Authentication of traditional Chinese prescriptions

One example for a preparation of several herbs used in CHM is Si-Wu-Tang. Si-Wu-Tang is composed of the four herbs Radix Angelicae Sinensis,Rhizoma Chuanxiong, Radix Paeoniae Alba, and Radix RehmanniaePreparata, and is widely used for the treatment of female disease5,6.The separate analysis of the herbs contained in Si-Wu-Tang and detectionby Q-TOF mass spectrometry enabled the selection and tentativeidentification of characteristic components of each herb. The selectedpeaks from each herb were matched to the peaks detected in Si-Wu-Tangin terms of first and second dimension retention times as well asagreement of the base peak in the respective mass spectra as a means ofauthentication of traditional Chinese prescriptions. In addition, changesfollowing the omission and replacement of one herb from Si-Wu-Tangwere investigated.Figure 3 shows the analysis of Rhizoma Chuanxiong with the selectedpeaks (A, B) as well as the analysis of Si-Wu-Tang with the matched peaksfrom Rhizoma Chuanxiong (C, D).

Instrumentation

The Agilent 1290 Infinity 2D-LC Solution used in this work was comprised of the following modules:• Two Agilent 1290 Infinity Binary Pumps • Agilent 1290 Infinity Autosampler with 1290 Infinity Thermostat • Agilent 1290 Infinity Thermostatted Column Compartment• Agilent 1290 Infinity Valve Drive with 2-position/4-port-duo valve

equipped with two 40-µL loops• Agilent 1290 Infinity Diode Array Detector with Max-Light Cartridge CellMass spectrometric detection was performed using an Agilent 6530 Accurate-Mass Q-TOF LC/MS System equipped with an Agilent Jet Stream ESI source.

Software

• Agilent OpenLAB CDS ChemStation Edition, revision C.01.07 [27] with Agilent 1290 Infinity 2D-LC Acquisition Software, revision A.01.02 [24].

• Agilent MassHunter Workstation Software, LC/MS data acquisition for Agilent 6200 series TOF/6500 series Q-TOF, revision B.05.01, qualitative analysis, revision B.06.00.

• GC Image LCxLC-HRMS Edition software, revision 2.5b0 for 2D-LC data analysis from GC Image LLC., Lincoln, NE, USA.

Samples and sample preparation

Samples of herbs used in Chinese herbal medicine were kindly providedby Patrick Kwik from Congress Pharmacy in Karlsruhe, Germany.Samples were prepared as decoctions, in the same manner as they areprepared for pharmaceutical use. The samples were weighed and atenfold amount of water was added. The samples were allowed to soak inthe cold water for 60 minutes and were then boiled for 30 minutes. Aftercooling, aliquots of the decoctions were centrifuged at 10,000 rpm for 15minutes. Aliquots of the supernatant phases were filtered using a 1-mLplastic syringe with Captiva Premium Syringe Filters RegeneratedCellulose, 15 mm, 0.45 μm before injection into the HPLC system.

Comprehensive 2D-LC method

Method development

Using a decoction from mulberry twigs (Mori Ramulus) as sample, acomprehensive 2D-LC method was developed. For this purpose, a suitablecombination of reversed-phase columns and eluents for the first- andsecond-dimension separation, providing maximum coverage of the two-dimensional separation space, was identified. To enlarge the accessibletwo-dimensional separation space, a shifted gradient was designed forthe second-dimension separation.Among the tested column and solvent combinations, the combination ofthe ZORBAX SB-Aq column with methanol as organic modifier in the firstdimension and the ZORBAX Bonus-RP column with acetonitrile as organicmodifier in the second dimension provided maximum coverage of the two-dimensional separation space. To further enlarge the accessible two-dimensional separation space a continuously shifted gradient wasdesigned for the second-dimension separation. Figure 1 compares theseparation obtained using the continuously shifted second-dimensiongradient (Figure 1B) to the separation obtained using repeating second-dimension gradients of 5 to 95 % acetonitrile (Figure 1A). A morecomplete coverage of the two-dimensional separation space wasobserved using the shifted gradient, and wrap-around was avoided.

Results and Discussion

Analysis and Authentication of Chinese Herbal Medicine

using Comprehensive 2D-LCSonja Krieger, Agilent Technologies, Waldbronn, Germany

Introduction

Conclusions

Traditional Chinese medicine (TCM) represents a holistic healthcaresystem that is based on human experience over the past 3,000 years andhas become increasingly popular in Western countries during the lastdecade. TCM includes various treatments, such as Chinese herbalmedicine (CHM), acupuncture, and moxibustion, among others1-3.In Chinese herbal medicine (CHM), the pharmaceutical efficacy is thoughtnot to result from the presence of a single bioactive component, but todepend on the synergistic effects of multiple components of the herbs1-3.The analysis and authentication of CHM is a challenging task because ofthe complexity of the herbs used and the natural variability of the plants4.Generally, one or a few main compounds or pharmaceutically activecomponents are currently used as marker compounds in the analysis ofCHM2,4. A more complete characterization of CHM can be achieved bychromatographic fingerprinting. The construction of a chromatographicfingerprint and subsequent comparison with the fingerprint of a clinicallyproven reference product is regarded as an effective method forauthentication of CHM1-4.For a complete characterization of CHM, one-dimensional liquidchromatography (1D-LC) does not provide enough resolving power.Therefore, comprehensive two-dimensional liquid chromatography (2D-LC)is the method of choice for their analysis.

Experimental

• Comprehensive two-dimensional liquid chromatography (2D-LC) is the method of choice for the analysis of complex samples such as CHM

• Characteristic components of the individual herbs can be detected in a traditional Chinese prescription

• One or a few marker compounds are not sufficient for authentication of traditional Chinese prescriptions

References1. Xie, P. S. and Leung, A. Y., Journal of Chromatography A, 1216, 1933-1940, 2009.2. Liang, X. M., et al., Journal of Chromatography A, 1216, 2033-2044, 2009.3. Yang, D. Z., et al., Journal of Chromatographic Science, 51, 716-725, 2013.4. Liang, Y. Z., et al., Journal of Chromatography B-Analytical Technologies in the

Biomedical and Life Sciences, 812, 53-70, 2004.5. Chen, X. P., et al., Asian Journal of Chemistry, 25, 6263-6266, 2013.6. Wang, Z. J., et al., Journal of Pharmaceutical and Biomedical Analysis, 50, 232-244, 2009.

Columns

First dimensionAgilent ZORBAX RRHT SB-Aq,

2.1 x 100 mm, 1.8 µm

Second

dimension

Agilent ZORBAX RRHT Bonus-RP,

3.0 x 50 mm, 1.8 µm, or

Agilent Poroshell 120 Bonus-RP,

3.0 x 50 mm, 2.7 µm

First dimension pump

Solvent A Water + 0.1 % formic acid

Solvent B Methanol + 0.1 % formic acid

Flow rate 0.05 mL/min

Gradient

0 minutes – 0 % B

10 minutes – 0 % B

70 minutes – 95 % B

80 minutes – 95 % B

Post time 30 minutes

Second dimension pump

Solvent A Water + 0.1 % formic acid

Solvent B Acetonitrile + 0.1 % formic acid

Flow rate 2.5 mL/min

Gradient Continuously shifted gradient

Thermostatted Column Compartment

First dimension column at 30 °C

Second dimension column at 50 °C

Modulation (2-position/4-port-duo valve)

The 2-position/4-port-duo valve was switched automatically

after each second dimension modulation cycle of 30

seconds. The loops were used in a cocurrent manner (filled

and eluted in the same flow direction).

Autosampler

Injection volume 5 µL

Sample temperature 6 °C

Needle wash10 seconds in methanol/water

(50/50; v/v)

Diode Array Detector

For MS detection, the effluent from the second dimension

column was split approximately 4:1 between the DAD and

the MS using a T-piece.

Wavelength 254 nm/4nm, Ref.: 380 nm/40 nm

Data rate 80 Hz

Mass Spectrometer

Ionization mode Positive/Negative

Acquisition rate 10 spectra/s

Jet stream ESI source conditions

Gas temperature 300 °C

Gas flow 8 L/min

Nebulizer 50 psi

Sheath gas

temperature300 °C

Sheath gas flow 9 L/min

Capillary 3,000 V

Nozzle 500 V

A B

Figure 1:Comprehensive 2D-LC analysis of a decoction from Mori Ramulus;

(A) repeating second-dimension gradients from 5-95 % ACN;

(B) continuously shifted second-dimension gradient.

The comprehensive 2D-LC method developed using a decoction from MoriRamulus was applied to the analysis of decoctions from four differentherbs used in CHM. The method provided good separation of thecompounds contained in the decoctions from all herbs analyzed, withoutany method adjustments (see Figure 2). The decoctions from the herbs allpresent very complex samples not amenable to comprehensive analysisusing 1D-LC. Furthermore, differences in the elution pattern (fingerprint)of components of the decoctions from the individual herbs can beobserved.

A B

C D

Figure 2:Comprehensive 2D-LC analysis of decoctions from herbs used in

CHM; (A) Mori Ramulus; (B) Lycii Fructus; (C) Radix Angelicae

Sinensis; (D) Rhizoma Atractylodis Macrocephalae.

A B

C D

Figure 3:Comprehensive 2D-LC analysis of decoctions from Rhizoma

Chuanxiong (A, B) and Si-Wu-Tang (C, D) with MS detection in

positive (A, C) and negative (B, D) ionization mode.

With two exceptions, 75 % or more of the selected peaks could bematched to peaks detected in Si-Wu-Tang (see Table 1). Generally,nonmatching of peaks could be explained by the fact that the peaks didnot show the same base peak in the mass spectrum, which can occurwhen partial coelution of peaks is observed in Si-Wu-Tang.

A

B

C

D

Figure 5:Comprehensive 2D-LC analysis of adulterations of Si-Wu-Tang;

(A, B) omission and (C, D) replacement of Rhizoma Chuanxiong with

MS detection in positive (A, C) and negative (B, D) ionization mode.

A

B

Figure 4:

Comprehensive 2D-LC analysis

of a decoction from Si-Wu-

Tang with MS detection in

positive (A) and negative (B)

ionization mode.

Peaks matched from the

individual herbs are marked:

Radix Angelicae Sinensis:

yellow

Rhizoma Chuanxiong: black

Radix Paeoniae Alba: white

Radix Rehmanniae Preparata:

red

Herb

MS positive MS negative

Selected

peaks

Matched

peaks

Selected

peaks

Matched

peaks

Radix Angelicae Sinensis 9 7 9 7

Rhizoma Chuanxiong 13 11 17 10

Radix Paeoniae Alba 9 5 10 10

Radix Rehmanniae Preparata 12 9 11 9

Sample

MS positive MS negative

Selected

peaks

Matched

peaks

Selected

peaks

Matched

peaks

Si-Wu-Tang 13 11 17 10

Si-Wu-Tang without Rhizoma Chuanxiong 13 6 17 5

Si-Wu-Tang with replacement of Rhizoma

Chuanxiong13 6 17 4

Table 1: Matching of selected peaks from each herb contained in Si-Wu-Tang.

Table 2: Matching of selected peaks from Rhizoma Chuanxiong in adulterations of

Si-Wu-Tang.

To investigate the possibility of detecting an adulteration of Si-Wu-Tang,an analysis of Si-Wu-Tang without Rhizoma Chuanxiong and of Si-Wu-Tangwith Rhizoma Chuanxiong replaced by the rhizome of another herb wasperformed (see Figure 5). In the adulterations of Si-Wu-Tang aconsiderably smaller number of selected peaks were matched (see Table2), which discloses that the analyzed samples did not contain RhizomaChuanxiong. The matching of characteristic peaks from RhizomaChuanxiong with peaks detected in the adulterations of Si-Wu-Tang can beexplained by the fact that not all selected peaks from Rhizoma Chuanxiongare unique to this herb. In the analysis of Si-Wu-Tang with replacement ofRhizoma Chuanxiong, additional peaks originating from the replacementherb were detected. The number of matched peaks did not increase,which shows that the replacement herb did not contain any of theselected characteristic components of Rhizoma Chuanxiong.

In positive ionization mode, 11 out of 13 selected peaks were matched andin negative ionization mode 10 out of 17 selected peaks were matched.This shows the possibility to detect characteristic components of anindividual herb in a traditional Chinese prescription.Figure 4 shows the analysis of Si-Wu-Tang with the peaks matched fromthe individual herbs. Several peaks detected in Si-Wu-Tang can beattributed to more than one individual herb, for example, senkyunolide A,Z-ligustilide and ferulic acid are contained in Radix Angelicae Sinensis andRhizoma Chuanxiong.