optimisation of ultra-performance lc conditions using response surface methodology for rapid...

Research Article

Optimisation of ultra-performance LCconditions using response surfacemethodology for rapid separation andquantitative determination of phenoliccompounds in Artemisia minor

A method that couples rapid, sensitive, reproducible and accurate ultra-performance LC

(UPLC) with quadrupole-TOF-MS was established for the first simultaneous qualitative

and quantitative analysis of phenolic compounds in Artemisia minor. Box–Behnken

designs (BBDs) were applied as an effective tool to optimise major parameters that

influence the resolution of UPLC, including three gradient steps and column tempera-

ture. Under optimal UPLC conditions, a total of 23 phenolic compounds in the crude

methanol extracts of A. minor were well separated on a Waters Acquity UPLC BEH C18

column (100� 2.1 mm, 1.7 mm particle size) within 16.5 min, and the compounds were

unequivocally or tentatively identified via comparisons with authentic standards and

literature. In this study, a total of six major phenolic compounds were quantified in A.minor and the method was validated to be sensitive, precise and accurate within the LOD

from 1.24 to 5.27 mg/mL, and the overall intra- and inter-day variations in detection were

less than 3.76%. The recovery of the method ranged from 97.9 to 103.8% with RSDs that

were less than 5.8%. These results demonstrate that this approach has the potential for

quality control of A. minor and other Tibetan herbal medicines.

Keywords: Artemisia minor / Box–Behnken designs (BBDs) / Phenoliccompounds / Quadrupole-TOF-MS / Ultra-performance LCDOI 10.1002/jssc.201000452

1 Introduction

The genus Artemisia is a large, diverse genus of plants with

nearly 400 species belonging to the family Asteraceae

(Compositae), which is widely distributed throughout the

temperate regions in Asia, Europe, North America

and South Africa [1–3]. There are approximately 200

Artemisia species in China, and some of them are

commonly prescribed as traditional Chinese medicines.

Artemisia minor Jacq. ex Bess. has long been used in Tibetan

folk medicine for treating fever, rheumatism, dysentery,

scabies and bruising; A. minor is located in the

Qinghai-Tibet Plateau of China at an altitude range of

3000–5800 m [4].

Our previous phytochemical study showed that the

main bioactive components of A. minor are phenolic

compounds that include euoniside, umbelliferone, isofrax-

idin, artemetin, arteminorin C, etc. [5]. Some of these

phenolic compounds, such as arteminorin C and caffeic

acid, exhibited inhibitory activity on xanthine oxidase or

protein tyrosine phosphatase 1B [5].

Several studies on the evaluation of the quality of

various species of Artemisia plants have been reported using

HPLC [6], LC-MS [7], GC-MS [8], and high-speed counter-

current chromatography [9]; however, these methods are

time-consuming and also suffer from lower separation

efficiency and insufficient resolution.

The use of ultra-performance LC (UPLC) has become

widespread, and UPLC has proven to be an efficient chro-

matographic separation tool, which not only provides higher

peak capacity with excellent peak shapes but also gives a

greater resolution and increased sensitivity within a short

detection time [10–12]. In addition, UPLC can be coupled to

a diode-array detector (DAD) and a quadrupole-TOF-MS,

which provide rapid separation of complicated samples and

abundant structural information. Thus, UPLC has the

potential to be used in the fields of drug metabolism,

metabolite profiling and quality control of herbal medicines.

Yan Zhou1

Franky Fung-Kei Choi2

Zhi Zhou He1

Jing-Zheng Song2

Chun-Feng Qiao2

Xin Liu1

Li-Sheng Ding1

Suo-Lang Gesang3

Hong-Xi Xu4

1Key Laboratory of MountainEcological Restoration andBioresource Utilization,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu, P. R. China

2Hong Kong Jockey Club Instituteof Chinese Medicine, Shatin,New Territories, Hong Kong,P. R. China

3Tibet Autonomous RegionInstitute for Food and DrugControl, Lhasa, P. R. China

4Shanghai University ofTraditional Chinese Medicine,Shanghai, P. R. China

Received June 22, 2010Revised July 27, 2010Accepted September 17, 2010

Abbreviations: BBD, Box–Behnken design; BPI, base peakintensity; DAD, diode-array detector; FA, formic acid; RSM,

response surface methodology; UPLC, ultra-performance LC

Correspondence: Dr. Hong-Xi Xu, Shanghai University ofTraditional Chinese Medicine, Shanghai, P. R. ChinaE-mail: [email protected]: 186-28-85223843

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 3675–3682 3675

Furthermore, optimisation techniques in analytical

chemistry, which are termed the ‘‘one-variable-at-a-time’’

method, are carried out by changing only one parameter

while other parameters are held constant [13]. Disadvan-

tages to this type of optimisation include incomplete

depiction of the effects of the parameter on the entirety of

the system response and an increase in the number,

time and expense of the experiments necessary to conduct

the research. Response surface methodology (RSM),

which is a widely used optimisation approach, has been

introduced to predict the optimised chromatographic

conditions. Among the most relevant multivariate statistic

techniques, Box–Behnken designs (BBDs) are a class of

rotatable or nearly rotatable second-order designs based on

three-level incomplete factorial designs. The theory and

applications of RSM and BBDs for the optimisation of

HPLC methods have been described in detail elsewhere

[14–16]. They are more efficient than other response surface

designs because they can be used to establish a quadratic

response surface.

To date, there has been no report on the evaluation of

quality and standardisation of A. minor. In this study, we

report a rapid, effective and sensitive method for the iden-

tification and quantification of the major phenolic compo-

nents in A. minor. Because the phenolic compounds in A.minor are structurally similar and their chromatographic

separation may be problematic, the BBDs were selected to

evaluate the chromatographic conditions that influence the

resolution of UPLC. The UPLC-DAD-Q-TOF-MS method

that was developed was then applied to the simultaneous

qualitative and quantitative analysis of the phenolic

compounds in A. minor.

2 Materials and methods

2.1 Reagents and materials

Methanol and ACN were of HPLC-MS grade from Fisher

Scientific UK (Loughborough, UK), and formic acid (FA)

and leucine–enkephalin was of spectroscopy grade from

Sigma-Aldrich (St. Louis, MO, USA). Deionised water was

purified using a Milli-Q SP Regent Water system from

Millipore (Bedford, MA, USA).

The standards of scopolin (2), syringin (3), caffeic acid

(4), euoniside (5), fraxin-8-O-b-D-glucopyranoside (6),

umbelliferone (8), scopoletin (9), isofraxidin (10), arte-

minorin C (16), 3,30-biisofraxidin (18), tricin (19), artemi-

norin A (20) and acacetin (21), 5,8-dihydroxy-7,40-dimethoxy-

flavone (22) and 5,7-dihydroxy-3,6,40-trimethoxy-flavone (23)

were isolated in our laboratory (over 95% purity by UPLC-

MS). Their structures were identified based on IR, UV and

NMR spectroscopic analysis [5].

The dried aerial part of A. minor was collected in the

Tibet region of China in 2007. The voucher specimen was

authenticated by Mr. Suolang Gesang, Tibet Autonomous

Region Institute for Food and Drug Control.

2.2 Preparation of standard solutions

Six major compounds, including syringin (3), euoniside (5),

fraxin-8-O-b-D-glucopyranoside (6), umbelliferone (8),

scopoletin (9) and isofraxidin (10), were selected for

quantitative analysis. Each accurately weighed standard

was dissolved in methanol to prepare individual stock

solutions. A series of working standard solutions were

prepared by appropriate dilution of the stock solution with

methanol to construct the calibration curve: syringing (3),

3.20–40.0 mg/mL; euoniside (5), 3.52–26.40 mg/mL; fraxin-8-

O-b-D-glucopyranoside (6), 4.59–34.40 mg/mL; umbellifer-

one (8), 2.69–16.80 mg/mL; scopoletin (9), 3.07–19.20 mg/

mL; isofraxidin (10), 10.99–82.40 mg/mL. All solutions were

stored at 41C in a refrigerator before analysis.

2.3 Preparation of sample solution

The dried plant sample of A. minor was ground into a fine

powder using a pulveriser, and 0.1 g of the powder was

accurately weighted and transferred to a 10 mL capped

conical flask; 2 mL of methanol was added to the flask, and

the powder was extracted using a supersonic washer at

50 Hz for 30 min. The extract was filtered, and the

remaining residue was extracted once more. The extraction

solutions were combined and diluted to 5 mL with

methanol in a volumetric flask. For quantitative analysis,

2 mL of the extract solution was accurately piped to a 25-mL

flask and was further diluted with methanol to produce

1.6 mg/mL of the crude extracts. The collected solution was

filtered through a 0.22-mm PTFE syringe filter. An aliquot of

filtrate (2 mL) was injected into the UPLC instrument for

analysis.

2.4 UPLC conditions

Analysis was performed on a Waters ACQUITYTM UPLC

system (Waters, Milford, MA, USA) equipped with a binary

solvent delivery system, an auto-sampler, and ACQUITY

photodiode-array detection system. The chromatography

was performed on a Waters ACQUITY UPLC BEH C18

column (2.1� 100 mm id, 1.7 mm, Waters). The raw data

were acquired and processed with MASSLYNX 4.1 Software.

UV spectra by DAD were recorded between 190 and 400 nm,

and the detection wavelength was set to 280 nm. The mobile

phase consisted of (A) water containing 0.1% FA v/v and (B)

ACN. The gradient conditions and the column temperature

were optimised by BBDs. The UPLC eluting conditions

were optimised as follows: linear gradient from 8 to 15% B

(0–6 min), linear gradient from 15 to 25% B (6–15 min),

linear gradient from 25 to 40% B (15–20 min), and linear

gradient from 40 to 100% B (20–21 min). The flow rate was

0.5 mL/min. The column and auto-sampler were main-

tained at 50 and 101C, respectively. Each wash cycle

consisted of 200 mL of strong solvent (80% ACN and

J. Sep. Sci. 2010, 33, 3675–36823676 Y. Zhou et al.


600 mL of weak solvent (10% ACN). The injection volume of

the standards and sample was 2 mL.

2.5 MS

MS was performed using a Waters Q-TOF Premier

(Micromass MS Technologies, Manchester, UK) equipped

with an ESI source operating in negative ion mode. The

desolvation gas was set to 600 L/h at a temperature of 401C,

and the source temperature was set to 1021C. The capillary

voltage and cone voltage were set to 3000 and 30 V,

respectively. Argon was employed as the collision gas at a

pressure of 7.066� 10�3 Pa. The instrument was operated

with the first resolving quadrupole in a wide pass mode with

the collision cell operating at 5.0 V. All MS data were

acquired using the LockSprayTM to ensure mass accuracy

and reproducibility. The reference compound leucine-

enkephalin (m/z 554.2615) was used as the lock mass in

negative ESI mode at a concentration of 100 pg/mL with an

infusion flow rate of 10 mL/min. The data were collected into

two separate data channels, and the instrument spent 0.2 s

on data acquisition for each channel with a 0.02 s inter-

channel delay.

2.6 Experimental design

Data analysis and desirability function calculations were

performed by Microsoft Excel 2002 (Microsoft). Experimen-

tal design was performed by SASs 9.0 (SAS Institute, Cary,

NC, USA). Optimisation criteria and desirability limits are

summarised in Table 1. Four factors of UPLC, which are (i)

the ratio of mobile phase B (G1_ACN (B%)) for the first

solvent gradient; (ii) the ratio of mobile phase B (G2_ACN

(B%)) for the second solvent gradient; (iii) the ratio of

mobile phase B (G3_ACN (B%)) for the third solvent

gradient and (iv) column temperature T (1C) were optimised

by BBDs.

2.7 UPLC method validation for quantitative

analysis of the phenolic compounds

To evaluate the method precision, the mixture of six

phenolic standards (compounds 3, 5, 6, 8–10) in solution

was analysed under the optimal conditions six consecutive

times in one day for intra-day variation, while the inter-day

variation test was conducted for the mixture of six phenolic

compounds on three successive days. The RSD was taken as

a measure of the precision in the results.

A recovery experiment was performed to confirm the

accuracy of the method; this experiment was carried out by

spiking accurate amounts of the six phenolic standards

(compounds 3, 5, 6, 8–10) into an A. minor sample, which

was then extracted and analysed as detailed previously.

Spiked samples were analysed in five replicates.

3 Results and discussion

3.1 Optimisation of chromatographic conditions by

BBDs

In these studies, the chromatographic conditions of UPLC

were optimised to obtain the best separation results by

analysing the methanolic extract of A. minor. The primary

challenge in this analysis was to achieve separation between

the peaks of the 15 authentic phenolic compounds. The

selection of the mobile phase was achieved by trying several

combinations of mobile phase systems of ACN–water and

methanol–water with or without FA, and the chromato-

graphic behaviour was explored using C8 and C18 columns

(Acquity BEH C8 2.1� 100 mm id, 1.7 mm column; Acquity

BEH C18 2.1� 100 mm id, 1.7 mm column, Waters). Results

from the preliminary experiment showed that a sufficient

separation was obtained in the gradient elution with a mobile

phase that consisted of 0.1% FA and ACN using a C18

Table 1. Experimental plan for BBD and the obtained global

response

Experiment Factorsa) Global Derringer

desirability

G1_ACN

(%)

G2_ACN

(%)

G3_ACN

(%)

T (1C)

1 10 25 50 37.5 0.0000

2 10 40 50 37.5 0.0000

3 20 25 50 37.5 0.0000

4 20 40 50 37.5 0.0000

5 15 32.5 40 25 0.0000

6 15 32.5 40 50 1.2502

7 15 32.5 60 25 0.0000

8 15 32.5 60 50 0.0000

9 10 32.5 50 25 0.0000

10 10 32.5 50 50 0.0000

11 20 32.5 50 25 0.0000

12 20 32.5 50 50 0.0000

13 15 25 40 37.5 0.0000

14 15 25 60 37.5 0.0000

15 15 40 40 37.5 0.0000

16 15 40 60 37.5 0.0000

17 10 32.5 40 37.5 0.0000

18 10 32.5 60 37.5 0.0000

19 20 32.5 40 37.5 0.0000

20 20 32.5 60 37.5 0.0000

21 15 25 50 25 0.0000

22 15 25 50 50 1.3815

23 15 40 50 25 0.0000

24 15 40 50 50 1.2927

25 15 32.5 50 37.5 0.0000

26 15 32.5 50 37.5 0.0000

27 15 32.5 50 37.5 0.0000

a) Factors of G1_ACN, G2_ACN and G3_ACN represent the

concentration of ACN at the first, second and third time points,

respectively. T represents the column temperature.

J. Sep. Sci. 2010, 33, 3675–3682 Liquid Chromatography 3677


column as compared to the other mobile phase systems, such

as the water–methanol and 0.1% FA–methanol system.

Although the addition of FA in the mobile phase was found

to suppress the peak tailing of the target compounds, the

resolution of certain peaks was unsatisfactory, which required

that the gradient profile be further optimised.

Based on the preliminary experiments, three-step

gradient profiles with two factors, consisting of the ratio of

mobile phase B and the column temperature were investi-

gated. The step gradient profiles and the column tempera-

ture (T) represent four parameters of UPLC that were

optimised with the aid of BBDs, which resulted in 27

experiments. The initial composition of the mobile phase

was 8% ACN, and the end point of the first linear gradient

(G1, 6 min) was optimised by varying the ratio of mobile

phase B from 8 to 15%; the second end point (G2, 15 min) of

the gradient step was optimised from 15 to 25% of mobile

phase B; the third end point (G3, 20 min) of the gradient

step was optimised from 25 to 40% of mobile phase B; and

the column temperature (T) was optimised from 25 to 501C.

The detailed experimental design is listed in Table 1. The

criteria selected for optimisation was resolution between the

15 target peaks (compounds 1–15) and their corresponding

adjacent peaks. The Derringer desirability function

D ¼ ðR1 � R2 � � � � � R16Þ1=16, where R1�R16 was the

smaller resolution between the 15 compounds and each

adjacent peak (R1�R16 was adjusted to 1.5 if their real values

were larger than 1.5). The optimal gradient conditions,

which resulted in the best resolution, could be obtained as

the global desirability response reached its maximum.

The effects of the composition of the mobile phase and

column temperature on the global desirability of the

separation of the 15 target compounds are illustrated in

Fig. 1. The predicted model for the optimisation was:

Y 5�9.08� 10�111.96� 10�1G1�1.89� 10�1G219.06�10�2G311.91� 103T�6.54� 10�2G1

213.04� 103G22�2.37�

103G2T�7.20� 104 G23. This equation was found to

adequately predict the experimental results; in the equation,

Y was the predicted global desirability, and G1, G2, G3 and Twere described above. Consequently, the optimal UPLC

elution conditions were obtained as follows: linear gradient

from 8 to 15% B (0–6 min), linear gradient from 15 to 25% B

(6–15 min), linear gradient from 25 to 40% B (15–20 min)

and the column temperature was set at 501C. Under these

UPLC conditions, the 15 target peaks were well separated

without interfering components, and co-elution with possi-

ble interfering species was not observed after 18 min.

3.2 Identification of the major phenolic compounds

in A. minor

The standard solutions and A. minor sample were analysed

using the optimised UPLC conditions. The chromatogram

Figure 1. The prediction profile plots (upperpanel) and the main effect plots (lowerpanel) of three gradient steps and columntemperature on the global desirability ofresolution between 15 target compoundsand their adjacent peaks. G1, G2, G3, T andY are the same as in the text. Circles indicatethe optimum desirability.



at 280 nm and the base peak intensity (BPI) chromatograms

in negative ionisation mode of the A. minor sample are

shown in Fig. 2. More than 20 peaks in the crude extracts of

A. minor were detected and well separated in the BPI

chromatogram of A. minor. Fifteen of these peaks were

unambiguously identified by comparing the retention time,

UV spectra, high-resolution ESI-MS and MS/MS spectra

with those of authentic standards, including scopolin (2),

syringin (3), caffeic acid (4), euoniside (5), fraxin-8-O-b-D-

glucopyranoside (6), umbelliferone (8), scopoletin (9),

isofraxidin (10), arteminorin C (16), 3,30-biisofraxidin (18),

tricin (19), arteminorin A (20) and acacetin (21), 5,8-

dihydroxy-7,40-dimethoxy-flavone (22), and 5,7-dihydroxy-

3,6,40-trimethoxy-flavone (23). In addition, nine components

without standards (peaks 1, 7, 11–15, 17) could be tentatively

identified by matching the empirical molecular formula of

the [M–H]� ion and the product ions in MS/MS with those

in the literature. The structures of these compounds are

depicted in Fig. 3, and the retention time, UV and mass data

of each compound are shown in Table 2.

All of the phenolic compounds exhibited abundant

[M–H]� ions as the base peak in the negative ESI-MS

spectra. The addition of FA to the mobile phase resulted in

[M1HCOO]� adduct ions that can also be observed in the

mass spectra of some components. As shown in Table 2, the

mass error for all of the assigned components was below

5 ppm, which is regarded as acceptable certainty.

Compound 1 displayed an [M–H]� ion at m/z 533.1864

that corresponds to the molecular formula C23H33O14. In

the MS/MS spectra, product ions at m/z 371.1348 and

209.0811 that form by the loss of one glucose unit

(162.0516 Da) and two glucose units (162.05161

162.0537 Da), respectively, were observed as abundant

peaks. This finding in combination with other supporting

evidence from the literature allowed compound 1 to be

tentatively identified as syringinoside [17].

Compound 7 exhibited an accurate mass [M–H]� ion at

m/z 163.0397 that corresponds to the molecular formula

C9H7O3, which underwent preferential losses of CO2 and

H2O to yield product ions at m/z 119.0495 and 145.0294,

respectively, in the MS/MS spectrum. Compound 7 was

then tentatively assigned as 2-propenoic acid [18].

Compounds 11–13 are quinic acid derivatives.

Compound 11 presented an [M–H]� ion at m/z 677.1481

that corresponds to the molecular formula C34H29O15. In

the MS/MS spectrum, fragment ions at m/z 515.1198,

353.0865 and 191.0552 were observed, which were formed

through progressive losses of caffeoyl residues (m/z162.0317, C9H6O3), respectively. The product ion at m/z179.0346 also indicated the presence of the caffeoyl acid

Figure 2. (A) UPLC-UV chromatogram of A. minor; (B) UPLC-Q-TOF BPI chromatogram of A. minor.



group. Thus, compound 11 was tentatively assigned as 3,4,5-

tricaffeoylquinic acid [7]. The quinic acid derivatives referred

to as compounds 12 and 13 are isomers that yielded the

same empirical molecular formula C25H23O12. In the

MS/MS spectra, 12 exhibited product ions at m/z 353.0862

and 191.0558; 13 exhibited product ions at m/z 353.0864

and 173.0453. According to literature [19], fragment ions

at m/z 191.0558 and 173.0453 were characteristic for

Figure 3. Structures assigned in the extractof A. minor.

Table 2. Identification of the major chemical constituents observed in online negative UPLC-MS spectra from A. minor

Peak

no.

tR (min) UV lmax (nm) Molecular

formula

Detected

mass

[M–H]– m/z

calculated mass

Error

(ppm)

Identification

1 0.60 220 C23H33O14 533.1864 533.1870 �1.1 Syringinoside

2a) 1.82 245, 320 C16H17O9 353.0870 353.0873 �0.8 Scopolin

3a) 2.06 221, 264 C17H23O9 371.1324 371.1342 �4.9 Syringin

4a) 2.23 242, 324 C9H7O4 179.0348 179.0344 2.2 Caffeic acid

5a) 2.52 228, 291 C17H19O10 383.0975 383.0978 �0.8 Euoniside

6a) 3.24 230, 288 C17H19O10 383.0977 383.0978 �0.3 Fraxin-8-O-b-D-glucopyranoside

7 3.47 234, 309 C9H7O3 163.0397 163.0395 1.2 2-Propenoic acid

8a) 3.98 240, 324 C9H5O3 161.0237 161.0239 �1.2 Umbelliferone

9a) 4.38 231, 341 C10H7O4 191.0349 191.0344 2.6 Scopoletin

10a) 5.06 231, 341 C11H9O5 221.0446 221.0450 �1.8 Isofraxidin

11 5.82 245, 325 C34H29O15 677.1501 677.1506 �0.8 3,4,5-Tricaffeoylquinic acid

12 7.08 248, 325 C25H23O12 515.1179 515.1190 �2.1 3,5-Dicaffeoylquinic acid

13 8.13 248, 325 C25H23O12 515.1182 515.1190 �1.6 4,5-Dicaffeoylquinic acid

14 8.64 285 C19H17O7 357.0971 357.0974 �0.8 Arteminorin D

15 8.78 240, 328 C20H19O8 387.1089 387.1080 2.3 Artemetin

16a) 9.33 240, 285 C20H13O9 397.0572 397.0560 3.0 Arteminorin C

17 10.83 230, 345 C17H13O8 345.0613 345.0610 0.9 5,7,30 ,40-Tetrahydroxy-6,50-dimethoxyflavone

18a) 11.39 189 C22H17O10 441.0811 441.0822 �2.5 3,30-Biisofraxidin

19a) 11.62 225, 347 C17H13O7 329.0665 329.0661 1.2 Tricin

20a) 11.92 230, 347 C22H17O10 441.0817 441.0822 �1.1 Arteminorin A

21a) 14.81 237, 267 C16H11O5 283.0615 283.060 3.2 Acacetin

22a) 15.27 235, 340 C17H13O6 313.0719 313.0712 2.2 5,8-Dihydroxy-7,40-dimethoxy-flavone

23a) 16.40 230, 345 C18H15O7 343.0824 343.0818 1.7 5,7-Dihydroxy-3,6,40-trimethoxy-flavone

a) Identified with authentic compounds.



3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid,

respectively. Moreover, 3,5-dicaffeoylquinic acid was more

easily eluted from the C18 reversed-phase column when

compared with 4,5-dicaffeoylquinic acid. Thus, compounds

12 and 13 were tentatively assigned as 3,5-dicaffeoylquinic

acid [19, 20] and 4,5-dicaffeoylquinic acid [19, 20], respectively.

The empirical molecular formula of compound 14 was

determined to be C19H17O7 with a [M–H]� ion at m/z357.0971. The main fragment ions were observed at m/z313.1079 and 283.0974, which were formed by the loss of

CO2 and a consecutive loss of CH2O in the MS/MS.

Compound 14 was tentatively identified as arteminorin D,

which has been previously reported for A. minor [5].Compounds 15 and 17 are close analogues with similar

UV absorption maxima around 240 nm and 330 nm, which

are typical of flavones. Compound 15 presented a [M–H]� ion

at m/z 387.1089 that corresponds to the molecular formula

C20H19O8. In the MS/MS spectrum, a characteristic fragment

ion was observed at m/z 195.0395, which was formed viaRetro-Diels-Alder rearrangement at the double bond of

C9–C10. In addition, fragment ions that were formed by

successive loss of a CH3 radical group were observed. A

similar fragmentation pattern was observed for compound

17, which showed extensive losses of H2O units, indicating

the presence of hydroxyl units in its structure. Thus, 15 and

17 were tentatively assigned as artemetin [5] and 5,7,30,40-

tetrahydroxy-6,50-dimethoxyflavone [21], respectively.

3.3 Validation of the UPLC method for quantitative

analysis of the designated phenolic compounds

Under the optimum UPLC conditions, the quantitative

method validation, which included linearity, the LOD, LOQ,

precision and accuracy, were performed by monitoring the

UV lmax at 280 nm.

3.3.1 Linearity, LOD and LOQ

Linear calibration curves were constructed using six

different concentrations of the six markers. All of the

calibration graphs were plotted based on linear regression

analysis of the integrated peak area (y) versus concentration

(x, mg/mL). Good linear correlation of these six standard

curves was obtained and confirmed by the correlation

coefficients (R2 values), which ranged from 0.9979 to 0.9994.

The sensitivity of the method was evaluated by determining

the LODs and LOQs, which were defined as the S/N of 3

and 10, respectively. These parameters were determined

empirically using triplicate analysis of a series of decreasing

concentrations of a standard solution. The developed

method was found to be sensitive: LODs and LOQs were

no greater than 5.27 and 10.99 mg/mL, respectively (Table 3).

3.3.2 Precision and recovery

To evaluate the precision of the developed method, the

standard solution mixture was analysed six consecutive

times in one day to observe intra-day variation, and the

mixture was analysed on three successive days to observe

inter-day variation. The RSD was taken as a measure of the

precision, and the developed method was found to be

precise, exhibiting intra-day variability RSD values between

0.43–1.14% and inter-day variability RSD values between

1.13 and 3.76% (Table 4).

To evaluate the accuracy of this method, recovery

experiments were carried out by spiking accurate amounts

Table 3. Regression equation, linearity ranges, correlation coefficients and LOD and LOQ for the six markers in A. minor

Peak no. Compounds Regression equation Linear range (mg/mL) R2 LOD (mg/mL) LOQ (mg/mL)

3 Syringin y 5 23.21x – 13.06 3.20 – 40.00 0.9992 1.28 3.20

5 Euoniside y 5 35.54x – 0.665 3.52 – 26.40 0.9988 1.69 3.52

6 Fraxin-8-O-b-D-glucopyranoside y 5 26.25x – 9.360 4.59 – 34.40 0.9983 2.20 4.59

8 Umbelliferone y 5 30.05x 1 0.780 2.69 – 16.80 0.9994 2.24 2.69

9 Scopoletin y 5 25.48x – 3.690 3.07 – 19.20 0.9979 1.24 3.07

10 Isofraxidin y 5 17.55x – 62.61 10.99 – 82.40 0.9988 5.27 10.99

Table 4. Precision, recovery and the contents of the six markers in A. minor

Peak no. Compounds Precision Mean recovery (%, n 5 6) %RSD Contents (mg/g)

Intra-day RSD% (n 5 6) Inter-day RSD% (n 5 3)

3 Syringin 0.45 3.76 99.02 3.79 16.13

5 Euoniside 0.43 2.16 98.88 5.84 11.57

6 Fraxin-8-O-b-D-glucopyranoside 1.14 2.45 100.23 1.46 16.78

8 Umbelliferone 0.51 1.94 97.90 2.17 5.43

9 Scopoletin 1.32 3.44 102.07 4.54 5.14

10 Isofraxidin 0.51 1.13 103.83 3.06 14.75



of the six standards into a measured amount of A. minormaterial (0.1 g); the sample was then extracted and analysed

with the method described above. Each sample was analysed

in six replicates. The total amount of each standard in each

analysis was calculated from the corresponding calibration

curve, respectively. The assay was satisfactory, resulting in

mean recovery from 97.90 to 103.83% with an RSD of less

than 2.17% for the six standards (Table 4).

The results of the validation experiments indicate that

the developed UPLC method was sensitive, repeatable and

accurate for the quantitative determination of phenolic

compounds in A. minor.

3.4 Sample analysis

The established analytical method was then subsequently

applied to a simultaneous determination of six major phenolic

compounds, including syringin (3), euoniside (5), fraxin-8-O-

b-D-glucopyranoside (6), umbelliferone (8), scopoletin (9) and

isofraxidin (10) in the methanol extracts of A. minor.The amounts of compounds 3, 5, 6, and 10 were gener-

ally high, ranging from 11.15 to 16.78 mg/g in the crude

extracts of A. minor, and the amounts of compounds 8 and 9were 5.43 and 5.14 mg/g, respectively (Table 4). These results

indicate that A. minor is a rich source of phenolic compounds.

4 Concluding remarks

The RSM BBDs were successfully applied to optimise three-

step gradient profiles as well as the column temperature

used in UPLC. Under optimal UPLC conditions, a high

degree of separation of the phenolic compounds in A. minorwas achieved. This optimised UPLC-Q-TOF-MS method has

been used for simultaneous qualitative and quantitative

analysis of the phenolic compounds in A. minor. Accurate

mass measurements (o5 ppm) and definite elemental

compositions were obtained for molecular ions and

fragment ions. A total of 23 phenolic compounds were

unequivocally or tentatively identified via comparisons with

authentic standards and the literature. In addition, this

UPLC method has been validated as a rapid, sensitive,

accurate and reproducible method, which may serve as an

effective standardisation technique for quality control of this

valued Tibetan medicine.

This research was supported by the National Key TechnologyR&D Program of China (2007BAI31B02) and the NationalNatural Science Foundation of China (30973634 & 21072185).

The authors have declared no conflict of interest.

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