
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12940-4
10.1016/j.heliyon.2024.e36909
e36909
Research Article
LC-MS based strategy for chemical profiling and quantification of dispensing granules of Ginkgo biloba seeds
Zhang Facheng zhangfc@polifarma.com.cn
a1
Fei Qingqing zingfei@163.com
bc1
Huang Xiaojun bc
Yu Sheng bc
Qiu Rongli bc
Guan Lan a
Wu Baoxiang a
Shan Mingqiu shanmingqiu@njucm.edu.cn
bc⁎
a Polifarma (Nanjing) Co., Ltd., Nanjing, 210038, PR China
b Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China
c School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China
⁎ Corresponding author. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China. shanmingqiu@njucm.edu.cn
1 Equal first authors.

29 8 2024
15 9 2024
29 8 2024
10 17 e3690917 6 2024
22 8 2024
23 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Ginkgo biloba seeds have been used as a traditional Chinese medicine for hundreds of years to treat diseases such as cervicitis, cough, asthma and other lung diseases. As a novel form, the dispensing granules (GSDG) of Ginkgo biloba seeds have been widely employed in clinic. However, its chemical profiling is not yet clear, which has restricted in-depth research in many fields.

In this study, a high performance liquid chromatography coupled with quardrupole time-of-flight mass spectrometry method was used for the component characteration with the help of accurate molecular weights, fragmentation pathways, reported data, literatures and even some reference standards. Furthermore, in multiple-reaction monitoring mode, a high performance liquid chromatography coupled with quadrupole linear ion trap mass spectrometry method was developed and applied for simultaneous determination of the bioactive phytochemicals.

As a result, a total of 56 components in GSDG were identified including 12 amino acids, 9 organic acids, 6 nucleosides and nucleobases, 6 flavonoids, 5 vitamins, 5 terpenoid lactones, 4 carbohydrates and 9 other compounds As for quantitative analysis, glutamic acid, asparatic acid, histidine, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, eucomic acid, N-(N-glucopyranosyl)-indoleacetylaspartate and N-(N-glucopyranosyl)-indoleacetylglutamate were selected as the analytes for quanlity marker of GSDG. After necessary validation tests, the developed quantitative method was successfully put into use for 10 batches of GSDG. In all batches, N-(N-glucopyranosyl)-indoleacetylaspartate was the richest phytochemical with the amount of 17.3–25.7 mg/g while ginkgolide J (0.0197–0.0335 mg/g) was determined to be the poorest.

The study is supposed to exhibit a comprehensive chemical profiling and to provide some strong basis for preparation technology, quality control and even for action mechanism of GSDG, this novel form of Chinese medicine.

Graphical abstract

Image 1

Keywords

Dispensing granules of Ginkgo biloba seeds
LC-MS
Qualitative analysis
Quantitative analysis
==== Body
pmc1 Introduction

It is well known that the decoction is one of the significant and common dosage forms of Chinese medicines. Clinically, a decoction preparation for administration is usually prepared by patients themselves by boiling all composition drugs as prescribed by doctors with water and then removing the dregs. However, due to lack of standardized procedures and specifications, such as regarding the frequency of decoction (twice or thrice), the decocting process seems to be time-consuming and difficult to provide a stable quality.

In recent decades, dispensing granules (DG), a novel dosage form of Chinese medicines, has been popularly accepted and used [[1], [2], [3]]. The DG is prepared from traditional decoction pieces as the raw material, which involves many technology procedures, including cleaning, washing, cutting, boiling, concentration, drying, granulation and packing [4]. Because of the use of water as the decocting solvent, many water-soluble substances are retained in DG, which means that several drugs can be mixed in DG form and dissolved in hot water, thus quickly and conveniently producing a target decoction. It is acknowledged that the DG has a stable quality relative to the traditional decoction and allows pharmacists to make up a prescription easily and patients to take conveniently. In our opinions, the DG is consistent with the theory of traditional Chinese medicine (TCM) and very near to traditional decoction with thousands of years of history.The DG has been gradually widely used in clinic and focused from researchers in China.

Ginkgo biloba seeds (GS), a famous traditional Chinese medicine, was firstly recorded in Compendium of Materia Medica by Li Shizhen in the Ming dynasty [5]. From then on, it has been used for treating phlegm, cough and asthma, vaginal discharge, and frequent enuresis and urination by exerting the effects of warming lung and supplementing qi, calming asthma, stopping tourniquet and urination [6,7]. Nowadays, it is still clinically used to treat asthma, pulmonary tuberculosis, vaginitis, and other conditions [8,9]. The DG form of GS (GSDG) has also been widely employed alone or in combination and has attracted increasing attention. However, similar to the majority of Chinese medicines, the complicated chemical composition of GSDG is still unclear and no publication is available on its full-scale qualitative and quantitative analysis. In many provinces in China, only ginkgolide B or ginkgolide C is included as the quality marker in the local GSDG's quality standard, which does not conform to the concept of “multi-component, multi-pathway, multi-target, multi-effect”. This has greatly limited further research work, especially in exploring its substantial basis and action mechanism.

In this study, we used high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) to characterize GSDG's complex and diverse chemical composition based on the observed retention times, high-resolution MS data, fragmentation pathways in the negative or positive ion mode, combined with related literature and available reference standards. In addition, a high performance liquid chromatography coupled with quadrupole linear ion trap mass spectrometry (HPLC-QTRAP-MS) was established for simultaneous assay of 10 major active phytochemicals in multiple-reaction monitoring (MRM) mode and was successfully applied for 10 batches of GSDG. To our knowledge, the present study is the first to qualitatively analyze GSDG and simultaneously determine multiple bioactive phytochemicals and it is expected to exhibit an integrated chemical profiling of GSDG, and help explore the material basis of this Chinese medicine for its further clinical therapy.

2 Methods

2.1 Materials and reagents

In the present study, both acetonitrile and methanol were of LC-MS grade and bought from Merck (Merck, Darmstadt, Germany), and formic acid (HPLC grade) was bought from Anaqua Chemicals Supply (ACS, Houston, USA). A Milli-Q system (Millipore, Bedford, MA, USA) was used to prepare ultrapure water.

The reference standards of glutamic acid (98.6 %), asparatic acid (99.3 %) and histidine (98.7 %) were provided by Shanghai Macklin Biochemical Technology Co., Ltd. The reference standards of ginkgolide A (98.0 %), ginkgolide B (99.4 %), ginkgolide C (99.0 %), and ginkgolide J (98.2 %) were purchased from Taizhou Dan Ding Biological Technology Co., Ltd. The reference standards of eucomic acid (98.5 %), N-(N-glucopyranosyl)-indoleacetylaspartate (98.6 %) and N-(N-glucopyranosyl)-indoleacetylglutamate (98.3 %) were prepared and isolated from GS in our laboratory. The purities of these standards were all determined by HPLC in our laboratory.

10 batches of GSDG (S1-S10) were made and provided by Polifarma (Nanjing) Co., Ltd.

2.2 Preparation standard and sample solutions

2.2.1 Preparation of standard solutions

For the 10 bioactive components, the individual standard solutions were prepared in 50 % methanol. An appropriate volume of each standard solution was added in a 50 mL volumetric flask and diluted with 50 % methanol to obtain the mixed stock standard solution containing 10 analytes as follows: aspartic acid at 4.10 μg/mL, glutamic acid at 3.96 μg/mL, histidine at 4.06 μg/mL, ginkgolide A at 1.09 μg/mL, ginkgolide B at 0.667 μg/mL, ginkgolide C at 0.520 μg/mL, ginkgolide J at 5.15 μg/mL, N-(N-glucopyranosyl)-indoleacetylaspartate at 99.9 μg/mL, N-(N-glucopyranosyl)-indoleacetylglutamate at 40.1 μg/mL, and eucomic acid at 19.9 μg/mL. All solutions were stored in a refrigerator at 4 °C and centrifuged at 10000 rpm for 5 min before injection.

2.2.2 Preparation of test solution

0.2 g of GSDG powder was accurately weighed and sonicated for 1h in 40 mL of 50 % methanol. The extract was replenished with 50 % methanol for loss and filtered through a 0.22 μm membrane. Then, the resulting GSDG test solution was treated as the above standard solutions.

2.3 Qualitative analysis

A Shimadzu HPLC system (Kyoto, Japan) coupled with an AB SCIEX QTOF 5600 mass spectrometer (Foster City, CA, USA) was used for component characterization. With its temperature at 30 °C, A Welch Ultimate AQ-C18 column (250 mm × 4.6 mm × 5 μm) was used for chromatographic separation. At the flow rate of 1.0 mL/min, the mobile phase was composed of acetonitrile (A) and 0.3 % formic acid (B) with the elution gradient: 0–5 min, 10 % A; 5–15 min, 10–14 % A; 15–20 min, 14 % A; 20–30 min, 14–25 % A; 30–35 min, 25–35 % A; 35–40 min, 35–85 % A; 40–45min, 85–10 % A. The injection volume was 10 μL. Electron spray ionization (ESI) was employed in the full scan mode with the range of m/z 50–1500. Information-dependent acquisition of ions was performed in both negative mode and positive mode with ion spray voltages set at −4500 V and 5500 V, respectively. Other mass parameters were set as follows: curtain gas at 40 psi, both ion source gas 1 and 2 at 60 psi, and ESI temperature at 600 °C.

2.4 Quantitative analysis

For simultaneous assay of the representative phytochemicals, an AB SCIEX QTRAP 5500 mass spectrometer (Foster City, CA, USA) was employed accompanying with a Shimadzu HPLC system (Kyoto, Japan). All 10 analytes were quantified in MRM mode. The Applied Biosystems/MDS Sciex Analyst software (version 1.5.2) was used to design and optimize compound-dependent mass parameters, shown in Table 1. Except the gradient elution program, the chromatographic separation was accomplished in the same conditions as the above in “2.3. Qualitative analysis” including the column and its temperature, flow rate, mobile phase system and injection volume. The gradient elution program was as follow: 0–5 min, 10 % A; 5–7min 10–14 % A; 7–8 min, 14 % A; 8–10 min, 14–25 % A; 10–13 min, 25–35 % A; 13–18 min, 35–85 % A; 18–20 min, 85–95 % A; 20–35min, 95 % A; 35–38 min, 95–10 % A.Table 1 MS parameters of the 10 analytes.

Table 1Analyte	Formula	Precursor ion (m/z)	Product ion (m/z)	Declustering potential (V)	Collision energy (eV)	Collision cell exit potential (eV)	
Histidine	C6H9N3O2	156.1	110.0	59	10	14	
Aspartic acid	C4H7NO4	134.1	88.0	59	10	14	
Glutamic acid	C5H9NO4	148.1	83.9	58	14	14	
N-(N-glucopyranosyl)-indoleacetylaspartate	C20H24N2O10	451.0	173.0	−80	−38	−15	
N-(N-glucopyranosyl)-indoleacetylglutamate	C21H26N2O10	465.0	128.1	−140	−36	−9	
Eucomic acid	C11H12O6	239.0	178.6	−85	−20	−13	
Ginkgolide J	C20H24O10	469.2	423.1	−38	−19	−15	
Ginkgolide C	C20H24O11	439.1	383.3	−150	−21	−15	
Ginkgolide B	C20H24O10	423.1	367.3	−150	−20	−15	
Ginkgolide A	C20H24O9	407.1	351.1	−150	−19	−15	

2.5 Quantitative method validation

To confirm the applicability of the quantitative method, validation investigation was performed by assessing linearity, LOQ and LOD, repeatability, precision, stability, and recovery.

In the linearity test, a series of standard solutions of at different dilutions (1, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64) were used to plot the calibration curves with their concentrations (X, μg/mL) and the corresponding peak areas (Y). The S1 sample solution was consecutively analyzed six times in the precision test and was then analyzed at 0, 1, 2, 4, 6, and 8 h in the stability test. Six sample solutions of S1 were quantified for the contents of the target compounds in the repeatability test. In addition, in the recovery test, the reference standard of each analyte was spiked into the S1 sample at 100 % of its amount in the S1 sample to prepare the sample solution in six copies and to calculate the recovery using the following formula: (determined amount−theoretical amount) × 100 %/spiked amount.

3 Result and discussion

3.1 Qualitative analysis

A GS chemical composition database was established on the basis of previous relevant studies. The chemical profiling analysis of GSDG was performed with the Peakview software using the data obtained from HPLC-QTOF-MS, including the exact molecular mass, retention time and other MS/MS data. The determination of chemical formula was based on the common quasi-molecular ion ([M+H]+ or [M−H]−) in the positive or negative mode, with a mass deviation ≤10 ppm. The structural identification was mainly based on MS/MS fragment ions and proposed cleavage pathways in conjunction with the available literature data, and where applicable, confirmed against with the available reference standards. A total of 56 compounds, which were classified into amino acids, organic acids, nucleobases and nucleosides, flavonoids, vitamins, terpenoid lactones, carbohydrates and other chemical classes, were identified. The total ion chromatograms (TICs) of GSDG are shown in Fig. 1 (A, in the positive and B, in the negative mode). The results of the structure identification for all compounds are listed in Table 2.Fig. 1 TICs of GSDG in the positive (A) and negative (B) mode.

Fig. 1

3.1.1 Amino acids

A total of 12 amino acids were identified in GSDG in the positive mode, the more favorable ionization mode than the negative mode for this class of phytochemicals. The characteristic loss of a NH3 (17 Da) or a HCOOH (46 Da) and the loss of a H2O (18 Da) from the hydroxyl group at the branch chain of the molecules were common in their full scan positive MS/MS spectra. Considering their accurate masses and similar fragmentation pattern, C2 (C5H9NO2), C11 (C5H11NO2) and C32 (C4H7NO4) were identified to be proline, valine and aspartic acid, respectively [[10], [11], [12]]. C18 and C19 were found to have almost identical protonated ions and fragment ions, indicating a common chemical formula (C6H13NO2) and the same breaking pattern. However, a much higher relative intensity was observed for the product ion of [M+H−HCOOH−NH3]+ of C19 (m/z 69.0739) than that of C18 (m/z 69.0738). In accordance with their fragment mechanism involving intermediary ion/neutral complexes, C18 and C19 were identified as leucine and isoleucine, respectively [13,14].

C1 showed a quasi-molecular ion [M+H]+ at m/z 175.1189, consistent with a molecular formula of C6H14N4O2. From this precursor ion, two fragment ions at m/z 116.0723 (C5H9NO2) and 60.0620 (CH5N3) were generated due to the losses of a NH=C(NH2)2 group and a CH2=CHCH2CHNH2COOH group, respectively. The first fragment ion further lost a HCOOH unit to produce the base peak ion at m/z 70.0703. The product ions at m/z 158.0925 [M+H−NH3]+ and 130.0086 [M+H−NH3−CO]+ arose from another fragmentation pathway of [M+H]+, i.e., the sequential losses of a NH3 moiety and a CO unit (28 Da). Based on the observed cleavage behaviors, C1 was identified to be arginine [15].

C21 showed its quasi-molecular ion [M+H]+ at m/z 182.0759, consistent with a molecular formula of C9H11NO3. The calculated degree of unsaturation of 5, supported the presence of a phenyl group in the molecule. The loss of a HCOOH moiety from [M+H]+ produced a product ion at m/z 136.0798, which further lost a NH3 unit to give [M+H−HCOOH−NH3]+ at m/z 119.0534. In addition, [M+H−HCOOH]+ underwent the sequential losses of a CH2=NH moiety and an oxygen atom followed by the rearrangement, resulting in a fragment ion at m/z 91.0572, which further produced a product ion [M+H−HCOOH−CH2=NH−O−CH2]+ at m/z 77.0407 by losing a CH2 group. C25 exhibited similar fragmentation pattern. In its MS/MS spectrum, the protonated ion, decarboxylation ion, and deamination ion were observed at m/z 166.0926 ([M+H]+), 120.0830 ([M+H−HCOOH]+), and 103.0571 ([M+H−HCOOH−NH3]+), respectively, each with a mass difference of 16 Da (O) from the corresponding ion of C21. Furthermore, additional two fragment ions were detected at m/z 91.0579 and 77.0434, similar to those of C21. Thus, C21 and C25 were identified to be tyrosine and phenylalanine, respectively [16,17].

The molecular formulas of C5 and C6 were determined to be C5H7NO3 and C5H9NO4, respectively, based on their protonated ions [M+H]+ at m/z 130.0514 and 148.0613, respectively. A difference of H2O between C5 and C6 indicated that C5 was the dehydration product of C6. In the MS/MS spectrum of C6 (Supplementary Fig. 1), another dehydration (between the amino group and the remote hydroxyl group) was observed, resulting in a product ion at m/z 130.0517 [M+H−H2O]+, which further underwent cleavages via two possible pathways. The first involved the elimination of a CO group, producing a fragment ion at m/z 102.0580 [M+H−H2O−CO]+, followed by the further elimination of a NH3 moiety, forming another fragment ion at m/z 85.0318 [M+H−H2O−CO−NH3]+. The second pathway was associated with the formation of the base peak ion [M+H−H2O−HCOOH]+ at m/z 84.0482 along with [M+H−H2O−HCOOH−CO]+ at m/z 56.0562. For C5, some similar product ions were detected, including [M+H−CO]+ (m/z 102.0623), [M+H−HCOOH]+ (m/z 84.0484) and [M+H−HCOOH−CO]+ (m/z 56.0568). By comparison with their reference standards, C5 and C6 were identified to be pyroglutamic acid and glutamic acid, respectively [12].

3.1.2 Organic acids

In GSDG, a total of 9 organic acids were identified in the negative ion mode. These components experienced cleavages by losing HCOOH, CO2 (44 Da), CO or H2O, consistent with the characteristic fragmentation of carboxylic groups which are the common structure of organic acids.

The molecular formula of C35 was determined to be C11H12O6, due to its precursor ion at m/z 239.0557 [M−H]−. Two initial fragment ions at m/z 221.0455 [M−H−H2O]− and 195.0660 [M−H−CO2]− resulted from the dehydration and decarbonation reactions of [M−H]−, respectively. A common and fundamental intermediate ion [M−H−H2O−CO2]− (or [M−H−CO2−H2O]−) was generated at m/z 177.0550 due to the further loss of a CO2 or a H2O moiety. After the individual elimination of a CO unit and a CO2 unit, the intermediate was converted into [M−H−H2O−CO2−CO]− (m/z 149.0606) and [M−H−H2O−CO2−CO2]− (m/z 133.0657), respectively. Then, a neutral loss of a CH2=C=CH2 unit happened to the latter ion, producing [M−H−H2O−CO2−CO2−C3H4]− at m/z 93.0361. Besides, a CH≡COH group was speculated to fall out of the benzene ring, which took place on [M−H−H2O]− and [M−H−CO2−H2O−CO]−, resulting in two product ions at m/z 179.0342 and 107.0506. Taken together, C35 was identified as eucomic acid [18,19]. Its MS/MS spectrum and proposed cleavage pathways are shown in Fig. 2.Fig. 2 MS/MS spectrum and proposed fragmentation pathway of eucomic acid.

Fig. 2

C52 exhibited its quasi-molecular ion [M−H]− at m/z 277.1439, consistent with chemical formula of C18H30O2. The two peaks at m/z 233.1537 and 121.0296 represented the two ions produced by the losses of a CO2 group and a C9H16O2 group on the benzene ring, respectively. Their common product ion was observed at m/z 77.0415 [M−H−C9H16O2−CO2]− (or [M−H−CO2−C9H16O2]−). Due to the ester bond cleavage and rearrangement of the precursor ion, the third product ion was observed at m/z 147.0084 [M−H−C8H18O]−. In addition, the loss of an alkyl side chain followed by the dehydrogenation resulted in a fragment ion at m/z 107.0510. Based on the published literature data, C52 might be monoethylhexyl phthalic acid [20,21].

C10 showed a deprotonated ion at m/z 191.0568 (base peak), suggesting its chemical formula was C7H12O6. Its characteristic fragment ions were detected at m/z 173.0469 [M−H−H2O]−, 155.0421 [M−H−2H2O]−, 137.0282 [M−H−3H2O]−, 127.0393 [M−H−H2O−HCOOH]−, 111.0463 [M−H−2H2O−CO2]−, and 93.0354 [M−H−3H2O−CO2]−. Obviously, dehydration, decarboxylation and decarbonation were its dominant fragmentation pathways. C28 produced a parent ion [M−H]− at m/z 193.0519 (C10H10O4), which further yielded diagnostic ions at m/z 178.0263 [M−H−CH3]−, 149.0579 [M−H−CO2]−, 134.0372 [M−H−CH3−CO2]−, and 106.0422 [M−H−CH3−CO2−CO]−. C31 provided its quasi-molecular ion [M−H]− at m/z 137.0244, consistent with a molecular formula of C7H6O3. Its base peak ion was observed at m/z 93.0355 [M−H−CO2]−, with a fragment ion at m/z 65.0401 [M−H−CO2−CO]−. Based on their MS/MS data, C10, C12, C27, C28 and C31 were identified to be quinic acid, shikimic acid, vanillic acid, ferulic acid and salicylic acid, respectively [[22], [23], [24], [25], [26], [27]], all of which were further confirmed by comparing with their reference standards.

3.1.3 Nucleosides and nucleobases

Similar to amino acids, both nucleosides and nucleobases are a group of nitrogenous substances common in botanicals. Three nucleobases and three nucleosides were identified in GSDG. The presence of the fragment ions, at m/z 268.1040 [M+H]+, 136.0628 [M+H−C5H8O4]+, and 119.0370 [M+H−C5H8O4−NH3]+ in the MS/MS spectrum of C15 indicated that it followed the characteristic cleavage pathways of the nucleosides, including the losses of a ribose unit (C5H8O4, 132 Da) and an ammonia unit in the positive mode. C4 showed its characteristic fragment ions at m/z 136.0617 [M+H]+, 119.0365 [M+H−NH3]+, and 92.0275 [M+H−NH3−HCN]+. C20 showed its characteristic fragment ions at m/z 152.0566 [M+H]+, 135.0325 [M+H−NH3]+, and 107.0461 [M+H−NH3−CO]+. C22 showed its characteristic fragment ions at m/z 153.0431 [M+H]+ and 136.0158 [M+H−NH3]+. The cleavage behaviors of the three components were consistent with those of nucleobases, involving the loss of an ammonia unit, a hydrocyanic acid (27 Da) or a carbonyl group. With these MS/MS information and the published data, C4, C15, C20, C22 were identified to be adenine, adenosine, guanine, xanthine, respectively [28].

C29 showed a protonated ion [M+H]+ at m/z 352.1718, which produced a base peak ion at m/z 220.1170 [M+H−C5H8O4]+ by losing a ribose. This aglycone ion further underwent fragmentation via two cleavage pathways. The first involved dehydration and deamination, producing two fragment ions [M+H−C5H8O4−H2O]+ and [M+H−C5H8O4−H2O−NH3]+ at m/z 202.1084 and 185.0847, respectively. The second was associated with the loss of a side chain on amidogen followed by the further deamination, contributing to [M+H−C5H8O4−C5H8O]+ at m/z 136.0649 and [M+H−C5H8O4−C5H8O−NH3]+ at m/z 119.0425. Based on the above information, C29 was tentatively identified to be ribosylzeatin [29], a common plant growth regulator. Fig. 3 exhibits its MS/MS spectrum and proposed cleavage pathways.Fig. 3 MS/MS spectrum and proposed fragmentation pathway of ribosylzeatin.

Fig. 3

3.1.4 Flavonoids

Flavonoids are the known fundamental substances in botanicals. The cleavage of the glycosidic bond which is characteristic to flavonoid glycosides and the losses of 146 Da, 162 Da, and 308 Da which are usually attributed to the rhamnose, glucose, and rutinose moieties, respectively [23,30,31], are important for structure identification of flavonoids. In GSDG, 6 flavonoids were identified as folows.

C38 showed its [M−H]− ion at m/z 609.0911, suggesting that its chemical formula was C27H30O16. An aglycone ion at m/z 301.0086 indicated the loss of a rutinose. As another fragment ion at m/z 300.0003 had a higher relative intensity than the ion at m/z 301.0086, C38 was speculated to be 3-O-rutinoside. RDA reaction and rearrangement were found to be the predominant cleavage pattern for this aglycone ion. Due to the two different RDA cleavages, two different fragment ions were yielded at m/z 150.9897 and 107.0038, respectively. The third pattern caused two product ions at m/z 178.9813 and 121.0172. These results revealed that the aglycone might be quercetin. In addition, three other fragment ions at m/z 271.0004, 255.0078 and 243.0183, corresponding to [M−H−Rut−H−CO−H]−, [M−H−Rut−H−CO−OH]− and [M−H−Rut−H−CO−H−CO]−, respectively, confirmed the above speculation. By comparison with its reference standard, C38 was identified to be rutin [32,33]. Its MS/MS spectrum and proposed cleavage pathways are presented in Supplementary Fig. 2. Both C39 and C40 were also 3-O-rutinosides due to the presence of ion pairs of the deprotonated ion and desugarization ion at m/z 593.0964 and 285.0151 and at m/z 623.1117 and 315.0248, respectively. Based on these accurate molecular weights and fragmentation behaviors, and in conjunction with the published data, C39 and C40 were identified to be nicotiflorin and narcissin, respectively [34,35].

C50 and C53 shared a common chemical formula C15H10O5 because their deprotonated ions [M−H]− were detected at m/z 269.0425 and 269.0446, respectively. The consecutive elimination of a CO group and an oxygen atom led to the product ions at m/z 241.0506 and 225.0520 for C50 and at m/z 241.0519 and 225.0551 for C53, respectively. In addition, the formation of a group of diagnostic ions at m/z 151.0718 and 117.0328 for C50 and at m/z 151.0555, 117.0346, and 107.0195 for C53 revealed RDA fragmentation pathways. Based on the published data, C50 and C53 were identified to be genistein and apigenin, respectively [[36], [37], [38]], both of which were confirmed by comparison with their reference standards.

3.1.5 Vitamins

In GSDG, 5 vitamins were detected, including vitamin B2, vitamin B3, vitamin B5, vitamin B6, and vitamin H.

C26 showed its quasi-molecular ion at m/z 220.1193 in the positive mode, consistent with a chemical formula of C9H17NO5. On account of several hydroxyl groups, several dehydration product ions were observed at m/z 202.1084 [M+H−H2O]+, 184.0968 [M+H−2H2O]+, and 166.0945 [M+H−3H2O]+ due to the losses of one, two, and three H2O units, respectively. The third product ion further lost a C6H7NO unit and a C5H8 unit to produce the fragment ions at m/z 57.0772 [M+H−3H2O−C6H7NO]+ and 98.0264 [M+H−3H2O−C5H8]+, respectively. Two fragment ions at m/z 156.1005 ([M+H−2H2O−CO]+) and 142.0851 ([M+H−2H2O−CO−CH2]+) arose from the sequential losses of a CO unit and a CH2 unit from [M+H−2H2O]+. For the latter, two downstream ions were observed at m/z 124.0780 and 70.0343, owing to the losses of a H2O moiety and a C4H8O group (72 Da), respectively. In addition, the production of two product ions at m/z 90.0593 and 72.0505 were due to the breaking of amido bond accompanied with the loss of a C6H10O3 (130 Da) group from [M+H]+ and [M+H−H2O]+, respectively. Based on these accurate molecular weights and proposed fragmentation pathways (Fig. 4), C26 was temporarily assigned as pantothenic acid (vitamin B5) [39,40]. C13 showed its protonated ion at m/z 124.0397, consistent with a chemical formula of C6H5NO2. Three cleavage pathways, i.e., dehydration, decarbonation and decarboxylation, resulted in three different product ions at m/z 106.0307 ([M+H−H2O]+), 80.0531 ([M+H−CO2]+) and 78.0383 ([M+H−HCOOH]+), respectively. All three ions further underwent decyanation to form their respective homologous products at m/z 79.0462 ([M+H−H2O−HCN]+), 53.0459 ([M+H−CO2−HCN]+) and 51.0313 ([M+H−HCOOH−HCN]+). According to its MS/MS spectrum and proposed fragmentation pathway as shown in Supplementary Fig. 3, C13 might be nicotinic acid (vitamin B3) [37,41]. Additionally, C14, C23 and C36 were speculated to be pyridoxal (a form of vitamin B6), biotin (vitamin H) and riboflavin (vitamin B2) because their quasi-molecular ions [M+H]+ were detected at m/z 168.0655 (C8H9NO3), 245.0954 (C10H16N2O3S) and 377.1483 (C17H20N4O6), respectively [[42], [43], [44]].Fig. 4 MS/MS spectrum and proposed fragmentation pathway of vitamin B5.

Fig. 4

3.1.6 Terpenoid lactones

As is known, terpenoid lactones are the major bioactive components of the plant Ginkgo biloba. These components are characterized with three lactonic rings and many hydroxyl groups in their chemical structures. Therefore, the opening of the lactonic rings and sequential elimination of CO2, CO, and H2O represent the typical fragmentation pathways in the MS/MS spectra. For example, C41 showed its deprotonated ion [M−H]− at m/z 439.1237, consistent with a chemical formula of C20H24O11. In the high-weight region of the MS/MS spectrum, the major fragment ions, which followed the breaking patterns above mentioned, were observed at m/z 411.1287, 383.1336, 365.1220, 321.1342, 303.1220, 277.1433, 259.1346 and 241.1244, corresponding to [M−H−CO]−, [M−H−2CO]− (base peak), [M−H−2CO−H2O]−, [M−H−2CO−H2O−CO2]−, [M−H−2CO−H2O−CO2−H2O]−, [M−H−2CO−H2O−2CO2]−, [M−H−2CO−H2O−2CO2−H2O]− and [M−H−2CO−H2O−2CO2−2H2O]−, respectively. In addition, there were several cleavage-induced residue ions at m/z 141.0191 ([C6H5O4−]−), 125.0246 ([C6H5O3−]−), 113.0244 ([C5H5O3−]−), 97.0301 ([C5H5O2−]−) and 72.9948 ([C2HO3−]−). Among them, the last one was the ring-opening product arisen from a five-membered lactonic ring and was also found in the spectrum of C42 (m/z 72.9959), C44 (m/z 72.9952), C45 (m/z 72.9950), and C46 (m/z 72.9941). Thus, the fragment ion at m/z 72.99 was considered as the diagnostic ion for this category of phytochemicals. As shown in Table 2, C44 (or C45) demonstrated a mass difference of 16 Da (O) from C41 between the precursor ion and many product ions. Hence, C41 was deduced to be ginkgolide C [[45], [46], [47]]. Its MS/MS spectrum and proposed fragmentation pathways are demonstrated in Fig. 5. C42, C44, C45, and C46 showed their protonated ions at m/z 325.0929, 423.1344, 423.1278 and 407.1347, respectively. According to their accurate molecular weights, the chemical formulas of C42, C44, C45 and C46 might be C15H18O8, C20H24O10, C20H24O10 and C20H24O9, respectively. Because of the similar fragmentation pathways and residue ions as above mentioned, C42, C44, C45, and C46 were identified as bilobalide, ginkgolide J, ginkgolide B, and ginkgolide A, respectively [[48], [49], [50]]. These terpenoid lactones were further confirmed by comparison with their reference standards.Table 2 Chemical constituents identified in GSDG by HPLC-QTOF-MS.

Table 2No.	RT (min)	Molecular formula	MS/MS fragments	Mode	Error (ppm)	Identification	
1	2.31	C6H14N4O2	175.1189, 158.0925, 130.0086, 116.0723,
70.0703, 60.0620	+	−3.0	Arginine	
2	2.34	C5H9NO2	116.0826, 70.0702	+	0.8	Proline	
3	2.37	C6H9N3O2	156.0767, 155.9004, 114.9519, 72.9433	+	−2.0	Histidinea	
4	2.68	C5H5N5	136.0617, 119.0365, 94.0426, 92.0275	+	−1.0	Adenine	
5	2.71	C5H7NO3	130.0514, 102.0623, 85.8761, 84.0484,
70.0705, 56.0568	+	0.6	Pyroglutamic acida	
6	2.73	C5H9NO4	148.0613, 130.0517, 102.0580, 85.0318,
84.0482, 56.0562	+	−1.0	Glutamic acida	
7	2.78	C18H32O16	503.1617, 425.1368, 383.1201, 341.1124,
281.0843, 263.0762, 221.0650, 179.0579,
161.0428, 143.0371, 101.0232, 89.0287,
73.0325, 59.0159	–	−0.8	Raffinose	
8	2.86	C12H22O11	341.1073, 263.0828, 179.0546, 161.0435,
143.0353, 119.0353, 113.0243, 101.0241,
89.0247, 85.0320, 71.0146, 59.0159	–	−0.8	Sucrose	
9	2.93	C6H12O6	179.0567, 161.0465, 143.0381, 131.0355,
129.0197, 113.0928, 101.0232, 89.0277,
85.0306, 83.0140, 71.0130, 59.0152,
57.0379	–	4.0	Glucose or Mannose or Fructose	
10	2.96	C7H12O6	191.0568, 173.0469, 155.0421, 137.0282,
127.0393, 111.0463, 93.0354, 87.0092,
85.0310, 59.0163	–	5.9	Quinic acida	
11	2.99	C5H11NO2	118.0862, 72.0857, 58.0709, 57.0631,
56.0557, 55.0608, 54.8879	+	−0.3	Valine	
12	3.13	C7H10O5	173.0455, 154.9999, 111.0092, 99.0093,
85.0313, 83.0123, 72.9930	–	5.1	Shikimic acida	
13	3.61	C6H5NO2	124.0397, 106.0307, 80.0531, 79.0462,
78.0383, 53.0459, 51.0313	+	−0.6	Nicotinic acid (Vitamin B3)	
14	3.71	C8H9NO3	168.0655, 122.0658, 94.9876	+	−0.7	Pyridoxal (Vitamin B6)	
15	3.79	C10H13N5O4	268.1040, 136.0628, 119.0370	+	0.4	Adenosine	
16	3.92	C5H11NO2S	150.0583, 133.0331, 102.0552, 90.9187,
84.0493, 74.0259, 61.0275, 56.0543	+	−7.1	Methionine	
17	4.08	C9H13NO3	184.1055, 152.0713, 134.0617, 124.0774,
106.0661, 94.0690, 80.0536, 77.0431,
65.0458	+	−1.5	4′-O-methylpyridoxine (Ginkgotoxin)a	
18	4.13	C6H13NO2	132.1017, 90.9121, 86.1002, 85.8403,
69.0738, 57.0719, 55.0211, 53.0063	+	−0.6	Leucine	
19	4.27	C6H13NO2	132.1016, 86.1002, 72.9421, 69.0739,
55.0676	+	−0.6	Isoleucine	
20	4.43	C5H5N5O	152.0566, 135.0325, 123.0716, 110.0351,
107.0461, 93.0130, 67.0391, 65.0644	+	1.4	Guanine	
21	4.97	C9H11NO3	182.0759, 136.0798, 122.0623, 119.0534,
93.0638, 91.0572, 77.0407, 67.0654	+	−1.2	Tyrosine	
22	5.02	C5H4N4O2	153.0431, 136.0158, 110.0364, 93.0082,
82.0487, 81.0113, 53.0199	+	−6.1	Xanthine	
23	5.05	C10H16N2O3S	245.0954, 124.6801, 98.0588, 80.0503,
68.0536	+	7.3	Biotin (Vitamin H)	
24	5.31	C6H13NO5	180.0866, 162.0771, 137.0823, 135.0303,
122.0618, 107.0379, 71.0657	+	4.0	Galactosamine	
25	5.66	C9H11NO2	166.0926, 120.0830, 107.0535, 103.0571,
93.0731, 91.0579, 79.0591, 77.0434,
51.0303	+	−1.7	Phenylalanine	
26	6.09	C9H17NO5	220.1193, 202.1084, 184.0968, 166.0945,
156.1005, 142.0851, 124.0780, 98.0264,
90.0593, 72.0505, 70.0343, 57.0772	+	0.1	Pantothenic acid (Vitamin B5)a	
27	6.33	C8H8O4	167.0319, 166.8347, 152.0107, 108.0201	–	7.8	Vanillic acida	
28	7.67	C10H10O4	193.0519, 178.0263, 149.0579, 134.0372,
106.0422	–	5.5	Ferulic acida	
29	11.27	C15H21N5O5	352.1718, 220.1170, 202.1084, 185.0847,
159.0744, 148.0547, 136.0649, 119.0425	+	−0.5	Ribosylzeatin	
30	13.12	C16H20O9	355.1034, 193.0502, 149.0603	–	−0.4	trans-Ferulic acid-4-O-β-D-glucoside	
31	13.67	C7H6O3	137.0244, 93.0355, 65.0401	–	5.9	Salicylic acida	
32	13.88	C4H7NO4	134.0445, 74.0323, 70.0421, 57.9431,
55.9573	+	−0.9	Aspartic acida	
33	13.90	C20H24N2O10	451.1347, 433.1241, 407.1451, 335.1234,
292.1181, 173.0711, 156.0452, 132.0297,
130.0660, 128.0504, 101.0250, 88.0411,
71.0155	–	−0.4	N-(N-glucopyranosyl)-indoleacetylaspartatea	
34	16.07	C21H26N2O10	465.1506, 447.1416, 421.1628, 336.1095,
292.1197, 259.1095, 173.0736, 156.0449,
146.0464, 128.0360, 101.0254	–	−2.8	N-(N-glucopyranosyl)-indoleacetylglutamatea	
35	16.73	C11H12O6	239.0557, 221.0455, 195.0660, 179.0342,
177.0550, 149.0606, 148.0526, 135.0453,
133.0657, 121.0303, 107.0506, 106.0431,
93.0361, 87.0100, 59.0166	–	2.3	Eucomic acida	
36	20.44	C17H20N4O6	377.1483, 359.1168, 243.0882, 198.0661,
172.0895, 145.0696, 118.0688, 77.0374,
57.0363	+	0.5	Riboflavin (Vitamin B2)	
37	21.26	C8H10	107.0855, 91.0593, 77.0401, 65.0474	+	1.4	Xylene	
38	25.90	C27H30O16	609.0911, 301.0086, 300.0003, 271.0004,
255.0078, 243.0183, 178.9813, 150.9897,
121.0172, 107.0038	–	−3.6	Rutina	
39	26.43	C27H30O15	593.0964, 285.0151, 284.0075, 255.0071,
229.0299, 187.0398	–	−4.6	Nicotiflorin	
40	26.50	C28H32O16	623.1117, 315.0248, 314.0170, 300.0026,
285.0165, 271.0022, 255.0085, 243.0096	–	−2.7	Narcissin	
41	26.70	C20H24O11	439.1237, 411.1287, 383.1336, 365.1220,
321.1342, 303.1220, 277.1433, 259.1346,
241.1244, 141.0191, 125.0246, 113.0244,
97.0301, 72.9948	–	−2.1	Ginkgolide Ca	
42	27.08	C15H18O8	325.0929, 307.0845, 219.1016, 193.1229,
191.1073, 175.1127, 165.0555, 136.0523,
135.0145, 118.0418, 107.0501, 99.0823,
72.9959	–	0.9	Bilobalidea	
43	27.82	C14H20O6	283.1187, 265.1099, 221.1147, 195.0462,
180.0253, 179.1038, 145.0253, 121.0297,
72.9954	–	0.1	2- Phenylethyl-β-D-glucopyranoside	
44	28.03	C20H24O10	423.1344, 395.1167, 367.1394, 349.1308,
331.1178, 287.1280, 261.1510, 243.1384,
215.1450, 186.0864, 149.0976, 99.0853,
83.0558, 72.9952	–	−2.6	Ginkgolide Ja	
45	28.41	C20H24O10	423.1278, 395.1331, 367.1381, 349.1279,
305.1385, 287.1275, 261.1492, 243.1390,
141.0186, 125.0247, 113.0247, 72.9950	–	−2.6	Ginkgolide Ba	
46	29.66	C20H24O9	407.1347, 379.1385, 351.1439, 333.1359,
307.1559, 289.1432, 263.1644, 245.1542,
72.9941	–	−2.8	Ginkgolide Aa	
47	30.28	C13H20O	193.1586, 107.0907, 67.0571, 55.0621	+	0.1	Ionone	
48	30.47	C9H13N3O4	226.0950, 93.0237	–	−0.2	2′-Deoxycytidine	
49	30.63	C30H26O13	593.1300, 165.0209, 121.0330	–	−4.4	Procyanidin	
50	30.80	C15H10O5	269.0425, 241.0506, 225.0520, 197.0574,
182.0352, 151.0718, 117.0328	–	−0.4	Genisteina	
51	31.13	C18H28O2	277.2162, 235.1667, 145.0991, 93.0696,
79.0613, 67.0541	+	0.5	5, 12-Octadecadiynoic acid	
52	31.96	C18H30O2	277.1439, 233.1537, 147.0084, 134.0373,
127.1128, 121.0296, 119.0135, 107.0510,
77.0415, 75.0256	–	1.0	Monoethylhexyl phthalic acid	
53	32.44	C15H10O5	269.0446, 241.0519, 225.0551, 197.0595,
181.1653, 151.0555, 117.0346, 107.0195	–	−0.4	Apigenina	
54	34.56	C13H22O	195.1743, 95.0887, 81.0726, 69.0826,
67.0576, 55.0576	+	−3.2	Nerylacetone	
55	37.11	C21H24O5	356.1623, 311.1666, 309.3121, 293.1486	–	0.9	Myricanone	
56	39.67	C15H24	205.1950, 149.0224, 121.0366, 107.0840,
69.0826, 57.0760	+	−1.3	Ylangene	
a compared with its reference standard.

Fig. 5 MS/MS spectrum and proposed fragmentation pathway of ginkgolide C.

Fig. 5

3.1.7 Carbohydrates

In the negative mode, C9 showed its quasi-molecular ion at m/z 179.0567, consistent with a molecular formula of C6H12O6, a typical saccharide with the unsaturation degree of 1. Due to the presence of some hydroxyl groups, the loss of a H2O moiety was often observed. The initial dehydration product ion [M−H−H2O]− (m/z 161.0465) seemed very important because it underwent further fragmentation through four possible downstream cleavage pathways. As for the first two, the further loss of a CH3OH unit led to a fragment ion at m/z 129.0197 [M−H−H2O−CH3OH]− and the further loss of a H2O moiety produced a fragment ion at m/z 143.0381 [M−H−2H2O]−. For the third pathway, an intermediate deformylation product ion [M−H−H2O−HCHO]− was detected at m/z 131.0355 and two end product ions were observed at m/z 85.0306 and 113.0928, corresponding to [M−H−H2O−HCHO−H2O−CO]− and [M−H−H2O−HCHO−H2O]−, respectively. The fourth pathway mainly involved the sequential losses of a C2H2O unit and a H2O molecule, generating an intermediate product ion [M−H−H2O−C2H2O−H2O]− with a four-membered ring at m/z 101.0232. Two end product ions at m/z 83.0140 and 59.0152 were attributed to the individual loss of a H2O moiety and a C2H2O unit, respectively. Therefore, C9 was speculated to be a hexose [51,52]. Based on the limited information about its configuration obtained from the MS/MS spectrum, C9 was temporarily identified to be glucose, fructose or mannose. Supplementary Fig. 4 exhibits the MS/MS spectrum and proposed cleavage pathways of C9 assumed to be glucose. According to their accurate molecular masses of 503.1617 and 341.1073 in the negative mode, C7 and C8 were regarded as C18H32O16 and C12H22O11, respectively. In their spectra, the fragment ions of a hexose, which were consistent with the cleavage pathways of a monosaccharide like C9, were also shown at m/z 179.0579, 161.0428, 143.0371, 101.0232, 89.0287, 73.0325, and 59.0159 for C7, and at m/z 179.0546, 161.0435, 143.0353, 101.0241, 89.0247, 85.0320, 71.0146, and 59.0159 for C8. In addition, a mass gap of 162 Da was common among m/z 503.1617, 341.1124 and 179.0579, among m/z 425.1368, 263.0762 and 101.0232, and among m/z 383.1201, 221.0650 and 59.0159 for C7, as well as between m/z 341.1073 and 179.0546, and between m/z 263.0828 and 101.0241 for C8. Accordingly, C7 and C8 were tentatively assigned as raffinose (a trisaccharide) and sucrose (a disaccharide) [[53], [54], [55]].

3.1.8 Other compounds

In addition to the categories listed above, another 9 compounds were identified in GSDG, as detailed below. As a special component, C33 showed its protonated ion [M−H]− at m/z 451.1347, consistent with a molecular formula of C20H24N2O10. The dehydration between two carboxyl groups produced a product ion containing succinic anhydride at m/z 433.1241 [M−H−H2O]−. Additional two initial fragment ions, corresponding to [M−H−CO2]− and [M−H−HOOCCH=CHCOOH]−, respectively, were observed at m/z 407.1451 and 335.1234 in the MS/MS spectrum. The further losses of a glucose group (162 Da) and a CONH group (43 Da) were also observed in the MS/MS spectrum. Depending on the loss sequence, two intermediate ions were produced at m/z 173.0711 (base peak) and 292.1181, respectively, and a common end product ion was obtained at m/z 130.0660 [M−H−HOOCCH=CHCOOH−C6H10O5−CONH]−., A fragment ion at m/z 156.0452 [M−H−HOOCCH=CHCOOH−C6H10O5−NH3]− was formed due to the loss of a NH3 moiety from the base peak. Furthermore, two product ions at m/z 128.0504 and 101.0250 arose from the sequential elimination of a CO unit and a HCN moiety. On the other hand, the quasi-molecular ion of C34 was detected at m/z 465.1506 in the negative mode, consistent with a chemical formula of C21H26N2O10. Two product ions, which resulted from two different fragmentation pathways, were observed at m/z 447.1416 [M−H−H2O]− and 421.1628 [M−H−CO2]−, respectively. These three ions were found to have a common distance of 14 Da (CH2) to those of C33, indicating that the two components might be the neighboring homologues. The third ion was further converted into a product ion at m/z 259.1095 after a further deglycosylation. Additionally, several fragment ions detected at m/z 292.1197 [M−H−CO2−C5H7NO3]−, 173.0736 [M−H−CO2−C6H10O5−CH3CH=CHCOOH]−, 156.0449 [M−H−CO2−C6H10O5−CH3CH=CHCOOH−NH3]−, 128.0360 [M−H−CO2−C6H10O5−CH3CH=CHCOOH−NH3−CO]−, and 101.0254 [M−H−CO2−C6H10O5−CH3CH=CHCOOH−NH3−CO−HCN]− were found to be similar to C33. C33 and C34 were temporarily deduced to be N-(N-glucopyranosyl)-indoleacetylaspartate and N-(N-glucopyranosyl)-indoleacetylglutamate, respectively [56]. The MS/MS spectrum and proposed fragmentation pathway of C33 are demonstrated in Fig. 6.Fig. 6 MS/MS spectrum and proposed fragmentation pathway of N-(N-glucopyranosyl)-indolylacetylaspartate

Fig. 6

As shown in Fig. 7, C17 exhibited its quasi-molecular ion at m/z 184.1055 [M+H]+ and an initial ion at m/z 152.0713 [M+H−CH3OH]+ in the positive mode. Two different fragmentation pathways were found to be responsible for two different intermediate ions (m/z 124.0774 [M+H−CH3OH−CO]+ and 134.0617 [M+H−CH3OH−H2O]+) and a common product ion [M+H−CH3OH−H2O−CO]+ at m/z 106.0661. This product ion further produced [M+H−CH3OH−H2O−CO−C2H2]+ at m/z 80.0536 by losing a C2H2 unit and [M+H−CH3OH−H2O−CO−HCN−H2]+ at m/z 77.0431 by decyanation and dehydrogenation. Accordingly, C17 was identified to be 4′-O-methylpyridoxine (ginkgotoxin) [57,58], which was confirmed by comparison with its reference standard and the published data.Fig. 7 MS/MS spectrum and proposed fragmentation pathway of ginkgotoxin.

Fig. 7

3.2 Quantitative analysis

3.2.1 Selection and justification of the analytes

Ten representative biological active components were selected for quantification by HPLC-QTRAP-MS as the quality indicators of GSDG. The terpenoid lactones have shown the considerable effects on the pulmonary diseases. Ginkgolide C was documented to alleviate acute lung injury caused by paraquat poisoning via regulating the Nrf2 and NF-κB signaling pathways and to mitigate tumourigenesis in non-small cell lung cancer through abrogation of STAT3 activation cascade [59,60]. Ginkgolide B could protect human A549 cells from lipopolysaccharide-induced inflammatory responses by reducing TRIM37-mediated NF-κB activation [61]. Ginkgolide A treatment was documented to be effective in a murine model of neutrophil-predominant asthma through inhibiting response in the immune Th17 cells [62]. Among amino acids, histidine could improve lung function and ameliorate lung inflammation by inhibiting the activation of the NLRP3 inflammasome of the mice with chronic obstructive pulmonary disease [63]. Glutamic acid was reported to inhibit free radical processes, protect and mitigate CCl4-induced oxidative stress in the lung tissues of male rats [64]. Both N-(N-glucopyranosyl)-indoleacetylaspartate and N-(N-glucopyranosyl)-indoleacetylglutamate were also reported to be antitussive compounds [56,65], while eucomic acid had the potential to increase the activity or expression of cytochrome c oxidase in human immortalized keratinocyte cell line (HaCaT) [66]. Furthermore, N-(N-glucopyranosyl)-indoleacetylaspartate, N-(N-glucopyranosyl)-indoleacetylglutamate and eucomic acid were selected because they contributed three dominating common peaks observed in the HPLC fingerprint chromatogram of GSDG (Supplementary Fig. 5).

3.2.2 Method validation

The regression equation and linear range derived for each analyte are summarized in Table 3, along with the limit of quantitation (LOQ) and limit of detection (LOD). The signal-noise ratios at LOQ and LOD levels were 10 and 3, respectively. With the coefficient of correlation (R2) ≥ 0.99 for all analytes, the method is linear for the determination of each analyte in the given range.Table 3 Calibration curves, LODs and LOQs of the 10 analytes.

Table 3Analyte	Regression equation	R2	Linear range (μg/mL)	LOD (μg/mL)	LOQ (μg/mL)	
Histidine	Y = 2.03 × 106X + 6.65 × 105	0.9946	0.317–10.2	1.20 × 10−3	1.95 × 10−2	
Aspartic acid	Y = 7.13 × 105X + 5.51 × 104	0.9990	0.256–16.4	3.13 × 10−2	6.25 × 10−2	
Glutamic acid	Y = 8.62 × 105X + 4.25 × 105	0.9976	0.619–19.8	9.80 × 10−3	7.81 × 10−2	
N-(N-glucopyranosyl)-indoleacetylaspartate	Y = 2.57 × 105X + 1.65 × 105	0.9990	0.313–99.9	9.80 × 10−3	3.91 × 10−2	
N-(N-glucopyranosyl)-indoleacetylglutamate	Y = 1.59 × 105X – 7.00 × 103	0.9997	0.313–40.1	4.90 × 10−3	9.80 × 10−3	
Eucomic acid	Y = 1.46 × 106X – 3.22 × 105	0.9992	0.311–19.9	2.40 × 10−3	1.95 × 10−2	
Ginkgolide J	Y = 5.64 × 106X – 1.07 × 105	0.9996	9.66 × 10−2–1.55	2.34 × 10−2	4.69 × 10−2	
Ginkgolide C	Y = 1.89 × 106X + 2.94 × 104	0.9992	1.25 × 10−2–0.400	2.44 × 10−4	1.95 × 10−3	
Ginkgolide B	Y = 1.01 × 107X + 3.66 × 106	0.9933	2.61 × 10−2–0.834	1.27 × 10−4	5.09 × 10−4	
Ginkgolide A	Y = 3.71 × 104X + 4.29 × 104	0.9971	0.686–11.0	1.05 × 10−4	8.37 × 10−4	

The results obtained from the precision, stability, repeatability, and recovery tests are present in Table 4. The RSD values for precision and stability were within 1.35 %–4.52 % and 1.49 %–5.54 %, respectively. The repeatability RSD values ranged from 1.90 % to 4.46 %. The average method recoveries ranged from 88.84 % to 104.74 % (RSDs ≤5.60 %). Taken together, this method is shown to be accurate, sensitive, reproducible and suitable for the determination of the 10 selected markers in GSDG.Table 4 Precision, repeatability, stability and recovery of the 10 analytes.

Table 4Analyte	Precision RSD (%)	Repeatability RSD (%)	Stability RSD (%)	Recovery (%)	Recovery RSD (%)	
Histidine	2.93	4.43	4.07	104.74	4.03	
Aspartic acid	1.35	4.3	2.96	88.84	3.21	
Glutamic acid	1.72	2.8	3.60	102.61	2.23	
N-(N-glucopyranosyl)-indoleacetylaspartate	1.97	2.79	2.46	97.29	3.79	
N-(N-glucopyranosyl)-indoleacetylglutamate	3.29	2.97	3.07	97.99	2.25	
Eucomic acid	2.83	1.90	1.49	97.62	2.61	
Ginkgolide J	4.52	3.29	5.54	99.73	5.52	
Ginkgolide C	3.82	3.49	5.37	98.00	3.27	
Ginkgolide B	2.2	4.46	2.60	96.84	4.02	
Ginkgolide A	2.39	2.13	2.37	96.01	5.24	

3.2.3 Method application

Ten batches of GSDG were quantitatively tested for 10 indicator components by the validated HPLC-QTRAP-MS. The MRM chromatograms are shown in Fig. 8 (A for the standard solution and B for the test solution). A large variation in contents was observed for 10 analytes in 10 batches, as shown in Table 5. N-(N-glucopyranosyl)-indoleacetylaspartate was richest in each batch with the amount within 17.3–25.7 mg/g, while ginkgolide B (0.0270–0.0794 mg/g), ginkgolide C (0.0654–0.107 mg/g), ginkgolide J (0.0197–0.0335 mg/g), and histidine (0.177–0.382 mg/g) were determined to have the lowest amounts, below 1 mg/g. On the other hand, the contents of another 5 components generally ranged between 1 mg/g and 10 mg/g in 10 batches of GSDG, including N-(N-glucopyranosyl)-indoleacetylglutamate (3.54–5.31 mg/g), eucomic acid (3.36–5.51 mg/g), ginkgolide A (0.904–2.39 mg/g), aspartic acid (0.957–1.82 mg/g), and glutamic acid (1.22–2.81 mg/g). The relative contents for individual analytes in each batch were calculated using the average content of 10 batches as the denominator, and used for analysis of dispersion degree. As shown in Supplementary Table and Supplementary Fig. 6, all data were found to fall between 50 % and 150 % and the largest difference in content was seen for ginkgolide B (2.94-fold) among the different batches, while the least for N-(N-glucopyranosyl)-indoleacetylaspartate (1.49-fold). These results revealed the relative stable quality of these 10 batches of GSDG in terms of 10 chemical markers evaluated.Fig. 8 MRM chromatograms of standard solution (A) and test solution (B).

Fig. 8

Table 5 Contents of the 10 analytes in GSDG (mg/g).

Table 5Analyte	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	X ± SD	
Histidine	0.382	0.290	0.296	0.177	0.244	0.325	0.284	0.261	0.186	0.343	0.279 ± 0.0649	
Aspartic acid	1.04	0.957	1.63	1.66	1.82	1.75	1.81	1.49	1.09	1.23	1.45 ± 0.337	
Glutamic acid	2.32	1.85	2.65	1.74	2.43	1.66	2.81	1.37	1.22	1.50	1.96 ± 0.558	
N-(N-glucopyranosyl)-indoleacetylaspartate	25.2	17.3	19.0	20.4	25.7	22.8	18.9	23.0	21.1	22.2	21.6 ± 2.74	
N-(N-glucopyranosyl)-indoleacetylglutamate	4.26	3.83	3.54	4.04	5.31	5.15	3.56	4.74	3.78	4.36	4.26 ± 0.633	
Eucomic acid	5.51	3.58	4.80	5.06	3.36	5.17	4.92	4.30	4.22	3.99	4.49 ± 0.713	
Ginkgolide J	0.0300	0.0238	0.0204	0.0335	0.0197	0.0318	0.0206	0.0295	0.0200	0.0254	0.0255 ± 0.00534	
Ginkgolide C	0.0709	0.0984	0.0824	0.0683	0.0654	0.107	0.0782	0.0932	0.0724	0.0858	0.0822 ± 0.0139	
Ginkgolide B	0.0576	0.0621	0.0794	0.0270	0.0392	0.0704	0.0726	0.0468	0.0298	0.0577	0.0543 ± 0.0181	
Ginkgolide A	2.39	1.18	1.49	1.89	0.904	2.06	1.71	1.57	1.17	2.22	1.66 ± 0.488	

4 Conclusion

As a novel kind of Chinese medicine, DG has drawn significant concerns on its pharmacological and clinical researches, due to its similarity to traditional decoction. However, few studies on its chemical composition have been carried out.

To our knowledge, the present study is the first to reveal GSDG's chemical profiling by HPLC-QTOF-MS. With the observed accurate molecular masses, proposed fragmentation pathways, published literature, comparison with reference standards, as well as a homemade database and some on-line databases, 56 natural products have been identified or temporarily speculated, including 12 amino acids, 9 organic acids, 6 nucleosides and nucleobases, 6 flavonoids, 5 vitamins, 5 terpenoid lactones, 4 carbohydrates and 9 other components. Based on these results, an assay method was established for simultaneous determination of 10 representative bioactive components by HPLC-QTRAP-MS, by which the quality of 10 batches of GSDG was successfully evaluated. The validation in terms of linearity, stability, repeatability, precision and recovery has proven this assay method to be rapid, accurate, sensitive and convenient to operate. These mentioned methods may offer powerful support for the in-depth researches on GSDG in the future, especially in the fields of pharmaceutics, quality evaluation, pharmacokinetics, pharmacodynamic material basis and corresponding action mechanism. The present study can increase the knowledge and understanding of this Chinese medicine, and help utilize and develop it efficiently.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Funding

Not applicable.

Data availability statement

Data will be made available on request.

CRediT authorship contribution statement

Facheng Zhang: Writing – original draft, Conceptualization. Qingqing Fei: Writing – original draft, Investigation. Xiaojun Huang: Resources. Sheng Yu: Project administration, Investigation. Rongli Qiu: Resources. Lan Guan: Supervision. Baoxiang Wu: Investigation, Conceptualization. Mingqiu Shan: Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article.Multimedia component 1

Multimedia component 1

figs1 figs1

figs2 figs2

figs3 figs3

figs4 figs4

figs5 figs5

figs6 figs6

Acknowledgements

Not applicable.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36909.
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