a method for the analysis of ginsenosides, malonyl ginsenosides

DIETARY SUPPLEMENTS

A Method for the Analysis of Ginsenosides, Malonyl Ginsenosides,and Hydrolyzed Ginsenosides Using High-Performance LiquidChromatography with Ultraviolet and Positive ModeElectrospray Ionization Mass Spectrometric Detection

BRIAN DUFF SLOLEY, YI-CHAN JAMES LIN, DOUGLAS RIDGWAY, HUGH ALEXANDER SEMPLE, and YUN KAU TAM

Novokin Biotech Inc., Analytical Department, 108 Advanced Technology Centre, 9650 20th Ave NW, Edmonton, Alberta,

Canada T6N1E5

RONALD THOMSON COUTTS

University of Alberta, Department of Psychiatry, Neurochemical Research Unit, Edmonton, Alberta, Canada T6G 2B7

RAIMAR L�BENBERG

University of Alberta, Faculty of Pharmacy and Pharmaceutical Science, Edmonton, Alberta, Canada T6G 2N8

NUZHAT TAM-ZAMAN1

Sinoveda Ltd., Room 1902, 19/F, Haleson Bldg, 1 Jubilee St, Central Hong Kong

A high-performance liquid chromatographic

separation coupled to diode array absorbance and

positive mode electrospray mass spectrometric

detection has been developed for the analysis of

ginsenosides, malonyl ginsenosides, and hydrolyzed

ginsenosides in extracts of Asian ginseng (Panax

ginseng) and American ginseng (P. quinquefolius).

The method is capable of separating, identifying, and

quantifying the predominant ginsenosides found in

heated alcoholic extracts of Asian and American

ginseng roots routinely sold as nutraceuticals. It also

separates and identifies the malonyl ginsenosides

often found in cold alcoholic extracts of ginseng root

and has the potential to quantify these compounds if

pure standards are available. Furthermore, it can

separate and identify ginsenoside hydrolysis products

such as those readily produced in situations

mimicking gastric situations, including those used for

dissolution studies (i.e., 0.1 N HCl, 37�C).

Extracts of Asian and American ginseng (Panax ginseng

and P. quinquefolius, respectively) contain an

extremely complex mixture of sugar conjugates of

dihydroxylated and trihydroxylated dammarane triterpenes

collectively known as ginsenosides. A typical hot alcoholic

extract of ginseng root may contain significant amounts of as

many as 12 ginsenosides and more than 12 additional minor

ginsenosides (see ref. 1 for a comprehensive review), as well

as other compounds including panaxynols and linoleic acid.

The number of ginsenosides present in a sample increases

with cold alcoholic extracts of ginseng root because malonyl

conjugates of several ginsenosides, which appear to be

hydrolyzed by hot extraction procedures, can be observed (2).

Furthermore, ginsenosides are readily hydrolyzed under

acidic conditions, including situations reminiscent of gastric

conditions (3), or by intestinal bacteria (4, 5). This hydrolysis

results in the production of a number of ginsenoside-related

products and, in the case of partial hydrolysis, it is not unusual

to see in excess of 40 ginsenoside-related compounds in a

sample.

Most assays directed towards ginseng confine themselves

to the determination of the major ginsenosides and use

high-performance liquid chromatography (HPLC) with

ultraviolet (UV) absorbance detection (6–10). Peak identity is

based on retention characteristics when compared to an

authentic standard, leaving considerable room for error

considering the number of closely eluting ginenosides and

related metabolites found in various ginseng extracts. More

recently, HPLC coupled to various forms of mass

spectrometry (MS) detection have become available, and

some of these techniques have been applied to the analysis of

ginseng (10–13). This permits some confirmation of peak

identity, although a number of ginsenosides have identical

molecular weights and very similar fragmentation patterns.

Usually, a combination of HPLC retention characteristics and

mass spectral signatures can unequivocally identify a

ginsenoside. The use of coupled HPLC/MS is also useful in

determining the presence of malonyl conjugates of the

ginsenosides, which have been previously neglected (14, 15).

Our aim was to develop a separation and analysis method

that could not only routinely quantify the 8 to 10 major

ginsenosides usually found in commercial ginseng extracts

using absorbance detection at 205 nm but could also, when

necessary, measure and identify minor ginsenosides, malonyl

ginsenosides, and hydrolysis products using positive-mode

16 SLOLEY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006

Received May 6, 2005. Accepted by AP June 23, 2005.Corresponding author's e-mail: [email protected]

Currently unaffiliated.

electrospray ionization mass spectrometry (ESI-MS).

Furthermore, the method was designed to be able to

distinguish between extracts of P. ginseng and

P. quinquefolius by accurately quantitating the ratio of

ginsenoside Rg1 to Re (the ratio of Rg1 to Re is higher in

P. ginseng) and by demonstrating the presence or absence of

quinquenoside R1, an acetylated form of ginsenoside Rb1 that

is more prevalent in P. quinquefolius (1).

Experimental

Ginseng Samples

Ginseng samples were obtained as either standardized dry

root extracts or dry root from commercial sources.

Standardized root extracts came with a certificate of analysis

that included the identification of the ginseng species from

which the extract was derived.

Chemicals and Reagents

Standards of pure ginsenosides (Rg1, Re, Rb1, Rc, Rb2, and

Rd) were obtained from Chromadex (Santa Ana, CA). All

chemicals used were reagent grade or better. Solvents were

HPLC grade.

Preparation of Ginseng Samples

Dried extracts of ginseng from various commercial sources

were placed in 70% methanol at a concentration of 10 mg/mL.

The samples were mixed in a Vortex mixer and sonicated to

provide samples in which the saponins were fully dissolved.

The samples were then clarified by centrifugation in a desktop

centrifuge to remove any residual plant material and insoluble

excipients or fillers. The supernatants were diluted 1:10 with

20% methanol to provide a final concentration representative

of 1 mg/mL, and 20 to 100 �L were then injected directly into

the HPLC system.

Dried ginseng roots were crushed and ground to a fine

powder. The powder was placed in 70% methanol at a

concentration of 10 mg/mL in 1.5 mL microcentrifuge tubes

with locking caps and solubilized at room temperature in a

sonicating water bath at room temperature for 30 min. The

extracts were centrifuged in a desktop centrifuge for 10 min

and the supernatants collected. The extraction was repeated

2 more times, and the supernatants were combined and dried

in a vacuum centrifuge. The dried extract was reconstituted in

an appropriate volume of 20% methanol (concentration

0.5–1 mg/mL) and injected directly into the HPLC system.

Hydrolyzed ginseng and ginsenoside samples were

prepared by dissolving ginseng extracts at a concentration of

10.0 mg/mL or pure ginsenosides at a concentration of

100 �g/mL in 0.1 N HCl and warming the solution at 37�C for

1 h. The solution was neutralized with 0.1 N NaOH and the

samples diluted with 50% methanol to a concentration of

1 mg/mL (extract) or 10 �g/mL (pure ginsenoside). The

samples were then directly applied to the HPLC system. A

further experiment involving dissolution studies of ginseng

capsules and tablets in simulated gastric fluid under 3 pH

conditions (0.1 N HCl, pH 1.2; 0.2 M potassium phthalate,

pH 4; and 0.2 M potassium phosphate, pH 6.8) at 37�C was

performed using the USP28 General Chapter 711, p. 3572,

method for gastric dissolution.

Chromatographic Conditions

Separation of the ginsenosides, malonyl ginsenosides, and

hydrolyzed ginsenosides was accomplished using a Hewlett

Packard (Palo Alto, CA) Series 1100 HPLC system equipped

with a diode array detector (DAD) and ESI-MS detection. The

column used was a Phenomenex (Torrance, CA) Luna 3 �

C18(2) (150 × 4.6 mm) equipped with a guard column

(Phenomenex Security Guard C8, 4 � 3 mm). Separation was

achieved using gradient elution (mobile phase A, 90% 10 mM

ammonium formate + 10% acetonitrile; mobile phase, B 90%

acetonitrile + 10% 100 mM ammonium formate; flow rate,

1.00 mL/min). The gradient program was time = 0 min,

solvent B = 10%; time = 25 min, solvent B = 15.6%;

time = 45 min, solvent B = 50%; time = 50 min,

solvent B = 90%; time = 55 min, solvent B = 90%; time =

60 min, solvent B = 10%. There was a 5 min reequilibration

time after the final gradient event. The column was

maintained at 40�C.

DAD Conditions

Elution of chemicals was monitored by a DAD set at

205 nm with reference at 220 nm. Signals at 254 nm

(reference 360 nm) and 280 nm (reference 360 nm) were also

monitored to provide information regarding chemicals

potentially originating from sources other than ginseng root.

ESI-MS Conditions

After passing through the DAD, eluate was fed directly to

the ESI mass spectrometer. The mass spectrometer was set in

positive mode, and the spray chamber conditions were gas

temperature, 350�C; drying gas, 13.0 L/min; and nebulizer

pressure, 50 psig. The mass spectrometer was operated in both

scan and selected-ion monitoring (SIM) modes. In the scan

mode, ions were monitored from 400 to 1250 amu with the

fragmentor set at 90 V, gain at 1.0, and threshold at

150 counts. The scan mode was operated 30% of the time. In

the SIM mode, 3 time intervals were used to monitor eluting

materials. The time period from 0 to 30 min was used to

selectively monitor ginsenosides Rg1 and Re and their major

hydrolysis products. The time period between 30 and

40.5 min was used to monitor ginsenosides Rb1, Rc, Rb2, and

Rd as well as their malonyl conjugates and some of their

minor hydrolysis products. The period between 40.5 and

60 min was used to monitor the major hydrolysis products of

ginsenosides Rb1, Rc, Rb2, and Rd. In the SIM mode, the MS

detector was set to detect particular ions related to the

ginsenosides eluting during the selected time periods. The

ions examined included protonated molecules (MH+),

sodium adducts (MNa+), and fragments reflecting the

loss of sugar molecules and/or sugar residues and/or water

in various combinations. For the time period 0 to 30 min, the

SLOLEY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006 17

ions examined (amu) were 423.3, 441.4, 442.4, 587.4, 603.4,

621.4 (hydrolysis product), 639.5 (hydrolysis product), 749.5,

785.5 (hydrolysis product), 801.5 (Rg1 MH+), 823.5

(Rg1 MNa+), 947.5 (Re MH+), and 969.5 (Re MNa+). For the

time period 30 to 40.5 min, the ions examined were 425.4,

587.4, 749.5, 785.5 (hydrolysis product and fragment),

947.5 (Rd MH+), 969.5 (Rd MNa+), 1079.5 (Rb2 and

Rc MH+), 1101.5 (Rb2 and Rc MNa+), 1109.5 (Rb1 MH+),

and 1131.5 (Rb1 MNa+) amu. Malonyl ginsenosides

provided rather small signals for the intact molecules but

produced more intense signals from many fragments

identical to those of the daughter ginsenosides; thus, it was

not necessary to use signals derived from the intact malonyl

molecules (MH+ and MNa+) to observe them. The time


Figure 1. Representative chromatograms obtained from DAD absorbance at 205 nm and positive-modeESI-MS-SIM analysis of Panax ginseng and P. quinquefolius root extracts. (A) DAD trace obtained from a 1 mg/mL

(100 �L) extract sample of P. ginseng root that was extracted at room temperature with 70% methanol. (B) DAD trace

obtained from a 1 mg/mL (100 �L) extract sample of P. quinquefolius root that was extracted at 80�C with 70%methanol. (C) LC/MS-SIM trace corresponding to A. (D) LC/MS-SIM trace corresponding to Figure 1B.

period from 40.5 to 60 min was used to examine ions

749.5, 785.5, and 789.5 amu derived from the hydrolysis

of Rb1, Rc, Rb2, Rd, and related ginsenosides.

Results

Separation and Detection of Ginsenosides

A chromatogram typical of a commercial extract of

P. ginseng root (room temperature, 85% methanol extract,

1 mg/mL, injection of 100 �L dissolved in 20% methanol)

obtained using the HPLC separation and diode array

absorbance detection at 205 nm is illustrated in Figure 1A. The

major ginsenosides (Rg1, Re, Rb1, Rc, Rb2, and Rd) were well

resolved, although some minor ginsenosides contributed small

peaks at the front of the Rb1 peak. The same conditions were

used to analyze an extract of P. quinquefolius (Figure 1B)

derived from a hot alcoholic extraction (70% methanol, 80�C)

using a 100 �L injection of a 1 mg/mL sample dissolved in 20%

methanol. Again, the important ginsenosides (Rg1, Re, Rb1, Rc,

Rb2, and Rd) were well resolved. Furthermore, the decreased

ratio of Rg1 to Re and the presence of quinquenoside R1 usually

found in P. quinquefolius extracts was demonstrated. The

presence of the major ginsenosides was confirmed by SIM

mode MS in both P. ginseng (Figure 1C) and P. quinquefolius

(Figure 1D) extracts.

Because ginsenosides Rg1 and Re were often very difficult

to resolve and many ginsenosides, including Rb1, Rc, Rb2, and

Rd as well as their malonyl conjugates, eluted in a rather

limited retention window, it was evident that there was little

room for improvement in run time without sacrificing

resolution. It was also determined that samples had to be

loaded on to the HPLC apparatus in solutions containing

50% or less methanol. Higher concentrations of methanol

resulted in loss of resoluton of ginsenosides Rg1 and Re.

The limit of detection (LOD; �g/mL) and limit of

quantification (LOQ; �g/mL) for the ginsenosides [3 times

background (LOD) and 10 times background (LOQ) with a

20 �L injection] using the DAD at 205 nm were Rg1 (1.7, 4.3),

Re (2.2, 7.0), Rb1 (2.5, 7.0), Rc (1.8, 5.9), and Rd (2.2, 6.8).

When positive-mode ESI detection was used, the LOD and

LOQ values (�g/mL, 20 �L injection) were Rg1 (0.01, 0.03),

Re (0.02, 0.06), Rb1 (0.12, 0.40), Rc (0.03, 0.10), and Rd

(0.07, 0.25). The standard graph for the DAD was linear over

a range of at least 5.0 to 250 �g/mL. The ESI detector

generally provided a quadratic response over a range of 0.1 to

100 �g/mL.

Separation and Detection of Malonyl Ginsenosides

Malonyl ginsenosides are easily hydrolyzed in hot

alcoholic extracts and are not often observed in commercial

extracts. This may change as the industry begins to use

supercritical carbon dioxide extraction procedures. The

presence of malonyl ginsenosides (malonyl Rb1, malonyl Rc,

malonyl Rb2, and malonyl Rc) was demonstrated in both


Figure 2. Representative chromatograms of hydrolysis products obtained by incubation of pure ginsenoside Rg1

and Rb1 (100 �g/mL) in 0.1 N HCl at 37�C for 60 min. (A) Four hydrolysis products obtained from ginsenoside Rg1.(B) Six hydrolysis products obtained from ginsenoside Rb1.

DAD and SIM-MS chromatograms of cold extracted

P. ginseng root (Figures 1A and 1C) and P. quinquefolius root

(spectral data not presented). Identification of the malonyl

conjugates was by mass spectral characteristics obtained from

the scan mode, which included their MH+ and MNa+ signals

as well as fragmentation patterns that contained numerous

ions common to their respective daughter ginsenosides.

Separation and Detection of Hydrolyzed

Ginsenosides

Hydrolyzed ginseng extracts and hydrolyzed pure

ginsenosides were produced by mimicking gastric conditions

(0.1 N HCl, 37�C) for 1 h. Intact ginsenosides hydrolyzed

readily and virtually completely under these conditions. When

the U.S. Pharmacopeia (USP) method for dissolution of

capsules or tablets under gastric conditions was used, almost

complete hydrolysis of ginsenosides occurred within 10 min of

the beginning of release of the material into the artificial gastric

fluid (data not presented). In contrast, no hydrolysis products

were observed when the dissolution study was performed at

neutral pH (50 mM KH2PO4 adjusted to pH 6.8 with NaOH,

37�C, up to 60 min; data not presented). Typical SIM-MS

chromatograms of hydrolyzed ginsenoside Rg1 (Figure 2A)

and hydrolyzed ginsenoside Rb1 (Figure 2B) are presented.

Ginsenoside Rg1 provides 2 molecules with MH+ 639 ions that

have almost no UV absorbance at 205 nm, and a further

2 molecules with MH+ 621 with UV absorbance at 205 nm.

Ginsenoside Re provides 1 molecule with MH+ 621 in common

with hydrolyzed ginsenoside Rg1 and 2 molecules with MH+

785 ions that are all almost completely invisible to absorbance

detection at 205 nm but are readily seen by ESI-MS.

Ginsenoside Re also provides 2 further molecules with MH+

ions with molecular weights 749, 785 (not common to

ginsenoside Re), and 789 amu were seen for 6 common

hydrolysis products from ginsenosides Rb1, Rc, Rb2, and Rd.

These compounds were detected with both the DAD and

ESI-MS.

Mass Spectral Interpretations

An exhaustive interpretation of all of the mass spectral

information accumulated by this method is beyond the scope

of this paper. However, generalized interpretation of the data

will provide some interesting observations. Mass spectral

information from all of the ginsenosides followed similar

patterns. MH+ and MNa+ ions were always present. All

fragments observed in the positive mode reflected either the

loss of one or more sugar molecules, sugar residues, or water

molecules from the ginsenoside, leaving a positively charged

ion containing the terpenoid backbone. This pattern is nicely

illustrated by the spectrum obtained for ginsenoside Rb1

(Figure 3). Additional signals relating to positively charged

sugar molecules and residues that had fragmented away from

the terpenoid backbone were also evident. A schematic of the

fragmentation of ginsenoside Rb1 is illustrated in Figure 4.

Interestingly, the fragmentation pattern for ginsenoside Re

indicates that a rhamnose residue or molecule is expelled only

after 2 glucose molecules have been expelled. This suggests

that rhamnose cannot be the terminal sugar in the 2-sugar

chain reported in the literature [see Shibata et al. (1) for an

example]. Whether this observation indicates a problem with

the published structure of ginsenoside Re or is an artifact of

ESI-MS remains to be determined. Further analysis using

dual-stage mass spectroscopy (MS/MS) and nuclear magnetic

resonance imaging are required to more exactly elucidate the

structure of ginsenoside Re.

Dissolution of Ginseng Products and Recovery of

Ginsenosides from Root Preparations

Extraction efficiency for ginsenosides in preparations

containing standardized ginseng root extracts was determined

for a range of methanol–water mixtures and extraction

volumes. Recovery of ginsenosides was determined to be

highest with 70% methanol and with concentrations of up to

10 mg/mL ginseng extract. Use of these conditions resulted in

complete recovery of the ginsensosides. Recovery of

ginsenosides was also determined by spiking 0.9 mL of an

8% ginsensoside content extract (10 mg/mL) with 0.1 mL of a

second extract containing 80% ginsenosides. Thus, to a

known amount of 8% ginsenoside extract was added a known

amount of 80% ginsenoside extract containing 100% of the

ginsenoside content of the original preparation. Recovery was

greater than 94% for each ginsenoside.

Extraction of ginseng root powder indicated greater than

80% recovery on the first extraction, greater than 92% recovery

after 2 extractions, and greater than 96% recovery after

3 extractions.

Discussion

A sensitive and comprehensive analysis of ginsenosides

obtained from aqueous alcoholic extracts of P. ginseng and

P. quinquefolius root is possible by using HPLC coupled to


Figure 3. Mass spectrum obtained from ginsenosideRb1.

diode array and positive-mode ESI-MS detection. Using a

single chromatographic separation procedure, all of the major

ginsenosides and their malonyl conjugates and hydrolysis

products can be separated sufficiently to provide quantitative

analysis. Provided appropriate standards are available and

sufficient chromatographic resolution is achieved,

quantitative analysis of the major ginsenosides and their

malonyl conjugates is possible using the DAD alone. This

requires less elaborate equipment and is sufficient for most

quality control analyses required by the nutraceutical industry.

Analysis of a number of the acid hydrolysis products of the

ginsenosides is also possible with the DAD, but several

hydrolysis products originating from ginsenosides Rg1 and Re

absorb little light energy between 200 and 380 nm and require

positive-mode ESI-MS for detection.

Depending on which ginsenoside is being evaluated, ESI-MS

is between 25 and 140 times more sensitive than the DAD at

205 nm. Furthermore, MS in the SIM mode is more selective

than the DAD. DAD can be expected to provide many other

signals if other plant materials and/or ginseng leaf materials are

used in combination with ginseng root extracts to provide the

final formulation of products being analyzed. Certainly, common

materials such as phenylpropanoids and flavonoids can be

expected to have similar reversed-phase retention characteristics

and will absorb substantially at 205 nm, and this will cause

interference in complex mixtures when the DAD is used to

quantify the ginsenosides. On the other hand, it is possible that

quantification by the MS detector could be compromised due to

possible matrix effects resulting from the many ginseng

formulations that are encountered. Ultimately, it may be

preferable to compare information obtained from both the DAD

and the MS detector in order to provide an accurate quantitative

assessment of ginseng preparations.

The potential for ginsenoside hydrolysis was tested by

examining the hydrolysis of pure ginsenosides and ginseng

extracts in simulated gastric fluid (0.1 N HCl) at 37�C. This was

performed in a small reaction vessel for 1 h and using the USP

conditions for dissolution studies for various periods up to 1 h.

In both situations, hydrolysis of all the major ginsenosides was

rapid, and the dissolution studies indicated that significant

hydrolysis occurred within 10 min after the capsule or tablet

began releasing its contents. Intact ginsenosides are poorly

absorbed, but readily hydrolyzed under physiological

conditions to smaller and presumably higher permeability

compounds, leading to possible questions about the clinical

relevance of in vitro pharmacological studies of intact

ginsenosides. Detailed pharmacological studies of the

hydrolyzed compounds would not only be interesting, but they

have the potential to contribute substantially to an

understanding of the clinical effects of this important herb.

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Figure 4. Schematic interpretation of thefragmentation pattern obtained from ginsenoside Rb1.

a method for the analysis of ginsenosides, malonyl ginsenosides

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