ORIGINAL PAPER

A sensitive liquid chromatography–mass spectrometrymethod for simultaneous determination of alisol A and alisolA 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma

Yue Yu & Qing Li & Kaishun Bi & Pu Xie &

Guoguang Yang & Xiaohui Chen

Received: 26 August 2010 /Revised: 8 October 2010 /Accepted: 7 November 2010 /Published online: 24 November 2010# Springer-Verlag 2010

Abstract A liquid chromatography–mass spectrometry(LC-MS) method was developed and validated for thesimultaneous determination of alisol A and alisol A 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma usingdiazepam as an internal standard. A 200-μl plasma samplewas extracted by methyl tert-butyl ether and the separationwas performed on Kromasil C18 column (150×4.6 mm,5 μm) with the mobile phase of acetonitrile (containing 0.1%of formic acid)–water (73:27, v/v) at a flow rate of 0.8 ml/min in a run time of 10 min. The two analytes weremonitored with positive electrospray ionization by selectedion monitoring mode. The lower limit of quantitation forboth alisol A and alisol A 24-acetate were 10 ng/ml. Thecalibration curves were linear in the measured range 10–1,000 ng/ml for alisol A and 10–500 ng/ml for alisol A 24-acetate. The mean extraction recoveries were above 74.7%for alisol A and above 72.4% for alisol A 24-acetate frombiological matrixes. The intra- and inter-day precision for allconcentrations of quality controls was lower than 14.1%(RSD %) for each analyte. The accuracy ranged from−12.3% to 9.8% (RE %) for alisol A, and −8.6% to 14.2%(RE %) for alisol A 24-acetate. The method was successfullyapplied to the study on the pharmacokinetics of alisol A andalisol A 24-acetate in rat plasma.

Keywords Alisol A . Alisol A 24-acetate . Alisma orientale(Sam.) Juz. . LC-MS . Pharmacokinetics

Introduction

Rhizoma Alismatis, the dried rhizome of Alisma orientale(Sam.) Juz., is a famous Traditional Chinese Medicinewhich has been widely used for diuretic, hypolipidemic,anti-nephrolithic, anti-atherosclerotic, anti-inflammatory,and anti-diabetic purposes, etc. in China for more than1,000 years [1, 2]. Rhizoma Alismatis is known to be richin protostane-type triterpenes [3–5]. Alisol A and alisol A24-acetate are two major active triterpenes isolated fromRhizoma Alismatis (Fig. 1), which were demonstrated topossess diuretic, anti-inflammatory and hypocholesterole-mic effects [6, 7]. Recently, much attention has been paid tothe pharmacological activity of these protostane-typetriterpenes [7–9], and a few papers dealing with theirpharmacokinetics have been published.

To elucidate the ADME process of Rhizoma Alismatis invivo, a liquid chromatography–mass spectrometry (LC-MS)method was developed for the simultaneous determination ofalisol A and alisol A 24-acetate in rat plasma. In this paper,pharmacokinetic study of these two active protostane-typetriterpenes in rat was investigated after single oral adminis-tration of Rhizoma Alismatis extract. This is the first report ondetermination of the major triterpenes in rat plasma after oraladministration of Rhizoma Alismatis extract. The results ofthe pharmacokinetics study provided a meaningful basis forevaluating the clinical applications of the herbal medicine.

Experimental

Materials

The dried rhizomes of A. orientale (Sam.) Juz. werepurchased from the Medical Material Co. of Liaoning

Y. Yu :Q. Li :K. Bi : P. Xie :G. Yang :X. Chen (*)Department of Pharmaceutical Analysis, ShenyangPharmaceutical University,Wenhua Road 103,Shenyang 110016, Chinae-mail: cxh_syphu@yahoo.com.cn

Anal Bioanal Chem (2011) 399:1363–1369DOI 10.1007/s00216-010-4426-9

province (Shenyang, China). Alisol A (purity>95%) wasisolated in the author’s laboratory (Department ofPharmaceutical Analysis, Shenyang Pharmaceutical Uni-versity, Shenyang, People’s Republic of China), and thestructure was characterized by spectral methods, includ-ing MS, 1H- and 13C-NMR spectra. The data wereconsistent with those reported in literatures [10–12].Alisol A 24-acetate was purchased from Nanjing Univer-sity of Traditional Chinese Medicine (Nanjing, People’sRepublic of China) (purity>98%). Diazepam (purity>98%) (Fig. 1), used as the internal standard (IS), waspurchased from the National Institute for Control ofPharmaceutical and Biological Products (Beijing, China).Acetonitrile (high-performance liquid chromatography(HPLC) grade) were obtained from Fisher Scietific (FairLawn, NJ, USA). Formic acid (HPLC grade) waspurchased from Kermel Chemical Reagent Co., Ltd.(Tianjin, China). Methyl tert-butyl ether (analytical grade)was purchased from Sinopharm Chemical Reagent Co.,Ltd. (Shanghai, China). All other reagents were ofanalytical purity. Distilled water, prepared with deminer-alized water, was used throughout the study.

Animals

Six male pathogen-free Wistar rats (220–250 g) wereprovided by the Experimental Animal Center of ShenyangPharmaceutical University (Shenyang, People’s RepublicChina). Animal experiments were performed in accordancewith the Regulations of Experimental Animal Administra-tion issued by the State Committee of Science andTechnology of the People’s Republic of China.

LC-MS instruments and conditions

The assay was performed on Shimadzu 2010 liquidchromatography–mass spectrometry (Japan) equipped withan electrospray ionization (ESI) interface. Liquid chromato-graphic separations of the analytes were performed byKromasil C18 column (150×4.6 mm, 5 μm) at roomtemperature with the mobile phase of acetonitrile (contain-ing 0.1% of formic acid)–water (73:27, v/v) at a flow rate of0.8 ml/min (25% of the eluent was splitted into the inlet ofthe mass spectrometer) in a run time of 10 min. Theinjection volume was 20 μl. The analytes and IS wereionized by ESI source in positive ion mode under thefollowing source conditions: nebulizing gas, 1.5 l/min;CDL temperature, 250 °C; heat block temperature, 200 °C;detector voltage, 1.75 kV, and the other parameters werefixed as the tuning file. Analysis was carried out by selectedion monitoring (SIM) for alisol A [M−H2O+H]+ m/z473.40, alisol A 24-acetate [M−H2O+H]+ m/z 515.40, andIS [M+H]+ m/z 284.95.

Preparation of Rhizoma Alismatis extract

The crude drug (30 g) was extracted twice by refluxingwith 95% ethanol (1:10, w/v), 1 h for each time. Afterremoving the ethanol under reduced pressure, the residuewas dissolved in aqueous solution of 0.5% CMC-Na to getthe Rhizoma Alismatis extract with a Rhizoma Alismatisconcentration of 1.5 g/ml (containing 0.5 mg/g alisol A and0.4 mg/g alisol A 24-acetate).

Standard solutions

The stock solutions of alisol A, alisol A 24-acetate anddiazepam (IS) were prepared by dissolving a proper amountof alisol A, alisol A 24-acetate and diazepam in acetonitrileto obtain nominal concentrations of 50 μg/ml for each. Aseries of mixture standard working solutions with concen-trations 100–10,000 ng/ml for alisol A and 100–5,000 ng/ml for alisol A 24-acetate were obtained by diluting themixture of the stock standard solutions with acetonitrile.The working solution of IS (125 ng/ml) was prepared bydiluting IS stock solution with acetonitrile. All solutionswere stored at 4 °C.

Sample preparation

Plasma samples (200 μl) were spiked with 20 μl of IS. Themixtures were extracted with 2 ml of methyl tert-butyl etherby vortexing for 5 min. After centrifugation at 1,000×g for5 min, the organic layer was transferred to a polypropylenetube and evaporated to dryness under nitrogen gas at roomtemperature. The residue was reconstituted in 100 μl of

O

OHHO

HO

OH

Alisol A

O

OH

HOHO

OCOCH3

Alisol A 24-acetate

NNH3C

O

ClDiazepam

a

b

c

Fig. 1 Chemical structures ofalisol A (a), alisol A 24-acetate(b), and diazepam (IS) (c)

1364 Y. Yu et al.

mobile phase and 20 μl was injected for the LC-MSanalysis.

Method validation

Calibration standards of alisol A (10, 25, 100, 250, 500, and1,000 ng/ml) and alisol A 24-acetate (10, 25, 50, 100, 250, and500 ng/ml) were prepared by spiking the appropriate amountof the mixture standard working solutions (20 μl) and ISworking solution (20 μl) into 200 μl drug-free rat plasma.Calibration was performed by least-squares linear regressionof the peak area ratios of the two analytes to the IS versus thenominal standard concentration with a weighted (one persquare of concentration) factor [13]. The calibration curveswere constructed by plotting the peak area ratio against thedrug concentration prepared.

Quality control samples (QC samples) at low, medium,and high concentrations (at 20, 400, and 800 ng/ml foralisol A and 20, 125, and 400 ng/ml for alisol A 24-acetate)and the lower limit of quantitation (LLOQ) samples (10ng/ml for both alisol A and alisol A 24-acetate), which wereprepared in the same manner as the calibration standards,were analyzed to validate the accuracy and precision of thedescribed method. The intra-day precision, inter-day preci-sion and accuracy were evaluated over a period of threeconsecutive days with six replications at each concentrationper day (n=18) and calculated with calibration curvesobtained daily. The precision was expressed as the relativestandard deviation (RSD) while the accuracy was evaluatedas the relative error (RE) [14].

The extraction recovery of internal standard diazepam,alisol A and alisol A 24-acetate was determined bycomparing the peak area of the analytes extracted fromreplicating QC samples of low, medium and high concen-trations (n=6, for each concentration level) with the peakarea of the analytes from non-extracted standard solutionsat equivalent concentration.

Stability testing

The stability of alisol A and alisol A 24-acetate in rat plasmawas studied by three replicates of QC samples at eachconcentrations under different storage conditions: three freeze(−20 °C)–thaw (room temperature) cycles, 24-h storage atroom temperature and frozen (−20 °C) for 30 days.

Method application

Drug administration and sampling

All of the six rats were fasted for 12 h, with free access towater prior to the experiments. After giving 18 g/kg ofRhizoma Alismatis extract (calculated by crude drug) toeach rat orally, the blood samples (0.3 ml) were obtainedfrom the oculi chorioideae vein at 0, 0.08, 0.17, 0.33, 0.75,1, 1.5, 2, 4, 6, 7, 8, 9, 10, 12, 16, and 24 h which wereplaced in heparinized tubes and separated by centrifugationat 1,000×g for 5 min. The separated plasma samples werefinally stored at −20 °C until analysis.

Pharmacokinetics analysis

The plasma concentrations of alisol A and alisol A 24-acetate at different times were calculated from the dailycalibration curve, which were expressed as means ± SD. Allthe pharmacokinetic parameters were processed by non-compartmental analysis using DAS 2.1 pharmacokineticprogram (Chinese Pharmacological Society). Maximumplasma concentration (Cmax) and the time to reach the

400 450 500 550 m/z

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556.60497.25454.25 532.60383.30

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284.95

453.05274.10318.00

208.00218.00

a

b

c

Fig. 2 The product ion mass spectra of alisol A (a), alisol A 24-acetate (b), and IS (c) (Please note: a [M+Na]+ (m/z 513.15) and[M−H2O+H]+ (m/z 473.40) of alisol A; b [M+Na]+ (m/z 555.35) and[M−H2O+H]+ (m/z 515.40) of alisol A 24-acetate; and c [M+H]+

(m/z 284.95) of IS)

A sensitive liquid chromatography–mass spectrometry method 1365

maximum plasma concentration (Tmax) were obtaineddirectly from the observed values.

Results and discussion

Sample preparation

The sample preparation was based on liquid–liquid extraction,optimized by testing the influence of different kinds of organicreagents (cyclohexane, diethyl ether, methyl tert-butyl ether,

ethyl acetate) on the recovery. In brief, the best result wasobtained with the procedure described above.

LC-ESI-MS optimization

Alisol A, alisol A 24-acetate, and IS were analyzed by MS inESI positive ion mode and the full-scan mass spectrums ofthem after direct injection in mobile phase are shown in Fig. 2.The sensitive ions in full-scan positive-ion mass spectra were[M−H2O+H]+ (m/z 473.40) and [M+Na]+ (m/z 513.15) foralisol A, [M−H2O+H]+ (m/z 515.40) and [M+Na]+ (m/z

0.0 5.0 10.0

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3.86

3

(A)

(B)

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1

1

2

2

3

3

Fig. 3 SIM chromatograms of ablank rat plasma sample (a), ablank rat plasma spiked withalisol A (10 ng/ml), alisol A 24-acetate (10 ng/ml) and IS (b),and a rat plasma sample 1 h afteroral administration of RhizomaAlismatis extract (c). 1, alisol A;2, alisol A 24-acetate; 3, IS

1366 Y. Yu et al.

555.35) for alisol A 24-acetate, and [M+H]+ (m/z 284.95) fordiazepam (IS). However, the response was more stable andshowed better linearity than [M+Na]+ (m/z 513.15) for alisolA in SIM mode when the ion [M−H2O+H]+ (m/z 473.40)was selected as detecting ion. So the quantitative analysis wascarried out by SIM for alisol A m/z 473.40, alisol A 24-acetate m/z 515.40, and IS m/z 284.95. Alisol A 24-acetatewas unstable when it was dissolved in methanol and heated[1]. So APCI was abandoned. In order to increase sensitivity,formic acid was tested as a modifier. Addition of 0.1% formicacid enhanced the sensitivity compared with that with noadditives.

Method validation

Specificity

The chromatograms for blank plasma, blank plasma spikedwith alisol A, alisol A 24-acetate and IS as well as the ratplasma sample after administration of Rhizoma Alismatisextract were presented in Fig. 3. There was no endogenousinterference at the retention times of alisol A (6.4 min),alisol A 24-acetate (8.3 min), and diazepam (IS, 3.9 min).

Linearity and LLOQ

The calibration curves showed good linearity over theconcentration range of 10–1,000 ng/ml for alisol A and

10–500 ng/ml for alisol A 24-acetate. The typicalcalibration curves were as follows: alisol A, y=1.21×10−3 x+1.68×10−3 (r=0.9940); alisol A 24-acetate,y=1.79×10−3 x−1.01×10−3 (r=0.9987). x referred to theconcentration of the analytes in plasma (ng/ml); y referredto the peak area ration of the analytes and IS. The LLOQfor alisol A and alisol A 24-acetate were 10 ng/ml withthe RSD of six replications being 7.01% and 5.76%,respectively.

Precision and accuracy

The intra- and inter-day precision (presented as RSD) andaccuracy (presented as RE) are shown in Table 1. All theresults indicated that overall reproducibility of the methodwas acceptable.

Extraction recovery and ionization

The mean extraction recoveries were determined using sixreplicates of QC samples at three concentration levels. Therecoveries of alisol A and alisol A 24-acetate were 70.7–79.5% and 70.1–79.7%, respectively (Table 1). The meanextraction recovery of internal standard Diazepam was80.5%.

As for ionization, the peak area ratios of the two analytesand IS after spiking evaporated plasma samples at threeconcentration levels compared to neat standard solutions

Table 2 Stability for analysis of alisol A and alisol A 24-acetate in rat plasma (n=6)

Concentration spiked (ng/ml) Three freeze–thaw cycles 24 h at room temperature 30 days at −20 °C

RE (%) RSD (%) RE (%) RSD (%) RE (%) RSD (%)

Alisol A 20 −5.9 1.1 6.8 5.9 −9.9 1.8

400 5.6 3.6 3.3 2.8 9.2 0.9

800 3.6 2.7 5.5 1.9 3.7 0.1

Alisol A 24-acetate 20 8.7 3.7 6.2 2.7 14.9 9.4

125 7.0 4.8 9.3 4.3 10.3 7.5

400 −4.2 2.3 5.4 3.9 6.2 4.3

Table 1 Precision, accuracy, and extraction recoveries of the LC-MS method for analysis of alisol A and alisol A 24-acetate in rat plasma

Concentration spiked (ng/ml)

Inter-day (RSD(%))

Intra-day (RSD(%))

Accuracy (RE(%))

Extraction recovery (mean ± SD(%))

Alisol A 20 13.6 4.4 −12.3 74.7±4.0

400 5.5 2.7 9.8 76.1±0.8

800 6.4 2.6 6.9 74.8±2.5

Alisol A 24-acetate

20 14.1 7.0 14.2 72.4±2.1

125 8.9 3.6 −8.6 75.7±1.3

400 10.0 2.4 8.7 75.7±3.7

A sensitive liquid chromatography–mass spectrometry method 1367

ranged from 86.4% to 96.4% for alisol A and from 88.8%to 92.5% for alisol A 24-acetate, suggesting that the methodwas free from matrix effect.

Stability

The stability study showed that the two activeprotostane-type triterpenes were stable after threefreeze–thaw cycles and showed no significant degrada-tion for 24 h at room temperature, even for 30 days at−20 °C (Table 2). Alisol A 24-acetate was reportedunstable, which might be transformed into alisol A 23-acetate and deacetylated to yield alisol A in methanol andby heating [1]. During the study, the temperature wasstrictly controlled and methanol was not used throughoutthe experiment. The results showed that the assaydescribed above is satisfactory with respect to accuracy,precision and stability.

Pharmacokinetic studies

The plasma drug concentrations of alisol A and alisol A 24-acetate can be determined by the established LC-ESI-MSmethod. The plasma concentrations versus time profileswere presented in Fig. 4. The pharmacokinetic parametersof alisol A and alisol A 24-acetate in rats calculated byDAS 2.1 software were shown in Table 3. The half-life (T1/2) is calculated based on the second peak. The Cmax andtime to Cmax (Tmax) were obtained from the observed datadirectly based on the second peak. The area under the curve(AUC0–t) from 0 to 24 h was calculated using the lineartrapezoidal rule with extrapolation to infinity (AUC0–∞)from the last measurable concentration using the terminalelimination rate constant (ke) calculated by linear regressionof the final log-linear part of the drug concentration–timecurve.

Both of the concentration–time curves of alisol A andalisol A 24-acetate in rat plasma after oral administration ofRhizoma Alismatis extract showed significant double-peakphenomenon, which was often occurred in the pharmaco-kinetics study of herb medicines [15–19]. Absorption ofdrugs (especially for herb medicines) from the gastrointes-tinal tract was a complex process and the phenomenonmight be caused by the following reasons: (1) RhizomaAlismatis mainly contained protostane triterpenes and mostof them could be inter-transformed [1, 3–5, 20], whichinduced the presence of two peaks with the second peaklarger than the first one. Alisol B might be changed to alisolA, and alisol B 23-acetate or alisol B 11, 23-diacetate mightbe changed to alisol A 24-acetate by the hydration in acidconditions [21]. (2) Variability of absorption withindifferent regions of the gut might be a probable reason forthe double-peak phenomenon; (3) Enterohepatic circulationmight be existed and the analytes were reabsorbed viaintestinal tract. Further studies are needed to investigate themechanism underlying the double-peak phenomenon foralisol A and alisol A 24-acetate.

Conclusions

A simple, rapid, and sensitive LC-MS method with highextraction efficiency for the simultaneous determination ofalisol A and alisol A 24-acetate in rat plasma after oraladministration of Rhizoma Alismatis extract was firstdeveloped and validated. It was applied successfully for

0

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0Time (h)

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cent

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(A)

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300

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cent

ratio

n (n

g/m

l) Alisol A 24-acetate

(B)

2420161284

0 2420161284

Fig. 4 The concentration–time curves of alisol A (a) and alisol A 24-acetate (b) in rat plasma after oral administration of RhizomaAlismatis extract

T1/2 (h) Cmax (μg/L) Tmax (h) AUC(0–t) (μg h/L) AUC(0–∞) (μg h/L)

Alisol A 3.1±1.6 476.1±188.6 9.7±0.5 2,945±1,024 3,453±1,623

Alisol A 24-acetate 2.9±0.9 233.1±55.4 9.2±1.0 1,820±464 1,874±450

Table 3 Pharmacokinetic param-eters of alisol A and alisol A24-acetate in rats after oraladministration of RhizomaAlismatis extract

1368 Y. Yu et al.

the simultaneous pharmacokinetic studies of protostane-type triterpenes in rat plasma.

Acknowledgment This work was financially supported by NaturalScience Foundation of China (No.20875064)

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