Determination of Differentiating Markers in Coicis Semen From Multi-Sources Based on Structural Similarity Classification Coupled With UPCC-Xevo G2-XS QTOF.

Coicis semen, a medicinal food, is derived from the dried and mature seeds of Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf, a member of the Gramineae family. Lipids are its main constituents. Previous literature reported that coicis semen contains twenty triglycerides and twelve diglycerides. However, we identified thirty-five triglycerides, sixteen diglycerides, four monoglycerides, and two sterols under the preoptimized conditions of UPCC-Xevo G2-XS QTOF combined with a personalized TCM database. Furthermore, we successfully determined glycerol trioleate content to evaluate quality differences. Finally, we identified the fatty acid compositions of seven out of nine differential markers via Progenesis QI using principal component analysis, orthogonal projection to latent structures-discriminant analysis, and the LipidMaps database. In addition, we applied a software-based classification, a method that was previously developed by our team, to verify and predict structurally similar compounds. Our findings confirmed that UPCC-Xevo G2-XS QTOF combined with software-based group classification could be used as an efficient method for exploring the potential lipid markers of seed medicine.

Introduction

Common sense dictates that various natural ingredients exist in TCM. However, most reports on the active components of TCM have focused on polysaccharides, alkaloids, and flavonoids. Fatty oils are widely available ingredients of herbs, and the limited attention that they have received may restrict their further development and application. Fatty oils can be obtained as an active ingredient from animals and plants (Liu et al., 2015; Chen et al., 2016; Hong et al., 2016). A number of TCM contain fatty oils, which are mainly derived from the seeds and fruits of herbs. Diverse fatty oils comprise of glycerol and different types of saturated, monounsaturated, and polyunsaturated fatty acids that each exert therapeutic effects.

Coicis semen (Job’s tears seed or adlay), which has been documented in the 2015 edition of the Chinese pharmacopoeia, is the dry and mature seed of Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf. It is not only a commonly used TCM, but it is also a commonly consumed food. Coicis semen has been proven to have numerous functions, such as detoxification and dampness and arthralgia removal; it also reduces cancer risk (Yu et al., 2011; Bai et al., 2018; Duan, 2018; Xu et al., 2018; Choi et al., 2019; Huang Q. et al., 2019; Huang Y. L. et al., 2019; Li et al., 2019; Liu H. Q. et al., 2019; Liu Y. N. et al., 2019; Manosroi et al., 2019; Qian et al., 2019; Zhang et al., 2019). Moreover, it has a wide range of anti-inflammatory, antioxidation, analgesic, and sedative effects, as well as pharmacological effects against gastric cancer, hepatocellular cancer, Lewis lung cancer, non-small cell lung cancer, pancreatic cancer, and pulmonary cancer (“Fuzheng” category represented by coicis semen oil) (Manosroi et al., 2016a; Manosroi et al., 2016b; Qian et al., 2016; Xi et al., 2016; Wang et al., 2016; Han et al., 2017; Qu et al., 2017a; Schwartzberg et al., 2017; Jang et al., 2018; Kim et al., 2018; Zhang et al., 2018). It has a variety of clinical dosage forms, such as microporous microspheres, microemulsions, and intravenous emulsions (Qu et al., 2016; Qu et al., 2017b; Qu et al., 2017c; Trinh et al., 2017; Chen et al., 2018; Guo et al., 2019). Discrepancies in curative effects can be attributed to the various types, contents, and other nutrients of different fatty oils.

Recent research shows that the variety and content of the active components of different fatty oils remarkably influence human health and have their own specific advantages in treatment. Near-infrared spectroscopy analysis showed that the lipid contents of forty-one polished coicis semen samples range from 5.14% to 9.40%. Coicis semen oils contain seven types of triglycerides (trilinolein, 1,2-linolein-3-olein, 1-palmitin-2-linolein-3-olein, 1-palmitin-2,3-linolein, 1-palmitin-2, 3-olein, triolein, and 1,2-olein-3-linolein). Hou et al. identified twenty triglycerides and twelve diglycerides in the lipid profile of coicis semen and developed a green quantification strategy for simultaneously determining the content of 7 TGs (LLL, LLP, LLO, POL, OOL, OOP, and OOO) by combining core–shell column technology and SSDMCs. Lin et al. found β-sitosterol and stigmasterol in the ethyl acetate fraction of an adlay hull extract. Dong et al. established a rapid and reliable m-SPE approach using magnetic multiwalled carbon nanotubes as the adsorbent for the purification of type A trichothecenes, including T-2 toxins, HT-2 toxins, diacetoxyscirpenol, and neosolaniol, in coicis semen (Dong et al., 2016; Hou et al., 2018a; Hou et al., 2018b; Lin et al., 2019).

However, several components, especially glycerides, of coicis semen oil still require analysis, and the identification of the active ingredients of this material needs in-depth research. Therefore, our team used Acquity UPCC-Xevo G2-XS QTOF coupled with software-based group classification to further excavate, identify, and visually classify active ingredients in coicis semen oils. Coicis semen oils have boundless development prospects and need to be explored in-depth to lay a foundation for its new preparation, development, and extensive clinical application.

Materials and Methods

Materials and Reagents

Glycerol trioleate with the purity of more than 99.9% as determined via HPLC–ELSD was purchased from the Nature Standard Co. Ltd (Shanghai, China). Acetonitrile and methanol (HPLC–MS grade) were purchased from Merck (Darmstadt, Germany). Ammonium formate (HPLC grade) was purchased from Sigma-Aldrich. High-purity CO2 (99.999%) was purchased from the Shanghai Yizhi Industry Gases Co., Ltd. (Shanghai, China). All other reagents used in sample preparation were of analytical grade. Seven batches of dried coicis semen were purchased from different TCM enterprises in China. The manufacturers and batch numbers of the samples were as follows: batch number 180901 (Zhejiang Chinese Medical University Medical Pieces Co., Ltd., Hangzhou); batch number 190101 (Jirentang Pharmaceutical Co., Ltd., Guiyang); batch number 190105 (Zuoli Baicao Herbal Pieces Co., Ltd., Jiangxi); batch number 181201 (Huadong Herbal Pieces Co., Ltd., Hangzhou); batch number 181206 (Zuoli Baicao Herbal Pieces Co., Ltd., Zhejiang); batch number 190122 (Haiyuan Prepared Slices of Chinese Crude Drugs Co., Ltd., Nanjing); and batch number 190216 (Haichang Chinese Medicine Group Co., Ltd., Nanjing).

Preparation of Reference and Sample Solutions

The appropriate amount of glyceryl trioleate, which was used as the reference substance, was weighed accurately and diluted with n-hexane to prepare a series of working solutions with concentrations of 0.0099, 0.0988, 0.9881, 4.9405, and 9.8810 μg/mL.

All samples were ground and passed through a No. 3 sieve (355 ± 13 μm). The passing rate of the particles was maintained at more than 80%. The sample preparation procedure was as follows:

A total of 50.0 mL of n-hexane was added to 0.6 g of powdered coicis semen. The seed powder was soaked for 2 h and then sonicated (50 kHz, 250 W, KQ-500DB) for 30 min. The supernatant was filtered to obtain the sample solution. The filtrate was diluted with n-hexane 5 times and 100-fold for the analysis of different components of different samples and the quantitative analysis of glyceride trioleate, respectively. A total of 200 μL solution of each batch was mixed together and used as a pooled QC sample solution for the analysis of free fatty acids.

UPCC-Xevo G2-XS QTOF Parameters

On the basis of preliminary experiments, the final experimental conditions were determined as follows: Liquid phase system: ACQUITY UPCC; column: Torus 2-PIC, 3.0 × 100 mm, 1.7 μm; mobile phase A: CO2, mobile phase B: methanol acetonitrile = 9:1; column temperature: 55°C, ABPR: 2600 psi; compensation solution: methanol solution with 0.5 mM ammonium formate; flow rate: 0.5 mL/min; and injection volume: 0.3 μL. The gradient program was used with a flow rate of 1.0 mL/min and was as follows: 0–2 min (1% B), 2–7 min (1%–5% B), 7–10 min (5% B), and 10–13 min (5%–1% B).

The conditions for mass spectrometry are as follows: Mass spectrometry system: Xevo G2-XS QTOF; ionization method: ESI+/−; data acquisition mode: MSE; MSE impact energy: low, off, high, 20–50 eV; quantitative ion: m/z 902.8177; collection mass range: 100–1200 Da; capillary voltage: 2.0 kV; cone-hole voltage: 40 V; ion source temperature: 120°C; atomization temperature: 500°C; cone-hole gas flow rate: 50 L/h; and atomized gas flow rate: 1000 L/h. Data calibration was performed by using an external reference (LockSpray) with the constant infusion of leucine–enkephalin solution (200 pg/μL) at a flow rate of 5 μL/min.

The data processing software included UNIFI 1.9.4 and Masslynx V 4.1.

Data Acquisition and Analysis

Xevo G2-XS QTOF uses the patented LockSpray technology to ensure the accuracy of the collected data in real time. High-accuracy mass numbers can be obtained, and the combination of high-accuracy mass numbers and isotope distribution and secondary fragment information accurately provides molecular formulas. Xevo G2-XS QTOF applies patented MSE technology to obtain the primary and secondary mass spectral information of the compounds for further structural confirmation with one injection at the same time.

The ESI+/− mode was used for the analysis of glycerides and free fatty acids. The first step involved understanding the fragmentation pattern of glycerides as a whole. In the second step, by combining the fragmentation patterns and consulting related literature, a UNIFI database for the glyceride analysis of coicis semen was established. In the third step, the self-built database was imported into UNIFI in addition to the ChemSpider online database, and the appropriate analysis method was set. Furthermore, the software automatically analyzed primary and secondary mass spectral information. Finally, it quickly screened out the target through a unique workflow.

Our team developed a classification program in the Visual Basic for Applications (VBA; Microsoft, USA) and MATLAB v7.1 (The Mathworks, Natick, USA) environments. The classification program consisted of three parts (Shan et al., 2012).

A total of 2,916 features were introduced into the SIMCA-P 13.5 software (Umetrics, Umeå, Sweden) for principal component analysis (PCA) and orthogonal projection to latent structures–discriminant analysis (OPLS–DA). The corresponding variable importance in the projection value (VIP value) was calculated in the OPLS–DA model. A potential differential marker was selected when its VIP value exceeded 2.00 and its S-Plot exceeded 0.95.

Results and Discussion

Qualitative Results of Glycerides and Free Fatty Acids in Coicis Semen Oils

In the ESI+ mode, fifty-six and fifty-seven compounds were identified in five (No. 180901, No. 181201, No. 181206, No. 190101, and No. 190122) and two (No. 190105 and No. 190216) batches of samples, respectively. These compounds were mainly composed of glycerides. The total ion chromatogram of all samples is provided in , and the corresponding identified glycerides are listed in . Among them, fifty-six common compounds, including thirty-five triglycerides, fifteen diglycerides, four glycerides, and two sterols, were identified in comparison with the corresponding results of twenty triglycerides and twelve diglycerides (Hou et al., 2018a). However, the OP of diglycerides was identified only in two batches of samples, namely 190105 and 190216, likely because of the different processing technologies of different medical enterprises. The QC sample mentioned in was overlaid in the following differential component analysis.

Figure 1

Total ion chromatogram of (A) (qualitative analysis of glyceride contents of seven batches of coicis semen oils in ESI+); (B) (overlay of the QC sample for the differential component analysis of different samples in ESI+); (C) (qualitative analysis of free fatty acids of QC sample in ESI−).

Table 1

The qualitative results of glycerides in coicis semen oils in ESI+.

No.	Component name	Formula	Neutral mass (Da)	Observed m/z	Mass error (mDa)	Observed RT (min)	Response	Adducts
1	β-Sitosterol	C29H50O	414.3862	397.3827	-0.20	2.65	175317	-H2O+H
2	PPP	C51H98O6	806.7363	824.7724	2.20	2.87	76543	+NH4, +Na
3	Stigmasterol	C29H48O	412.3705	413.3782	0.40	2.98	280236	+H, +Na, -H2O+H
4	PMO	C51H96O6	804.7207	822.7572	2.70	3.01	40426	+NH4, +Na
5	PPS	C53H102O6	834.7676	852.8045	3.10	3.11	19271	+NH4, +Na
6	PML	C51H94O6	802.7050	820.7414	2.50	3.18	31125	+NH4, +Na
7	PPO	C53H100O6	832.7520	850.7880	2.20	3.26	4297765	+NH4, +H, +Na, -H2O+H
8	PSS	C55H106O6	862.7989	880.8372	4.40	3.38	5232	+NH4, +Na
9	PPL	C53H98O6	830.7363	848.7720	1.90	3.46	4668112	+NH4, +H, +Na, -H2O+H
10	POS	C55H104O6	860.7833	878.8203	3.20	3.54	1031215	+NH4, +H, +Na
11	PPOL	C53H96O6	828.7207	846.7580	3.40	3.64	344524	+NH4, +H, +Na
12	POO	C55H102O6	858.7676	876.8048	3.40	3.71	20321224	+NH4, +H, +Na, -H2O+H
13	OPC20:0	C57H108O6	888.8146	906.8520	3.60	3.80	323033	+NH4, +H, +Na
14	LLM	C53H94O6	826.7050	844.7426	3.70	3.83	49997	+NH4, +H, +Na
15	PLO	C55H100O6	856.7520	874.7887	2.90	3.89	21370394	+NH4, +H, +Na, -H2O+H
16	OOS	C57H106O6	886.7989	904.8357	2.90	3.96	3378949	+NH4, +H, +Na, -H2O+H
17	OLC17:0	C56H102O6	870.7676	888.8046	3.20	4.01	273530	+NH4, +H, +Na
18	OPC22:0	C59H112O6	916.8459	934.8839	4.10	4.03	51651	+NH4, +Na
19	LLP	C55H98O6	854.7363	872.7728	2.60	4.06	8367471	+NH4, +H, +Na, -H2O+H
20	OOO	C57H104O6	884.7833	902.8202	3.10	4.11	21932960	+NH4, +H, +Na, -H2O+H
21	OOO20:0	C59H110O6	914.8302	932.8674	3.30	4.19	590110	+NH4, +H, +Na
22	OOC19:1	C58H106O6	898.7989	916.8371	4.30	4.21	28860	+NH4
23	LLPo	C55H96O6	852.7207	870.7575	3.00	4.23	335763	+NH4, +H, +Na, -H2O+H
24	OOL	C57H102O6	882.7676	900.8044	2.90	4.27	26267878	+NH4, +H, +Na, -H2O+H
25	OLC20:0	C59H108O6	912.8146	930.8517	3.30	4.34	795167	+NH4, +H, +Na, -H2O+H
26	OLC19:1	C58H104O6	896.7833	914.8206	3.40	4.37	39779	+NH4, +Na
27	LLO	C57H100O6	880.7520	898.7884	2.60	4.43	22285984	+NH4, +H, +Na, -H2O+H
28	LOC20:1	C59H106O6	910.7989	928.8353	2.50	4.51	663937	+NH4, +H, +Na
29	LLC19:1	C58H102O6	894.7676	912.8009	-0.60	4.53	34245	+NH4
30	LOC22:0	C61H112O6	940.8459	958.8826	2.80	4.54	183383	+NH4, +Na
31	OOO24:0	C63H118O6	970.8928	988.9309	4.20	4.57	53885	+NH4
32	LLL	C57H98O6	878.7363	896.7721	2.00	4.60	8926747	+NH4, +H, +Na, -H2O+H
33	LLC22:0	C61H110O6	938.8302	956.8666	2.60	4.69	163391	+NH4, +Na
34	LOO24:0	C63H116O6	968.8772	986.9141	3.10	4.73	113189	+NH4, +Na
35	LLLn	C57H96O6	876.7207	894.7569	2.40	4.76	375488	+NH4, +H, +Na, -H2O+H
36	LLC24:0	C63H114O6	966.8615	984.8983	2.90	4.87	76278	+NH4, +Na
37	OLS	C57H104O6	884.7833	885.7856	-4.90	5.21	3683	+H
38	PP	C35H68O5	568.5067	551.5035	0.10	5.43	414186	-H2O+H, +Na
39	PS	C37H72O5	596.5380	579.5348	0.10	5.63	579793	-H2O+H, +Na
40	PO	C37H70O5	594.5223	577.5192	0.10	5.78	924986	-H2O+H, +H, +Na
41	PL	C37H68O5	592.5067	575.5031	-0.30	5.95	728564	-H2O+H, +H, +NH4, +Na
42	OS	C39H74O5	622.5536	605.5500	-0.30	5.98	150906	-H2O+H, +H, +Na
43	OO	C39H72O5	620.5380	603.5344	-0.30	6.11	2014873	-H2O+H, +H, +NH4, +Na
44	PP	C35H68O5	568.5067	551.5030	-0.40	6.19	17291	-H2O+H, +Na
45	OL	C39H70O5	618.5223	601.5188	-0.20	6.28	2010772	-H2O+H, +H, +NH4, +Na
46	LL	C39H68O5	616.5067	617.5130	-1.00	6.48	681857	+H, +NH4, +Na, -H2O+H
47	OP	C37H70O5	594.5223	617.5130	1.40	6.48	681857	+Na, +H, +NH4, -H2O+H
48	LP	C37H68O5	592.5067	575.5037	0.40	6.67	311434	-H2O+H, +H, +NH4, +Na
49	OS	C39H74O5	622.5536	645.5432	0.30	6.70	44689	+Na, +H, +NH4, -H2O+H
50	OO	C39H72O5	620.5380	603.5350	0.30	6.81	707855	-H2O+H, +H, +NH4, +Na
51	LO	C39H70O5	618.5223	601.5193	0.20	6.98	697567	-H2O+H, +H, +NH4, +Na
52	LL	C39H68O5	616.5067	639.4962	0.30	7.13	505275	+Na, +H, +NH4, -H2O+H
53	LLn	C39H66O5	614.4910	637.4809	0.60	7.30	13264	+Na, +H, +NH4, -H2O+H
54	MG(16:0/0:0/0:0)	C19H38O4	330.2770	353.2667	0.50	7.97	239151	+Na, -H2O+H
55	MG(17:0/0:0/0:0)	C20H40O4	344.2927	367.2841	2.20	8.08	632	+Na, -H2O+H
56	MG(18:0/0:0/0:0)	C21H42O4	358.3083	381.2977	0.20	8.19	259875	+Na, -H2O+H
57	MG(20:0/0:0/0:0)	C23H46O4	386.3396	409.3288	0.00	8.40	2705	+Na, -H2O+H

A total of thirty free fatty acids were identified in the ESI− mode, and some unsaturated fatty acids could have had isomers, which needed to be confirmed further by using a reference substance. The total ion chromatogram of the QC sample is given in , and the corresponding identified free fatty acids are listed in .

Table 2

The qualitative results free fatty acids in coicis semen oils in ESI-.

No.	Component name	Formula	Neutral mass (Da)	Observed m/z	Mass error (mDa)	Observed RT (min)	Response	Adducts
1	Oleic acid	C18H34O2	282.2559	281.2486	0.0	4.74	7887221	-H, +HCOO
2	Linoleic acid	C18H32O2	280.2402	279.2330	0.1	4.92	4629367	-H, +HCOO
3	Palmitic acid	C16H32O2	256.2402	255.2329	0.0	4.32	2866760	-H, +HCOO
4	Stearic acid	C18H36O2	284.2715	283.2642	-0.1	4.58	940078	-H, +HCOO
5	Arachidic acid	C20H40O2	312.3028	311.2956	0.0	4.82	131638	-H, +HCOO
6	cis-Vaccenic acid	C18H34O2	282.2559	281.2484	-0.2	6.02	126409	-H
7	9, 10- EPOXYOCTADECANOIC ACID	C18H34O3	298.2508	297.2432	-0.3	5.79	88969	-H
8	9, 10- EPOXYOCTADECANOIC ACID	C18H34O3	298.2508	297.2432	-0.3	5.92	79177	-H
9	11-Eicosenoic acid	C20H38O2	310.2872	309.2798	-0.1	4.97	75288	-H
10	Linolenic acid	C18H30O2	278.2246	277.2172	-0.1	5.09	61260	-H
11	Lignoceric acid	C24H48O2	368.3654	367.3579	-0.2	5.23	58141	-H
12	Behenic acid	C22H44O2	340.3341	339.3265	-0.3	5.03	55860	-H
13	Palmitoleic acid	C16H30O2	254.2246	253.2173	0.0	4.50	38739	-H
14	Margaric acid	C17H34O2	270.2559	269.2486	0.0	4.45	29580	-H
15	Arachidonic acid	C20H32O2	304.2402	349.2359	-2.5	4.74	22657	+HCOO
16	TRICOSANOIC ACID	C23H46O2	354.3498	353.3422	-0.3	5.13	17977	-H
17	Myristic acid	C14H28O2	228.2089	227.2017	0.1	4.05	15915	-H
18	10-Heptadecenoic acid	C17H32O2	268.2402	267.2327	-0.3	4.62	11423	-H
19	Eicosapentaenoic acid	C20H30O2	302.2246	347.2200	-2.8	4.92	11013	+HCOO, -H
20	Hexacosanoic acid	C26H52O2	396.3967	395.3887	-0.7	5.42	8914	-H
21	Pentacosanoic acid	C25H50O2	382.3811	381.3733	-0.5	5.33	8708	-H
22	Pentadecylic acid	C15H30O2	242.2246	241.2173	0.0	4.20	8677	-H
23	NONADECANOIC ACID	C19H38O2	298.2872	297.2798	-0.1	4.69	6204	-H
24	HENEICOSANOIC ACID	C21H42O2	326.3185	325.3111	-0.1	4.93	5972	-H
25	Octacosanoic acid	C28H56O2	424.4280	423.4200	-0.7	5.60	5361	-H
26	Laurie acid	C12H24O2	200.1776	199.1704	0.0	3.71	5056	-H
27	Erucic acid	C22H42O2	338.3185	337.3115	0.3	5.19	4770	-H
28	Nervonic acid	C24H46O2	366.3498	411.3476	-0.4	6.38	3848	+HCOO
29	11-14-Eicosadienoic acid	C20H36O2	308.2715	307.2643	0.1	5.14	2012	-H
30	Homo gamma linolenic acid	C20H34O2	306.2559	351.2511	-3.0	4.58	1984	+HCOO

As shown in , PLO (tR 3.90 min) provided a precursor ion ([M+NH4]+) at m/z 874.7874 with a double-bond equivalent. The MS/MS product ions at m/z 601.5184 ([M-P+H]+ palmitic acid), m/z 577.5175 ([M-L+H]+ linoleic acid), and m/z 575.5025 ([M-O+H]+ oleic acid) resulted from the sn-1, sn-2, and sn-3 cleavages of the ester groups, respectively. shows the secondary fragment matching diagram for OOO generated by the UNIFI software.

Figure 2

Fragmentation patterns of PLO (A) and OOO (B) with MS1 and MS2 spectra.

Software-Based Group Classification of Glycerides

Our teams previously developed a program in VBA and MATLAB for the classification of multiple complex components. This program successfully grouped the constituents in the n-hexane extract of coicis semen. Through the comprehensive analysis of herbal samples, fifty-seven peaks were identified and divided into four groups as shown in , . Three of these groups consisted of triglycerides and diglycerides. The remaining group was composed of four monoglycerides, two diglycerides, and two sterols. The chemical structures and special MS fragmentation pathways of these compounds indicated that the same group might have similar features, and unknown ingredients could be identified through the comprehensive software-based group classification of these compounds.

Figure 3

Affinity diagram of the mass spectra of glycerides with software-based group classification.

Figure 4

Network diagram of the mass spectra of glycerides with software-based group classification.

Differential Component Analysis of Different Samples

The Progenesis QI omics analysis software was used for differential component research. Before exploring quality markers, the analytical system was first validated for repeatability upon the injection of six QC samples. A total of 2,916 features were extracted and then imported into EZinfo for multivariate statistical analysis. PCA was used to study the variations in the oils of seven batches of coicis semen (). The differences between the groups of samples, namely No. 181216 and No.190122, were large. Furthermore, No. 190105 and No. 190101, which showed the largest differences, were subjected to OPLS–DA analysis (). These two groups were clearly distinct. Furthermore, we selected compounds with S Plot ≥ 0.95 and VIP ≥ 2 as markers (), and transferred them back to the QI for identification. Finally, nine markers were found (). Then, the LipidBlast, LipidMaps, and Chemspider databases were searched in QI for further identification. Among the nine markers found, seven were identified (five diglycerides, one triglyceride, and one stigmasterol), and the molecular formulas of the remaining two unknown compounds were estimated using an elemental composition tool. The abundance distribution of the nine markers in all the samples is shown in . The abundance of markers, except diglycerides (16:0/18:0/0:0), in No. 190105 were remarkably higher than that in No. 190101. This result could be related to the largest differences between No. 190105 and No. 190101 and indicated that massive differences in resources and processing technologies existed among medical enterprises.

Figure 5

Differential component analysis of different samples. (A): PCA classification of seven batches of samples; (B) OPLS–DA analysis of No. 190101 and No. 190105 with significant differences; (C) S-Plot of No. 190101 and No. 190105; (D) VIP diagram of No. 190101 and No. 190105.

Table 3

The identified results of nine differential markers between No. 190101 and No. 190105.

No.	m/z	Retention time (min)	Identity	Formula	Neutral mass (Da)
1	413.3781	2.99	Stigmasterol	C29H48O	412.3708
2	579.5337	5.64	DG(16:0/18:0/0:0)	C37H72O5	596.5370
3	963.7297	5.69	unknown	C53H102O14	962.7270
4	962.7224	5.69	TG(18:4/20:5/22:6)	C63H92O6	944.6894
5	961.7175	6.18	unknown	C53H100O14	960.7113
6	577.5188	6.52	DG(18:1/16:0/0:0)	C37H70O5	594.5220
7	603.5345	6.82	DG(18:1/18:1/0:0)	C39H72O5	620.5378
8	641.5118	6.99	DG(18:2/18:1/0:0)	C39H70O5	618.5222
9	639.4958	7.14	DG(18:2/18:2/0:0)	C39H68O5	616.5069

Figure 6

Abundance distribution of nine markers in all samples.

Quantitation of Glycerol Trioleate in Coicis Semen Oils

Investigation of Linear Relations

Reference solutions with concentrations of 0.0099, 0.0988, 0.9881, 4.9405, and 9.8810 μg/mL were used to perform three consecutive injections. The results showed that glyceryl trioleate had a good linear relationship in the range of 0.0090–9.8810 μg/mL, r> 0.9990.

Quantitative Limit Investigation

The reference solution was diluted stepwise at certain multiples until glyceride trioleate presented S/N ≈ 10. The results showed that the quantitative limit was 4.94 ng/mL.

Instrument Precision Inspection

The low, middle, and high concentrations (0.0099, 0.9881, and 9.8810 μg/mL) of the reference solution on the calibration curve were taken and used in six consecutive injections to check instrument precision. The RSD value was less than 3%, which indicated good precision.

Repeatability Test

Six powder samples (0.6 g each) of the same batch (No. 180901) were weighed and prepared via the sample solution preparation method. The average content of glyceryl trioleate was determined and calculated as 0.91%. The results showed that the RSD value was 4.12% (n = 6).

Recovery Rate Test

ine powder samples (0.3 g each) of the same batch of known content were weighed, and then low, medium, and high levels of the three different concentrations of the reference substance were added precisely. The reference substance/sample ratio was controlled at 0.5:1, 1:1, 1.5:1, and each concentration level was tested in triplicate. The results showed a high average recovery of 102.28%.

Sample Measurement Results

The content of glyceryl trioleate in seven batches of the samples was determined using the established method above. The representative chromatogram is shown in . Each batch was replicated in triplicate. The average content ranged from 0.84% to 1.05%. The RSD value was less than 5%.

Figure 7

(A) Chromatogram of reference (glycerol trioleate); (B) EIC diagram of representative sample (No. 180901).

Conclusion

The established analytical method fully demonstrated that ACQUITY UPCC enabled the fast and efficient chromatographic separation of lipids in coicis semen. Xevo G2-XS QTOF combined with LockSpray real-time external standard mass calibration technology ensured mass accuracy. The data collection method based on MSE tandem mass spectrometry without content discrimination ensured the full collection of information, and one-shot collection could obtain precursor ion and fragment ion information simultaneously with convenient, fast, and high-throughput characteristics.

By using the ACQUITY UPCC/Xevo G2-XS QTOF system combined with the UNIFI software, fifty-seven compounds of glycerides were identified and divided into four groups on the basis of their similar features via software-based group classification in the ESI+ mode. Moreover, thirty free fatty acids were identified in ESI−. In addition, QI omics analysis software found nine differential compounds between No. 190101 and No. 190105, and seven of these compounds were identified. Finally, the quantitative analysis of glyceryl trioleate (quality control component in the 2015 Edition of the Chinese Pharmacopoeia) and methodological verification were performed, and the results showed that the linearity, precision, reproducibility, recovery, and other parameters of the method were good. The established quantitative method determined that the glyceryl trioleate contents of the seven batches of samples ranged from 0.84% to 1.05%.

In summary, we identified additional glycerides and free fatty acids in coicis semen oils. Our results could supplement corresponding component research. Furthermore, nine differential components were found to be potential markers of quality for differentiating coicis semen with different origins. Finally, glyceryl trioleate was determined to evaluate its pros and cons. This approach might be useful for assessing the quality of TCM.

Data Availability Statement

All datasets presented in this study are included in the article/.

Author Contributions

Conceptualization: XW and GC. Data curation: RZ, XX, and QS. Formal analysis: KW. Funding acquisition: GC. Investigation: RZ, XX, and KW. Writing—original draft: RZ and XX. Writing—review and editing: XW and GC. All authors contributed to the article and approved the submitted version.

Funding

This research was supported by the Zhejiang Public Welfare Technology Application Research Project (2017C33175) and the National Natural Science Foundation of China (No. 81703707).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.