Simultaneous determination of 15 components in Radix Glehniae by high performance liquid chromatography–electrospray ionization tandem mass spectrometry

Food Chemistry 120 (2010) 886–894

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Simultaneous determination of 15 components in Radix Glehniae by high performance liquid chromatography–electrospray ionization tandem mass spectrometry Wei Yang, Chao Feng, Dezhi Kong, Xiaowei Shi, Xuguang Zheng, Yang Cui, Man Liu, Lantong Zhang, Qiao Wang * Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, PR China

a r t i c l e

i n f o

Article history: Received 7 January 2009 Received in revised form 22 October 2009 Accepted 24 October 2009

Keywords: HPLC–ESI–MS MRM Radix Glehniae Quantification

a b s t r a c t A novel qualitative and quantitative method using high performance liquid chromatography coupled with tandem mass spectrometry was developed for simultaneous analysis of 15 components including 10 coumarins, four phenolic acids and adenosine in Radix Glehniae, an important traditional Chinese medicine. The separation was performed on a C18 column with isocratic elution consisted of 0.1% formic acid and methanol (30:70, v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring (MRM) scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). The results indicated that the method was simple, rapid, specific and reliable. And we successfully applied it to analyze 20 Radix Glehniae samples from different sources. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Glehnia littoralis (G.) Fr. Schmidt ex Miq. (Umbelliferae) is a perennial herb native to northern Pacific countries, such as China, Japan, Canada and American (Li, Zhang, & Guan, 2008; Noboru, Chang, Lesley, & Bruce, 2002). Radix Glehniae, the dried roots of Glehnia littoralis, is widely used in these countries. It is an important traditional Chinese medicine called ‘‘Beishashen” in Chinese. Usually it was used as tonic, antiphlogistic and mucolytic medicine for the treatment of respiratory and gastrointestinal disorders in China (Lei & Zhang, 1998). More than 20 Chinese medicinal preparations containing Radix Glehniae are listed in China national drug standard, such as Zixinyin oral solution, Shenmaidihuang pills and Fuzhengyangyin pills, etc. Apart from mentioned above, Radix Glehniae is also a famous nutritional and health-care food usually used in Chinese cooking (Liu, 2006). In Japan, Radix Glehniae demonstrated its diaphoretic, antipyretic, and analgetic actions (Sasaki, Taguchi, Endo, & Yosioka, 1980) and was also welcomed as an edible plant (Satoh, Narita, Endo, & Nishimura, 1996). In Canada, Haida people applied it to remedying bladder infections and other ailments (Matsuura, Saxena, Farmer, Hancock, & Towers, 1996). Modern pharmacological studies suggested that Radix Glehniae

* Corresponding author. Tel.: +86 0311 86265625. E-mail address: [email protected] (Q. Wang). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.10.063

could mitigate, eliminate phlegm, relieve pain (Okuyama et al., 1998), as well as displayed outstanding antibacterial and antifungal bioactivities (Takahiro, Mitsuo, & Masaki, 1998). Recently, it was reported that Radix Glehniae was able to be a cheaper substitute for Panax quinquefolium with regard to its significant antioxidant effects (Ng, Liu, & Wang, 2004). A number of compounds including coumarins (Sasaki et al., 1980), phenolic acids (Yuan, Tezuka, Fan, Kadota, & Li, 2002), adenosine (Yuan et al., 2002), coumarin glycosides (Kitajima, Okamura, Ishikawa, & Tanaka, 1998), flavonoids (Yuan et al., 2002), etc. had been isolated from Radix Glehniae. Among them, the first three type of compounds including coumarins, phenolic acids and adenosine were proved to be responsible for the similar biological activities of the herb. Coumarins showed anti-tumor, anti-microbial, anti-inflammatory and anti-oxidative effects (Hao, Wang, Fu, & Yang, 2008; Mohd, Maryati, Asmah, & Jeffrey, 2009). The phenolic acids also exhibited the same effects (Delaquis, Stanich, & Toivonen, 2005; Gulcin, 2006; Wang et al., 2005). It was worth noting that adenosine acted as an important regulator of the inflammatory function of various cell types belonging to the immune system (Sitkovsky et al., 2004). Therefore, quantification of those compounds in Radix Glehniae would be of great significance for the quality evaluation of the herb. Unfortunately, assay method for Radix Glehniae was not described even in Chinese Pharmacopoeia (2005). Only a few studies

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on quantitative determination of chemical constituents in Radix Glehniae using reverse phase high performance liquid chromatography coupled with ultraviolet detector (HPLC–UV) have been reported (Li & Shi, 2005; Noboru et al., 2002). The constituents in Radix Glehniae are complex, and some of them usually are of low content. It is particularly difficult to simultaneously determine much more active constituents by HPLC–UV method in a short analysis run. Therefore, a more sensitive, selective and rapid method is demanded. The emergence of high performance liquid chro-

matography connected with tandem mass spectrometry (HPLCMS/MS) makes the determination possible. It can not only provide adequate structural information but also perform accurate quantification of multiple compounds. It is a powerful approach to solve the problems encountered in conventional means for quality control of traditional Chinese medicine such as HPLC–UV, TLC. In the present study, we firstly developed and validated a simple and accurate HPLC–ESI–MS method for simultaneous determination of 15 components in Radix Glehniae, including adenosine

NH2 N

HO

N

NH CH3O

N

O O

OH HO

O

O

O

O OH

OH

1. Adenosine

2. Scopoletin

3. Xanthotoxol OCH3

O

O

O

O

O

O

O

O

OCH3

O

OCH3

4. Xanthotoxin

5. Psoralen

6. Isoimpinellin O

OCH3

O

O

O

O

O

O

O

O

O

O

O

8. Oxypeucedanin

7. Bergapten

9. Imperatorin

O

O O

O COOH

O

O

HO

O

OCH3

OH

HO

O O

O

OH

11. Isoimperatorin

10. Cnidilin COOH

HO

12. Chlorogenic acid

COOH COOH

HO

OCH3

HO

OH

OH

13. Caffeic acid

14. Vanillic acid

OCH3

15. Ferulic acid

Fig. 1. Chemical structures of the 15 compounds in Radix Glehniae.

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(1), 10 coumarins (scopoletin, 2; xanthotoxol, 3; xanthotoxin, 4; psoralen, 5; isoimpinellin, 6; bergapten, 7; oxypeucedanin, 8; imperatorin, 9; cnidilin, 10; isoimperatorin, 11) and 4 phenolic acids (chlorogenic acid, 12; caffeic acid, 13; vanillic acid, 14; ferulic acid, 15). Their structures are listed in Fig. 1. During method development, multiple-reaction monitoring (MRM) was employed and an electrospray ionization source was operated in positive and negative mode at the same time. Furthermore, an informationdependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold so that the 15 components could be identified through enhanced product ion (EPI) scans. In addition, 20 batches of Radix Glehniae from different sources were compared using the developed method. 2. Experimental 2.1. Materials and reagents All Radix Glehniae samples were collected from their habitat plantations or purchased from the drug stores. Samples No. 1–17 were unpeeled crude herb collected from Neimeng, Shandong and Hebei province of China, respectively. Samples No. 18–20 were peeled commercial decoction pieces bought from the Factory of Traditional Chinese Medicine Sliced Tablets in the three provinces described above, respectively. Voucher specimens were deposited in the herbarium of School of Pharmacy, Hebei Medical University. Adenosine (110879–200202), scopoletin (110768–200504), psoralen (110739–200613), imperatorin (110826–200511), isoimperatorin (110827–200407), chlorogenic acid (110753–200413), caffeic acid (110885–200102), vanillin acid (110776–200402) and ferulic acid (110773–200611) were purchased from the China Institute for Control of Pharmaceutical and Biological Products. Bergapten, xanthotoxin, xanthotoxol, isoimpinellin were obtained from Shanghai Tauto Biotech Co., Ltd, China. Cnidilin and oxypeucedanin were isolated and purified from the roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. in our laboratory. The dried roots of Angelica dahurica were extracted with 75% ethanol under reflux. The extract was partitioned with petroleum ether and acetate. The acetate extract was evaporated to dryness under reduced pressure and then subjected to silica gel column chromatograph eluted with petroleum ether/acetate (3:1) to give fractions containing cnidilin and oxypeucedanin. Subsequently, the fractions were further separated and purified by preparative HPLC using an electrolyte-free mobile phase H2O/ CH3OH (25:75) as mobile phase to yield cnidilin and oxypeucedanin. The two compounds were identified by comparison of their 1H NMR, 13C NMR and MS data with the literature data (Gu et al., 2008; Harkar, Razdan, & Waight, 1984; Yang, Deng, Zhou, & Wu, 2002). The purities of all constituents were more than 98% according to HPLC analysis. HPLC grade methanol (Fisher, USA) was used for HPLC analysis. Deionized water was produced by Heal Force-PWVF Reagent Water System (Shanghai CanRex Analyses Instrument Corporation Limited, China). Analytical grade methanol (Tianjin Chemical Corporation, China) was used for sample preparation. Formic acid was HPLC grade purchased from Diamond Technology Incorporation.

column (150 mm  4.6 mm, 5 lm), and the column temperature set at 25 °C. The mobile phase consisted of (A) methanol and (B) 0.1% aqueous formic acid. Elution was performed by means of an isocratic gradient 30:70 (A:B, v/v) for 11 min. The flow rate was 1.0 ml/min. 2.2.2. Mass spectrometer Determination was performed using a 3200 QTRAPTM system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer equipped with Turbo V sources and TurboIonspray interface. The instrument operated using electrospray ionization source in positive and negative mode simultaneously. The ion spray voltage was set to 5500 V and 4500 V, respectively. The turbo spray temperature was maintained at 500 °C. Nebulizer gas (gas 1) and heater gas (gas 2) was set at 40 and 50 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. Multiple-reaction monitoring (MRM) mode was employed for quantification. The precursor-to-product ion pair, declustering potential (DP) and collision energy (CE) for each analyte are described in Table 1. The dwell time of each ion pair was 10 ms. In IDA criteria, the former target ions were excluded for 15 s and 3 most intense fragment ions of each analyte were select to perform product

Table 1 The retention time, MS/MS fragment ions, declustering potential (DP) and collision energy (CE) of the 15 components in Radix Glehniae.

2.2. Instrumentation and conditions 2.2.1. Liquid chromatography An Agilent 1200 liquid chromatography system (Agilent, USA), equipped with a quaternary solvent delivery system, an auto-sampler and a column compartment were used. The chromatographic separation was performed on an Agilent Zorbax Eclipse XDB-C18

a

Compounds

Retention times (min)

MS1 (m/z)

MS2 (m/z)

DP (V)

CE (eV)

Adenosine Scopoletin

1.47 1.76

268.3a 193.1a

10 15

20 30

Xanthotoxol

2.02

203.2a

20

32

Xanthotoxin

2.47

217.1a

10

25

Psoralen

2.51

187.2a

35

30

Isoimpinellin

2.87

247.1a

30

35

Bergapten

3.11

217.1a

100

40

Oxypeucedanin

3.59

287.2a

60

24

Imperatorin

5.26

271.1a

25

15

Cnidilin

6.37

301.1a

15

15

Isoimperatorin

9.10

271.1a

100

40

Chlorogenic acid Caffeic acid Vanillic acid

1.48

353.2a

136.1a 178.1 150.1 133.0a 175.1 147.1a 119.1 202.0a 174.0 161.1 143.1 131.1a 115.1 232.1 217.0a 189.2 161.2 202.0 174.0a 146.2 203.1a 159.2 147.2 203.2a 175.2 147.1 233.1a 218.1 173.1 203.2 159.2 147.1a 191.2a

1.61 1.68

a

179.3 167.3a

a

Ferulic acid

1.77

193.3a

Monitored MRM transitions.

135.3 152.2 108.1a 178.2 149.2 134.2a

35

25

30 30

25 20

30

20

W. Yang et al. / Food Chemistry 120 (2010) 886–894

ion scan. All instrumentations were controlled and synchronized by Analyst software (versions 1.4.2) from Applied Biosystems/ MDS Sciex. 2.3. Preparation of standard solutions The appropriate amount of standards were accurately weighted and dissolved in methanol to make 15 kinds of stock solutions, respectively. Then, each stock solution was diluted and mixed with methanol–water (75:25) to prepare a final mixed standard solution containing 0.513 lg/mL of adenosine, 0.425 lg/mL of scopoletin, 0.0390 lg/mL of xanthotoxol, 2.16 lg/mL of xanthotoxin, 2.27 lg/mL of psoralen, 0.336 lg/mL of isoimpinellin, 1.68 lg/mL of bergapten, 0.0155 lg/mL of oxypeucedanin, 1.33 lg/mL of imperatorin, 0.186 lg/mL of cnidilin, 1.47 lg/mL of isoimperatorin, 0.500 lg/mL of chlorogenic acid, 0.161 lg/mL of caffeic acid, 0.229 lg/mL of vanillic acid, 0.985 lg/mL of ferulic acid, respectively. 2.4. Sample preparation All crude drug samples were powdered to a homogeneous size by a mill, sieved through a No. 40 mesh sieve and further dried at 37 °C until constant weight. The dried powder of samples (0.2 g) was accurately weighed and extracted with 5.0 mL of 75% methanol in an ultrasonic ice-water bath for 30 min. The extracted solution was adjusted to the original weight by adding 75% methanol, and then the aliquot of the supernatant was filtered through a 0.45 lm microporous membrane before HPLC injection of 5 lL. 2.5. Peak identification Exact identification of each analyte is a prerequisite for successful quantification. In structural identification experiment, the information-dependent acquisition (IDA) method was employed to trigger the enhanced product ion (EPI) scans by analyzing MRM signals. All the peaks of target compounds in Radix Glehniae samples solution were unambiguously identified by comparison of retention time, parent and product ions with standards in MRM– IDA–EPI spectra. The retention time, characteristic MS/MS fragment ions data of each constituent are listed in Table 1. 2.6. Statistical analysis Data for content of samples are expressed as mean ± standard deviation. The effect of sample collecting time and processed procedure on the total amount of the analytes was analyzed using Nonparametric ANOVA, one-sided test of Wilcoxon Two-Sample Test (SAS Institute Inc., Cary, NC). Differences were considered to be significant when the P value was <0.05. 3. Results and discussion 3.1. Optimization of HPLC-MS/MS conditions The 15 analytes were at first characterized according to their mass spectra from syringe pump infusion analysis to ascertain their precursor ions and select product ions for use in MRM, respectively. The electrospray interface was used and good sensitivity and fragmentation were obtained. Atmospheric pressure ionization interface had been tested, but no obvious improvement was observed. It was also found that all the analytes could be ionized under positive and negative electrospray ionization conditions. According to sensitivity and reproducibility of dominated ions in full scan mass spectra, positive mode was finally selected for the

889

detection of adenosine and coumarins (compounds 2–11), while negative mode for phenolic acids (compounds 12–15). In the full scan mass spectra, the protonated molecular ions [M+H]+ and deprotonated molecular ions [M H] were stable and exhibited higher abundance, thus [M+H]+ and [M H] were chosen as the precursor ions for MS/MS fragmentation analysis of compounds 1–11 and 12–15, respectively. Declustering potential (DP) is one of the most important mass spectrometer parameters impacting ion response. Therefore DP was optimized in order to obtain the maximum sensitivity of [M+H]+ and [M H] . In MS/MS analysis, only precursor ion was isolated and then dissociated into product ions. Several fragment ions of the analytes were observed in the product ion spectra of [M+H]+ and [M H] from the analytes and the predominant fragment ions were chosen in MRM acquisition for quantification except bergapten and isoimperatorin. There were two pairs of isomers, bergapten and xanthotoxin, isoimperatorin and imperatorin, which could produce similar precursor and product ions. Therefore different MRM transitions were selected in order to recognize and calculate those isomers more easily. The most suitable collision energy was also determined by observing the maximum response for the MS/MS monitoring fragment ion. Both xanthotoxin and bergapten could produce ion [M+H]+, m/z 217.1, their product ions were similar which include ions m/z 174.0 and 202.0 (Fig. 2). Thus, when transition m/z 217.1/174.0 and 217.1/202.0 were determined in samples, the peaks of xanthotoxin and bergapten could be found simultaneously. Likewise, imperatorin and isoimperatorin could be monitored at the same time in samples with m/z 271.1/203.2 and m/z 271.1/147.1. Furthermore, both imperatorin and isoimperatorin could produce fragment ion m/z 203.2 in ion source, and fragment m/z 147.1 was the daughter ion of m/z 203.2. Thus, the peaks of imperatorin and isoimperatorin could also be detected when scanning with transition m/z 203.2/ 147.1 used for determining xanthotoxol. Different types of chromatographic columns were tested including Agilent Zorbax SB-C18, Zorbax Eclipse XDB-C18 column and Zorbax Extend-C18. There were no significant difference in separation efficiency on these columns, but the peak shapes were distinct. On Zorbax SB-C18, the peaks were more symmetric and sharp, so it was selected for analysis. Different mobile phase compositions were compared in view of achieving higher peak responses and shorter analysis time of target compounds in chromatograms. Moreover, the complete separation of xanthotoxin and bergapten, imperatorin, isoimperatorin and xanthotoxol must be achieved, for they could be monitored with the same transitions as described above. As a result, acetonitrile was not used due to its resemblance with methanol in ionization of the analytes. It was also found that an acidic eluent (adding 0.1% formic acid) was benefit for enhancing the ionization of adenosine and coumarins detected in positive electrospray interface mode and could guarantee sharp peak shape and reproducible retention time for phenolic acids. Although ionization of the phenolic acid was suppressed owing to existing of formic acid in mobile phase, it could not influence their quantification, which could be proved by good sensitivity and accuracy of analysis. The mobile phase of methanol and 0.1% formic acid (70:30, v/v) was performed so that all the 15 analytes could be eluted in 11 min and could not be interfered by other compounds. The typical extract ions chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of standards and sample (No. 8) are shown in Fig. 2. 3.2. Optimization of extraction method Variables involved in the procedure such as extraction method, solvent and time were investigated so as to obtain satisfactory extraction efficiency and quantitative results. It was showed that

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W. Yang et al. / Food Chemistry 120 (2010) 886–894

Positive ion mode

A

C

B 1.47

271.1

1.47

136.1

3.6e8

1.4e5

Adenosine 268.3

0.0

2.0

4.0

6.0

8.0

0.0

10.0

2.0

4.0

6.0

8.0

50

10.0

100

150

200

250

1.76 1.76

7.2e4

6700

133.0

8.5e7

Scopoletin 193.1

0.0

2.0

4.0

6.0

8.0

10.0

0.0

2.0

4.0

6.0

8.0

5.26

2.02

100

150

200

147.1

1.4e7

2.0 4.0 2.47

6.0

8.0

10.0

0.0

203.2

2.0

4.0

6.0

8.0

50

10.0

100

150

3.11

2.0

4.0

8.0

217.1

4.0

0.0

10.0

6.0

8.0

10.0

50

100

150

200

250

300

2.51

2.51

9.5e5

Intensity, cps

6.0

250

Xanthotoxin

2.47

0.0

200 202.0

7.0e6

6.5e4

1.5e4

300

Xanthotoxol

2.02 0.0

250

9.10

3.0e4

2.4e4

50

10.0

1.9e4

131.1

2.5e6

Psoralen 187.2

0.0

2.0

4.0

6.0

8.0

2.87

4.4e5

10.0

0.0

2.0 4.0 2.87

6.0

8.0

10.0

50

150

100

1.6e9

4.6e4

200

217.0 Isoimpinellin 247.1

0.0

2.0

4.0 3.11

6.0

8.0

10.0

0.0

4362

2.7e4

2.0

4.0 3.11 2.47

6.0

8.0

10.0

50

100

150

200

250

300

2.1e8 174.0 217.1 Bergapten

0.0

2.0

4.0

6.0

8.0

10.0

0.0

2.0

4.0

6.0

8.0

50

10.0

100

150

200

250

300

3.59 4600

3.59

4600

203.1 Oxypeucedanin

5.6e6

287.2 0.0

2.0

4.0

6.0

8.0

10.0

0.0

2.0

4.0

6.0

8.0

10.0

50

100

150

200

250

300

5.26 5.26

9.10

1.20e5

5.2e4

203.2

2.6e6

Imperatorin 271.1

0.0

2.0

4.0

5.8e4

6.0 6.37

8.0

10.0

0.0

2.0

4.0

6.0

8.0

10.0

50

100

150

5.4e4

2.0

4.0

6.0

Cnidilin 301.1

8.0

10.0

0.0

2.0

4.0

6.0

8.0

100

10.0

200

300

5.26

400

Isoimperatorin 1.7e7

9.10 2.8e4

1.4e5

300

233.1

9.4e6 6.37

0.0

250

200

8.01

9.10 147.1 0.0

2.0

4.0

6.0

8.0

10.0

Time, min

0.0

2.0

4.0

6.0

8.0

10.0

Time, min

50

100

150

271.1 200

250

300

m/z, amu

Fig. 2. Representative extract ions chromatograms (XIC) of multiple-reaction monitoring (MRM) chromatograms of adenosine, scopoletin, xanthotoxol, xanthotoxin, psoralen, isoimpinellin, bergapten, oxypeucedanin, imperatorin, cnidilin, isoimperatorin, chlorogenic acid, caffeic acid, vanillic acid and ferulic acid: (A) standards, (B) Radix Glehniae sample (No. 8) and (C) product ion scan spectra of 15 standards.

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W. Yang et al. / Food Chemistry 120 (2010) 886–894

Negative ion mode A

C

B 1.48

1.48

3600

1.09e4

191.2

7.9e7

Chlorogenic acid 353.2

0.0

1.0

Intensity, cps

5.5e4

2.0

3.0

0.0

4.0 120

1.61

2.0

1.0

3.0

100

4.0

1.61

3.0e7

200

300

400

Caffeic acid

135.3 179.3

0.0

1.0

2.0

3.0

0.0

4.0

1.68

1.0

2.0 1.68

3.0

4.0

650

100

150

200

250

5183

108.1

0.0

1.0

2.0

3.0

0.0

4.0

1.0

2.0

3.0

50

4.0

167.3

100

150

200

1.77 7540

9.5e7

1.77

2.2e4

300

Vanillic acid

7.5e7

Ferulic acid

134.2 193.3

2.02 0.0

1.0

2.0

3.0

4.0

0.0

1.0

Time, min

2.0

3.0

4.0

50

100

Time, min

150

200

250

300

m/z, amu

Fig. 2 (continued)

ultrasonic extraction was a preferred method, because its extraction efficiency was similar to refluxing extraction, as well as it was a very convenient method. Different concentrations of solvents such as 30%, 50%, 75% and 100% methanol were also compared. The results demonstrated that 75% methanol was the most effective solvent. Then, the samples were extracted with 75% methanol by ultrasonic extraction for 15, 30, 45 and 60 min to screen optimal extraction time. Consequently, 30 min was the most appropriate for all the compounds could be entirely extracted within this period of time.

MS conditions mentioned above versus their injected mass. As seen in Table 2, linearity of analytical response was good with square of correlation coefficients higher than 0.997, offering a dynamic range of about two orders of magnitude. Limit of detection (LOD) is the lowest mass of a compound that can be detected, while limit of quantification (LOQ) is the lowest mass of a compound that can be accurately and precisely qualified. Typically, they are three times and ten times noise level, respectively. For each target constituent, the LOD and LOQ were determined by serial dilution of standard solution under the described HPLC-MS/MS conditions. Detailed data are presented in Table 2.

3.3. Validation of the assay 3.3.1. Linearity, limit of detection and limit of quantification The mixed standard solution of different volume (1, 2, 4, 8, 16 and 32 lL) was injected to HPLC, respectively. Each calibration curve consisted of 6 different injected mass and was set up with the peak areas of the standards determined under the HPLC–ESI–

3.3.2. Precision Intra-day precision was examined by analyzing the standard solution within 1 day, and inter-day precision was determined for 3 independent days. Both assays were determined by performing three different concentration levels (high, medium and low) of the standards. The results showed that the intra- and inter-day

Table 2 Linear regression data, LOD and LOQ of the 15 active components in Radix Glehniae. Compounds Adenosine Scopoletin Xanthotoxol Xanthotoxin Psoralen Isoimpinellin Bergapten Oxypeucedanin Imperatorin Cnidilin Isoimperatorin Chlorogenic acid Caffeic acid Vanillic acid Ferulic acid

Regression equation 5

4

Y = 1.45e X + 6.51e Y = 9.32e4X + 3.11e4 Y = 1.37e5X + 3.69e3 Y = 1.25e5X + 3.71e5 Y = 2.05e5X + 6.99e5 Y = 6.37e5X + 2.47e5 Y = 2.46e4X + 3.17e4 Y = 2.76e5X + 797 Y = 4.44e5X + 5.48e5 Y = 3.32e5X + 5.40e4 Y = 4.67e4X + 3.60e4 Y = 3.40e4X + 5.36e3 Y = 1.23e5X + 5.85e3 Y = 7.02e3X + 1.42e3 Y = 3.16e4X + 2.76e4

r2

Linear range (ng)

LOD (ng)

LOQ (ng)

0.9997 0.9985 0.9979 0.9987 0.9986 0.9984 0.9977 0.9999 0.9989 0.9998 0.9983 0.9996 0.9998 0.9991 0.9999

0.513–16.400 0.425–13.600 0.039–1.248 2.160–69.100 2.270–72.600 0.336–10.752 1.680–53.800 0.015–0.496 1.330–42.600 0.186–5.950 1.470–47.000 0.500–16.000 0.161–5.152 0.229–7.328 0.985–31.520

0.002 0.042 0.019 0.159 0.024 0.007 0.136 0.008 0.036 0.046 0.147 0.004 0.008 0.012 0.008

0.007 0.109 0.061 0.312 0.071 0.032 0.337 0.026 0.118 0.181 0.433 0.024 0.030 0.035 0.030

In the regression equation Y = aX + b, X refers to the sample injection amount, Y the peak area, and r2 is the correlation coefficient of the equation. LOD, limit of detection; LOQ, limit of quantification.

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precisions (RSD) for the investigated components were less than 3.6% and 4.6%, respectively. 3.3.3. Accuracy Accuracy was determined by adding the mixed standard solutions with three different concentration levels (high, middle and low) to the known amounts of Radix Glehniae samples. Then the resultant samples were extracted and analyzed with the proposed method and triplicate experiments were performed at each level. The percentage recoveries were calculated according to the following equation: (total detected amount original amount)/added amount  100%. The results showed the average recovery were in the range of 96.6–104.6% with RSD ranging from 1.1% to 4.3%. The concentration levels and the detailed results are summarized in Table 3. 3.3.4. Repeatability and stability Six replicates of samples from the same batch were extracted and analyzed with the proposed method. The RSD value was calcu-

Table 3 Recoveries of the 15 active components in Radix Glehniae (n = 6). Compounds

Amount added (lg)

Amount founda (lg)

Recoveryb (%)

RSDc (%)

Adenosine

0.513 2.050 4.100 0.425 1.700 3.400 0.039 0.155 0.312 2.160 8.640 17.300 2.270 9.080 18.500 0.336 1.344 2.700 1.680 6.720 13.400 0.016 0.062 0.124 1.330 5.320 10.600 0.186 0.744 1.490 1.470 5.880 11.800 0.250

0.519 ± 0.020 2.046 ± 0.052 3.960 ± 0.085 0.434 ± 0.015 1.773 ± 0.044 3.530 ± 0.104 0.039 ± 0.001 0.153 ± 0.003 0.310 ± 0.007 2.258 ± 0.096 8.913 ± 0.207 17.663 ± 0.259 2.324 ± 0.047 9.428 ± 0.331 19.160 ± 0.218 0.346 ± 0.015 1.352 ± 0.035 2.734 ± 0.090 1.715 ± 0.072 6.655 ± 0.172 13.253 ± 0.262 0.016 ± 0.000 0.061 ± 0.001 0.129 ± 0.005 1.352 ± 0.036 5.430 ± 0.125 10.321 ± 0.157 0.192 ± 0.003 0.747 ± 0.029 1.507 ± 0.035 1.536 ± 0.055 6.014 ± 0.180 11.932 ± 0.265 0.260 ± 0.007

101.2 99.8 96.6 102.1 104.3 103.8 100.9 98.4 99.4 104.6 103.2 102.1 102.4 103.8 103.6 102.8 100.6 101.7 102.1 99.0 98.9 102.5 98.9 104.4 101.6 102.1 97.4 103.1 100.4 101.3 104.5 102.3 101.5 104.0

3.9 2.5 2.1 3.5 2.5 3.0 2.1 1.9 2.3 4.3 2.3 1.5 2.0 3.5 1.1 4.3 2.6 3.3 4.2 2.6 2.0 3.1 1.3 3.9 2.7 2.3 1.5 1.6 3.9 2.3 3.6 3.0 2.2 2.7

1.000 4.000 0.081 0.322 1.288 0.115 0.458 1.832 0.493 1.970 7.880

1.045 ± 0.021 4.032 ± 0.148 0.079 ± 0.002 0.332 ± 0.005 1.330 ± 0.041 0.111 ± 0.004 0.444 ± 0.011 1.882 ± 0.037 0.515 ± 0.019 1.991 ± 0.040 7.921 ± 0.109

104.5 100.8 98.7 103.4 103.1 97.3 96.9 102.7 104.6 101.1 100.5

2.1 3.7 2.3 1.5 3.1 3.3 2.4 1.9 3.7 2.0 1.4

Scoploetine

Xanthotoxol

Xanthotoxin

Psoralen

Isoimpinellin

Bergapten

Oxypeucedanin

Imperatorin

Cnidilin

Isoimperatorin

Chlorogenic acid

Caffeic acid

Vanillic acid

Ferulic acid

a b c

Mean ± standard deviation. Recovery (%) = amount found/amount added  100. RSD (%) = (SD/mean)  100.

lated as a measurement of method repeatability. The RSD values of the 15 compounds in these samples were less than 4.9%, which showed high repeatability of the method. In order to investigate the stability of the samples, the sample solution was analyzed every 12 h within 3 days and all analytes were found to be stable within 72 h (RSD < 4.7%) when the solution was stored at 4 °C. 3.4. Advantages of HPLC-MS/MS Customarily, the herbal chemical constituents involving conjugation groups in structures were analyzed with HPLC–UV method. Yet, converting the method from HPLC–UV to HPLC-MS/MS had some potential advantages. The most important one was that the HPLC-MS/MS could determinate the 15 compounds simultaneously when complete separation was not achieved, so that the analysis time was reduced to 11 min by switching the ion source polarity between positive and negative modes in a single chromatographic run. Moreover, MRM scanning mode offered good sensitivity for its significantly decreased level of noise and accordingly the enhanced response of analytes. Thus, the minor constituents in Radix Glehniae such as xanthotoxin and oxypeucedanin also could be accurately measured. In addition, MS detector possessed higher specificity and selectivity than UV detector. It not only cut down the interference of impurity peaks but also further identified the analytes on the base of fragment ions produced from the MRM–IDA–EPI mode. HPLC-MS/MS was also an economical method due to low consumption of solvents and time. In short, although HPLC-MS was apparently more expensive in comparison to other analytical techniques, this could be counterbalanced by its reliability, efficiency, applicability and rapidness. 3.5. Sample analysis The established analytical method was applied to determine the content of the 15 compounds in the 20 batches of Radix Glehniae samples, and the analytical results were summarized in Table 4. As seen from the data of sample No. 1–17 (crud herb), the contents of 15 compounds especially psoralen, xanthotoxin and bergapten varied considerably, which could be demonstrated more clearly with Table 4, the ratios of the maximum content to minimum content of each compound in sample No. 1–17. This result strengthened the reported concept that the amounts of above three coumarins could be remarkably raised in conditions such as bacterial infection and gradual drying at room temperature (Anetai, Masuda, & Takasugi, 1996; Anetai, Masuda, & Takasugi, 1997). It was indicated that besides different locality and collecting time, the storage circumstance was the essential reason for the variation of the three compounds in crud herb of Radix Glehniae. In Table 4, the total amounts of 15 characteristic compounds in each batch of Radix Glehniae samples were described. It was found that the total content of samples No. 1–6, 8 collected at the same time but from different origin varied from 214.726 to 344.038 lg/g, which was only a 1.6-fold variation. Diversely, the samples No. 8–17 collected in different times but from the same origin varied from 344.038 to 1141.378 lg/g, with 3.3-fold variation. The total contents of 15 constituents in samples collected in Oct. 2007 (sample No. 9–17) were higher than that in samples collected in Oct. 2008 (sample No. 1–6, 8), which has statistical significance by controlling P = 0.0005 < 0.05. The results again demonstrated that a number of reasons such as plant origin, growth circumstance and storage time might contribute to the differences in the level of active constituents among various Radix Glehniae samples and storage time was the principal reason. These all suggested that each collecting procedure involved should be standardized in the future to assure the quality of Radix Glehniae.

Table 4 Content of the 15 active components in Radix Glehniae (n = 3). Collection province

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Shandong-1 (2008.10) Shandong-2 (2008.10) Neimeng-1 (2008.10) Neimeng-2 (2008.10) Neimeng-3 (2008.10) Neimeng-4 (2008.10) Neimeng-5 (2008.09) Hebei-1 (2008.10) Hebei-2 (2007.10) Hebei-3 (2007.10) Hebei-4 (2007.10) Hebei-5 (2007.10) Hebei-6 (2007.10) Hebei-7 (2007.10) Hebei-8 (2007.10) Hebei-9 (2007.10) Hebei-10 (2007.10) Shandong (without bark) Hebei (without bark) Neimeng (without bark) Ratiob

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a b c

Shandong-1 (2008.10) Shandong-2 (2008.10) Neimeng-1 (2008.10) Neimeng-2 (2008.10) Neimeng-3 (2008.10) Neimeng-4 (2008.10) Neimeng-5 (2008.09) Hebei-1 (2008.10) Hebei-2 (2007.10) Hebei-3 (2007.10) Hebei-4 (2007.10) Hebei-5 (2007.10) Hebei-6 (2007.10) Hebei-7 (2007.10) Hebei-8 (2007.10) Hebei-9 (2007.10) Hebei-10 (2007.10) Shandong (without bark) Hebei (without bark) Neimeng (without bark) Ratiob

Contenta (lg/g) Adenosine

Scopoletin

43.439 ± 0.916 56.659 ± 0.911 92.366 ± 2.905 98.499 ± 1.790 70.969 ± 1.114 108.857 ± 3.003 29.810 ± 0.830 103.814 ± 2.003 28.175 ± 0.979 27.357 ± 0.728 20.951 ± 0.621 29.674 ± 0.601 21.224 ± 0.696 44.666 ± 1.144 35.807 ± 1.101 17.817 ± 0.591 19.997 ± 0.601 17.209 ± 0.393 10.122 ± 0.200 14.347 ± 0. 315 6.1

22.580 ± 0.598 23.873 ± 0.710 21.855 ± 0.801 19.734 ± 0.444 18.595 ± 0.401 22.217 ± 0.500 28.478 ± 0.601 13.577 ± 0.460 104.538 ± 3.690 50.209 ± 1.102 46.794 ± 1.599 80.737 ± 2.399 34.635 ± 0.601 57.971 ± 0.602 59.523 ± 0.705 51.762 ± 0.688 44.259 ± 1.398 0.168 ± 0.007 0.307 ± 0.012 0.252 ± 0.007 7.7

Imperatorin

Cnidilin

5.093 ± 0.132 4.890 ± 0.141 11.158 ± 0.311 11.484 ± 0.411 20.500 ± 0.612 22.332 ± 0.445 40.038 ± 1.368 30.065 ± 0.657 91.529 ± 2.646 40.242 ± 1.440 78.504 ± 2.779 55.099 ± 2.144 19.828 ± 0.316 42.887 ± 1.143 55.913 ± 1.856 32.508 ± 1.267 53.674 ± 1.338 1.645 ± 0.040 2.701 ± 0.061 1.737 ± 0.052 11.4

2.479 ± 0.043 3.674 ± 0.051 6.739 ± 0.192 7.463 ± 0.160 9.889 ± 0.151 21.894 ± 0.420 7.621 ± 0.141 5.006 ± 0.095 13.166 ± 0.390 4.628 ± 0.176 5.857 ± 0.186 4.911 ± 0.135 3.777 ± 0.081 4.974 ± 0.090 4.722 ± 0.125 3.903 ± 0.134 7.180 ± 0.214 0.765 ± 0.014 1.424 ± 0.053 0.898 ± 0.031 5.3

Xanthotoxol

Xanthotoxin

Psoralen

Isoimpinellin

Bergapten

Oxypeucedanin

0.952 ± 0.029 0.538 ± 0.014 0.798 ± 0.016 0.367 ± 0.008 0.609 ± 0.020 0.538 ± 0.015 2.563 ± 0.068 0.674 ± 0.011 6.737 ± 0.153 1.654 ± 0.051 3.529 ± 0.085 7.378 ± 0.155 4.135 ± 0.134 5.097 ± 0.149 8.768 ± 0.170 5.810 ± 0.162 5.168 ± 0.182 0.240 ± 0.006 0.499 ± 0.007 0.232 ± 0.007 16.3

28.309 ± 0.545 24.435 ± 0.424 8.712 ± 0.143 4.838 ± 0.105 10.079 ± 0.215 43.121 ± 1.343 294.010 ± 8.294 18.054 ± 0.518 424.899 ± 9.112 157.514 ± 3.047 162.983 ± 4.326 412.505 ± 11.124 124.700 ± 3.062 171.186 ± 5.039 183.491 ± 5.037 194.429 ± 4.253 173.009 ± 5.173 5.132 ± 0.162 6.272 ± 0.150 5.110 ± 0.137 87.6

7.999 ± 0.282 6.420 ± 0.176 5.148 ± 0.121 4.326 ± 0.030 2.447 ± 0.086 0.420 ± 0.007 76.810 ± 2.223 10.907 ± 0.360 163.986 ± 4.863 90.854 ± 2.618 69.579 ± 1.886 167.311 ± 2.467 66.006 ± 1.320 86.699 ± 2.309 89.192 ± 3.073 71.823 ± 2.290 85.868 ± 1.434 5.088 ± 0.120 5.138 ± 0.114 4.124 ± 0.138 398.7

3.420 ± 0.090 3.260 ± 0.107 3.260 ± 0.091 3.059 ± 0.120 5.710 ± 0.082 7.638 ± 0.125 78.412 ± 1.539 11.253 ± 0.302 29.971 ± 1.430 17.519 ± 0.338 27.802 ± 0.533 58.329 ± 1.323 15.390 ± 0.301 19.286 ± 0.439 25.030 ± 0.650 50.697 ± 0.910 14.065 ± 0.342 1.046 ± 0.041 3.584 ± 0.092 1.110 ± 0.030 17.9

16.818 ± 0.617 16.891 ± 0.505 12.007 ± 0.324 10.112 ± 0.297 10.623 ± 0.411 4.486 ± 0.134 106.244 ± 4.011 27.677 ± 0.494 99.685 ± 1.940 55.883 ± 1.315 63.900 ± 1.519 133.211 ± 4.053 56.393 ± 1.456 63.171 ± 1.913 65.284 ± 2.065 80.736 ± 2.292 61.932 ± 1.825 1.667 ± 0.032 2.637 ± 0.040 1.441 ± 0.031 29.7

0.558 ± 0.011 0.526 ± 0.014 1.529 ± 0.032 1.593 ± 0.040 3.831 ± 0.122 4.213 ± 0.055 2.738 ± 0.095 4.118 ± 0.130 1.136 ± 0.021 0.444 ± 0.014 1.603 ± 0.046 2.463 ± 0.082 0.895 ± 0.017 1.656 ± 0.062 1.975 ± 0.092 1.412 ± 0.051 1.624 ± 0.042 0.071 ± 0.002 0.112 ± 0.003 0.046 ± 0.001 9.5

Isoimperatorin

Chlorogenic acid

Caffeic acid

Vanillic acid

Ferulic acid

Total amountsc (lg/g)

9.932 ± 0.190 12.347 ± 0.227 19.787 ± 0.520 20.098 ± 0.560 17.920 ± 0.451 15.154 ± 0.475 29.054 ± 0.329 71.723 ± 1.436 61.004 ± 1.231 41.986 ± 0.942 65.153 ± 1.233 75.872 ± 1.723 59.275 ± 1.630 73.798 ± 2.315 68.265 ± 1.926 60.658 ± 2.112 65.153 ± 1.767 1.232 ± 0.040 2.380 ± 0.051 1.387 ± 0.020 7.6

35.045 ± 1.204 25.524 ± 0.319 20.187 ± 0.578 17.951 ± 0.650 21.702 ± 0.635 8.935 ± 0.172 16.653 ± 0.130 20.187 ± 0.564 46.296 ± 0.809 51.778 ± 1.552 68.366 ± 1.334 62.247 ± 1.937 47.146 ± 1.023 40.928 ± 0.987 43.001 ± 1.120 46.850 ± 1.540 53.869 ± 0.847 0.235 ± 0.008 0.261 ± 0.007 0.184 ± 0.005 6.0

16.540 ± 0.460 8.843 ± 0.193 9.672 ± 0.350 9.911 ± 0.210 9.868 ± 0.190 3.937 ± 0.070 1.800 ± 0.041 4.373 ± 0.147 16.387 ± 0.378 20.508 ± 0.521 12.550 ± 0.217 17.554 ± 0.259 9.146 ± 0.167 12.562 ± 0.390 13.613 ± 0.390 9.408 ± 0.265 13.749 ± 0.330 0.219 ± 0.007 0.949 ± 0.016 0.261 ± 0.008 9.2

4. 728 ± 0.090 3.091 ± 0.113 4.809 ± 0.105 4.826 ± 0.138 3.760 ± 0.097 6.332 ± 0.093 13.346 ± 0.150 1.302 ± 0.034 4.559 ± 0.080 2.781 ± 0.101 3.841 ± 0.090 3.203 ± 0.070 2.536 ± 0.049 3.268 ± 0.068 3.639 ± 0.121 2.838 ± 0.081 4.668 ± 0.117 1.908 ± 0.035 3.028 ± 0.077 1.712 ± 0.051 3.7

21.009 ± 0.651 23.755 ± 0.453 18.143 ± 0.379 16.472 ± 0.371 20.233 ± 0.320 14.621 ± 0.340 15.875 ± 0.198 21.308 ± 0.452 27.756 ± 0.537 24.293 ± 0.612 32.651 ± 0.712 30.884 ± 0.980 18.022 ± 0.430 38.902 ± 1.290 27.376 ± 0.797 27.209 ± 0.649 24.651 ± 0.537 0.166 ± 0.002 0.324 ± 0.011 0.092 ± 0.002 2.7

218.900 214.726 236.171 230.733 226.735 284.694 743.453 344.038 1118.824 587.648 664.063 1141.378 483.107 667.051 685.600 657.860 628.867 36.791 39.736 32.934

W. Yang et al. / Food Chemistry 120 (2010) 886–894

Sample No.

Content = Mean ± standard deviation (n = 3). The ratio of the maximum contents to minimum contents of the 15 compounds in Radix Glehniae samples No. 1–17. The total amounts of the 15 compounds in Radix Glehniae samples No. 1–20.

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Traditionally, the root of Glehnia littoralis can be used as Radix Glehniae after being processed including being boiled in water, skinned and dried. This processed procedure is also specified in Chinese Pharmacopeia (2005). However, it was found that the content of 15 compounds in unpeeled Radix Glehniae samples was extremely higher (about 6 times) than that in peeled ones comparing the results of samples No. 1–17 to that of No. 18–20, which is shown in Table 4. The comparison has statistical significance by controlling P = 0.0041 < 0.05. This means that many active components were in the cortex of the root and the peeling lead to reduction of active components. So we proposed that drying root of Glehnia littoralis could be directly used as medicine or pulmentum not needing to remove the barks.

4. Conclusion A novel sensitive and selective HPLC–ESI–MS/MS method operating both negative and positive scanning modes in single analysis process was developed and validated to simultaneously determinate and identify 15 constituents in 20 batches of Radix Glehniae. Among these constituents, scopoletin, xanthotoxol, isoimpinellin and oxypeucedanin in Radix Glehniae were quantitated for the first time. The proposed method showed appropriate accuracy and repeatability, and was successfully utilized to analyze 20 batches of Radix Glehniae samples from different sources. The satisfactory results demonstrated that the HPLC-MS/MS method offered a good alternative for routine analysis due to its rapidness, sensitivity and specificity and could be applied as a reliable quality evaluation method for Radix Glehniae. In the future, HPLC-MS/MS method would be more and more popular in analyzing herb medicine. Acknowledgement The work received financial support from the Significant Foundation of Pharmacy of Heibei Province, China. References Anetai, M., Masuda, T., & Takasugi, M. (1996). Preparation and furanocoumarin composition of Glehnia root produced in Hokkaido. Natural Medicines, 50(4), 399–403. Anetai, M., Masuda, T., & Takasugi, M. (1997). Preparation and chemical evaluation of Glehnia root prepared from Glehnia littoralis cultivated in Tottori prefecture. Natural Medicines, 51(4), 442–446. Delaquis, P., Stanich, K., & Toivonen, P. (2005). Effect of pH on the inhibition of Listeria spp. by vanillin and vanillic acid. Journal of Food Protection, 68(7), 1472–1476.

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