512,382.1499,310.1361,220.1011,163.0423

C37H51N3O17

-2.03

hydroxy-N1-caffeoyl-N3-caffeoyl-spermidine-di-hex

51

82.781

810.3698

648.3167,486.2650,472.2733,310.2177,234.1185,220.1025,163.0432

C37H51N3O17

-4.23

hydroxy-caffeoyl-N3-caffeoyl-spermidine-di-hex

26

Highlights 1. Chemical profiling of spermidines in goji berry was carried out by by UPLC-DAD-Q-TOF/MS. 2. The comprehensive fragmentation rules of four different groups of spermidines were carefully discussed and summarized by the MS/MS fragment ions analysis. 3. An effective sample pretreatment method was developed for the selective enrichment of spermindines based on a strong cation exchange-solid phase extraction(SCX-SPE). 4. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry.

27

Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

Hasanjan Ahad 1, a, b, c, Hongli Jin 1, b, Yanfang Liu b, Jixia Wang b, Guangying Sun a, Xinmiao Liang b, *, Haji Akber Aisa a, *

1: these authors contributed equally to this work a

Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi,

Xinjiang 830011, People`s Republic of China b

Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

c

University of Chinese Academy of Sciences, Beijing 100039, China

Fax: +86-411-84379539 Email: [email protected]

Tel.: +86-411-84379519 [email protected]

28

Author Statement

Hasanjan Ahad: Methodology, Validation, Investigation, Writing-Original draft Hongli Jin: Resources, Methodology, Review and Editing Yanfang Liu: Resources, Review and Editing, Supervision, Funding acquisition Jixia Wang: Methodology, Review and Editing Guangying Sun: Methodology, Review and Editing Xinmiao Liang: Conceptualization, Resources, Supervision, Funding acquisition Haji Akber Aisa: Conceptualization, Resources, Supervision, Funding acquisition

29

Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

Hasanjan Ahad 1, a, b, c, Hongli Jin 1, b, Yanfang Liu b, Jixia Wang b, Guangying Sun a, Xinmiao Liang b, *, Haji Akber Aisa a, *

1: these authors contributed equally to this work a

Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi,

Xinjiang 830011, People`s Republic of China b

Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

c

University of Chinese Academy of Sciences, Beijing 100039, China

Fax: +86-411-84379539 Email: [email protected]

Tel.: +86-411-84379519 [email protected]

30

Abstract

Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.

31

Chemical profiling of spermidines in Goji Berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

Hasanjan Ahad 1, a, b, c, Hongli Jin 1, b, Yanfang Liu b, Jixia Wang b, Guangying Sun a, Xinmiao Liang b, *, Haji Akber Aisa a, *

1: these authors contributed equally to this work a

Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi,

Xinjiang 830011, People`s Republic of China b

Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

c

University of Chinese Academy of Sciences, Beijing 100039, China

Fax: +86-411-84379539 Email: [email protected]

Tel.: +86-411-84379519 [email protected]

32

Conflict of Interest The authors have declared no conflict of interest.

33