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Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 271– 277

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om epage: www.elsev ier .com/ locate / jpba

PLC–MS/MS determination of paeoniflorin, naringin, naringenin andlycyrrhetinic acid in rat plasma and its application to a pharmacokinetictudy after oral administration of Si Ni San decoction

ing Wen, Ying Qiao, Jie Yang, Xinyu Liu, Yang Song, Zhigang Liu, Famei Li ∗

epartment of Analytical Chemistry, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China

r t i c l e i n f o

rticle history:eceived 27 October 2011eceived in revised form 17 March 2012ccepted 19 March 2012vailable online 28 March 2012

a b s t r a c t

A UPLC–MS/MS method was developed for the simultaneous determination of paeoniflorin, naringin,naringenin and glycyrrhetinic acid in rat plasma. A Waters BEH C18 column was used with a gradientmobile phase system of methanol–water containing 2 mM ammonium acetate. The analysis was per-formed on a positive ionization electrospray mass spectrometer via multiple reaction monitoring (MRM).

eywords:harmacokineticsi Ni San decoctionPLC–MS/MS

One-step protein precipitation with acetonitrile was used to extract the analytes from plasma. The limitsof quantification were 9.800 ng/ml for paeoniflorin, 5.100 ng/ml for naringin, 5.200 ng/ml for naringeninand 10.60 ng/ml for glycyrrhetinic acid, respectively. The intra- and inter-day precision (relative stan-dard deviation, RSD) ranged 4.9–12% and 2.8–13%, respectively. The accuracy (relative error, RE) was from−7.3% to 7.5% at all quality control (QC) levels. The validated method was applied to a pharmacokineticstudy in rats after oral administration of Si Ni San decoction.

Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

. Introduction

Si Ni San is a traditional Chinese medicine composed ofadix Bupleuri, Radix Paeoniae Alba, Fructus Aurantii Immatu-us and Radix Glycyrrhiza (1:1:1:1). The Chemical constituentsf Si Ni San mainly include saponins (including saikoside andaeoniflorin, etc.), flavonoid glycosides (including naringin, hes-eridin and liquiritin, etc.) and organic acids (including glycyrrhiziccid and benzoic acid, etc.), etc. Pharmacological studies havehown that Si Ni San had significant therapeutic effect on variousxperimental liver injury models [1]. It has been reported that thectivities of Si Ni San were stronger than those of single drugs,hich was the result of each constituent affecting the different

arget of liver injury [2]. Several reports [3–6] have indicated thataikosaponin, paeoniflorin, naringin and glycyrrhizin had playedn important role in Si Ni San. Our previous work has shown thataeoniflorin, naringin, naringenin and glycyrrhetinic acid, activeomponents of Si Ni San decoction were absorbed into the bloodnd could be detected after oral administration.Paeoniflorin is

ne of the main active ingredients in Radix Paeoniae which haseen used as a phytochemical marker for the quality control ofadix Paeoniae in Chinese Pharmacopoeia [7]. Pharmacological

∗ Corresponding author. Tel.: +86 24 2398 6289; fax: +86 24 2398 6289.E-mail address: lifamei@syphu.edu.cn (F. Li).

731-7085/$ – see front matter Crown Copyright © 2012 Published by Elsevier B.V. All rigoi:10.1016/j.jpba.2012.03.040

studies have exhibited that paeoniflorin has liver protecting [8],anti-inflammatory [9], anti-coagulant [10], antioxidant [11] andneuronprotective [12] effects. Naringin is a flavonoid present inmany Citrus fruits and traditional Chinese medicines. Like mostflavonoids, naringin has anti-inflammatory [13], anti-ulcer [14]and antioxidant [15] activities. Early research has shown thatorally administered naringin can be metabolized into naringeninand naringenin glucuronide [16–18]. Naringenin, the aglycone ofnaringin, has also been found to exhibit anti-ulcer [19,20], antiox-idant [15] and anticancer [21] effects. Glycyrrhizic acid (GL) isone of the bioactive ingredients in Radix Glycyrrhiza. GL has beenused in the treatment of hepatitis and chronic hepatitis [22–24].GL also has anti-inflammatory, antiviral and antioxidant activities[25]. After orally administered, GL is converted to glycyrrhetinicacid (GA) by intestinal bacteria in rats [26,27]. It was foundthat GL was subjected to presystemic metabolism and entero-hepatic circulation [28,29]. For the identification of metabolitesof Si Ni San see [30]. Despite the number of pharmacologi-cal studies of the four components, there is no report on LC orLC–MS methods for the pharmacokinetic study of the four com-ponents.

In the present paper, we developed and validated a sensitive

and selective UPLC–MS/MS method for the simultaneous determi-nation of paeoniflorin, naringin, naringenin and glycyrrhetinic acidin rat plasma. The method was applied to a pharmacokinetic studyin rats after oral administration of Si Ni San decoction.

hts reserved.

272 J. Wen et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 271– 277

genin

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Fig. 1. Chemical structures of paeoniflorin, naringin, narin

. Experimental

.1. Chemicals and reagents

The reference standards of paeoniflorin, naringin, glycyrrhetiniccid and lysinopril (internal standard, I.S.) (purity >98%) were pur-hased from the National Institute for the Control of Pharmaceuticalnd Biological Products (Beijing, China). The reference standard ofaringenin (purity >98%) was purchased from Baozetang Medicalechnology Co. Ltd. (Jiangsu, China). The structures of these com-ounds are shown in Fig. 1. Radix Bupleuri was purchased from

ortheast Big Pharmacy (Shenyang, China). Radix Paeoniae Alband Fructus Aurantii Immaturus were purchased from Weikangig Pharmacy (Shenyang, China). Radix Glycyrrhiza was purchased

rom Beijing Tongrentang Pharmacy (Shenyang, China). The above

, glycyrrhizic acid, glycyrrhetinic acid and lysinopril. (I.S.)

four medicinal materials were authenticated by Professor Jincai Lu(College of Traditional Chinese Materia Medica, Shenyang Pharma-ceutical University). Methanol of HPLC grade was obtained fromTedia (Fairfield, OH, USA). Ammonium acetate of HPLC grade wasobtained from Dikma (Richmond Hill, NY, USA). All other reagentswere of analytical grade.

2.2. Instrumentation and analytical conditions

2.2.1. Liquid chromatographyChromatography was performed on an ACQUITY UPLCTM

system (Waters Corp., Milford, MA, USA) with cooling autosam-pler and column oven. An ACQUITY UPLCTM BEH C18 column(100 mm × 2.1 mm, 1.7 �m) was employed. The column tempera-ture was maintained at 35 ◦C and a gradient elution with methanol

and B

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J. Wen et al. / Journal of Pharmaceutical

A)–water (2 mM ammonium acetate) (B) was used. The gradientrogram was as follows: 0–2.5 min, 25–90% A, 2.5–4.5 min, 90% A.he flow rate was set at 0.25 ml/min. The autosampler was condi-ioned at 4 ◦C and the injection volume was 10 �l for analysis.

.2.2. Mass spectrometryTriple-quadrupole tandem mass spectrometric detection was

arried out on a Micromass Quattro microTM API mass spectrometerWaters Corp, Milford, USA) with an electrospray ionization (ESI)nterface. The ESI source was operated in positive ionization mode.uantification was performed using MRM of the transitions of m/z97.9 → 178.9 for paeoniflorin, m/z 581.2 → 273.0 for naringin, m/z72.9 → 152.8 for naringenin, m/z 470.9 → 134.8 for glycyrrhetiniccid and m/z 406.2 → 246.1 for I.S., respectively.

The optimal MS parameters obtained were as follows: capillaryoltage 3.0 kV; cone voltage for paeoniflorin, naringin, naringenin,lycyrrhetinic acid and I.S. was 13, 15, 30, 20 and 30 kV, respec-ively; source temperature 100 ◦C and desolvation temperature50 ◦C. Nitrogen was used as desolvation and cone gas with a flowate of 500 and 30 l/h. Argon was used as the collision gas at aressure of approximately 2.8 × 10−3 mbar. The optimized colli-ion energy for paeoniflorin, naringin, naringenin, glycyrrhetiniccid and I.S. was 20, 18, 25, 37 and 23 eV, respectively. All data col-ected in centroid mode were processed using MassLynxTM NT 4.1oftware with a QuanLynxTM program (Waters Corp., Milford, MA,SA).

.3. Preparation of standards and quality control (QC) samples

The standard stock solutions (196.0, 204.0, 208.0 and12.0 �g/ml) of paeoniflorin, naringin, naringenin and gly-yrrhetinic acid were prepared by dissolving required amount ofhe reference standards in methanol. The working solutions of cal-bration curve were prepared by mixing and diluting the stockolutions of each compound with methanol. Standard solution of.S. was prepared by dissolving lysinopril in methanol at a finaloncentration of 11.30 �g/ml.

The standard plasma samples of calibration curve were pre-ared by spiking 20 �l of the above working solution into 100 �lf blank plasma to yield the concentration at 9.800, 19.60, 98.00,90.0, 980.0, 1960, 3920 ng/ml for paeoniflorin, 5.100, 10.20, 51.00,55.0, 510.0, 1020, 2040 ng/ml for naringin, 5.200, 10.40, 52.00,60.0, 520.0, 1040, 2080 ng/ml for naringenin and 10.60, 21.20,06.0, 530.0, 1060, 2120, 4240 ng/ml for glycyrrhetinic acid, respec-ively. Low, medium, and high concentrations of QC samples werehosen to be 19.60, 313.6, 3136 ng/ml for paeoniflorin, 10.20, 163.2,632 ng/ml for naringin, 10.40, 166.4, 1664 ng/ml for naringeninnd 21.20, 339.2, 3392 ng/ml for glycyrrhetinic acid, respectively.

.4. Plasma sample preparation

I.S. solution (20 �l) was pipetted into 1.5 ml EP tubes and evap-rated to dryness under a gentle stream of nitrogen. The residueas vortex-mixed with 100 �l of plasma samples for 30 s and then

50 �l of acetonitrile was added. The mixture was vortex-mixedor 60 s and centrifuged at 13,000 rpm for 10 min to separate therecipitated protein, 10 �l of the supernatant were injected intohe UPLC–MS/MS system for analysis.

.5. Method validation

.5.1. SelectivityThe selectivity was investigated by comparing the chro-

atograms of blank plasma obtained from six rats with those oforresponding standard plasma sample spiked with paeoniflorin,

iomedical Analysis 66 (2012) 271– 277 273

naringin, naringenin, glycyrrhetinic acid and I.S. and plasma sampleafter oral administration of Si Ni San decoction.

2.5.2. Linearity and lower limit of quantification (LLOQ)Calibration curves were prepared using the standard plasma

samples described in Section 2.3, and constructed from the peak-area ratios of each analyte to I.S. versus plasma concentrations usinga 1/x2 weighted linear least-squares regression model. The LLOQis defined as the lowest concentration on the calibration curve atwhich an acceptable accuracy (RE) within ±20% and a precision(RSD) below 20% can be obtained.

2.5.3. Precision and accuracyFive replicates of QC samples of each analyte at three QC levels

were included in each run to determine the intra- and inter-day (3days) precision of the assay. Accuracy was determined as the per-centage difference between the mean concentrations determinedand the nominal concentrations. According to the guidance of FDA,the RSD determined at each concentration level is required notexceeding 15% and RE within ±15% of the actual value.

2.5.4. Extraction recovery and matrix effectExtraction recovery of the four analytes was evaluated at three

QC levels and for the I.S. at one concentration by comparing thepeak areas obtained from plasma samples with the analytes spikedbefore and after extraction. Matrix effect was evaluated by compar-ing the peak areas of the analytes obtained from plasma sampleswith the analytes spiked after extraction to those from the neatstandard solutions at the same concentration. An endogenousmatrix effect was implied if the ratio is less than 85% or more than115%.

2.5.5. StabilityStability of the four analytes in plasma was assessed by ana-

lyzing QC samples of three levels during the sample storageand processing procedures. Short-term stability was assessed byanalyzing QC samples kept at room temperature for 4 h, whichexceeded the routine preparation time of samples. Long-term sta-bility was evaluated by keeping QC samples at −20 ◦C for 15 days.Freeze–thaw stability was investigated after three freeze (−20 ◦C)and thaw (room temperature) cycles. Post-preparation stabilitywas assessed by analyzing the extracted QC samples kept in theautosampler at 4 ◦C for 12 h. All stability testing QC samples weredetermined using calibration curve of freshly prepared.

2.6. Pharmacokinetic study

Healthy male SD rats, weighing 200–240 g, were obtainedfrom the Animal Center of Shenyang Pharmaceutical Univer-sity (Shenyang, China). All protocols of animal experiments wereapproved in accordance with the Regulations of Experimental Ani-mal Administration issued by the State Commission of Science andTechnology of the People’s Republic of China. The rats were fastedfor 12 h but with free access to water prior to the experiments.

Si Ni San extractum was dissolved with 0.5% CMC-Na yieldinga concentration of 2.5 g/ml (expressed in grams of crude drugs perml). The rats were given orally with 40 g/kg Si Ni San (equivalentto 2.2 mg/g for paeoniflorin, 21.5 mg/g for naringin and 5.8 mg/gfor glycyrrhizic acid). Blood samples (0.3 ml) were collected in1.5 ml heparinized polythene tubes before dosing and 0.17, 0.33,0.50, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 12, 14, 24, 30, 48, 60 and 72 hafter dosing. The blood samples were immediately centrifuged at

13,000 rpm for 10 min and the plasma was stored at −20 ◦C untilanalysis.

The maximum plasma concentrations (Cmax) and the time toreach the maximum concentrations (Tmax) were obtained directly

274 J. Wen et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 271– 277

Fig. 2. Representative MRM chromatograms of paeoniflorin (channel 2), naringin (channel 3), naringenin (channel 4), glycyrrhetinic acid (channel 5) and I.S. (channel 1) inrat plasma: (A) blank plasma sample; (b) Blank plasma sample spiked with paeoniflorin, naringin, naringenin, glycyrrhetinic acid at the LLOQ and I.S.; (C) plasma samplef g.

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rom a rat 8.0 h after oral administration of Si Ni San decoction at a dose of 40 g/k

rom the observed data. The elimination rate constant (ke) was cal-ulated on the slope of the linear regression of log-transformedoncentration versus time using the last four measurable points.he elimination half-life (t1/2) was calculated using 0.693/ke.he area under plasma concentration–time curve (AUC0–t) tohe last measurable plasma concentration (Ct) was estimated bysing the linear trapezoidal rule. The area under the plasmaoncentration–time curve to time infinity (AUC0–∞) was calculateds AUC0–∞ = AUC0–t + Ct/Ke. The mean residence time (MRT) was cal-ulated as AUMC0–∞/AUC0–∞. Data were expressed in mean andtandard deviation (SD) for each group. The significance of differ-nce was assessed by Student’s t-test.

. Results and discussion

.1. Analysis method optimization

In our study, various mobile phase conditions were tried tobtain optimized responses, suitable retention times and good peakhapes for the analytes. Methanol, acetonitrile, 5 mM ammoniumcetate, 0.1% formic acid and 100% water were tested as potential

obile phases. Finally a gradient elution with methanol and water

ontaining 2 mM ammonium acetate was chosen to obtain satis-actory sensitivity and good peak shapes. The gradient programSection 2.2.1) was used for short analytical time (4.5 min) and

avoiding “cross-talk” peaks. Lysinopril was selected as the inter-nal standard for its similarity in the retention and ESI ionization tothose of analytes. For the MS condition, both positive and negativescan modes were tested and the positive mode was selected dueto higher sensitivity. MRM mode was used to monitor both quasi-molecule and fragment ions, which made the method more specific.ESI source temperature, capillary and cone voltage, flow rate ofdesolvation gas and cone gas were optimized to obtain highestintensity of protonated molecules of the four analytes.

3.2. Sample extraction

Liquid–liquid extraction (LLE) was initially developed with dif-ferent solvents, however, it could not eliminate the interferencesfrom the sample matrix. Protein precipitation (PPT) was exploredto extract the four analytes from plasma samples. The recovery ofPPT with acetonitrile was higher than with methanol. Satisfactoryrecovery and higher responses were obtained with 150 �l acetoni-trile.

3.3. Method validation

3.3.1. SelectivityThe selectivity was evaluated by extracting blank rat plasma

from six different matrices and comparing the MS/MS responses

J. Wen et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 271– 277 275

Table 1Precision and accuracy data for the determination of paeoniflorin, naringin, naringenin and glycyrrhetinic acid in rat plasma (n = 3 days and five replicates per day).

Component Added (ng/ml) Found (ng/ml) (mean ± SD) Intra-day RSD (%) Inter-day RSD (%) Relative error (%)

Paeoniflorin 9.800 9.437 ± 0.660 4.8 14 −3.719.60 19.19 ± 1.57 6.9 13 −2.1

313.6 315.8 ± 20.2 5.5 9.7 0.73136 3371 ± 185 5.1 8.0 7.5

Naringin 5.100 5.125 ± 0.431 5.7 15 0.510.20 10.68 ± 1.22 12 13 4.7

163.2 167.6 ± 12.7 6.9 10 2.71632 1647 ± 86.0 5.2 4.4 0.9

Naringenin 5.200 5.425 ± 0.522 8.9 13 4.110.40 10.12 ± 0.67 5.1 12 −2.7

166.4 177.1 ± 10.7 6.0 7.7 6.41664 1753 ± 89 5.4 4.4 5.3

Glycyrrhetinic acid 10.60 10.21 ± 1.00 9.9 9.2 −3.721.20 20.98 ± 1.29 4.9 11 −1.0

339.2 332.0 ± 24.2 7.7 6.7 −2.13392 3145 ± 217 7.4 2.8 −7.3

Table 2Pharmacokinetic parameters of paeoniflorin, naringin, naringenin and glycyrrhetinic acid after oral administration of Si Ni San decoction at a dose of 40 g/kg (n = 6,mean ± SD).

Component Paeoniflorin Naringin Naringenin Glycyrrhetinic acid

Cmax (ng/ml) 870.9 ± 238.3 368.9 ± 195.3 1034 ± 402.0 3972 ± 342.0Tmax (h) 0.70 ± 0.11 0.75 ± 0.17 13.6 ± 0.89 27.6 ± 3.28Ke (h) 0.25 ± 0.11 0.13 ± 0.04 0.15 ± 0.04 0.07 ± 0.01t1/2 (h) 3.19 ± 1.54 5.76 ± 2.12 5.00 ± 1.46 9.40 ± 1.15

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AUC0–∞ (ng h/ml) 2489 ± 817.1 1492MRT0–∞ (h) 4.11 ± 2.34 9.96

t the retention times of the four analytes to the responses of theLOQ. The representative chromatograms of blank plasma, blanklasma spiked with LLOQ and plasma sample obtained after oraldministration of Si Ni San decoction are shown in Fig. 2. Underhe established chromatographic condition, no endogenous inter-erence from the plasma was observed and all the four analytes asell as I.S. were not interfered with each other.

.3.2. Linearity and LLOQTypical equations for the calibration curves and correlation

oefficients (r) were y = 3.74 × 10−3x + 1.07 × 10−2, r = 0.9953 foraeoniflorin, y = 1.31 × 10−3x + 4.97 × 10−3, r = 0.9967 for naringin,

= 5.39 × 10−3x + 2.85 × 10−3, r = 0.9988 for naringenin, and = 8.09 × 10−4x + 1.90 × 10−3, r = 0.9949 for glycyrrhetinic acid.he calibration curves of the four analytes were linear in theanges of 9.800–3920 ng/ml, 5.100–2040 ng/ml, 5.200–2080 ng/mlnd 10.60–4240 ng/ml, respectively. The LLOQ of the four analytesere 9.800 ng/ml, 5.100 ng/ml, 5.200 ng/ml and 10.60 ng/ml,

espectively, with precision (RSD) below 20% and accuracy (RE)ithin ±20% (Table 1).

.3.3. Precision and accuracyThe intra- and inter-day precision and accuracy were deter-

ined by measuring five replicates of QC samples at threeoncentration levels. The precision and accuracy are shown inable 1. The intra- and inter-day precision ranged 4.9–12% and.8–13%, respectively. The accuracy derived from QC samples wasithin −7.3 to 7.5% for three QC levels.

.3.4. Extraction recovery and matrix effect

The extraction recoveries from QC samples at three concen-

rations levels were 87.50 ± 3.75%, 90.45 ± 6.98%, 84.94 ± 1.56%or paeoniflorin, 84.55 ± 8.32%, 87.83 ± 5.50%, 86.65 ± 1.34% foraringin, 95.62 ± 7.14%, 90.51 ± 8.75%, 90.90 ± 1.76% for naringenin

.8 12,242 ± 6381 77,481 ± 16,674

.8 12,284 ± 6377 78,817 ± 16,707 16.25 ± 1.64 32.85 ± 4.07

and 89.98 ± 5.73%, 93.35 ± 1.61%, 91.76 ± 8.06% for glycyrrhetinicacid, respectively, whereas 83.11 ± 5.34% for I.S..

With regard to matrix effect, all the calculated values werebetween 90.93% and 109.8%. No significant matrix effect forpaeoniflorin, naringin, naringenin, glycyrrhetinic acid and I.S. wasobserved, indicating that no co-eluting substance influenced theionization of the analytes and I.S..

3.3.5. StabilityThe concentrations of paeoniflorin, naringin, naringenin and

glycyrrhetinic acid measured in the stability study were between89.5% and 102.9% of the initial values, indicating that the analytesin rat plasma were stable for 4 h at room, 15 days at −20 ◦C, threefreeze–thaw cycles and 12 h after pretreatment.

3.4. Pharmacokinetic application

The validated UPLC–MS/MS method was applied to a phar-macokinetic study of four major components in rats afteroral administration of Si Ni San decoction. The mean plasmaconcentration–time curves of paeoniflorin, naringin, naringeninand glycyrrhetinic acid are presented in Fig. 3 and the pharma-cokinetic parameters of the four components are shown in Table 2.

As seen from Table 2, the Tmax are 0.70 h for paeoniflorin, 0.75 hfor naringin, 13.6 h for naringenin and 27.6 h for glycyrrhetinic acid,respectively. Naringin is quickly absorbed into the body and alsoquickly eliminated after oral administration of Si Ni San decoc-tion. Another small peak can be seen at 12 h (Fig. 3B), which may bedue to the enterohepatic circulation of naringin in rats. Its metabo-lite, naringenin is produced slowly after oral administration ofSi Ni San decoction. Glycyrrhetinic acid could not been detected

in Si Ni San decoction in vitro. However, the Cmax of glycyrrhetinicacid is 3972 ng/ml after oral administration of Si Ni San decoc-tion. Therefore, the pharmacokinetic studies of the four analytesneed to be further investigated.

276 J. Wen et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 271– 277

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cknowledgment

This work is supported by grant from the National Foundationf Natural Sciences of China (no. 81102786).

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