Accepted Manuscript

Title: Qualitative and quantitative analysis of an alkaloid

fraction from Piper longum L. using ultra-high performance
liquid chromatography-diode array detector-electrospray
ionization mass spectrometry

Author: Kuiyong Li Yunpeng Fan Hui Wang Qing Fu Yu Jin

Xinmiao Liang

PII: S0731-7085(15)00107-7
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2015.02.012
Reference: PBA 9953

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 20-12-2014

Revised date: 5-2-2015
Accepted date: 6-2-2015

Please cite this article as: K. Li, Y. Fan, H. Wang, Q. Fu, Y. Jin, X. Liang, Qualitative
and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-
high performance liquid chromatography-diode array detector-electrospray ionization
mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2015),
http://dx.doi.org/10.1016/j.jpba.2015.02.012

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 1 Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L.
2 using ultra-high performance liquid chromatography-diode array
3 detector-electrospray ionization mass spectrometry

4 Kuiyong Li, Yunpeng Fan, Hui Wang, Qing Fu, Yu Jin∗ and Xinmiao Liang∗

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5 Engineering Research Center of Pharmaceutical Process Chemistry, Ministry of

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6 Education, School of Pharmacy, East China University of Science and Technology,

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7 Shanghai 200237, PR China

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21 Corresponding author: Yu Jin, Xinmiao Liang

22 School of Pharmacy,
23 East China University of Science and Technology,
24 Shanghai 200237, China
25 Tel: +86-021-64250633
26 Fax: +86-021-64250622

Page 1 of 25
27 E-mail: Jiny@ecust.edu.cn, liangxm@ecust.edu.cn
28

29

30 Abstract
31 In a previous research，an alkaloid fraction and 18 alkaloid compounds were
32 prepared from Piper longum L. by series of purification process. In this paper, a

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33 qualitative and quantitative analysis method using ultra-high performance liquid
34 chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS) was

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35 developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid
36 fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid

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37 compounds were identified. A further qualitative analysis of the alkaloid fraction was
38 accomplished by UHPLC-MS/MS method. Another 25 amide alkaloids were
39
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identified according to their characteristic ions and neutral losses. At last, a
40 quantitative method for the alkaloid fraction was established using four marker
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41 compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,
42 4E-octadecadienamide. After the validation of this method, the contents of above four
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43 marker compounds in the alkaloid fraction were 57.5 mg/g, 65.6 mg/g, 17.7 mg/g and
44 23.9 mg/g, respectively. Moreover, the relative response factors of other three
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45 compounds to piperine were calculated. A comparative study between external

46 standard quantification and relative response factor quantification proved no
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47 remarkable difference. UHPLC-DAD-MS method was demonstrated to be a powerful

48 tool for the characterization of the alkaloid fraction from P. longum L. and the result
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49 proved that the quality of alkaloid fraction was efficiently improved after appropriate
50 purification.
51 Key words: qualitative; quantitative; alkaloid fraction; Piper longum L. ultra-high
52 performance liquid chromatography-diode array detector-electrospray ionization mass
53 spectrometry (UHPLC-DAD-MS)
54 1. Introduction
55 Traditional Chinese Medicines (TCMs) play an indispensable role in the prevention
56 and treatment of diseases. Nowadays, TCMs are attracting more and more attention in

Page 2 of 25
57 the world not only for its clinic application, but also as one of the most important
58 sources in drug discovery [1,2]. Usually, hundreds of chemical components are
59 contained in one herb. Hence, the complexity and variability of herb extracts bring
60 significant challenges in identifying their chemical composition.
61 The crude extractfrom the fruits of Piper longum L. has various biological activities,
62 such as anti-inflammatory, analgesic, anti-amoebic, anti-depressant, hepatoprotective,

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63 etc. [3]. Most of the earlier works on Piper species suggested that the major bioactive
64 constituents were amide alkaloids [4-6]. In order to identify the chemical composition

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65 of P. longum L., lots of purification work have been done by column chromatography
66 (CC), thin-layer chromatography (TLC) [7] and high-speed counter-current

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67 chromatography (HSCCC) [8]. Recently, the advantages of preparative high
68 performance liquid chromatography (HPLC) on high performance separation, online
69
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detection with ultraviolet (UV) and/or mass spectrum (MS), and auto fraction
70 collection make them successfully applied in the isolation and preparation of
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71 compounds from a complex system [9]. In our previous research, an two dimensional
72 (2D) preparative HPLC combing normal phase and reversed phase method has been
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73 developed and applied to the purification of alkaloid fraction from P. longum L..
74 Totally, 28 amide alkaloids have been obtained [10]. All isolation work has been
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75 accumulatedto allow a deep understanding of the chemical compositions of P. longum

76 L.. While, the complexity of TCMs has been now disclosed with the developing of
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77 analysis techniques, and more unknown components have been separated and
78 detected. So, more effective purification methods and technologies are still necessary.
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79 The most common method for analyzing TCMs is chromatography [11]. For
80 example, TLC is used for identification of P. longum L. in the China pharmacopeia
81 (ChP).HPLC has been used to analysis P. longum L. due to its high efficiency, high
82 stability and good sensitivity [12]. Nowadays, thanks to the development of
83 ultra-high performance liquid chromatography (UHPLC), fast and ultra-fast
84 separation can be achieved by working with columns packed with sub-2 μm particles
85 operating at ultra-high pressure. In our previous work [10], excellent separation for P.
86 longum L. on UHPLC, especially for the purity determination of compounds has been

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 87 illustrated. Sun et al. [4] have successfully applied HPLC-MS in the analysis of amide
88 alkaloids in P. longum L.. Most amide compounds from P. Longum L. can be
89 classified into four types. The characteristic MS fragments and neutral losses of
90 different types have been summarized, and 43 amide alkaloids were successfully
91 identified. Furthermore, Amide alkaloids in P. longum L. can be separated with
92 supercritical fluid chromatography (SFC), which showed good orthogonality with

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93 HPLC. Hence, an off-line 2D chromatography combining SFC and HPLC was
94 developed to separate alkaloid fraction of P. longum L. [13].

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95 As discussed above, quality control of TCMs benefits from the development of
96 analytical technique. Generally, as an important part of quality control, qualitative

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97 analysis using standards is most accurate. However the identification work always
98 encounted for the problems of lacking standard compounds and information of the
99
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unknown compounds. Meanwhile, HPLC-MS, a powerful technique for qualitative of
100 chemicals in complex matrices, has been widely used in the identification of the
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101 minor components in TCMs when standards are not available.
102 Another important part of quality control for this alkaloid fraction was quantitative
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103 analysis. Taking P. longum L. as an example, the content determinations of active

104 ingredients is carried out using piperine as the only marker compound in the 2010
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105 ChP. For such a complex matrices, it seems not enough to use single marker
106 compound in quantitative analysis to control the TCMs' quality because there contains
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107 multiple active components according to the theory of TCMs. At present, the
108 determination of multiple active components has attracted more and more attentions.
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109 In previous study [14], relative response factor method, which was practical to solve
110 the lack of so many chemical standards in TCMs, was applied for the quality control
111 of TCMs.
112 The aim of this study is to carry out the qualitative and quantitative analysis for
113 quality evaluation of an alkaloid fraction from P. longum L. Here，the alkaloid fraction
114 was purified from P. longum L. after a series of purification processes for removing
115 other unknown components, such as pigment. 18 standards prepared in previous study
116 were firstly used in the qualitative analysis of alkaloid fraction. The MS rules were

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117 verified firstly based on 18 standard components, and then other alkaloid compounds
118 in thefraction were identified and speculated according to their MS2 fragments. Four
119 marker compounds were used in a rapid UHPLC - UV quantitative analysis method to
120 evaluated alkaloid fraction. Furthermore, piperine, the most easily available
121 compound, was used as the reference and the relative response factors of other three
122 marker compounds were calculated. A comparative study between external standard

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123 quantification and relative response factor quantification was researched.
124 2. Experiment

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125 2.1 Reagents and materials
126 Acetonitrile (ACN) of HPLC grade was purchased from J&K (Beijing, China).

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127 Water used in this study was purified with a Milli-Q water purification system
128 (Millipore, Bedford, MA, USA).
129
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P. longum L. was purchased from Anguo Herb Market, Hebei province (China).
130 The herb was authenticated by the Institute of Medication, Xiyuan Hospital of China
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131 Academy of Traditional Chinese Medicine.
132 The amide alkaloid fraction and 18 compounds (shown in Fig.1) investigated in this
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133 study were prepared by our lab [10]. All samples were stored at -20◦C before used.
134 2.2 Instruments and methods
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135 The UHPLC-DAD analysis was performed on a Waters ACQUITY UHPLC®

136 H-Class system including a quaternary solvent manager, a sample manager-FTN, a
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137 column manager and a PDA detector. The separation was performed on an Acquity
138 UHPLC HSS T3 column (100× 2.1 mm i.d., 1.8 µm, Waters, USA), abbreviated as
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139 HSS T3. The mobile phase consisted of water (A) and acetonitrile (B). The linear
140 gradient was as follows: 0-10 min, 65-80 % B; 10-20 min, 80-95% B; 20-30 min,
141 95% B at a flow rate of 0.2 mL/min. The injection volume, column temperature and
142 UV detection wavelength were set at 4 µL, 25 ◦C and 254 nm, respectively. Data
143 acquisition and processing of UHPLC were conducted using Waters Empower 3
144 software.
145 The UHPLC-MS analysis of alkaloid fraction was performed on a Waters
146 ACQUITY UPLC system with a Quattro Micro MS (triple quadrupole) operating in

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147 ESI+ mode (Waters, USA). The column and chromatographic conditions were the
148 same as described above. ESI-MS and MS2 parameters were as follows: capillary
149 voltage was set to 2.5 kV, and cone voltage to 25 V, nebulization gas was set to 550
150 L/h, source temperature and desolvation temperature were 120 ◦C and 380 ◦C,
151 respectively. Argon was employed as collision gas, and the collision energy was 20V
152 to obtain MS2 data. UV and MS data were processed using Masslynx (v 4.1) software.

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153 2.3 Standard solution and sample preparation
154 Individual stock solutions of 18 standard compounds were prepared by dissolving

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155 them in ACN. A mixed solution of four standard compounds was prepared by diluting
156 of stock solution with 50% ACN (v/v), and their concentration were as follows:

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157 piperine (compound 1) 0.515 mg/mL， pipernonatine (compound 6) 0.500 mg/mL,
158 guineensine (compound 10) 0.510 mg/mL, N-isobutyl-2E, 4E-octadecadienamide
159
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(compound 15) 0.500 mg/mL, respectively. And then a series of mixed solution were
160 prepared for plotting standard curve. Mixed solution at lowest concentration was
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161 further diluted to determine the limits of quantification (LOQ) of the respective
162 compounds.
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163 The alkaloid fraction used for UHPLC-UV and UHPLC-MS analysis was
164 accurately weighed and dissolved with ACN, and diluted with 50% ACN (v/v) to
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165 0.403 mg/mL. All prepared solutions were stored at 4 ◦C when not in use.
166 3. Result and discussion
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167 3.1 Qualitative analysis of the alkaloid fraction by standard compounds and
168 UHPLC-MS
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169 A simple and stable UHPLC-DAD method was developed for qualitative analysis
170 of the alkaloid fraction. 18 alkaloid compounds that were purified from P. longum L.
171 were used as standards (Fig. 2A). By comparing their retention times and MS date, all
172 18 compounds were found in the alkaloid fraction (Fig. 2B).
173 After compared with standard compounds, the main peaks in the alkaloid fraction
174 were confirmed. But some minor components were not identified due to the lack of
175 standard references. The structures of most amide alkaloids are varied from the
176 unsaturated fatty acids and the piperidine or isobutylamine substitution groups.

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177 According to previous study [4], the amide alkaloids obtained from P. longum L. can
178 be clasfied into types A, B, C, D and others, as shown in Fig.1. Types A and B have
179 the same benzodioxole group at R1 with the difference lies in piperidine or
180 isobutylamine at R2 group, while types C and D have the same alkyl group at R1 with
181 R2 substituted by piperidine or isobutylamine. So the chemical structures of amide
182 compounds in this alkaloid fraction can be speculated according to the characteristic

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183 fragments of different types.
184 In UHPLC-MS analysis, the alkaloids showed generally good response in positive

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185 mode, and [M+H]+ ions and/or [M+Na]+ adduct ions can be detected stably with
186 sufficient abundance. The collision energy was also optimized in order to obtain

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187 suitable ion fragments in MS2 for further identification of minor components. 18
188 standard compounds were divided into four types according to the above rule.
189
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Compounds 1, 3, 4, 6, 8, 9 and 11 belong to type A, compounds 7, 10 and 12 belong
190 to type B, compounds 2, 5, 13, 14, 15, 16 and 18 belong to type C and only compound
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191 17 belong to type D. Firstly, the R2 group is mainly justified by neutral loss in
192 high-mass region. The unique neutral losses of 85 and 113 Da，due to the loss of
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193 piperidine with subsequent loss of CO, would be characteristic for the presence of
194 piperidine in type A(compound 9, Fig. 3A) and type D (compound 17, Fig. 3D).
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195 Another acyl piperidine ion at m/z 112 could also verify the presence of piperidine.
196 Similarly, the unique loss of isobutylamine with subsequent loss of CO, producing the
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197 neutral losses at 73 and 101 Da related to the existence of isobutylamides in type
198 B(compound 10, Fig. 3B) and type C (compound 14, Fig. 3C). Secondly, the
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199 low-mass fragments in the MS2 can be used for the elucidation of the R1 group. For
200 type A (compound 9, Fig. 3A) and type B (compound 10, Fig. 3B), the fragmentation
201 at double bond produced the characteristic methylbenzodioxole and
202 propenylbenzodioxole ions at m/z 135 and 161, respectively. These two ion fragments
203 proved the R1 group is benzodioxole. If R1 group is pentyl group in type C (compound
204 14, Fig. 3C) and type D (compound 17, Fig. 3D), a series of alkyl ions such as m/z
205 119 and 133 or 121 and 135 could be observed. The laws described above were
206 verified by 18 standard compounds. After R1 and R2 group were confirmed, the n1, n2

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207 and n3 could be calculated. The detailed formula can refer to the literature [4].
208 According to the above fragmentation rule, 25 minor components in the alkaloid
209 fraction were identified (shown in Fig. 2B). An overview of 25 minor compounds was
210 given in Table 1, including the precursor ions ([M+H]+), the ion fragments and the
211 structure types, and deduced structures [4, 5, 10, 15-20].The compound number in
212 Table 1 was given according to the UHPLC retention time. All of 25 compounds, 2', 6',

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213 8' and 14' belong to type A, 1', 3', 4' and 7' belong to type B , 5', 9, '10', 12', 20', 21 and
214 24' belong to type C, 11',15', 17', 18', 19', 23' and 25' belong to type D, and only

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215 compounds 16' and 22' belong to other types. The R2 group in these two compounds
216 are pentylamine group, so the unique neutral loss in the high-mass region was 87 Da.

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217 Taking compound 16' for example, its molecular weight (MW: 348) was the same as
218 compound 23' (MW: 348), but MS2 fragmentation of them produced fragment ion at
219
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[M+H-85]+ (compound 23', Fig. 4A)and [M+H-87]+ (compound 16’, Fig. 4B),
220 respectively. Interestingly, characteristic ion fragments in low-mass region of
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221 compound 16' and compound 18'(MW: 346, Fig. 4C) were the same. Thus, R1 group
222 of compound 16' was an alkyl group and R2 group was a pentylamine group with
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223 branched or straight chain. Similarly, compound 22' was also attributed to other type
224 (Fig. 4D).
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225 In summary, a total of 43 amide alkaloids were identified, including 18 amide

226 alkaloids confirmed with standard compounds and 25 amide alkaloids identified by
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227 UHPLC-MS. Characterization of the minor components resulted in a better

228 illustration of the chemical components in the alkaloid fraction.
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229 3.2 quantitative analysis of the alkaloid fraction

230 3.2.1 Validation of the quantitative method
231 After qualitative analysis of the alkaloid fraction, piperine (compound 1) ，
232 pipernonatine (compound 6), guineensine (compound 10) and N-isobutyl-2E,
233 4E-octadecadienamide (compound 15), were chosen for quantitative analysis. Four
234 marker compounds were in P. longum L. well separated with reasonable peak shape
235 and resolution (Fig. 2A).
236 Stock solutions containing four reference compounds were prepared and diluted to

Page 8 of 25
237 six different concentrations for construction of calibration curves. Each concentration
238 was analyzed in triplicates, and then the calibration curves were constructed by
239 plotting the peak area versus the concentration of each compound. Good linear
240 correlation at this chromatographic conditions were confirmed by the correlation
241 coefficients (r2> 0.9992), and the linearity ranges of compound 1, 6, 10 and 15 were
242 5.15 - 103.00 μg /mL, 5.00 - 100.00 μg /mL, 5.10 - 103.00 μg/mL, and 5.00 - 100.00

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243 μg/mL, respectively.
244 The LOQ were calculated as ten times of the signal-to-noise ratios. LOQs for

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245 compound 1, 6, 10 and 15 were 0.50 μg/mL, 0.43 μg/mL, 0.13 μg/mL, 0.11 μg/mL,
246 respectively.

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247 The mixture standard solution (compound 1, 20.60 μg/mL; compound 6, 20.00
248 μg/mL; compound 10, 20.40 μg/mL; compound 15, 20.00 μg/mL) was analyzed under
249
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the optimal conditions and intra- and inter-day variations were chosen to determine
250 the precision of the developed method. The precision was examined by RSD of five
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251 repetitive injections in the same day and in three consecutive days, respectively. The
252 intra- and inter-day precisions were within 1.0% and 1.5%, respectively.
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253 According to the procedure in 2.4, six independently sample solutions were
254 prepared and analyzed in parallel in order to test the repeatability. The RSDs were
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255 1.2%, 1.2%, 1.2% and 1.4%, respectively.

256 For stability test, sample solution stored at room temperature was investigated by
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257 replicate injection of the sample solution at 0, 2, 4, 8, 12 and 24 h. The RSD of

258 contents of the four reference compounds in the same sample ranged between 1.3%
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259 and 1.8% (Table 2), indicating that the sample solution was stable at room
260 temperature for at least 24 h.
261 Recovery test was carried out to investigate accuracy of this method. Four standard
262 solutions at low, medium and high levels (50%, 100% and 150%) were added to 1 mL
263 the sample solution (2.014 mg/mL) described in 2.4 and diluted to 5 mL with 50%
264 ACN (v/v) in volumetric flask. Each level was prepared three times in parallel. The
265 results showed that the assay was satisfactory with the mean recovery and RSD less
266 than 4.5% for the four components (Table 2).

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267 All these values within acceptable limits indicated this UHPLC-DAD method is
268 reliable with excellent repeatability, recovery rate and precision.
269 3.2.2 Quantitative determination of the alkaloid fraction
270 After the method validation，determination of the alkaloid fraction was carried out
271 using external standard method. The content of four compounds in the alkaloid
272 fraction was as follows: piperine, 57.5 mg/g; pipernonatine, 65.6 mg/g; guineensine,

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273 17.7 mg/g; and N-isobutyl-2E, 4E-octadecadienamide, 23.9 mg/g. The percent content
274 of four marker compounds in the alkaloid fraction reached 16.47%. It indicated that

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275 some uncertain ingredient in P. longum L. was efficiently removed after appropriate
276 purification.

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277 However, the availabilities of most standard compounds for multi-components
278 quantitative analysis in TCMs are limited. Thus, using an easily available single
279
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component contained in complex sample as reference compound to determine
280 multiple analogues had been applied by calculating relative response factors. In this
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281 study, piperine (compound 1) was used as the reference compound. Relative response
282 factors were obtained for compounds 6, 10 and 15, by calculating the ratios of peak
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283 area of unit concentration to that of piperine (Table 3). To validate the external/
284 relative response factor method, the amounts of four maker compounds were
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285 calculated (Table 3). The differences between these two methods (external standard
286 and relative response factors) were acceptable. Although the potential fluctuation in
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287 relative response factors in different laboratories should be paid attention. Single
288 chemical standards as a reference to determine the ingredients which have similar
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289 chemical structures might be an alternative approach if chemical standards in complex

290 samples are not available.
291 4. Conclusions
292 In this study，quality evaluation of the alkaloid fraction purified from P. longum L.
293 was researched by a comprehensive qualitative and quantitative analysis. Quantitative
294 analysis of the alkaloid fraction was performed by UHPLC-DAD and UHPLC-MS
295 methods. 18 major compounds and 25 minor compounds were found in the alkaloid
296 fraction. A UHPLC-UV method was employed using four marker compounds as

Page 10 of 25
297 external standards for the quantitative analysis of alkaloid fraction. The total amount
298 of four marker compounds in the alkaloid fraction reached 16.47%, indicating that
299 large amounts of unknown or uncontrolled components, such as pigment and volatile
300 oil, was removed in the purification of alkaloid fraction from P. longum L. A method
301 based on relative response factor was also used for quantitative analysis. There were
302 not remarkable differences observed comparing with the external standard method.

t
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303 The result illustrated that the material basis of TCMs could be explained clearly and
304 the quality control could be improved using modern analysis methods and technology,

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305 relatively. If comprehensive pharmacology be researched in the further, this alkaloid
306 fraction with systematic quality evaluation could be as an import source in drug R&D.

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307 Acknowledgments
308 This work was supported by the Natural Science Foundation of Shanghai, China
309
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(Grant No. 13ZR1453200) and the Specialized Research Fund for the Doctoral
310 Program of Higher Education of China (Grant No.20130074120017).
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us
367 Planta Med. 72(2006) 527-532.
368 [20] H. Kikuzaki, M. Kawabata, E. Ishida, Y. Akazawa, Y. Takei N. Nakatani, LC-MS
369
an
Analysis and Structural Determination of New Amides from Javanese Long Pepper
370 (Piper retrofractum), Biosci. Biotech. Biochem. 57 (1993) 1329-1333.
M
371
372
373 FIGURE CAPTION
ed

374 Fig. 1 Structure information of 18amide alkaloids used for qualitative and quantitative
375 analysis of an alkaloid fraction from P. longum L.
pt

376 Fig. 2 Chromatograms of 18 standard compounds (A) and all 43 alkaloids were
ce

377 identified by UHPLC-MS in alkaloid fraction (B).

378 Fig. 3 Ion fragments spectra of [M+H]+ ions of (A) compound 9, type A; (B)
379 compound 10, type B; (C) compound 14, type C and (D) compound 17, type D.
Ac

380 Fig.4 Ion fragments spectra of [M+H]+ ions of (A) compound 23', type D; (B)
381 compound 16', other type; (C) compound 18', type D and (D) compound 22', other
382 type.
383 Table 1 Characterization of compounds 1'-25' in the alkaloid fraction
384 Table 2 Summary of calibration curves, linear range, LOQ, repeatability, intra-day and
385 inter-day precisions, repeatability, stability and recoveries for four marker
386 compounds.

Page 13 of 25
387 Table 3 Contents of four marker compounds in alkaloid fraction purified from P.
388 longum L.

t
ip
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M
ed
pt
ce
Ac

Page 14 of 25
*Graphical Abstract

t
ip
cr
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ed
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Ac

Page 15 of 25
*Highlights (for review)

1. Comprehensive qualitative and quantitative analysis of an alkaloid fraction

purified from piper longum L.
2. 18 standard compounds and 25 minor amide alkaloids were identified.
3. A method based on relative response factor was used for quantitative analysis
4. The quality of alkaloid fraction was efficiently improved after appropriate
purification.

t
ip
cr
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an
M
ed
pt
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Ac

Page 16 of 25
Table(s)

t
ip
cr
Table 1. Characterization of compounds 1'-25' in alkaloid fraction

us
Compounds [M+H]+ Fragment ion in MS2 n1 n2 n3 Type Deduced structure
Netrual loss of Alkyl ion produced

an
R2 group/Da by R1 group
1' 276 73,101 135,161 0 2 0 B dihydropiperlonguminine
2' 314 85,113 135,161 0 2 1 A piperdardine

M
3' 328 73,101 135,161 1 2 1 B Retrofractamide A
4' 358 73,101 135,161 1 6 0 B piperchabamide D
5' 252 73,101 119,133 121,135 0 2 1 C N-isobutyl-2E,4E-dodecadienamide
6' 370 85,113 135,161 1 6 0 A piperchabamide B

d
7' 386 73,101 135,161 0 8 1 B 13-(1,3-benzodioxol-5-yl)-N-(2-methylpropyl)-(2E,4E)-

8'
9'
398
306
85,113
73,101te 135,161
119,133 121,135
1
1
8
4
0
1
A
C
Tridecadienamide
piperchabamide C
(2E,4E,10Z)-N-isobutylhexadeca -2,4,10-trienamide
ep
10' 282 73,101 119,133 121,135 0 6 0 C (2E)-N-isobutyltetradec -2-enamide
11' 342 85,113 119,133 121,135 3 2 1 D Unidentified
12' 332 73,101 119,133 121,135 2 4 1 C Unidentified
c

13' 318 85,113 119,133 121,135 1 4 1 D 1-[(2E,4E,10Z)-hexadeca trienyl]-piperidine

14' 424 85,113 135,161 1 8 1 A Unidentified
Ac

15' 336 85,113 135,161 0 9 0 D (2E)- heptadec -2-enoic acid piperidine

16' 348 87,115 119,133 121,135 1 6 1 O (2E,4E,12E)- N-pentyl-2,4,12-octadecatrienamide
17' 320 85,113 119,133 121,135 0 6 1 D 1-[(2E,4E)-1-oxo-2,4-hexadecadienyl]-piperidine
18' 346 85,113 119,133 121,135 1 6 1 D 1-(2E,4E, 12Z)-octadecatrienoylpiperidine
19' 348 85,113 119,133 121,135 1 8 0 D Unidentified
20' 360 73,101 119,133 121,135 2 6 1 C Unidentified
21' 364 73,101 119,133 121,135 0 10 1 C N-isobutyl-2E,4E-decyldecadienamide

Page 17 of 25
 t
ip
cr
22' 376 87,115 119,133 121,135 1 8 1 O (2E,4E,14E)- N-pentyl-2,4,14-icosatrienamide

us
23' 348 85,113 119,133 121,135 0 8 1 D 1-(2E,4E)-octadecadienoylpiperidine
24' 390 73,101 119,133 121,135 1 10 1 C (2E,4E,14Z)-N-Isobutyldecosene-2,4,14-trienamide
25' 376 85,113 119,133 121,135 0 10 1 D 1-[(2E,4E)-1-oxo-2,4-eicosadienyl)]-piperidine

an
Note: O, other type.

M
d
te
c ep
Ac

Page 18 of 25
Table(s)

t
ip
cr
Table 2. Summary of calibration curves, linear range, LOQ, repeatability, intra-day and inter-day precisions, repeatability, stability and recoveries for four marker

us
compounds.

an
Compound Calibration r2 Linear Range LOQb Intra-day Inter-day Repeatability Stability Recoveries % (n = 3)
Curvea (μg /mL) μg/mL (RSD, %) (RSD, %) RSD (%) RSD (%)
(n = 6) (n = 3) (n = 6) (n = 6)

M
50%c 100%c 150%c RSD(%)d
y=20364x-
1 0.9999 5.15 ~103.00 0.50 0.9 0.9 1.2 1.6 97.9 101.3 106.4 4.2
6.8277

d
y=26035x-
6 0.9997 5.00 ~100.00 0.43 0.9 1.2 1.3 1.7 99.5 94.7 96.9 2.5
6.8604

10
y=61781x-
1.9828
0.9998
te
5.10 ~103.00 0.13 0.8 1.2 1.3 1.8 105.5 105.0 110.1 2.6
ep
y=40817x+
15 0.9992 5.00 ~100.00 0.11 0.4 0.3 1.4 1.3 104.3 103.3 108.3 2.5
1.9530
a
y is the peak area, x is the concentration of standard solutions (μg /mL).
b
LOQ refers to the limits of quantity, S/N = 10.
c

c
average of three determinations
Ac

d
average of nine determinations

Page 19 of 25
Table(s)

Table 3. Contents of four marker compounds in alkaloid fraction purified from P. Longum L...

a
Compound F Contents/% Relative deviation
external standard relative response factor
method method
1 1.00 5.75 5.75 0.00
6 1.28 6.56 6.55 0.15
10 3.03 1.77 1.78 -0.56

t
15 2.00 2.39 2.40 -0.42

ip
a Relative deviation = (content determined by the present method-content determined
by relative response factor method)*100/content determined by relative response

cr
factor method.

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Page 20 of 25
Figure(s)

Fig.1 Structure information of 18 amide alkaloids used for qualitative and quantitative analysis of
the alkaloids fraction from P. Longum L.

t
Type A

ip
cr
Type B

us
Type C

Type D

Compound Name Type

an n1 n2 n3 [M+H]+
M
1 piperine A 1 0 1 286
2 pellitorine C 1 0 1 224
3 piperanine A 0 6 0 288
ed

4 dehydropipernonaline A 2 0 1 340
pt

N-isobutyl-2E,4E-
5 C 0 1 1 238
undecadienamide
ce

6 pipernonatine A 1 2 1 342
7 retrofractamide B B 1 4 1 356
8 Pipernonatine A 1 4 0 344
Ac

(2E,4E,10E)-N-11-(3,4-Met
9 hylenedioxyphenylhmdecatr A 1 4 1 368
ienoylpiperidine

10 Guineensine B 1 6 1 384
(2E,4E,12E)-13-(benzo[d]
[1,3]dioxol-6-yl)-1-
11 A 1 6 1 396
(piperidin-1-yl)trideca-2,4,1
2-trien-1-one
12 brachyamide B B 1 8 1 412
N-isobutyl-2E,4E-
13 C 0 6 1 308
hexadecadienamide
Page 21 of 25
 (2E,4E,12Z)-N-Isobutylocat
14 C 1 6 1 334
adeca-2,4,12-trienamide
N-isobutyl-2E,4E-
15 C 0 8 1 336
octadecadienamide
(2E,4E,14Z)-N-Isobutyleico
16 C 1 8 1 362
sa-2,4,14-trienamide
1-[(2E,4E,14Z)-1-oxo-2,4,1

t
17 D 1 8 1 374

ip
4-eicosatrienyl]-piperidine
N-isobutyl-2E,4E-
18 C 0 10 1 364

cr
decyldecadienamide

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Page 22 of 25
Figure(s)

Fig. 3 Ion fragments spectra of [M+H]+ ions of (A) compound 9, type A; (B) compound 10, type
B; (C) compound 14, type C and (D) compound 17, type D.
120510

135
100 O
112 (A)
O (CH2)4 N
%

O
[M+H-113]+ [M+H-85]+
*

t
133 368
140 *
* 265 283

ip
161 185 187 215 246 255 300314 350 372
120510 119 340
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375
135

cr
100
O
(B)
O (CH2)6 N

us
H
%

O
[M+H-101]+ [M+H-73]+
131 161 * *
201 283 * 384
120510
0
100 125 150
187

175 200
215
225
257
250
an
275 300
311 313 342
325 350
356

375
*
334
400
m/z
400
100
O
(C)
M
H3C (H C) (CH2)6 N
2 4
H
%

109
ed

135 [M+H-101]+ [M+H-73]+

123 149 166 *
177
194 222 *
233 236 261 278 292 316 329 338
346
120510
0 m/z
100 125 150 175 200 225 250 275 300 325 350
pt

*
374
100
O
(D)
112 H3C (H C)
ce

2 4 (CH2)8 N
%

[M+H-113]+ [M+H-85]+
Ac

121 123 149 166 * *

289 332
178 194 222 261 304 346 356 376 389
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375

Page 23 of 25
Figure(s)

Fig 4 Fragment ions spectra of [M+H]+ ions of (A) compound 23', type D; (B) compound 16',
other type; (C) compound 18', type D and (D) compound 22', other type.

120510
*
348
100 (A)
112
%

t
ip
[M+H-85]+
119 123 *
138 166 180 194 208 222 245 263 280 294 320 330 358360
120510
0

cr
m/z
100 125 150 175 200 225 250 275 300 325 350
109
100
* (B)

us
348

121
%

123

120510
142
168 180
196
[M+H-87]+
* 278
210 236 261
an 320
306 371 375389
0 m/z
M
100 112
125 150 175 200 225 250 275 300 325 350 375 400
100
(C)
*
ed

346
%

121
138
[M+H-85]+
150 152 166
* 318 328
pt

180 194 208 222 261 278 304 357 360

120510
0 m/z
100 125 150 175 200 225 250 275 300 325 350
*
350
ce

100
(D)
Ac
%

112
[M+H-87]+
111
121 140 149 167 201 *
283 337
213 227 241 259 276 295 328 358360
0 m/z
100 125 150 175 200 225 250 275 300 325 350

Page 24 of 25
Figure(s)

Fig. 2 Chromatograms of 18 standard compounds (A) and all 43 alkaloids

were identified by UHPLC-MS in alkaloid fraction (B)

(A)
8

t
13

ip
14
15 18
4

cr
3 9 16
2 57
17

us
6 11
1 10 12

an
16
(B)
M
14' 14
17
9 13'
2 5' 10' 11' 13 21', 22' 18
6' 9' 12 12'
ed

4'
19'20' 23' 24' 25'
6
1
18'
pt

5 10 17'
4 7' 11 15'
8' 15
3' 3 16'
2' 78
ce

1'
Ac

Page 25 of 25