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Journal of Chromatography B, 949– 950 (2014) 7– 15

Contents lists available at ScienceDirect

Journal of Chromatography B

j ourna l h om epage: www.elsev ier .com/ locate /chromb

imultaneous determination of caffeic acid derivatives byPLC–MS/MS in rat plasma and its application in pharmacokinetic

tudy after oral administration of Flos Lonicerae–Fructus Forsythiaeerb combination

ei Zhoua,b,c, Jinjun Shand, Shouquan Wangd, Wenzheng Jue, Minxin Mengf,aochang Caia, Liuqing Dia,b,c,∗

College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, PR ChinaJiangsu Engineering Research Center for Efficient Delivery System of TCM, PR ChinaNanjing Engineering Research Center for Industrialization of Chinese Medicine Pellets, PR ChinaFirst Medicine College, Nanjing University of Chinese Medicine, Nanjing 210046, PR ChinaDepartment of Clinical Pharmacology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210093, PR ChinaWaters Corporation, United States

r t i c l e i n f o

rticle history:eceived 26 September 2013ccepted 26 December 2013vailable online 7 January 2014

eywords:los Lonicerae–Fructus Forsythiae herbombinationaffeic acid derivativesPLC–MS/MSral deliveryharmacokinetics

a b s t r a c t

The current study aims to investigate the pharmacokinetic study of eight caffeic acid derivatives(forsythoside A, isoforsythoside, forsythoside B, neochlorogenic acid, chlorogenic acid, cryptochloro-genic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid) following oral administration ofFlos Lonicerae–Fructus Forsythiae herb combination in rats. A rapid and sensitive ultra performanceliquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to deter-mine the eight caffeic acid derivatives simultaneously in rat plasma. After mixing with the internalstandard (IS) tinidazole, plasma samples were pretreated by liquid–liquid extraction with n-butyl alco-hol/ethyl acetate (7:3, v/v). The separation was performed on an Acquity UPLC HSS T3 C18 column(100 mm × 2.1 mm, 1.8 �m) at a flow rate of 0.4 mL min−1, and acetonitrile/methanol (4:1, v/v)–0.4%formic acid was used as mobile phase. The detection was performed on a triple quadrupole tandemmass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source withpositive and negative ionization modes. All calibration curves had good linearity (r > 0.991) over theconcentration ranges of 1.097–2246 ng mL−1 for neochlorogenic acid, 6.535–6692 ng mL−1 for chloro-genic acid, 2.103–2153 ng mL−1 for cryptochlorogenic acid, 0.5058–129.5 ng mL−1 for 3,5-dicaffeoylquinicacid, 0.3205–82.05 ng mL−1 for 3,4-dicaffeoylquinic acid, 1.002–512.8 ng mL−1 for isoforsythoside,0.4795–982.1 ng mL−1 for forsythoside A and 0.7587–776.9 ng mL−1 for forsythoside B, respectively.The intra- and inter-batch precisions were all within 15% and the accuracy (relative error, RE%) allranged from 85.68% to 114.7%. It was shown from pharmacokinetic parameters that the rank order ofAUC0–t, Cmax and T1/2k for phenolic acids was chlorogenic acid > neochlorogenic acid ≥ cryptochlorogenicacid > 3,4-dicaffeoylquinic acid ≥ 3,5-dicaffeoylquinic acid (most of them had significant differences),which corresponded to their administration dosages to rats, but that of MRT0–t and T1/2z were oppo-

site. Besides, the AUC0–t, Cmax, MRT and T1/2z except T1/2k of isoforsythoside and forsythoside B had nosignificant difference, compared to that of forsythoside A though their administration dosages weresignificantly lower than that of forsythoside A. All results showed that the method was applied to thepharmacokinetic study of the eight caffeic acid derivatives in rat plasma successfully after oral admin-istration of Flos Lonicerae–Fructus Forsythiae herb combination, and there were significant differences ofcaffeic acid derivatives even isomers in the pharmacokinetic parameters.

∗ Corresponding author at: College of Pharmacy, Nanjing University of Chineseedicine, Nanjing 210046, PR China. Tel.: +86 25 86798226; fax: +86 25 83271038.

E-mail address: diliuqing@hotmail.com (L. Di).

570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2013.12.035

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Herbs used together in combination are the basic composi-tion units of Chinese herbal formulas and have special clinicalsignificance in traditional Chinese Medicine (TCM). The herb

8 W. Zhou et al. / J. Chromatogr. B

Fig. 1. Product ion mass spectra of [M−H]− ions of (1) forsythoside B, (2) iso-forsythoside and (3) forsythoside A in negative mode (A1 and A2) and [M+H]+ ions of(4) 3,5-dicaffeoylquinic acid, (5) 3,4-dicaffeoylquinic acid, (6) neochlorogenic acid,

949– 950 (2014) 7– 15

combinations (mixture of two herbs) are much simpler thancomplicated formulations in composition but retain the basictherapeutic features. Flos Lonicerae possesses wide pharmacolog-ical actions, such as antibacterial, anti-inflammatory, antiviral,antiendotoxin, blood fat reducing, antipyretic, etc. [1]. And Fruc-tus Forsythiae has the effects of antibacterial, antiviral, antioxidant,anti-inflammatory, anti-obesity, antiemetic, etc. [2]. The two herbsare the basic components of Chinese herbal preparations suchas Shuang-Huang-Lian tablet, Yin-Qiao-Jie-Du tablet, Fufang Jin-Huang-Lian Granule, Qin-Re-Jie-Du oral liquid and Fufang Qin-Lanoral liquid, which are extensively used in clinical practice [3]. It wasreported that the caffeic acid derivatives [4], e.g. phenylethanoidglycosides with one coffee acyl group such as forsythoside A,isoforsythoside and forsythoside B, caffeoylquinic acids with onecoffee acyl group such as chlorogenic acid, neochlorogenic acidand cryptochlorogenic acid, and dicaffeoylquinic acids with twocoffee acyl groups such as 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid (Fig. 1) were all the main components inthe commercial Flos Lonicerae–Fructus Forsythiae herb combinationpreparations [3–8].

There were several studies concerning the quantification ofchlorogenic acid (a major component of Flos Lonicerae) andforsythoside A (a major component of Fructus Forsythiae) in plasma,respectively [8–22]. However, apart from chlorogenic acid andforsythoside A, simultaneous pharmacokinetic studies of othercaffeic acid derivatives especially forsythoside B and isoforsytho-side have not been reported after oral administration of FlosLonicerae–Fructus Forsythiae herb combination in rats, and verylittle attention has been devoted to the pharmacokinetic stud-ies of these components. In the previous pharmacokinetic studiesof chlorogenic acid and forsythoside A, the concentrations ofchlorogenic acid and forsythoside A in plasma were analyzedby liquid chromatography with UV or MS [8–22]. However, FlosLonicerae–Fructus Forsythiae herb combination contains a series ofcaffeic acid derivatives that possess not only isomer properties, butalso a wide range of concentrations in plasma. Therefore, it is desir-able to develop an analytical method to allow eight analytes to bequantified simultaneously in rat plasma.

In this study, a rapid and sensitive ultra performance liq-uid chromatography–tandem mass spectrometry (UPLC–MS/MS)method was developed for the simultaneous determination offorsythoside A, isoforsythoside, forsythoside B, chlorogenic acid,neochlorogenic acid, cryptochlorogenic acid, 3,4-dicaffeoylquinicacid and 3,5-dicaffeoylquinic acid in rat plasma. The method wasfully validated and applied to the pharmacokinetic study of caffeicacid derivatives in rat plasma following oral administration of FlosLonicerae–Fructus Forsythiae herb combination.

2. Experimental

2.1. Chemicals and materials

Flos Lonicerae (bud of Lonicera japonica Thunb.) and FructusForsythiae (fruit of Forsythia suspense) were purchased from Yi-Feng drug store (Nanjing, China) and were authenticated by Prof.Wu (Department of Pharmacognosy, Nanjing University of ChineseMedicine). Gauzes (1 mm pore size) were purchased from ShanghaiYinjing Health and Medical Materials Co., Ltd. (Shanghai, China).

Chlorogenic acid and tinidazole (used as internal standard, IS) werepurchased from National Institute for the Control of Pharmaceuticaland Biological Products (Beijing, China). Neochlorogenic acid, cryp-tochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic

(7) chlorogenic acid, (8) cryptochlorogenic acid and tinidazole (IS) in positive mode(B1–B3), respectively.

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W. Zhou et al. / J. Chroma

cid and forsythoside B (98% pure) were purchased from Sichuaneikeqi Bio-tech Co., Ltd. (Sichuan, China). Forsythoside A (98%

ure) was purchased from Shanghai Nature Standard Co., Ltd.Shanghai, China). Isoforsythoside (98% pure) was purchased fromhengdu Herbpurify Co., Ltd. (Sichuan, China).

.2. Apparatus and operation conditions

.2.1. Liquid chromatographyChromatographic analysis was performed on a Waters Acquity

PLC system (Waters Co., Milford, MA, USA), consisting of ainary pump solvent management system, an online degasser,nd an autosampler. An Acquity UPLC HSS T3 C18 column100 mm × 2.1 mm, 1.8 �m) was employed and the column temper-ture was maintained at 40 ◦C. The mobile phase was composed of A0.4% formic acid) and B (acetonitrile/methanol 4:1, v/v) using a gra-ient elution of 10–11% B at 0–0.5 min, 11–13% B at 0.5–0.75 min,3–15% B at 0.75–1.5 min, 15–10% B at 1.5–2 min, 10–30% B at–2.8 min, 30–30% B at 2.8–3.37 min, 30–10% B at 3.37–4 min, andold for 1.5 min. The flow rate was set at 0.4 mL min−1. The auto-ampler was conditioned at 4 ◦C and the injection volume was 5 �L.

.2.2. Mass spectrometryMass spectrometry detection was performed using Xevo Triple

uadrupole MS (Waters Co., Milford, MA, USA) equipped withn electrospray ionization source (ESI). The ESI source was setn positive ionization mode for neochlorogenic acid, chloro-enic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and,4-dicaffeoylquinic acid and in negative ionization mode for

soforsythoside, forsythoside A and forsythoside B, respectively.he analyte detection was performed by using multiple reac-ion monitoring (MRM) mode at m/z transitions of 354.8 → 162.9or cryptochlorogenic acid, chlorogenic acid and cryptochloro-enic acid, 516.9 → 162.9 for 3,5-dicaffeoylquinic acid and,4-dicaffeoylquinic acid, 622.9 → 160.9 for isoforsythoside andorsythoside A, 754.9 → 160.9 for forsythoside B and 247.8 → 120.8or IS, respectively (Fig. 1). The parameters in the source wereet as follows: capillary voltage, 3.3 kV; source temperature,50 ◦C; desolvation temperature, 500 ◦C; cone gas flow, 50 L h−1;esolvation gas flow, 1000 L h−1; cone voltage, 18 V for cryp-ochlorogenic acid, chlorogenic acid and cryptochlorogenic acid,4 V for 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid,4 V for isoforsythoside and forsythoside A, 68 V for forsythoside Bnd 36 V for IS, respectively; collision energy, 12 V for cryptochloro-enic acid, chlorogenic acid and cryptochlorogenic acid, 20 V for,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid, 50 V for iso-orsythoside and forsythoside A, 66 V for forsythoside B and 16 V forS, respectively. Dwell time was automatically set by the software.

.3. Preparation of calibration standard and quality control (QC)amples

Stock solutions were separately prepared by dissolving theccurately weighed eight standard reference compounds with aixture of methanol/water (60:40, v/v) containing 0.1% formic acid.

mixed stock solution was obtained by mixing all the eight stockolutions above, and given a final concentration of 33.69 �g mL−1

or cryptochlorogenic acid, 100.4 �g mL−1 for chlorogenic acid,2.31 �g mL−1 for neochlorogenic acid, 1.942 �g mL−1 for 3,5-icaffeoylquinic acid, 1.230 �g mL−1 for 3,4-dicaffeoylquinic acid,.692 �g mL−1 for isoforsythoside, 14.73 �g mL−1 for forsytho-

ide A and 11.65 �g mL−1 for forsythoside B, respectively. Theixed stock solution was serially diluted with a mixture ofethanol/water (60:40, v/v) containing 0.1% formic acid to provideorking standard solutions of desired concentrations. The internal

949– 950 (2014) 7– 15 9

standard solution of tinidazole was prepared to the concentrationof 120.0 ng mL−1 in methanol.

Calibration standards and quality control (QC) samples wereprepared as the following: 10 �L of standard working solu-tion was evaporated to dryness by a gentle stream of nitrogen,and then 150 �L of blank rat plasma was added. The sampleswere prepared prior to use during validation and pharmacoki-netic study. The final calibration concentration ranges were1.097–2246 ng mL−1 for neochlorogenic acid, 6.535–6692 ng mL−1

for chlorogenic acid, 2.103–2153 ng mL−1 for cryptochloro-genic acid, 0.5058–129.5 ng mL−1 for 3,5-dicaffeoylquinicacid, 0.3205–82.05 ng mL−1 for 3,4-dicaffeoylquinic acid,1.002–512.8 ng mL−1 for isoforsythoside, 0.4795–982.1 ng mL−1

for forsythoside A and 0.7587–776.9 ng mL−1 for forsythoside B,respectively. The QC samples were prepared at concentrations of2.000, 55.00, 1500 ng mL−1 for neochlorogenic acid, 13.00, 240.0,4500 ng mL−1 for chlorogenic acid, 4.000, 75.00, 1400 ng mL−1

for cryptochlorogenic acid, 1.000, 9.000, 86.00 ng mL−1 for3,5-dicaffeoylquinic acid, 0.7000, 6.000, 55.00 ng mL−1 for3,4-dicaffeoylquinic acid, 2.000, 26.00, 340.0 ng mL−1 for iso-forsythoside, 1.000, 25.00, 650.0 ng mL−1 for forsythoside A and1.500, 28.00, 510.0 ng mL−1 for forsythoside B, respectively in drug-free plasma. The standards and quality controls were extracted oneach analysis day with the same procedures for plasma samples asdescribed below.

2.4. Sample preparation

An aliquot of plasma, 75 �L for 10 and 20 min samples and150 �L for the rest of the samples collected during the pharma-cokinetic study were vortex mixed with 10 �L of 5% formic acid,10 �L of IS solution (120 ng mL−1) and 20 �L of hydrochloric acid(2 mol L−1) in an eppendorf tube, and 1000 �L of n-butyl alco-hol/ethyl acetate (7:3, v/v) was added to extract the eight caffeicacid derivatives from the plasma. The samples were vortexed for1.5 min, and centrifuged at 9659 × g for 5 min. The 800 �L of super-natant was transferred into another eppendorf tube and driedunder a flow of nitrogen gas. The residue was re-constituted in100 �L acetonitrile/methanol (4:1, v/v)–0.4% formic acid (1:9, v/v),and centrifuged (9659 × g for 10 min). The supernatant was trans-ferred to an autosampler vial and an aliquot of 5 �L was injectedinto the UPLC–MS/MS system for analysis.

2.5. Method validation

The method was validated in terms of specificity, selectivity,calibration curve, sensitivity, matrix effect, accuracy, precision andstability, in accordance with the USA Food and Drug Administration(FDA) bioanalytical method validation guidance [23].

2.5.1. Specificity and selectivityThe specificity of the method was evaluated by comparing the

chromatograms of six different batches of blank rat plasma sam-ples, plasma samples spiked with the analytes and IS, and plasmasamples after an oral dose. Blank rat plasma samples were ana-lyzed for endogenous interference, followed by spiking with IS forthe interference of IS.

2.5.2. Linearity and lower limits of quantification (LLOQ)The linearity of each calibration curve was determined by plot-

ting the peak area ratio (y) of analytes to IS vs the nominalconcentration (x) of analytes with weighted (1/x) least square lin-

ear regression. The LLOQ was defined as the lowest concentrationon the calibration curve with an acceptable accuracy (relative error,RE) within ±20% and a precision (relative standard deviation, RSD)below 20%.

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.5.3. Precision and accuracyThe intra- and inter-batch precisions and accuracy were carried

ut through quantifying three concentration levels of QC samplessix samples for each concentration level) on the same analyti-al run and on three different analytical runs, respectively. Therecisions and accuracy were evaluated by relative standard devi-tion (RSD%) and by nominal concentration = (average measuredalue/nominal value) × 100%, respectively.

.5.4. Recovery and matrix effectThe extraction recoveries of analytes at three QC levels were

etermined by comparing the peak areas obtained from sixxtracted QC samples with those obtained from pure referencetandards spiked in post-extracted blank rat plasma at the sameoncentration. The matrix effects were evaluated by comparing theeak areas obtained from samples where the extracted matrix waspiked with standard solutions to those obtained from the pureeference standard solutions at the same concentration.

.5.5. Stability experimentThe stability of caffeic acid derivatives in rat plasma was

ssessed using QC samples, which were freshly prepared andmmediately mixed with 10 �L of 5% formic acid followed by stor-ng at −70 ◦C for one month to evaluate the long-term stability.he post-preparation stability was tested by determination of thextracted QC samples stored at room temperature for 24 h. Thereeze and thaw stability was determined using QC samples afterhree freeze–thaw cycles (−70 to 20 ◦C).

.6. Pharmacokinetic study

Flos Lonicerae combined with Fructus Forsythiae (1:1, w/w) wereecocted twice with boiling water (1:10, w/v) for 45 min, and thextracted solution was filtered through 5 layer gauzes. The filtra-ions were concentrated to a thick solution with the concentrationf 1 g raw medicine per milliliter [24].

The contents of neochlorogenic acid, chlorogenic acid, cryp-ochlorogenic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquiniccid, forsythoside A, isoforsythoside and forsythoside B were deter-ined to be 3.78, 13.4, 4.15, 1.73, 2.70, 5.90, 0.65 and 1.08 mg/mL of

xtracts, respectively, using the same chromatography conditionss described above.

This validated method was applied to monitor the plasmaoncentrations of neochlorogenic acid, chlorogenic acid, cryp-ochlorogenic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquiniccid, forsythoside A, isoforsythoside and forsythoside B in rats afteringle oral administration of Flos Lonicerae–Fructus Forsythiae herbombination extracts.

Sprague Dawley rats (250 g, male) obtained from Experimen-al Animal Center of Nanjing University of Chinese Medicine wereasted for 12 h, but were given access to water prior to the oraldministration of extract prepared above with concentration of7.8 mg/kg for neochlorogenic acid, 134 mg/kg for chlorogeniccid, 41.5 mg/kg for cryptochlorogenic acid, 17.3 mg/kg for 3,5-icaffeoylquinic acid, 27.0 mg/kg for 3,4-dicaffeoylquinic acid,9.0 mg/kg for forsythoside A, 6.5 mg/kg for isoforsythoside and0.8 mg/kg for forsythoside B, respectively. After dosing for 0, 5, 10,0, 30, 40, 55, 70, 100, 160, 250, 600 and 1440 min, 400 �L bloodas collected from the pre-intubated catheter and put into tubesith heparin sodium injection (10 �L) and ascorbic acid (2 �g) atredetermined time points. Subsequently, plasma was prepared byentrifugation at 1816 × g for 7 min and immediately analyzed or

tored at −70 ◦C for further analysis after being mixed with 10 �Lf 5% formic acid.

The pharmacokinetic parameters of forsythoside A, iso-orsythoside forsythoside B, neochlorogenic acid, chlorogenic

949– 950 (2014) 7– 15

acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid, including the peak plasma concentration(Cmax), the time to Cmax (Tmax), the AUC from 0 to infinity (AUC0–∞),the AUC from 0 to time (AUC0–t), mean residence time (MRT), andterminal elimination half-life (T1/2z) were calculated by the non-compartmental analysis of plasma concentration vs time data usingthe “DAS 2.1.1” software (Mathematical Pharmacology ProfessionalCommittee of China, Shanghai, China) and rapid elimination half-life (T1/2k) was calculated from the formula T1/2k = 0.693/kel, wherethe elimination rate constant (kel) was calculated by linear regres-sion of the rapid elimination points of the semi-log plot of plasmaconcentration against time. The comparison of pharmacokineticparameters was accomplished by SPSS 16.0 (Statistical Package forthe Social Science).

3. Results and discussion

3.1. Method development

3.1.1. Optimization of mass spectrometryThe stock solutions of the analytes and IS diluted with a

mixture of methanol/water (60:40, v/v) containing 0.1% formicacid were directly infused along with the mobile phase into themass spectrometer with electrospray ion source. The responseobserved in the positive ionization mode was higher than thatin the negative ionization mode for neochlorogenic acid, chloro-genic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and3,4-dicaffeoylquinic acid, owning to their ion enhancement effects,though we found that the response in negative ion mode with-out acid addition in the process of the whole analyzing procedurewas better than that in positive ion mode. However, it wasfound that, whether acid in the process of the whole analyz-ing procedure was added or not, the response for forsythosideA, isoforsythoside and forsythoside B in the negative ionizationmode was higher than that in the positive ionization mode. Inthe precursor ion full-scan spectra, the most abundant ions wereprotonated molecules ions [M+H]+ for neochlorogenic acid, chloro-genic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and3,4-dicaffeoylquinic acid, respectively and deprotonated molecules[M−H]− for forsythoside A, isoforsythoside and forsythoside B,respectively (Fig. 1). Parameters such as desolvation temperature,ESI source temperature, capillary voltage, cone voltage, flow rateof desolvation gas and cone gas were optimized to obtain thehighest intensity of protonated and deprotonated molecules ionsof analytes. The ion pairs of precursor → product for MRM detec-tion were generated by the intellistart procedure (Fig. 1), whichwas embedded in the Masslynx software. The MRM transitionsat 622.9 → 160.9, 622.9 → 160.9, 754.9 → 160.9, 354.8 → 162.9,354.8 → 162.9, 354.8 → 162.9, 516.9 → 162.9, 516.9 → 162.9 and247.8 → 120.8 were selected to analyze forsythoside A, isoforsytho-side, forsythoside B, cryptochlorogenic acid, chlorogenic acid, cryp-tochlorogenic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinicacid and IS, respectively.

3.1.2. Optimization of chromatographyFig. 2 showed the representative UPLC–MS/MS chromatograms

of blank plasma (A), blank plasma spiked with the eight ana-lytes and IS in LLOQ (B), and the plasma sample at 20 minafter oral administration of Flos Lonicerae–Fructus Forsythiaeherb combination (C). No interfering peak was observed inblank plasma under the assay conditions. The retention time

was 3.59 min, 3.63 min, 3.56 min, 1.61 min, 2.24 min, 2.38 min,3.93 min, 4.11 min and 2.98 min for forsythoside A, isoforsythoside,forsythoside B, neochlorogenic acid, chlorogenic acid, cryp-tochlorogenic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic

W. Zhou et al. / J. Chromatogr. B 949– 950 (2014) 7– 15 11

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ig. 2. Representative MRM chromatograms of compounds 1–8 and tinidazole (IS)n LLOQ; (C) plasma sample at 20 min following oral administration of Flos Lonicera

cid and IS, respectively. All analytes were eluted rapidly with.5 min.

Phenylethanoid glycosides containing forsythoside A andsoforsythoside as isomers, and phenolic acids containing

plasma: (A) blank plasma; (B): blank plasma spiked with the eight analytes and IStus Forsythiae herb combination.

neochlorogenic acid, chlorogenic acid and cryptochloro-genic acid as isomers and 3,5-dicaffeoylquinic acid and3,4-dicaffeoylquinic acid as isomers had the same precursor andproduct ions in mass spectrometry, respectively. Therefore, it was

12 W. Zhou et al. / J. Chromatogr. B 949– 950 (2014) 7– 15

Table 1Regression data and LLOQs of forsythoside B, isoforsythoside, forsythoside A, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and3,4-dicaffeoylquinic acid.

Compound Range (ng mL−1) Linear regression equation Correlation coefficient LLOQ (ng mL−1)

Forsythoside B 0.7587–776.9 Y = 0.0005531x + 0.0003568 0.9996 0.7587Isoforsythoside 1.002–512.8 Y = 0.001179x − 6.350e−005 0.9983 1.002Forsythoside A 0.4795–982.1 Y = 0.002148x + 0.001663 0.9990 0.4795Neochlorogenic acid 1.097–2246 Y = 0.02321x + 0.06481 0.9980 1.097Chlorogenic acid 6.535–6692 Y = 0.02786x + 0.4591 0.9924 6.535

5x + 095x +

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Cryptochlorogenic acid 2.103–2153 Y = 0.02323,5-Dicaffeoylquinic acid 0.5058–129.5 Y = 0.00993,4-Dicaffeoylquinic acid 0.3205–82.05 Y = 0.0104

ndispensable to separate the isomers in the three groups withPLC.

The bridged ethylsiloxane/silica hybrid (BEH) C18 columns50 mm × 2.1 mm, 1.7 �m) and (100 mm × 2.1 mm, 1.7 �m) all didot separate the three groups of isomers from each other well evennder the highly aqueous mobile phase, because of their similar,igh water solubility. It was reported in the previous study that hightrength silica (HSS) T3 (C18) 1.8 �m bonded phase was fabricatedo retain and separate small water-soluble polar organic com-ounds [25], and we found from Fig. 2 that an Acquity UPLC HSS T318 column (100 mm × 2.1 mm, 1.8 �m) elicited a suitable reten-ion and a base-line separation between the analytes, especiallyicaffeoylquinic acids, compared with the previous reports [8],hough approximately six peaks as the isomers of dicaffeoylquiniccids in dicaffeoylquinic acids channel were unidentified (Fig. 2C).esides, since forsythoside A, isoforsythoside, forsythoside B,eochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-icaffeoylquinic acid and 3,4-dicaffeoylquinic acid are caffeic aciderivatives [4], formic acid in the UPLC mobile phase at a concentra-

ion of 0.4% was added so as to overcome the peak-tailing effect, andmprove the resolution for isomers in three groups. Interestingly,he response in ESI–MS/MS for five phenolic acids in the positiveonization mode was improved because of ion enhancement effects,

able 2ntra-batch, inter-batch precision and accuracy of forsythoside B, isoforsythoside, foricaffeoylquinic acid and 3,4-dicaffeoylquinic acid.

Compound Concentration (ng mL−1) Inter-batch (

Precision (RS

Forsythoside B 1.500 14.92

28.00 14.51

510.0 9.980

Isoforsythoside 2.000 11.01

26.00 9.240

340.0 11.35

Forsythoside A 1.000 10.05

25.00 8.010

650.0 5.900

Neochlorogenic acid 2.000 10.37

55.00 3.710

1500 6.270

Chlorogenic acid 13.00 13.07

240.0 10.67

4500 8.160

Cryptochlorogenic acid 4.000 14.18

75.00 6.090

1400 10.58

3,5-Dicaffeoylquinic acid 1.000 10.69

9.000 14.83

86.00 4.640

3,4-Dicaffeoylquinic acid 0.7000 10.50

6.000 10.61

55.00 6.090

.1009 0.9970 2.1030.01791 0.9995 0.5058.05129 0.9929 0.3205

and ion suppression effects on phenylethanoid glycosides in thenegative ionization mode could not be ignored, but had no differ-ence no matter how much acid was added. [26]. And we found thata mixture solvent system containing acetonitrile/methanol (4:1,v/v), not acetonitrile or methanol alone, could be utilized as organicphase to separate the three groups of isomers well. In addition, itwas difficult to separate chlorogenic acid and cryptochlorogenicacid as isomers and forsythoside A and isoforsythoside as isomersin the chromatography owning to their high similar polarity unlessan optimized gradient with seven different segments as shownabove was selected, and the content of mobile phase B was loweredfrom 15 to 10% at the time of 1.5–2 min. The results (Fig. 2B and C)showed that the gradient elution of the mobile phase was suitablefor the phenylethanoid glycosides and phenolic acids separation,and the method described above could achieve symmetric peakshape, high resolution (Rs > 1.5) among peaks and short run timefor the simultaneous analysis of the eight compounds in plasma.

3.1.3. Optimization of sample preparation

Phenylethanoid glycosides and phenolic acids are caffeic acid

derivatives and susceptible to oxidation. However, it was foundthat samples were stable in acidic conditions and unstable in neu-tral and basic conditions (data not shown). Therefore, the acidic

sythoside A, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-

n = 3) Intra-batch (n = 6)

D, %) Accuracy (%) Precision (RSD, %) Accuracy (%)

101.7 12.90 113.1108.7 11.43 105.0104.3 7.74 105.84

89.09 11.87 90.0095.64 5.12 86.36

111.9 7.490 105.9

103.0 6.970 112.6107.4 7.000 97.88106.0 5.02 103.70

98.26 10.83 99.60111.6 1.410 114.67

98.76 3.390 92.42

95.13 13.96 97.6195.38 3.390 102.1

103.8 3.600 100.73

100.27 12.41 85.9398.34 2.140 104.285.68 10.84 86.21

99.33 10.61 111.596.93 8.62 86.6596.51 1.20 92.20

99.24 7.040 99.58102.8 2.18 90.31103.2 6.07 102.71

W. Zhou et al. / J. Chromatogr. B 949– 950 (2014) 7– 15 13

Table 3Recoveries, matrix effects and stability of forsythoside B, isoforsythoside, forsythoside A, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinicacid and 3,4-dicaffeoylquinic acid (n = 6).

Compound Concentration(ng mL−1)

Recovery Matrix effect Freeze–thaw cycles At −70 ◦C for 1 month At roomtemperaturefor 24 h

Average(%)

RSD (%) Average(%)

RSD (%) Remain(%)

RSD (%) Remain(%)

RSD (%) Remain(%)

RSD (%)

Forsythoside B 1.500 44.17 8.440 97.78 12.45 111.1 11.17 93.01 13.54 113.9 6.32028.00 55.96 13.80 97.35 10.85 88.36 4.410 100.4 11.40 88.09 10.89

510.0 51.76 10.53 103.4 6.250 110.4 6.150 106.8 5.650 114.0 5.950

Isoforsythoside 2.000 52.27 11.91 112.5 11.11 104.3 11.46 114.3 11.58 103.6 8.88026.00 63.96 9.560 103.2 7.550 100.9 5.990 109.9 14.08 93.14 10.42

340.0 72.34 7.400 95.58 6.090 113.5 3.480 99.67 3.430 86.93 6.110

Forsythoside A 1.000 78.13 13.61 91.11 5.450 87.40 10.80 98.30 9.530 99.04 13.1525.00 80.31 8.290 97.03 8.430 107.4 6.950 113.6 6.610 87.28 5.970

650.0 73.65 1.720 105.2 5.560 103.6 7.110 114.3 3.050 95.66 7.760

Neochlorogenic acid 2.000 78.67 12.14 102.7 6.410 113.1 10.80 102.4 12.22 101.7 13.0655.00 83.29 7.130 95.19 2.260 94.50 5.130 96.69 6.820 103.1 1.660

1500 80.30 4.370 94.52 3.530 96.70 7.190 85.22 4.430 92.29 4.910

Chlorogenic acid 13.00 82.45 10.93 92.49 3.920 91.38 10.94 95.17 12.85 85.53 7.330240.0 80.09 8.940 89.96 6.170 106.2 6.730 106.8 13.90 99.39 5.030

4500 82.63 2.750 95.58 9.540 99.39 4.910 92.31 2.780 90.45 3.460

Cryptochlorogenic acid 4.000 70.07 13.88 95.65 2.790 90.88 10.66 113.4 3.140 85.35 14.0075.00 84.07 7.490 94.82 4.120 92.29 5.850 112.8 5.990 86.18 1.940

1400 78.28 4.190 94.94 6.410 97.41 3.440 85.58 10.37 85.79 3.740

3,5-Dicaffeoylquinic acid 1.000 80.77 14.60 100.4 5.320 101.6 13.09 109.5 11.31 111.3 6.6309.000 69.72 4.350 106.6 5.580 112.49 2.82 114.2 6.620 85.59 5.780

86.00 64.32 6.480 110.7 7.010 90.38 5.920 97.58 2.800 93.47 9.980

3,4-Dicaffeoylquinic acid 0.7000 83.97 14.21 96.00 9.770 112.7 12.05 108.3 14.49 99.98 13.04

siftiaa

datwBws

rtaatmonnsupenpha

6.000 65.25 14.47 101.655.00 85.25 9.430 105.8

olvents were used throughout the sample preparation, includ-ng collection, treatment and reconstitution procedures. To preventrom potential degradation of caffeic acid derivatives in the blood,he fresh collected blood samples were stored on ice and thenmmediately centrifuged at 4 ◦C for separation of plasma, thoughscorbic acid (2 �g) as antioxidant was added in the eppendorf tubet predetermined time points.

Due to wide ranges of plasma concentrations of caffeic aciderivatives especially chlorogenic acid, plasma at 10 and 20 minfter i.g. dosing were diluted 1-fold with blank plasma and we foundhat the concentration of diluted plasma for caffeic acid derivativesas 1 time lower than that of undiluted plasma (data not shown).esides, the regression data and relative recovery of QC samplesere satisfactory (Tables 1 and 2). All evidence indicated that the

ample preparation above was reasonable.In order to extract the analytes and IS with high, stable

ecoveries and no endogenous interference at the retentionime, five types of reagents using methanol, acetonitrile, ethylcetate, n-butyl alcohol or a mixture of n-butyl alcohol/ethylcetate were tried for precipitation of protein or solvent extrac-ion in rat plasma. It was found that protein precipitation using

ethanol or acetonitrile gave an approximately 90% recoveryf the eight analytes whilst there was poor repeatability andon-negligible matrix effects from endogenous plasma compo-ents on the ionization of the analytes and the IS (data nothown). Besides, it was shown with liquid–liquid extractionsing n-butyl alcohol that the extraction was consistent, com-ared with protein precipitation, but also showed serious matrixffects; while using ethyl acetate that the matrix effects were

egligible, however, there was extremely low recovery forhenylethanoid glycosides. As a result, a mixture of n-butyl alco-ol/ethyl acetate (7:3, v/v) was utilized to extract the eight analytesnd the IS, with consistent extraction efficiency and precision

7.580 114.9 1.970 109.0 10.76 114.5 9.0407.900 99.00 6.960 90.36 3.360 88.65 4.200

(Table 3). In addition, the matrix effects were negligible, and extrac-tion recovery of analytes was stable and satisfactory (Table 4).Therefore, it was demonstrated that n-butyl alcohol/ethyl acetate(7:3, v/v) was the best extraction solvent for all the analytes and IS.

3.2. Method validation

3.2.1. Selectivity and specificityThe representative chromatograms of blank plasma, blank

plasma spiked with the standard solutions and plasma samplesobtained following oral administration of caffeic acid derivatives inrats were shown in Fig. 2. Under the established chromatographiccondition, there were no endogenous interference in the plasmasand all the eight analytes as well as IS could be well separated fromeach other.

3.2.2. Linearity and calibration curveThe regression equation, correlation coefficients and linearity

ranges for the eight analytes were shown in Table 1. The resultsshowed that they all exhibited good linearity. The LLOQs forforsythoside B, isoforsythoside, forsythoside A, neochlorogenicacid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinicacid and 3,4-dicaffeoylquinic were 0.7587 ng mL−1, 1.002 ng mL−1,0.4795 ng mL−1, 1.097 ng mL−1, 6.535 ng mL−1, 2.103 ng mL−1,0.5058 ng mL−1, and 0.3205 ng mL−1, respectively.

3.2.3. Precision and accuracyThe results of the intra- and inter-batch precisions and accuracy

of all the analytes in LLOQ and QC samples were summarized inTable 2. The intra- and inter-batch precisions ranged from 6.970to 14.35% and 0.7800 to 11.74%, respectively. The accuracy derivedfrom QC samples was between 90.00% and 113.8% for each QC level

14 W. Zhou et al. / J. Chromatogr. B

Tab

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=

6,

mea

n

±

SD).

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3,5-

dic

affe

oylq

uin

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acid

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C max

(ng

mL−

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949– 950 (2014) 7– 15

of the eight analytes. The assay values on both intra- and inter-batchwere all within the acceptable range.

3.2.4. Extraction recovery and matrix effectThe mean recoveries of all the analytes at different concentra-

tions were shown in Table 3. The extraction recoveries of threelevel QC samples were stable. The extraction recoveries of IS was75.38 ± 2.750%. The matrix effect of blank plasma of all the analyteswas found to be within the acceptable range, and all values were inthe range from 85.0% to 115% (Table 3). The matrix effect of IS was95.82 ± 2.560%. Thus, it was demonstrated that the plasma matrixeffect was negligible for the assay.

3.2.5. StabilityStability of the eight analytes during the sample storing and

processing procedures was fully evaluated by analysis of QC sam-ples. The results (Table 3) indicated that these analytes in acidifiedplasmas were all stable for one-month storage at −70 ◦C, 24 h atroom temperature and three freeze–thaw cycles with accuracy allin the range from 85.22 to 114.9%.

3.3. Pharmacokinetic study

This validated UPLC–MS/MS method reported for the first timeby us was successfully applied to the pharmacokinetic study offorsythoside B, isoforsythoside, forsythoside A, neochlorogenicacid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinicacid and 3,4-dicaffeoylquinic acid in rat plasma following oraladministration of Flos Lonicerae–Fructus Forsythiae herb combi-nation. The assay was proved to be sensitive enough for thedetermination of these analytes in rat plasma.

It was found from Fig. 3 and Table 4 consistently thatthe values of Tmax for phenolic acids had little significanceand the rank order of AUC0–t, Cmax and T1/2k of pheno-lic acids in Flos Lonicerae–Fructus Forsythiae herb combinationwas chlorogenic acid > neochlorogenic acid ≥ cryptochlorogenicacid > 3,4-dicaffeoylquinic acid ≥ 3,5-dicaffeoylquinic acid (most ofthem had significant differences), which corresponded to theiradministration dosages to rats, but that of MRT0–t and T1/2z wereopposite, and the values were higher largely than that of T1/2k,which demonstrated that the total elimination in vivo of dicaf-feoylquinic acids might be slower than that of caffeoylquinic acids,influenced by plasma protein binding rate possibly. It was con-sistent with the previous report [27] that dicaffeoylquinic acidsespecially 3,4-dicaffeoylquinic acid with two coffee acyl groupshad higher binding abilities with human serum albumin (HSA)than caffeoylquinic acids with one coffee acyl group. Besides, theadministration dosages of isoforsythoside and forsythoside B inFlos Lonicerae–Fructus Forsythiae herb combination were 10 and 5times approximately lower than that of forsythoside A as isomer ofisoforsythoside, respectively, but the values of T1/2k except that ofAUC0–t, Cmax, MRT, T1/2z and Tmax were higher significantly than thatof forsythoside A, which indicated that the intestinal absorption ofisoforsythoside and forsythoside B might be higher significantlyand the metabolism might be lower significantly than that offorsythoside A. It was consistent with their chemical structuresthat two substituents of isoforsythoside and forsythoside B (O-1, 3positions) except that of forsythoside A (O-1, 4 positions) lay in thesame plane (Fig. 1), which resulted in the steric effects, and possiblythe low influence of the efflux transporters (P-gp and MRP2) andenzyme on the absorption and metabolism in vivo. Interestingly,the AUC0–t, Cmax and T1/2k of forsythoside A were lower signifi-

cantly than that of neochlorogenic acid and cryptochlorogenic acid,though the administration dosages were higher significantly thanthat of neochlorogenic acid and cryptochlorogenic acid, which wasconsistent with our previous report that, the apparent permeability

W. Zhou et al. / J. Chromatogr. B 949– 950 (2014) 7– 15 15

F B), cryi stratio

(mhavcmn

4

wfadpbetdFtp

A

uLSJpg

R

[

[

[

[

[

[

[

[

[

[

[

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ig. 3. Mean pharmacokinetic profiles of neochlorogenic acid (A), chlorogenic acid (soforsythoside (F), forsythoside A (G) and forsythoside B (H) following oral admini

Papp) in vitro of phenylethanoid glycosides such as forsythoside Aight be lower significantly and the metabolism in vivo might be

igher significantly compared with that of caffeoylquinic acids suchs neochlorogenic acid and cryptochlorogenic acid. In addition, thealue of T1/2z for 3,5-dicaffeoylquitnic acid was the highest amongaffeic acid derivatives, which indicated that there might be enor-ous differences among caffeic acid derivatives in pharmacoki-

etic parameters, although they had similar chemical properties.

. Conclusion

In this study, a rapid and reliable UPLC–MS/MS methodas developed for simultaneous analysis of forsythoside B, iso-

orsythoside, forsythoside A, chlorogenic acid, neochlorogeniccid, cryptochlorogenic acid, 3,4-dicaffeoylquinic acid and 3,5-icaffeoylquinic acid in rat plasma for the first time. Samplereparation was carried out by liquid–liquid extraction with n-utyl alcohol/ethyl acetate (7:3, v/v) and the data acquisition ofach sample was 5.5 min. The method has been successfully appliedo the pharmacokinetic study of the eight bioactive caffeic aciderivatives in rats following oral dose of Flos Lonicerae–Fructusorsythiae herb combination. The results also elucidated effectivelyhat there were significant differences in the pharmacokineticarameters of isomers of caffeic acid derivatives.

cknowledgements

The present study is supported financially by the National Nat-ral Science Foundation of China (81073071, 81273655), “Qingan” Project from Jiangsu Provincial Technology Innovation Teamupport Scheme, the priority Academic Program Development ofiangsu Higher Education Institution (No. ysxk-2010) and 2012rogram sponsored for scientific innovation research of collegeraduate in Jiangsu province (623).

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[[

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