Comparative analysis of steroidal saponins in four Dioscoreae herbs by high performance liquid chromatography coupled with mass spectrometry

Accepted Manuscript Title: Comparative analysis of steroidal saponins in four Dioscoreae herbs by high performance liquid chromatography coupled with mass spectrometry Author: Long Guo Su-Ling Zeng Yu Zhang Ping Li E-Hu Liu PII: DOI: Reference:

S0731-7085(15)30137-0 http://dx.doi.org/doi:10.1016/j.jpba.2015.08.038 PBA 10238

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

5-7-2015 25-8-2015 27-8-2015

Please cite this article as: Long Guo, Su-Ling Zeng, Yu Zhang, Ping Li, E-Hu Liu, Comparative analysis of steroidal saponins in four Dioscoreae herbs by high performance liquid chromatography coupled with mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.08.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Comparative

analysis

ofsteroidal

saponins

in

fourDioscoreae

herbsbyhigh

performance

chromatographycoupled withmass spectrometry

Long Guo1,Su-Ling Zeng1, Yu Zhang, Ping Li*, E-Hu Liu*

State Key Laboratory of Natural Medicines,China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing 210009, China

1

These authors contributed equally to this work.

liquid

*

Corresponding authors. Tel.: +86 25 83271379; fax: +86 25 83271379.

E-mail addresses:[email protected] (E.-H. Liu), [email protected] (P. Li).

Graphical abstract

Qualitative and Quantitative Analysis Dioscoreae Nipponica Rhizome

Dioscoreae Hypoglaucae Rhizome

Dioscoreae Spongiosae Rhizome

Dioscoreae Rhizome

Highlights

Chemometrics Analysis



An HPLC-MS method was established for qualitative and quantitative analysis of steroidal saponins in four Dioscoreae herbs.



A total of 11 saponins were characterized by HPLC-QTOF MS and 7 major saponins were quantified by HPLC-QQQ/MS.



PCA and HCA were performed to compare and discriminate the samples.



The results would be helpful inthe quality control of herbal medicines from Dioscoreaespecies.

Abstract Steroidal

saponins,

whichexhibit

multiple

pharmacological

effects,are

the

major

bioactive

constituentsin

herbal

medicines

fromDioscoreaespecies.In this study, a sensitivemethod based on highperformance liquid chromatography-mass spectrometry (HPLC-MS) was established

and

validatedfor

qualitative

and

fourDioscoreaeherbsincludingDioscoreaeNipponicaRhizome(DNR)and

quantitative

analysis

of

steroidal

saponins

in

DioscoreaeHypoglaucaeRhizome(DHR),

DioscoreaeSpongiosaeRhizome(DSR) and DioscoreaeRhizome(DR).A total of eleven steroidal saponins were identified byhigh performance liquid chromatography coupled with quadrupoletime-of-flight mass spectrometry (HPLC-QTOF/MS).Furthermore, seven major steroidal saponins wassimultaneous quantified using ahighperformance liquid chromatography coupled withtriple quadrupole mass spectrometry (HPLC-QQQ/MS).The qualitative and quantitativeanalysis results indicated that the chemical composition of DNR, DHR and DSR samples exhibited a high level of global similarity,while the ingredients in DR varied greatly from the other three herbs.Moreover, principal component analysis (PCA) and hierarchical clustering analysis (HCA) were performed to compare and discriminate theDioscoreaeherbs based on the quantitative data.The results demonstratedthe qualitative and quantitative analysis of steroidal saponinsbased on HPLC-MS is a feasible method for quality control of Dioscoreaeherbs. Keywords:Dioscoreaeherbs;Steroidal saponins;HPLC-QTOF/MS;HPLC-QQQ/MS

1. Introduction Herbs derived from Dioscoreaespecieshave been widely used in traditional medicine and cuisine in China and other countries since the ancient time.A number of Dioscoreaeherbs, including DioscoreaeNipponicaeRhizome (DNR, the driedrhizome of Dioscorea nipponica), DioscoreaeHypoglaucaeRhizome(DHR, the driedrhizome ofDioscoreaehypoglauca), DioscoreaeSpongiosaeRhizome(DSR, the driedrhizome ofDioscoreaspongiosa) and DioscoreaeRhizome (DR,the driedrhizomeofDioscoreaopposita) have been officially specified in Chinese Pharmacopoeia [1].Pharmacological andclinical studies revealed thatherbal medicines fromDioscoreaespecieshave multiplebiological activities, such as anti-inflammatory, anti-tumour,immunoregulatory,hormone-like,hypoglycaemicand cardiovascularproperties [2].Intriguingly, the four herbal medicines derived from the Dioscoreaespecies have similar morphologicalcharacteristics,but theirpharmacological effects and clinical applicationsare different according to the theory of traditional Chinesemedicine.DNR has remarkable effects on boneinjuries and dilating coronary arteries [3].DHR wasreported to possess anti-inflammatoryand anti-tumor activities and DSR was used for the treatment of rheumatism, urethral and renal infections, while DR coulddecrease damage in renal tubules as well as alleviate inflammation in the liver tissue[4].Phytochemical studies indicated that steroidal saponinsare the major bioactive constituentsin Dioscoreae species [2,5]. Steroidal saponins showed a wide range of biological activities and often prepared as botanical productsfor treating coronary heart disease and rheumatoid

arthritis [6,7].We believe thatdifferent pharmacological effects and clinical applications of these four herbal medicines may be correlated with the contents of steroidal saponins. In Chinese Pharmacopoeia (2010 version), thin layer chromatography (TLC) method was applied to identify the authenticity of DNR using a single marker compound (diosgenin)[1].For the other three Dioscoreaeherbs,limited investigations have been conducted for comprehensive analysis of steroidal saponins inthese herbal medicines.To our knowledge, there has been no work and little discussion on comparative analysis of steroidal saponinsinmedicinal herbsfrom Dioscoreae species.In view of the current situation, development of a sensitive method for qualitative and quantitative analysis of saponins inthese fourDioscoreaeherbswas urgently needed for their quality control and clinicalpractice[8]. However, the identification and quantification of saponins in crude extracts are challenging because of their similar structures and weak ultraviolet absorption[9,10]. Thoughevaporative light scattering detector (ELSD) could be employed as an alternative,it has low sensitivity and the quantitative results by HPLC-ELSD are seriously interfered by solvents[11,12]. Recently, high performance liquid chromatography-mass spectrometry (HPLC-MS) methodtechniquehas been widely accepted to be the predominanttool for qualitative and quantitative analysis of chemical constituents in complex samplesforit providesfavorable specificity and sensitivity[13-15].The HPLC-MS method has been

demonstrated to be useful forcomprehensive analysis of multiple components in herbal medicines [16-20]. Tomake a comprehensive comparison of Dioscoreae herbs and evaluate their quality,in this work,a high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) method was utilized for identification of the steroidalsaponins in these four Dioscoreae herbs. Later, ahighperformance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ/MS) method using multiple reaction monitoring (MRM) mode was applied to quantify the seven major saponins in 21 batches of Dioscoreaesamples.Furthermore, multivariate statistical analysis, includingprincipal component analysis (PCA) and hierarchical clustering analysis (HCA) were performedto provide more information about the chemical differences of the Dioscoreaeherbs. The present study will facilitate the comprehensive quality control of Dioscoreaeherbs and lay the foundation for future researches on their pharmacological activities.

2. Experimental

2.1. Chemicals, materials and reagents

Authentic standards of protodioscin(1), protogracillin (2), pseudoprotodioscin (5), pseudoprotogracillin(6), dioscin (8), gracillin (9), trillin (11) and the internal standard (IS)ginsenoside Rb1were purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu,China).The purities of these compounds were determined to be higher than95%. Thefour herbs from Dioscoreawere collected from different regions in China(Table S1).The voucher specimens, identified by Prof. Ping Li from Department of Pharmacognosy in China Pharmaceutical University, have been deposited in the State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China. Acetonitrile and methanol, both MS grade, were purchased from Merck (Darmstadt, Germany). Ultrapure water was prepared using a MilliQ system (Millipore, Bedford, MA, USA). HPLCgrade ethanol (Nanjing Chemical Reagent Factory, China) was used for sample preparation.

2.2. Instrument and chromatographic conditions Qualitativeanalyses were carried out on high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (Agilent Technologies, Santa Clara, CA, USA).Chromatographic separation was conducted on an Agilent ZorBax Extend-C18 column (4.6×50mm, 1.8μm,Santa Clara, CA, USA).The mobile phase was delivered at a flow rate of 0.5 mL/min and consisted of 0.1% formic acid water (A) and

acetonitrile using a gradient elution as follows: 0-2 min,20-20% B; 2-12 min, 20-28% B; 12-20 min, 28-45% B; 20-28 min, 45-45% B; 28-35 min, 45-100% B. The MS spectra were acquired in both positive and negative ion mode.Chromatographic separation was conductedon Sepax BR-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was delivered at a flow rate of 0.8 mL/min and consisted of water (A) and acetonitrile (B) using a gradient elution as follows: 15-30% B from 0-20 min, 30-50% B from 20-40 min, 50-55% B from 40-55 min, 55-100% B from 55-85 min, and 100% B from 85-100 min. The MS spectra were acquired in both positive and negative ion mode. Quantitative analysis was performed on a Shimadzu LC-30AD HPLC system (Kyoto, Japan) coupled to a Shimadzu LCMS-8050 triple quadrupole mass spectrometer (Kyoto, Japan). Chromatographic separationwas performed on anWaters (Wexford, Ireland)CORTECSTM UPLC C18(2.1×50 mm, 1.6 m) with an on-line filter in front of the column. Themobile phase was composed of water (A) and acetonitrile (B), with a gradient elution as follows: 0-15 min, 24-30% B; 15-20 min, 30%-65% B; 20-23 min, 65-95% B. The flow rate was set at 0.4 mL/min and the column temperature was 30°C. Sample injection was 1 L. The operation parameters of MS experiments were set as follows: drying gas (N2) temperature, 300°C; drying gas flow rate, 10.0 L/min; nebulizer gas (N2) flow, 3 L/min; capillary voltage, 4000V. Quantification was performed in the negative ionization by MRM mode.All the data of HPLC-QQQ/MS analysis were processed by using Shimadzu LabsolutionsLCMS Version 5.65 (Kyoto, Japan).

2.3. Preparation of standard solutions Reference standards was accurately weighed and thendissolved in ethanol-water (65:35, v/v)to yield the standard stocksolutions for the seven reference compounds at the concentrations of 500μg/mL.Then the working solutions wereprepared by appropriate dilution of the stock solutions with ethanol-water (65:35, v/v)to yield seven concentrations, from 0.5 μg/mL to100μg/mL.The working solutions containing seven reference standards wereprepared by appropriate dilution with ethanol-water (65:35, v/v).To each standardworkingsolution, an aliquot of stock solution ofginsenoside Rb1(IS) was added to make up a final concentration of 112ng/mL. The workingsolutions were stored at 4°C for furtheranalysis.

2.4. Preparation of sample solutions 7 batches of DNR, 4 batches ofDHR, 5 batches of DSRand 5 batches of DRwere collected from different provinces in China for the experiment. All the samples werecrushed until uniformly mixedand passed through a 60-mesh sieve. Preparation of sample solutions for qualitativeanalyseswas as follows: an aliquot (0.5 g) was accurately weighed and immersed in

methanol65% ethanol(25 mL). The mixture was then sonicated for 60 min. The methanol extract was centrifuged at 13000 rpm for 10 min. The supernatant was transferred to an autosampler vial for HPLC-DAD-TOF/MS analysis. For preparation of sample solutions used to determine the seven steroid saponins, each sample powder (0.05 g) was weighted accuratelysoaked in 25 mL of 65% ethanol. An appropriate amount of the internal standard solution was added. After the extracted solution was cooled to room temperature and centrifugedat 13000 rpm/min for 10 min. For quantification of protodioscin, protogracillin, pseudoprotodioscin, pseudoprotogracillin, dioscin andgracillin, the supernatant was diluted 100 times with 65% ethanol before theHPLC analysis. An aliquot of 1μLof the sample was injected for analysis.

2.5. Method validation The calibration curves were plotted with a series of concentrations of standard solutions and constructed from the peak area ratio of thetested reference peak to that of the internal standard versus their concentrations. The limit of detection (LOD) and limit of quantification (LOQ) for each analyte were expressed by 3- and 10-fold ofthe ratio of the signal-to-noise (S/N), respectively. The intra-day precision of the developed method was determined by repeated injectionof the sample solution six times within the same

day.While for inter-day test, the samples were examined in duplicates for consecutivethree days. The relative standard deviations (RSDs) werecalculated as the measure of precision. For the test of repeatability,six replicates of the same samples were prepared and analyzed.To confirm the stability, the same sample was stored at room temperature and analyzed with themethod described by replicate injection at 0, 2, 4, 8, 12 and 24 h.The RSDswere used to evaluate themethod repeatability and stability. Recovery of all thequantified constituents was determined to further evaluate the accuracy of the method.Aknown amount of theseven standards were added into a 0.25 g powder of6 batches of same samples, andthen extracted and analyzed with the same procedures.

3. Results and discussion

3.1. Qualitative analysis by HPLC-QTOF/MS 3.1.1. Optimization of chromatographic conditions In order to achieve a rapid and efficient analysis, a short chromatographiccolumn packed with 1.8μm porous particles was employed in

HPLC analysis. Different mobile phase (including methanol-water, acetonitrile-water, methanol-formic acid water solution, and acetonitrileformic acid water solution),flow rate (0.4, 0.5 and 0.6 mL/min) and the column temperatures(20, 25 and 30°C) were optimized. The acetonitrileformic acid water system at 25°Cwith a flow rate of 0.5mL/min was finally selected for the shorter analysis duration, greater separation ability and better peak shapes. In order to achieve rapid and efficient analysis,different mobile phase (including methanol-water, acetonitrile-water, methanol-formic acid solution, and acetonitrile-formic acid solution),flow rate (0.8 and 1.0 mL/min) and the column temperatures(25, 30 and 35°C)were optimized. The acetonitrile-water system in aqueous phase at 30°Cwitha flow rate of 0.8mL/min was finally selected for the shorter analysis duration, greater separation ability and better peak shapes.

3.1.2.Global characterization of steroid saponins in the four Dioscoreae herbs Steroid saponins, which consist of steroidaglycones and several sugar moieties,are reported to be themajor group of bioactive compounds in herbs fromDioscoreaespecies [21-24].In this work, a rapid identification of eleven steroid saponins was achieved by HPLC-QTOF/MS. The total ion current (TIC) profiles of the extract of the four herbs are presented in Fig 1.The retentiontime and the MSdata of the characterized

compounds are summarized in Table 1. Deprotonated ions of saponins tend to produce signals with losses of several sugar moieties in the negative mode.Protodioscin was used as an example to explore thefragmentation patterns for thesesaponins in detail.Compound 1 readily produced a strongdeprotonated molecular ion [MH](m/z 1047.5366) in negative mode.The main fragment ions [MHRha], [MH2Rha] and[MH2RhaGlu]produced by the further loss of glucosyl and rhamnosyl units were widely observed.Coelution with the reference standard and comparisonwith literature data [22], compound 1was unequivocally identified as protodioscin. Finally, a total of 11 steroid saponinswere identified, of which eight steroid saponins were confirmed by comparing the retention time and accurate massdata between samples and authentic standards. The qualitative analysis demonstrated that significant differences existed in chemical profiles among thefour Dioscoreaeherbs, and all the 11 steroid saponinswere detected in DNR, DHR and DSR, while only protodioscinwas observed in DR.

3.2. Quantitative analysis of seven major steroidal saponinsin fourDioscoreae herbs 3.2.1. Optimization of HPLC-QQQ/MSconditions The mass spectrometric conditions were optimized inboth positive and negative ion modes. The negative ionmode was selected sincethe

ionization of steroid saponins was more efficientcompared with the positive ion mode.For optimization of MRM conditions, the seven major steroidal saponinsincluding protodioscin, protogracillin, pseudoprotodioscin, pseudoprotogracillin, dioscin, gracillin andtrillin,werecharacterized by MS scan and MSn product ions to ascertaintheir precursor ions and to select product ions for use in MRMmode.The parameters ofcollision energy (CE) were optimized to get the richest relative abundance.The retention time (RT) and MS information for each analyte including [MH],precursor and product ions and CE are shown inTable 2.

3.2.2. Method validation The calibration curves obtained had good linearity (R2> 0.9993)and a relatively wide range of concentrations (from 0.5 μg/mL to 100μg/mL). The LOQs and LODs were less than375 ng/mL and 187.5 ng/mL, respectively. The RSD values forprecision were in the range of 2.89-7.23% for intra-day assaysand 3.90-5.68% for inter-day assays. The repeatability presented was in the range from 2.80-5.15%, and the stability was less than 5.09%. Therecovery of the method was in the range of 89.24-116.67%, withRSD < 7.45%. All these valuesindicated that the system was suitable for the quantitative analysis(Table 3).

3.2.3.Quantitative analysis The validated HPLC-QQQ/MSmethod was applied to analyze 21 batchesof samples, including 7 batches of DNR, 4 batches of DHR, 5 batches of DSR and 5 batches of DR. Thesevenmajor steroidal saponinswere quantified with the internal standard method based on respective calibration curves. The structures of these compounds are shown in Fig.2 and the typical MRM chromatograms are shown in Fig. 3.The results suggested thatHPLC-QQQ/MS was a very powerful technique for quantitative analysis of multicomponent of herbal medicines in terms of time saving and sensitivity. The quantitative results are presented in Table 4. Among the examined constitutes, protodioscin was the most abundant constituent in thesefour herbs. The average contents of protodioscin in DNR, DHR, DSR and DR samples were 17.84, 0.17, 4.11 and 0.11 mg/g, respectively. While the contents of trillinwere significantlylower than the other determined steroidal saponinsand it was hardly detected in DHR and DR. The total contents of the seven saponins were calculated, and it is clear thatDNR has the mostabundant steroidalsaponins(31.50-51.16 mg/g) among the four Dioscoreaeherbs. It should be noted that the contents of saponins were quite low(about 0.10 mg/g) in DR, and most saponin compounds could not be detected.The content distributions of each compound in each sample were displayed inFig. S1.

3.2.4.Comparison of Dioscoreaeherbs by principal component analysis (PCA) and hierarchical clustering analysis (HCA) According to the qualitative analysis and quantitative analysis results, we could find that the chemical profile ofDNR, DHR and DSR samples showed high level of similarity, whileDR samples were quite different with the other three Dioscoreaeherbs samples.Todiscriminateand evaluate the quality ofDNR, DHR and DSR, multivariate statistical methods such asPCA and HCA were employed based on the contents of the seven quantified saponins. PCA is an unsupervised pattern recognition method used for analyzing, classifying and reducing the dimensionality of numerical datasets, and it has been widely used to discriminate and compare the quality ofherbal medicines [25-27]. In PCA analysis, the contents of sevensteroidal saponins were set as variables, while different batches of samples were set as observations. The PCAscore scatter plot(Fig. 4) showed that samples were successfullyclassified into three groups,indicating that the samples areindeed different in the levels or occurrences of the constituents. The samples of DHR clustered in a small region, which indicating a closer relationship among this group samples.Comparatively, DNR samples were relatively discrete, which suggested that the quality of DNRwasless stable compared withthat of DHR and DSR. To compare and classify the three similar herbs(DHR, DNR and DSR), HCA was further used to cluster the samples. As shown in Fig. 5, the results were similar with PCA analysis. Sixteen samples were also classified into three groups, corresponding to DHR, DSR and DNR,

respectively.The PCA and HCA results demonstrated analysis of steroidal saponins based on HPLC-MS is a feasible method for quality control of Dioscoreae herbs.

4. Conclusion

In the presentstudy, we establishedan efficient and accurate HPLC-MS method for comparative analysis of the steroidal saponinsinfour medicinal herbsfrom Dioscoreae species. Qualitativeanalyses were performed onHPLC-QTOF/MSand eleven steroidal saponins were identified. Moreover, simultaneousquantification of seven major saponinswas carried out on HPLC-QQQ/MS. The established methodology displayed acceptable levels of linearity, precision, repeatability and accuracy.Thequalitative andquantitative results indicated that the chemical compositions ofDNR, DHR and DSR samples havea great similaritywhile the ingredients in DRwere significantly different from other three Dioscoreae herbs.The PCA and HCA analysis demonstrated that DNR, DHR and DSR samples could be easily discriminated from each other based on the contents of the steroidal saponins. The proposed method could be employed for quality controlofherbal medicinesfrom Dioscoreaespecies and would be helpful forfurtherpharmacological studies.

Acknowledgments This work was supported by a Project of the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAI29B07), the National Natural Science Foundation of China (81202898)and NaturalScience Foundation of Jiangsu Province (BK20130652).

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Legend to figures Fig. 1. Total ion chromatogramsof DNR, DHR, DSR and DRinpositive(A) and negative(B) ion modes by HPLC-QTOF MS.

(A) DNR

DHR

DSR

DR

(B) DNR

DHR

DSR

DR

1

1

8

2 34

5

6

7

2 34

56

7

10

56

9 7 8

10

9 10

11

8 9 11

2

1

34

11

1

8

2

1

3

4

9 56

7

3

4

56

7 8

1

11

10

11

10

11

8 9

2

1

10

2 34

56

9

7

1

Fig.2. Chemical structures of the sevenquantified saponins.

HO

OGlc

OGlc

O R

O R

O

1 Protodioscin: R = -β-D-Glc (4←1)-α-L-Rha

O

5 Pseudoprotodioscin: R = -β-D-Glc (4←1)-α-L-Rha

α-L-Rha (2←1)

α-L-Rha (2←1)

2 Protogracillin: R = -β-D-Glc (3←1)-β-D-Glc

6 Pseudoprotogracillin: R = -β-D-Glc (2←1)-α-L-Rha

α-L-Rha (2←1)

β-D-Glc (3←1)

O

8 Dioscin: R = -β-D-Glc (4←1)-α-L-Rha α-L-Rha (2←1)

O

9 Gracillin: R = -β-D-Glc (3←1)-β-D-Glc α-L-Rha (2←1)

R

O

11 Trillin: R = -β-D-Glc

Fig. 3. The typical MRM chromatograms of thesample(A) andstandard solution (B) obtained in negative mode. (1)protodioscin,

(2)protogracillin, (5)pseudoprotodioscin, (6)pseudoprotogracillin, (8)dioscin, (9)gracillin, (11)trillin.

4000000

(A)

3500000

(B)

12500

3000000

10000

2500000

7500

2000000 1500000

5000

1000000

2500

500000 0

0

1

500000 0

0 2500

2

250000

1

1000

0

2

0

5

10000

5

1000

0

0

IS

10000

IS

1000

0 0

2000

6

1000

6

2500

0

0

1000000

8

0.0

8

5000

0 0

1000000

9

500000

10000

0.0

9

0 0 0.0 2500

11

1000

0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

0 0.0

11 2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

Fig. 4.The score scatter plot ofprincipal component analysis(PCA) ofthe DNR, DHR and DSRsamples. The samplecodes in score scatter plot were the same as shown in Table 4.

R2X[1] = 0.521

R2X[2] = 0.185

Fig. 5.Dendrograms of hierarchical cluster analysis (HCA) using between-groups linkage of the DNR, DHR and DSRsamples.The samplecodes were the same as shown in Table 4.

No.

Identification

tR (min)

Formula

Proposal ions

Rescaled Distance Cluster Combine

DHR

DSR

DNR

Experimental m/z

Diff (ppm)

Fragmentation ions

Source

1 2 3 4 5 6 7 8 9 10 11

Protodioscin* Protogracillin* Zingiberogenin-2glcrha Protobioside Pseudoprotodioscin* Pseudoprotogracillin* Deltonin* Dioscin* Gracillin* Progenin II Trillin*

9.50 10.27

C51H84O22 C51H84O23

[MH] [MH]

1047.5366 1063.5328

1.48 0.25

901.4801, 755.4217, 593.3673 901.4849, 755.4432, 593.3758

DNR, DHR, DSR, DR DNR, DHR, DSR,

10.54

C45H73O18

[MH]

901.479

0.16

-

DNR, DHR, DSR,

11.43 15.18 15.56 23.33 24.36 24.58 27.64 30.47

C45H73O18 C51H82O21 C51H82O22 C45H74O19 C45H72O16 C45H72O17 C39H62O12 C33H54O8

[MH] [MH] [MH] [MHCOO] [MH] [MH] [MH] [MH]

901.4791 1029.5328 1045.5251 929.4752 867.4774 883.4717 721.4147 577.3738

1.26 1.83 -2.49 -0.05 -3.04 -2.29 2.98 -0.53

755.4218, 593.3699 883.4666, 737.4313 883.4782, 737.4295 883.4710, 721.4140, 575.3450 867.4721, 721.4198, 575.3626 721.4177, 575.3544 575.3843 -

DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR, DNR, DHR, DSR,

Table 1HPLC-QTOF/MS data for identification of steroidal saponinsin four Dioscoreaeherbs. *Confirmed by reference compounds

Table 2 Retention time (RT), MS data and collision energy (CE) of the seven determined steroidal saponins. NO.

Compound

RT (min)

[MH] (m/z)

Precursorion

Product ion

CE (V)

1

Protodioscin

2.20

1047.4

1047.4

901.3

44

2

Protogracillin

2.69

1063.4

1063.4

901.3

43

5

Pseudoprotodioscin

9.67

1029.3

1029.3

883.3

41

6

Pseudoprotogracillin

10.89

1045.4

1045.4

883.3

40

8

Dioscin

18.65

867.3

867.3

721.25

34

9

Gracillin

18.69

883.3

883.3

721.3

33

11

Trillin

19.70

621.4

621.4

45.2

23

IS

Ginsenoside Rb1

10.15

1107.5

1107.5

179.25

54

Table 3 Calibration curves, linear ranges, LOD and LOQ, precision, repeatability, stability and recovery of the seven investigated saponins. Precision RSD (%) NO

Compounds

Regression equation

r

2

LOQ (ng/ml)

LOD (ng/ml)

Intra-day

Intra-day

(n=6)

(n=6)

Recovery (n=6)

Repeatability RSD (%) (n =6)

Stability RSD (%)

Original

Spiked

Detected

Recovery

RSD

(μg)

(μg)

(μg)

(%)

(%)

1

Protodioscin

y = 34.51x - 0.007

1.000

2.4

1.2

5.70

5.13

3.73

4.52

604.60

610

1266.14

108.45

5.46

2

Protogracillin

y = 72.88x + 0.166

0.9994

4.5

1.8

7.23

5.68

3.99

4.96

81.09

80

152.48

89.24

5.01

5

Pseudoprotodioscin

y = 14.82x + 0.039

0.9994

375

187.5

2.89

3.90

4.51

5.09

36.58

42

79.83

102.98

7.45

6

Pseudoprotogracillin

y = 45.35x - 0.071

0.9999

137.5

55

2.52

5.77

2.80

3.34

9.33

9

19.05

108.00

4.16

8

Dioscin

y = 93.90x + 0.461

0.9993

2.2

1.1

6.31

5.42

5.15

5.04

40.15

40

84.45

110.76

5.46

9

Gracillin

y = 121.80x + 0.152

0.9993

2.6

1.3

4.78

4.96

3.61

4.97

15.68

18

33.88

101.11

4.02

11

Trillin

y = 162.70x + 0.026

0.9994

13.75

2.75

5.13

4.86

4.53

5.01

0.04

0.042

0.09

116.67

5.98

1 2 3

Table 4 The contents of the seven steroidal saponins in DNR, DHR, DSR and DR (mg/g, n=3).

NO.

Protodioscin

Protogracillin

Pseudoprotodioscin

Pseudoprotogracillin

Dioscin

Gracillin

Trillin

DNR-1

12.3816

0.5900

4.1210

0.2765

25.7599

2.3337

0.0036

DNR-2

18.6551

3.2841

1.0667

0.3296

20.2902

7.5329

0.0042

DNR-3

23.9939

5.0233

0.4661

0.2388

12.2283

6.2010

0.0050

DNR-4

15.4129

2.4427

0.4833

0.1851

14.7064

5.7157

0.0014

DNR-5

23.1962

4.5450

0.6131

0.2462

7.5537

4.2739

0.0009

DNR-6

24.1840

3.2435

1.4633

0.3731

1.6059

0.6271

0.0017

DNR-7

7.0811

1.4497

0.8835

0.2866

28.4994

12.5816

0.0049

DHR-1

0.2296

0.0378



0.0145

0.0893

0.0230



DHR-2

0.1482

0.0065

0.0019



0.0151

0.0143



DHR-3

0.1467

0.0032





0.0929

0.0071



DHR-4

0.1590

0.0013





0.0145

0.0029



DSR-1

7.7251

1.4218

0.6565

0.2449

2.0312

1.0323

0.0086

DSR-2

3.0118

0.2408

0.3962

0.2004

3.5021

1.4324

0.0141

DSR-3

2.9362

0.3658

0.5032

0.2101

3.0377

1.6241

0.0177

DSR-4

4.3559

0.5497

0.3530

0.1762

1.5941

0.7014

0.0170

DSR-5

2.5410

0.3877

0.3449

0.1530

4.9294

3.1974

0.0135

DR-1

0.1085













DR-2

0.1111













DR-3

0.0822













DR-4

0.1138













DR-5 4 5

0.1403













6

 : Not detected

7

37