CHEM. RES. CHINESE UNIVERSITIES 2011, 27(4), 550—556

Comparison of Different Cartridges of Solid Phase Extraction for Determination of Polyphenols in Tobacco by UPLC/MS/MS and Multivariate Analysis ZHANG Xia1*, LIU Wei1, XU Yong1, YANG Liu2, KONG Wei-song1, RUI Xiao-dong1, YANG Shuai1, CHEN Yong-kuan1 and MIAO Ming-ming1 1. Key Laboratory of Tobacco Chemistry, Yunnan Academy of Tobacco Science, Kunming 650106, P. R. China; 2. Research and Development Center of Hongta Tobacco(Group) Corporation Limited, Yuxi 653100, P. R. China Abstract The comparison of solid phase extraction(SPE) for the preconcentration and isolation of polyphenols in tobacco samples was carried out by ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and multivariate analysis. Several adsorbing materials of SPE(C18, NH2, SAX and OASIS) were investigated. It was found that the C18 and OASIS cartridges can not only speed up the purification process, but also simplify the SPE operation. A UPLC/MS/MS was used for the determination of polyphenols in tobacco samples after purification. All analytes were separated and determined in 2 min. The limit of detection was 0.05 ng/mL. Cluster analysis(CA) and principal component analysis(PCA) were used for the analysis of 4 varieties(flue-cured tobacco, oriental tobacco, sun-cured tobacco and burley) in order to interpret the effect of planting and machining process on the concentration of polyphenols. The different types of tobacco samples could be easily clustered by CA. PCA on the chemical composition of tobacco resulted in two principal components(PCs) that take 84.2% of the total variance. The PCA and CA indicate that the polyphenols can be used for distinguishing tobacco types. Keywords Solid phase extraction; Polyphenol; Ultra-high performance liquid chromatography/tandem mass spectrometry(UPLC/MS/MS); Tobacco; Cluster analysis; Principal component analysis Article ID 1005-9040(2011)-04-550-07

1

Introduction

The tobacco polyphenol, mainly including tannin, coumarin, flavonoids and proanthocyanidins, is one of the most important category compounds in tobacco. The concentration of polyphenols, to a certain extent, reflects the inherent quality of tobacco. In addition, it is responsible for the color of tobacco leaves and has an important contribution to the sensory quality. So studying the tobacco polyphenols and determining their relative contents can help to identify tobacco, tobacco products and compartmentalize the grade of tobacco[1]. In the present study, we focused our attention on determining the concentration and the bioactivities of tobacco polyphenols[2―4]. Several pretreatment methods have been applied in preconcentration for polyphenols analysis, such as pressed solution extraction[5], ultrasonic extraction[6,7] and solid phase extraction[8―10]. Solid phase extraction(SPE) is one of the simplest, effective and versatile methods for sample preparation[11], it has been widely used in the environment, drugs, clinical medicine and food chemistry[12―17]. Although several authors have tested the different adsorbing materials of SPE(Amberline XAD[18], Dowes 50WX8[19] and Bond Elut C18[20]) to remove the matrix in honey, however, it is difficult to compare the per-

formance of solid sorbents used for the removal and enrichment of the analytes from different matrices. Systematic study for SPE, especially based on the different mechanisms of extraction in tobacco chemistry, has not been reported. In relation to testing the different adsorbing materials, the work of Michalkiewicz[8] should be mentioned. The difference of adsorbing materials(C18, OASIS HLB, Strata-X and Amberlite XAD-2) for the separation of phenolic acid compounds and some flavonols in honey has been compared, but this work was just focused on the recoveries of the eluate of tested compounds. Thus, one of the aims of this work was to investigate in more details about the recoveries of sample load step and elution step. Usually high performance liquid chromatography (HPLC)[21], ultra-high performance liquid chromatography (UPLC)[22] and HPLC/MS/MS[23] are the most widely employed analytical methods for detecting and quantifying polyphenols in tobacco. Whereas the compositions of tobacco are very complicated and the concentrations of many tested compounds are very low and these tested compounds, are sometimes, not displayed in chromatograms or not separated fully by HPLC due to the interference of matrix effect, it maybe leads

——————————— *Corresponding author. E-mail: [email protected] Received February 11, 2011; accepted May 30, 2011. Supported by the Foundation of State Tabacco Monopoly Bureau(China) in Research Project(No.110200902008).

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to quantitative inaccurate. In addition, a shorter analytical time is also necessary to be considered. Although UPLC can evidently reduce the analytical time and improve the separation efficiency[24], it will also encounter the same problems as HPLC when the concentrations of tested compounds are trace. Compared with HPLC/MS/MS, UPLC/MS/MS is a recently developed new analytical technique. The analytical time is greatly reduced because of the chromatographic column with the small-diameter packing (99.999%) was used as curtain and auxiliary gas. MS/MS parameters were optimized by direct infusion at 10 µL/min of 1 µg/mL individual standard solution. Declustering potential(DP), Table 1

Precursor ion,

Production ion,

Rutin

m/z([M–H]+) 609.6

m/za 300.0Q 271.2C

Esculetin

177.0

Compound

1.91 1.5

1.52 1.79 1.23

entrance potential(EP), collision cell entrance potential(CEP), collision energy(CE) and collision cell exit potential(CXP) were established for each analyte. The values are displayed in Table 1. Data acquisition was carried out with different retention time window(Table 1); a dwell time of 100 ms was used. The chromatograms of polyphenols at the above conditions are shown in Fig.2.

UPLC/MS/MS parameters established for MRM acquisition mode(quantitation confirmation)

Time/min

1.33

Vol.27

Chlorogenic acid Caffeic acid Scopoletin

353.2 179.0 191.0 339.0b

Quercitrin

Parameter DP/V –87.0 –87.0

EP/V –7.8 –7.3

CEP/V –24 –40

CE/V –51 –75

CXP/V –1 –1

133.2Q

–44.6

–9.5

–17

–27

–1

105.0C

–46.0

–8.0

–10

–29

–0.9

191.0Q

–34.1

–5.0

–16

–25

–0.5

353.2C

–40.0

–4.0

–38

–14

–0.9

134.8Q

–45.0

–6.0

–12

–24

–1

106.8C

–45.0

–6.5

–12

–32

–1

176.0Q

–35.0

–6.0

–10

–22

–1

147.8C

–40.0

–6.0

–10

–31

–1

177.0Q

–45.0

–4.0

–11

–32

–0.5

133.0C

–40.0

–4.0

–24

–55

–0.5

a. Q: Quantitation; C: confirmation; b. [M+K]+

Fig.2 Extracted ion chromatograms of an extract in MRM acquisition mode (A) Esculetin; (B) chlorogenic acid; (C) caffeic acid; (D) scopoletin; (E) quercitrin; (F) rutin.

2.4

Statistics Analysis

Concentration of polyphenol compounds was evaluated by statistical method. For classification of 30 tobacco samples (4 varieties), CA and PCA were performed with the help of NTSYSpc(Ver.2.1) and Minitab statistical program.

3 3.1

Results and Discussion Optimization of UPLC/MS/MS Method

The influence of different mobile phase B content on analyte retention was investigated to improve sensitivity and obtain sharper peak shapes. In initial experiments, the separation for polyphenols was investigated with different

volume fractions of ammonium acetate(0.002%, 0.008%, 0.01%, 0.08%, and 0.1%). It was found that with the increase of the volume fraction of ammonium acetate in mobile phase, the separation for polyphenols became poor, and the peak shape was tailing seriously. MS sensitivity for chlorogenic acid and caffeic acid was also sharply lower. It was also found that at the lower volume fraction (0.002%, 0.008%) of ammonium acetate in the mobile phase, some flavonoid polyphenols(rutin, quercitrin and esculetin) had high sensitivity. The results agreed with the literature[32] that there were “LC-electrolyte effects” when the LC mobile phase contained low volume fraction of ammonium acetate for some flavonoid compounds. Based on the profiles of above-mentioned cases, the influence of acetic acid volume fraction(0.1%, 0.2% and 0.3%) on the retention of polyphenols and intensity of response were also investigated

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when the mobile phase changed from ammonium acetate to acetic acid. As the volume fraction of acetic acid increased, the intensity of response for all tested compounds increased with the sharp peak shape obtained. At the same time, the bad peak shape was obtained when the mobile phase was pure water. At last, 0.3% acetic acid was selected as optimum mobile phase. Column temperature was also investigated to improve sensitivity and reduce the analytical time. Temperatures from 30 °C to 55 °C were studied and 45 °C was selected as optimum temperature because of sharper peaks and higher peak heights. Finally, the extracted ion chromatogram of solution in the MRM mode was obtained under these optimized conditions that allowed the separation of the selected compounds in less than 2 min. It substantially decreased the analytical time and obtained higher efficiency.

3.2 Performance of Different SPE Cartridges Based on Different Extraction Mechanism For UPLC/MS/MS analysis of polyphenols in tobacco, a preliminary removal of sugars, pigments and xylogen must be performed. Expect for removing matrix components, isolation and concentration of analytes could be achieved. Degreasing usually not only increases the analytical time, but also leads to the loss of some of tested compounds[33]. The conventional analytical method previously developed is combine’s solid liquid extraction(SLE) to extract tested compounds followed by SPE to degrease and preconcentrate. In order to find out the most suitable cartridge to purify the tobacco samples, the performances of different SPE cartridges(C18 reverse phase cartridge, NH2 normal phase cartridge, SAX anion exchange cartridge and OASIS cartridge) were investigated for separating and enriching the polyphenols of tobacco with the standard solution[3 mL of 80%(volume fraction) methanol-0.3%(volume fraction) acetic acid with appropriate concentration of each analyte]. First of all, the preliminary optimization procedure for C18 and OASIS cartridges was: sample load(3 mL)→washing step [pure water or 0.3%(volume fraction) acetic acid water solution]→elution[80%(volume fraction) methanol or 80%(volume fraction) methanol-0.3% acetic acid]. Finally the optimization for C18 column was: sample load(3 mL)→washing step[0.3% (volume fraction) acetic acid, 3 mL]→elution[80%(volume fraction) methanol, 3 mL]; for OASIS was: sample load(3 mL)→washing step(pure water, 3 mL)→elution[80%(volume fraction) methanol-0.3% acetic acid, 3 mL]. The optimization routine for NH2 and SAX column was: sample load(3 mL)→ washing step(pure water, 3 mL)→elution[0.1%(volume fraction) acetic acid-80%(volume fraction) methanol, 0.3%(volume fraction) acetic acid-80%(volume fraction) methanol, 0.5% (volume fraction) acetic acid-80%(volume fraction) methanol, 1%(volume fraction) acetic acid-80%(volume fraction) methanol, 3 mL]. The extract of sample load(SL) step and elution(E) step were collected, respectively, for each of SPE cartridge.

3.2.1

NH2 Normal Column

NH2 column is a normal phase extract cartridge with a polar functional group(―NH2) of the short alkyl chain that is

553

bonded in the silica gel, and the interactions with analytes include hydrogen bonds, dipole-dipole interactions, π-π interactions and dipole-induced dipole interactions. In fact, the NH2 packing can be seen as a weak anion exchange resin when the strong polar solvent was used. It is appropriate to isolate and preconcentrate weak anion compounds and organic acid compounds. The recoveries of sample load step and elution step for NH2 cartridge were tested. All of the tested compounds except scopoletin were well retained on the NH2 cartridge. The elution ability was gradually decreased for rutin, caffeic acid and quercitrin with the increased volume fraction of acetic acid. For scopoletin and esculetin, the elution ability was maintained unchangeably. But it was gradually improved for chlorogenic acid. It can be explained that the acid radical ions in solution are neutralized with polar functional group(―NH2) of silica, so the retention of chlorogenic acid on the solid phase was weakened.

3.2.2 SAX Anion Exchange Column SAX cartridge is a strong anion exchange column, the fatty acids quaternary ammonium salt is bonded in the silica gel. The anion in the solution can be exchanged or adsored for its positive charges. The interaction with analytes is mainly electrostatic attraction. Some strong anion compounds(low pH2) are appropriate to be separated and pretreated on an SAX column. The recoveries of sample load step and elution step of SAX were compared, the SAX anion exchange column has the strong adsorption for chlorogenic acid, caffeic acid and esculetin. It was maybe that chlorogenic acid and caffeic acid were easily to be ionized to hydrogen ions and thus to be negatively charged, which made them adsorbed to the positively charged SAX-packing and retained on the column. To some other tested compounds such as rutin, quercitrin, and scopoletin, SAX column has the poor ability to retain them. It could be explained that the interaction with the positive charge of SAX-packing was weak because such substances were relatively more difficult to be ionized. Therefore, such compounds were not retained on SAX column. The elution ability for chlorogenic acid, caffeic acid and esculetin was improved with the volume fraction of acetic acid increasing. It was maybe due to the increased volume fraction of acetic acid(pH value of eluant from 4.1 to 3.5) restraining the ionization of chlorogenic acid, caffeic acid and esculetin and, therefore, weakening the electrostatic attraction with SAX-column. Changing the volume fraction of acetic acid showed less effect on rutin, quercitrin and scopoletin.

3.2.3

C18 And OASIS Cartridges

The retention mechanism of C18 cartridge depends on dispersion or London force. It is appropriate for the isolation and preconcentration of the compound with non-polarity to middle polarity. The retention mechanism of OASIS cartridge depends on van der Waals force, hydrogen bonds or dipole-dipole interactions[34]. OASIS HLB sorbent, a macroporous copolymer made from divinylbenzene and N-vinylpyrrolidone, exhibits both hydrophilic and liphophilic retention characteristics[35].

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CHEM. RES. CHINESE UNIVERSITIES

Fig.3 shows the recoveries of sample load step and elution step on C18 and OASIS cartridges. As can be seen from Fig.3, weak polar lipids, wax and pigment, and other substances were kept on the column and in these cases, polyphenols were not retained. Over 86% of recoveries of all tested compounds were obtained on C18 and OASIS columns. Comparison of NH2, SAX, C18 and OASIS columns makes it clear that C18 and OASIS were suitable for the extraction of polyphenols. It not only makes the tobacco samples purified rapidly, but also realizes the purposes of eliminating the washing step and elution procedure, simplifying

the operation and saving time. Finally, the C18 column was selected to rapidly extract and purify the tobacco samples after taking prices into consideration. But it had greater differences with the literature[8] report in that the recovery for eluate of quercitrin was more than 90% with C18 column. It could be interpreted that the differences of matrix between honey and tobacco are responsible for this phenomenon and the diversities of tested compounds are also to be considered, at the same time, the different pH values of extract play an important role in relating extract efficiency to absorb material.

3.3

Fig.3

Rutin(RU), chlorogenic acid(CLA), esculetin(EL), caffeic acid (CEA), scopoletin(SPL), quercitrin(QR). ―●―Oasis- SL; ―○―oasis-E; ―△―C18-SL; ―▲―C18-E.

Table 2 b

Calibration curves and detection limits for UPLC/MS/MS

LOD / (ng·mL–1)

LOQ / (ng·mL–1)

Linearity range/ (μg·mL–1)

Rutin

0.1

0.33

2―10 0.05―2.0

Esculetin

0.2

0.67

0.001―0.10

Chlorogenic acid

0.5

1.67

Compound

Validation of the Analytical Method

In order to evaluate the performance of the SPE-UPLC/ MS/MS method for the analysis of tobacco samples, the calibration curves, correlation factors(r) of the calibration curves and linearity range, intra-day and inter-day precisions, accuracies and recoveries were studied. Table 2 displays the results obtained. All the polyphenols tested have correlation factors r>0.998. Limit of detection(LOD), limit of quantification(LOQ) and linearity for each compound are also reported in Table 2. The LOD, defined as the lowest analyte concentration with a signal-to-noise(S/N) ratio of 3, and the LOQ, defined as the concentration with an S/N ratio of 10, were evaluated by injecting 5 μL of diluted polyphenols solutions.

Recoveries of sample load step(SL) and elution step(E) on C18 and OASIS SPE cartridges

a

Vol.27

Calibration curve y=135x+1.16×105 y=422x+7.28×103

rc

Recovery±RSDd(%)

0.9990 0.9987

101±6.3 92±3.6

Intra-day RSDe (%)

Inter-day RSDe (%)

6.5 4.3

7.8 5.82 0.39

y=1.19×103x

0.9999

88±0.36

0.17

2.0―8.0

y=302x+3.3×105

0.9987

96±7.2

7.4

8.5

0.05―3.0

y=185x-1.65×103

0.9997

86±4.1

6.38

7.64 7.1

Caffeic acid

1.0

3.33

0.002―0.40

94±5.7

5.1

0.05

0.17

0.002―0.40

y=1.19×103x-1.04×103 y=2.38×103x+2.7×103

0.9997

Scopoletin

0.9993

94±3.8

3.9

7.8

Quercitrin

0.2

0.67

0.001―0.10

y=1.93×103x+534

0.9993

94±0.33

0.46

0.91

a. LOD: limit of detection; b. LOQ: limit of quantification; c. the correlation coefficient. d. n=4; e. n=6.

The obtained LODs were lower than 1 ng/mL for all target 3.4 Application to Tobaccos compounds and four of them lower than 0.2 ng/mL. Precision, accuracy and the recoveries of target compounds were evaUnder the conditions above mentioned, the developed luated by spiked tobacco samples with analytes and labeled analytical method was used to determine polyphenols in standards at a low concentration(10 ng/mL or 100 ng/mL) and tobacco samples. Four varieties of thirty tobacco samples were extracted by the described SPE method. The intra-day precicollected and analyzed(Table 3). The most abundant polyphesions(n=6) were RSD≤7.4% for all target compounds. For nol compounds in all the studied tobacco samples were chlorointer-day assays, the extraction and analysis were performed in genic acid(8%―66%) and rutin(32%―83%). On the one hand, 6 different days(n=6) and the RSD(%) were lower than 8.5 for the average content of chlorogenic acid, rutin and caffeic acid all of the analytes. Recoveries(≥86%) are satisfactory for most in flue-cured tobacco(No.1―10) was higher than those of of the target compounds. Table 3 Polyphenols composition of tobacco samples(mg/g) Number

Item

103 Esculetin

Chlorogenic acid

Rutin

102 Caffeic acid

102 Scopoletin

102Quercitrin

Flue-cured tobacco

1―10

scope mean

5.78―17.56 9.88

9―12.34 10.72

7.28―10.30 8.98

10.72―33.4 19.93

11.72―27.60 19.44

0.83―2.14 1.24

Sun-cured tobacco

11―17

scope

3.5―17.12 12.65

0.65―8.52 4.74

1.68―7.7

0.94―11.44

12.84―23.2

0.19―1.59

3.45

7.17

18.21

0.78

0.87―2.08 1.21

0.04―0.37 0.18

0.18―1.42

0.16―0.61

4.32―10.62

0.06―0.08

0.59

0.38

6.44

0.07

12.52―14.82 13.39

8.32―10.52 9.59

6.44―9.92

11.1―18.18

0.59―1.32

0.52―1.36

8.67

15.54

0.87

0.87

Variety

mean Burley

18―24

Oriental tobacco

25―30

scope mean scope mean

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ZHANG Xia et al.

sun-cured tobacco(No.11―17) and burley(No. 18―24). On the other hand, scopoletin was the most abundant polyphenols compound in the sun-cured tobacco, as much as that in flue-cured tobacco. However, the contents of all tested polyphenols expect scopoletin in burley were at much lower level than those of other types. Scopoletin was detected in low concentration in oriental tobacco(No. 25―30), only about 1/8 of burley. These results show that there were evident differences in polyphenols of tobacco tested because of the distinction through their planting, production processing and the gene of tobacco.

3.5

Statistical Analysis of Data

To ascertain the significance of concentration differences of polyphenols among different tobacco samples, the data were statistically evaluated by the analysis of vafiance(ANOVA). The ANOVA performed on data expressing polyphenols concentration, showed that at 0.05 level, all the tested compounds, varied significantly in different tobacco samples. CA was used for disclosing the natural groups of tobacco samples characterized by the content values of six polyphenols. PCA was used for reducing the dimensionality of the data to a small number of components to examine the possible grouping of samples according to variety.

3.5.1

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information to attain the kinds of tobacco differentiation according to the established classes.

3.5.2

PCA Result

The differences among the 30 tobacco samples, according to the variety, were emphasized by the PCA. Fig.5(A) and (B) show the result. For polyphenols of tobacco, PCA yields two principal components, accounting for 84.2% of total variance. Fig.5(B) shows the corresponding loading plots that establish the relative importance of each variable and it is therefore useful for the study of relations among the variables and tobaccos. The first PC, which accounts for 68.7% of the variance, correlates negatively with rutin and chlorogenic acid and positively with the rest of the polyphenol compounds. The second PC correlates positively with chlorogenic acid, caffeic acid, quercitrin and esculetin, and negatively with rutin and scopoletin.

CA Result

Four varieties of thirty tobacco samples were analyzed by software NTSYs 2.1. The results achieved by CA are presented as a dendrogram(Fig.4). Four clusters were observed: fluecured tobacco samples(No. 1―10) were in a cluster together; samples 11 to 15 were sun-cured tobacco; samples 25―30 were made up of oriental tobacco and samples 18―24 were made up of burley and two sun-cured tobacco samples (No.16 and 17) in which the concentrations of six polyphenols were as close as those of burley. These results disclose that there were notable differences among four categories of tobacco samples and the polyphenols data may contain adequate

Fig.5

Score plot of first two principal components(PC) for classification of 30 tobacco samples of 4 varieties(A) and relation between the polyphenols compounds(B)

Note: ○: Flue-cured tobacco(No.1―10); ◆: sun-cured tobacco(No. 11―17); △: burley(No.18―24); ■: oriental tobacco(No.25―30).

Fig.4

Dendrogram of cluster analysis of 30 tobacco samples(4 varieties)

FCT: flue-cured tobacco; OT: oriental tobacco; BL: burley; SCT: sun-cured tobacco.

As can be seen from Fig.5(A), the first PC accounts for 68.7% of total variance; however, the second PC is only 15.5%. It is indicated that there was evident distribution difference in polyphenols of different kinds of tobacco for the first PC (variance 4.41, others