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Simultaneous determination of three bufadienolides in rat plasma after intravenous administration of bufadienolides extract by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry
a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 224–231
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journal homepage: www.elsevier.com/locate/aca
Simultaneous determination of three bufadienolides in rat plasma after intravenous administration of bufadienolides extract by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry Yu Zhang, Xing Tang ∗ , Xiaoliang Liu, Fang Li, Xia Lin Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
A novel, rapid and specific ultra performance liquid chromatography electrospray ion-
Received 24 November 2007
ization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed for
Received in revised form
simultaneous determination and pharmacokinetic studies of three bufadienolides (bufalin,
26 December 2007
cinobufagin and resibufogenin) in rat plasma. The analytes, bufalin, cinobufagin, resibufo-
Accepted 10 January 2008
genin and the internal standard, diphenhydramine were extracted from rat plasma samples
Published on line 18 January 2008
by a one-step liquid–liquid extraction and separated on an ACQUITY UPLCTM BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water (containing
Keywords:
0.1% formic acid) at a flow rate of 0.20 mL min−1 . Detection was carried out on a triple-
Bufadienolides
quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode
Ultra performance liquid
via an electrospray ionization (ESI) interface. The three bufadienolides could be simul-
chromatography–tandem mass
taneously determined within 3.0 min. Linear calibration curves were obtained over the
spectrometry
concentration ranges of 1.0–200 ng mL−1 for all the analytes. The intra- and inter-day pre-
Pharmacokinetics
cisions (relative standard deviation (R.S.D.)) were less than 11.35 and 10.87%, respectively.
Rat plasma
The developed method was applied for the first time to the pharmacokinetic studies of bufadienolides in rats following a single intravenous administration of 2.10 mg kg−1 bufadienolides. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Toad venom, also called “Chan Su” in China, is obtained from the postauricular and skin glands of Bufo bufo gargarizans Cantor and the other species of the same genus, family bufonidae [1]. It is often found in traditional Chinese medicinal ingredients, such as Liu-Shen-Wan [2] and Niuhuangxiaoyan tablets [3]. These Chinese medicines have been widely used in China, Japan and other Asian countries for a long time, and
∗
Corresponding author. Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (X. Tang). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.01.029
over the last decade, they have gained considerable favor in the United States and elsewhere [4]. Toad venom in small doses is used for stimulation of myocardial contraction, to produce an anti-inflammatory effect and for pain relief. Due to its anesthetic and antibiotic actions, it is also used for a variety of other purposes including the treatment of tonsillitis, sore throat, and palpitation [5]. Recently, toad venom has been used for the treatment of cancer [6–9]. The principal biologically active components of toad
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venom are bufadienolides, a class of cardioactive C-24 steroids with a characteristic ␣-pyrone ring at C-17 [10–11]. However, cardioactive steroids, including bufadienolides, usually have a narrow therapeutic index, and intentional therapeutic intoxication is well documented [12]. Therefore, the detection and accurate quantitation of bufadienolides in bio-fluids play a very important role in therapeutic drug monitoring and pharmacokinetic investigations. Up to now, most investigations have been based on the pharmacodynamics or pharmacology of bufadienolides and very little attention has been devoted to the pharmacokinetic study of bufadienolides in vivo. Also, one essential difference between traditional medicines and synthetic drugs is that the therapeutic effects of the former are due to the joint contribution of multi-components, not only the major ones [13]. Therefore, it is necessary to develop a more comprehensive and global assay to fully evaluate the pharmacokinetics of bufadienolides. Several HPLC/UV methods have been developed to determine bufadienolides in rat serum [14–15], in rat plasma [16] and human liver [12]. However, these HPLC/UV methods were not sensitive enough to determine bufadienolides in biological matrix for pharmacokinetics study after intravenous administration. The lower limit of quantitation (LLOQ) was more than 6.8 ng mL−1 . Recently, two LC/MS methods have been established for determination of bufadienolides. One LC/TOF-MS method has been reported for the determination of bufalin, cinobufagin and resibufogenin in dog plasma after oral administration of Liu-Shen-Wan [17]. This method was sensitive enough for pharmacokinetic study, but the run time was about 30 min which did not meet acquirement of high-throughput determination of biosamples. Another LC/MS/MS method has been developed for determination of five bufadienolides in rat plasma after intragastric administration of 100 mg kg−1 Chan Su [18]. The purpose of this study was to develop a rapid, selective and specific UPLC-ESI-MS/MS method, which enables simultaneous determination of three bufadienolides (bufalin, cinobufagin and resibufogenin) at 1.0 ng mL−1 in rat plasma. The total run time of the method per sample was only 3.0 min which was almost 10 times shorter than the reported method [17]. To our knowledge, this is the first report of the development, validation and application of a UPLC-ESI-MS/MS method for simultaneous determination of three bufadienolides in rat plasma and a study of their pharmacokinetics after a single intravenous administration of 2.10 mg kg−1 bufadienolides.
2.
Experimental
2.1.
Materials and reagents
Bufadienolides were extracted from toad venom (Bufo bufo gargarizans Cantor) by the Department of Pharmaceutics, Shenyang Pharmaceutical University, China. The bufadienolides extract was mainly composed of bufalin, cinobufagin and resibufogenin, with a content of 7.09%:9.37%:11.85%, respectively. The reference standards: cinobufagin (Lot #803-9202) and resibufogenin (Lot #0718-9306) were purchased from the
Table 1 – Gradient condition of UPLC Time (min)
Flow rate (mL min−1 )
A (%)a
B (%)b
Curve
Initial 1.0 2.5 3.0
0.20 0.20 0.20 0.20
10.0 55.0 55.0 10.0
90.0 45.0 45.0 90.0
Initial 6c 6 1d
a b c d
Acetonitrile. 0.1% Fomic acid water. Linear. Pre-step.
National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and bufalin (Lot #1032050314) was obtained from National Engineering Research Center for Traditional Chinese Medicine (Nanchang, Jiangxi, China). Diphenhydramine (internal standard, IS) was a kind gift of the Department of Analytical Chemistry of Shenyang Pharmaceutical University (Shenyang, China). The structures of the three bufadienolides and IS are shown in Fig. 1. Acetonitrile and formic acid (HPLC grade) were supplied by Dikma (Richmond Hill, NY, USA). Water was purified in a Barnstead EASYpure® II RF/UV ultrapure water system (Dubuque, Lowa, USA) and passed through a 0.22 m filter prior to use in all the studies. Other chemicals were of analytical grade.
2.2.
Instrumentation and conditions
2.2.1.
Ultra performance liquid chromatography (UPLC)
Chromatography was performed on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a conditioned autosampler at 4 ◦ C. The separation was carried out on an ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm i.d., 1.7 m; Waters Corp., Milford, MA, USA). The column temperature was maintained at 35 ◦ C. The analysis was achieved with gradient elution using (A) acetonitrile and (B) water (containing 0.1% formic acid) as the mobile phase. The gradient conditions are shown in Table 1. The injection volume was 5 L and the partial loop mode was used for sample injection.
2.2.2.
Mass spectrometer
The Waters ACQUITYTM TQD triple-quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) was connected to the UPLC system via an electrospray ionization (ESI) interface. The ESI source was operated in positive ionization mode with the capillary voltage set at 3.8 kV. The extractor and RF voltages were 2.0 and 0.1 V, respectively. The temperature of the source and desolvation was set at 80 and 400 ◦ C, separately. Nitrogen was used as the desolvation gas (500 L h−1 ) and cone gas (50 L h−1 ). For collision-induced dissociation (CID), argon was used as the collision gas at a flow rate of 0.20 mL min−1 (approximately 2.81 × 10−3 mbar). The multiple reaction monitoring (MRM) mode was used for quantification. Transition reactions of the analytes and internal standards are given in Table 2. All data collected in centroid mode were acquired using MasslynxTM NT4.1 software (Waters Corp., Milford, MA,
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Fig. 1 – The structures of bufalin (a), cinobufagin (b), resibufogenin (c) and diphenhydramine (d).
USA). Post-acquisition quantitative analyses were performed using a QuanLynxTM program (Waters Corp., Milford, MA, USA).
2.3.
Animals and blood sampling
Male Wistar rats (8 weeks old, 200 ± 20 g) were obtained from the Laboratory Animal Center of Shenyang Pharmaceutical University. The experimental protocol was approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. Rats were housed in groups of two or three with 12-h light/12-h dark cycle at a temperature of 22 ± 3 ◦ C, relative humidity of 45–60%, for 1 week. Immediately before the day of administration, the rats were fasted for 12 h but were allowed water ad libitum. Then, 2.10 mg kg−1 aqueous solution of bufadienolide extract containing 10% propylene glycol was administered to the rats
(equivalent to 149.95 g kg−1 bufalin; 198.20 g kg−1 cinobufagin; 250.65 g kg−1 resibufogenin) intravenously via the femoral vein. Blood samples from each rat were collected into heparinized Eppendorf tubes (2.0 mL) by puncture of the retroorbital sinus. This was performed at 0 (predose), 5, 10, 15, 20, 30, 45, 60, 90 and 120 min after administration. As soon as possible, the heparinized blood was centrifuged for 10 min at 2000 × g, and the plasma obtained was stored frozen at −20 ◦ C until analysis.
2.4. Preparation of standard and quality control (QC) solutions Stock solutions of bufalin, cinobufagin and resibufogenin were prepared by dissolving the accurately weighed standard compounds in methanol to give final concentrations of 10 g mL−1 for each analyte in the same volumetric flask. The mixed solution was then further diluted with methanol
Table 2 – Transition reactions of the analytes and internal standards Molecule Bufalin Cinobufagin Resibufogenin I.S.
Transition 387.2 → 255.1 443.2 → 365.1 385.2 → 367.1 256.1 → 167.0
Dwell (s) 0.05 0.05 0.05 0.05
Cone voltage (V) 45.0 50.0 45.0 30.0
Collision energy (eV) 25.0 20.0 20.0 30.0
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to achieve standard working solutions of desired concentrations. The internal standard working solution (120 ng mL−1 ) was similarly prepared by diluting a stock standard solution of diphenhydramine with water. All the working solutions were kept refrigerated (4 ◦ C) and brought to room temperature before use.
2.5. Preparation of calibration standards and quality control samples (QCs) The standard solutions were used to spike 200 L of blank plasma samples either for calibration standards of bufadienolides or for QCs in a prestudy and during the pharmacokinetic studies. Calibration standards were prepared at plasma concentrations of 1.0, 2.0, 5.0, 10, 20, 50, 100 and 200 ng mL−1 for each analyte, while QCs were prepared with blank plasma at LLOQ, low, medium and high concentrations of 1.0, 2.0, 50 and 180 ng mL−1 .
2.6.
Plasma sample preparation
Frozen plasma samples were thawed at room temperature and thoroughly vortexed prior to extraction. To a 200 L aliquot of plasma, 50 L of the IS and 150 L of water were added, then vortexed for 30 s. The mixture samples were then extracted with 3.0 mL ethyl acetate–diethyl ether (4:1, v/v) by shaking for 10 min in a test-tube shaker. After centrifugation for 10 min at 3000 × g, the supernatant organic layer was transferred to a polyethylene tube (5.0 mL) and evaporated to dryness at 40 ◦ C in a centrifugal concentrator (Labconco Corp., Missouri, USA). The residue was reconstituted in 100 L acetonitrile–water (55:45, v/v) and a 5.0 L aliquot of the solution was injected into the UPLC-ESI-MS/MS system for analysis.
2.7.
Method validation
Typical method development and establishment for a bioanalytical method include determination of selectivity, accuracy, precision, recovery, construction of a calibration curve, and measurement of the analyte stability in spiked samples [19]. Validation runs were conducted on 3 consecutive days. Each validation run consisted of a minimum of one set of calibration standards and six sets of QC plasma samples at three concentrations [20].
2.7.1.
Selectivity and matrix effect
To investigate the selectivity, chromatograms of rat blank plasma from six different donors were compared with those of QC plasma samples and plasma samples after intravenous administration. Matrix effects on the ionization of analytes were evaluated by comparing the peak area of analytes in the samples spiked post-extraction (A) with that of bufadienolides standard solutions dried directly and reconstituted with the same volume of acetonitrile–water (55:45, v/v) (B). Three concentrations of bufadienolides, each in triplicate, were studied. The ratio (A/B × 100)% was used to evaluate the matrix effect. The same assay method was also applied to the internal standard.
2.7.2.
227
Linearity and LLOQ
To evaluate linearity, plasma calibration curves were prepared and assayed at plasma concentrations ranging from 1.0 to 200 ng mL−1 for bufadienolides on 3 successive days. The calibration curves were fitted by least-square regression using 1x−2 as the weighting factor of the peak area ratio of bufadienolides to IS versus individual bufadienolide plasma concentrations. The concentrations of bufadienolides in QCs or unknown samples were calculated by interpolation from the calibration curves. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve with acceptable precision (relative standard deviation (R.S.D.)) below 20% and accuracy (R.E.) within ±20%.
2.7.3.
Accuracy, precision and recovery
Accuracy and precision were assessed by determining QCs using six replicates at three concentration levels on 3 different validation days. Precision was calculated as the R.S.D. within a single run and between different runs. The accuracy was expressed as the relative error (R.E.), i.e. (calculated concentration − nominal concentration)/(nominal concentration) × 100%. The accuracy was required to be within ±15%, and the intra- and inter-run precisions should not exceed 15%. The extraction recoveries of bufadienolides at three QC levels were determined by comparing the mean peak areas of analytes obtained from plasma samples with bufadienolides spiked before extraction with those spiked after extraction, which represented 100% recovery. The extraction recovery of the IS was determined in a similar way using the medium QC as a reference.
2.7.4.
Stability
QC plasma samples at two concentrations (low and high) were subjected to the conditions below. Bench top stability was assessed by analyzing QC plasma samples left at room temperature for 2.0 h which was longer than the routine preparation time of the samples. Autosampler rack stability was determined by analyzing the extracted QC plasma samples kept in auotsampler at 4 ◦ C for 6.0 h. Freeze–thaw stability was investigated after three freeze (−20 ◦ C)–thaw (room temperature) cycles. Storage stability was investigated by analyzing QC plasma samples after storage at −20 ◦ C for 7 days.
3.
Results and discussion
3.1.
IS and extraction solvent
A stable isotope-labeled analyte is the ideal IS for LC–MS/MS assay. However, sometimes it is difficult to obtain such a reference standard. In fact, analogs of the analyte are usually used as the IS because they exhibit similar behavior to the analyte during the sample extraction, chromatographic elution and mass spectrometric detection. However, the components of Traditional Chinese Medicines (TCM) are extremely complex. The identities of some are even unknown. It is difficult to decide whether the analyte analogs to be present or not. Thus, it is more difficult to select a suitable
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IS for simultaneous determination of three bufadienolides. To solve the problem, a chemical reference standard may be more suitable as IS, if it has a high response and rapid elution under the same LC–MS/MS conditions as the analytes. Therefore, diphenhydramine was used as the IS throughout the investigations and in the final pharmaceutical study. As bufadienolides are lipophilic compounds, a liquid–liquid extraction (LLE) method was applied to extract the analytes. Several extraction solvents, such as Ter butyl methyl ether (TBME), ethyl acetate, diethyl ether–ethyl acetate (5:1, v/v), n-hexane–diethyl ether (3:2, v/v), and nhexane–dichlormethane–isopropanol (20:10:1, v/v/v), were considered. The mean extraction recovery of bufadienolides was in the order of ethyl acetate > diethyl ether–ethyl acetate (5:1, v/v) > TBME ≈ n-hexane–dichlormethane–isopropanol (20:10:1, v/v/v) > n-hexane–diethyl ether (3:2, v/v). A pilot experiment showed that ethyl acetate was the most efficient for the extraction of bufadienolides, but it had a lower efficiency for the IS (ca. 50%). A high extraction recovery for the IS was obtained with both diethyl ether–ethyl acetate (5:1, v/v) and n-hexane–diethyl ether (3:2, v/v). This showed that diethyl ether was a good choice for the extraction of the IS. Hence, another extraction solvent, ethyl acetate:diethyl ether (4:1, v/v), was investigated. Addition of a small quantity of diethyl ether could improve the recovery of IS. While, owing to the lower boiling point of diethyl ether, it could be evaporated to dryness more quickly.
3.2.
Fig. 2 – Product ion spectra of [M+H]+ of bufalin (a), cinobufagin (b), resibufogenin (c) and diphenhydramine (d).
Mass spectrometry
The UPLC-MS/MS method for simultaneous determination of the three bufadienolides in rat plasma was investigated. Initially, bufadienolide responses to electrospray ionization (ESI) were evaluated by acquiring direct inlet MS-scan mass spectra under positive ionization. In positive ESI mode, the bufadienolides and internal standard formed predominately protonated molecules [M+H]+ in MS-scan mass spectra. Fig. 2 shows the product ion spectra of [M+H]+ ions from bufadienolides and IS. Bufalin gave a higher signal at m/z 255.1, formed by the loss of two water molecules ([M+H-36]+ ) then this was followed by the elimination of a 1,2-pyrone group ([M+H-132]+ ). Meanwhile, the product ion at m/z 351.2 ([M+H-36]+ ) was also clearly apparent in the spectrum of bufalin, which was adopted in Xu et al. report [18]. Cinobufagin showed an intense product ion at m/z 365.1, sequentially lost an acetic acid plus a water molecule ([M+H-78]+ ). In the case of resibufogenin, one water molecule characteristically disappeared, leading to the main fragment ion at m/z 367.1. Diphenhydramine showed a major fragment ion at m/z 167.0 corresponding to a neutral loss of [HOCH2 CH2 N(CH3 )2 ]. During the development of the MRM method, it was found that the product spectra of [M+H]+ ions of bufadienolides were dependent on the collision energy (CE). As far as bufalin was concerned, at a low-collision energy (CE, below 25 eV), the response of the fragment ion at m/z 255.1 was enhanced with the increase in CE. The greatest response of the m/z 255.1 ion was obtained with a CE value of 25 eV, although the protonated molecule (m/z 387.2; [M+H]+ ) was still the base peak. On steadily increasing the CE value, both the abundance of precursor ion (m/z 387.2) and the product ion (m/z 255.1)
was reduced to zero. When the CE value reached to 60 eV, another unknown fragment ion at m/z 90.9 appeared, but this exhibited a low response and much noise. Similar results were found with cinobufagin and resibufogenin. In addition, they all had a common fragment ion at m/z 90.9 which is shown in Fig. 3. During the early stage of the method development, attempts were also made to use atmospheric pressure chemical ionization (APCI) as an alternative ionization method for bufadienolide analysis. However, no obvious improvement was obtained. The selected ion recording (SIR) mode of operation has a better sensitivity than the multiple reaction monitoring (MRM) mode, but it is not suitable for the simultaneous analysis of three bufadienolides by virtue of poor specificity. Therefore, the multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) was used as a quantitation mode for bufadienolides. The mass transitions chosen for quantification were m/z 387.2 → 255.1 for bufalin, m/z 443.2 → 365.1 for cinobufagin, m/z 385.2 → 367.1 for resibufogenin, and m/z 256.1 → 167.0 for IS.
Fig. 3 – The common product ion spectrum of bufadienolides.
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3.3.
229
Chromatography
The chromatographic conditions were optimized. It was found that gradient elution was important for UPLC. It not only increased the sensitivity but also improved the chromatographic peaks and shortened the analysis time significantly. A mobile phase composed of acetonitrile–water (containing 0.1% formic acid) was used for chromatographic separation by gradient elution. The presence of a small amount of formic acid in the mobile phase improved the ionization of the analytes in positive ion mode of the UPLC-ESI-MS/MS, and subsequently improved the sensitivity. A diverting valve, between the analytical column and the mass spectrometer, was used to reduce contamination of the mass spectrometer. It directed the UPLC flow to a waste container during the first 1.5 min of the chromatographic acquisition, and then allowed the eluate to pass through the mass spectrometer only during analyte elution (1.5–3.0 min). In addition, during sample preparation, it was usual practice to reconstitute the residues with the starting mobile phase to prevent deterioration in the chromatographic behavior of the analytes. However, bufadienolides have a very poor water-solubility. To reconstitute the residues completely and improve the response of bufadienolides indirectly, the acetonitrile–water (55:45, v/v) was used to reconstitute the residues without compromising the chromatographic peaks. Furthermore, better chromatographic peaks were obtained with an injection volume of 5 L compared with 10 L. As the sample was dissolved in 55% organic phase, an injection onto a gradient starting with mainly aqueous composition (90% water) would cause bandbroadening. The greater the injection volume, the worse the effect. Therefore, a 5.0 L aliquot of the sample was injected by partial loop mode for analysis. As shown in Fig. 4, four channels were used for recording the response, channel 1 for cinobufagin with a typical retention time of 2.40 min, channel 2 for bufalin with a typical retention time of 2.17 min, channel 3 for resibufogenin with a typical retention time of 2.44 min, and channel 4 for IS with a typical retention time of 1.83 min. Strangely, in Fig. 4c, a peak with a retention time of 2.68 was also detected in channel 3 of all the plasma samples from rats. In terms of peak area, the maximum was reached at 5.0 min after intravenous administration, while it was decreased with the passage of time. In this context, the dosing solution was analyzed to determine whether the peak observed in the rat plasma was dosed or a metabolite. However, no similar peak was found in channel 3, so it appeared that the peak (V) could not be an unknown constituent of the bufadienolides, but a metabolite.
3.4.
Method validation
3.4.1.
Selectivity and matrix effect
Fig. 4 shows the typical MRM chromatograms of a blank rat plasma sample (a), a blank rat plasma sample spiked with bufadienolides at the LLOQ (1.0 ng mL−1 ) and IS (30 ng mL−1 ) (b), and a plasma sample from a rat at 5.0 min after a single intravenous administration of 2.10 mg kg−1 bufadienolides (c). No significant interferences were observed at the retention times of the bufadienolides and IS. Typical retention times for
Fig. 4 – Representative MRM chromatograms for bufalin (peak II, channel 2), cinobufagin (peak III, channel 1), resibufogenin (peak IV, channel 3) and IS (peak I, channel 4) in rat plasma samples: (a) a blank plasma sample; (b) a blank plasma sample spiked with bufadienolides at the LLOQ (1.0 ng mL−1 ), and IS (30 ng mL−1 ); (c) a plasma sample from a rat at 5 min after a single intravenous administration of 2.10 mg kg−1 bufadienolides.
bufalin, cinobufagin, resibufogenin and IS are 2.17, 2.40, 2.44 and 1.83 min, respectively. While the corresponding retention factors (k) are 3.25, 3.70, 3.77 and 2.58, which are between 2 and 4. Both the analytes and IS seem to be robust in the region. With regard to a matrix effect, all the ratios (A/B × 100)% were between 85% and 115%, which means that no significant co-eluting “unseen” endogenous substances interfered with the ionization of the analytes and IS.
3.4.2.
Linearity and LLOQ
Linear calibration curves were obtained over the concentration ranges of 1.0–200 ng mL−1 for the three bufadienolides in rat plasma. Typical equations of calibration curves are as
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Table 3 – Summary of precision and accuracy from QC samples of rat plasma extracts (in pre-study, n = 18) Analyte
Added C
Found C
Intra-run R.S.D. (%)
Inter-run R.S.D. (%)
Relative error (%)
Bufalin
2.0 50.0 180.0
2.04 52.83 185.10
5.96 4.53 1.41
10.87 5.72 7.01
1.79 5.67 2.83
Cinobufagin
2.0 50.0 180.0
1.84 53.24 181.19
10.71 10.65 8.39
5.50 7.09 6.42
−8.00 6.47 0.66
Resibufogenin
2.0 50.0 180.0
2.10 53.58 178.59
7.65 11.35 7.55
8.34 4.11 7.81
4.90 7.17 −0.78
follows: Bufalin :
Y = 2.450 × 10−1 x + 3.002 × 10−1 ,
Cinobufagin :
r = 0.9953
Y = 2.062 × 10−1 x + 2.715 × 10−1 ,
Resibufogenin :
Y = 1.129 × 10−1 x + 3.998 × 10−1 ,
r = 0.9915 r = 0.9952
The lower limit of quantification (LLOQ) was 1.0 ng mL−1 for three bufadienolides, respectively. The precision and accuracy at this concentration level were acceptable, with 5.19% of the R.S.D. and 2.10% of the R.E. for bufalin, 6.02% of the R.S.D. and 3.47% of the R.E. for cinobufagin, and 2.65% of the R.S.D. and 1.17% of the R.E. for resibufogenin.
3.4.3.
Accuracy, precision and recovery
The method showed good precision and accuracy. Table 3 summarizes the intra- and inter-run precision and accuracy for bufadienolides from QCs. The intra-run R.S.D., calculated from QCs, was less than 11.35%. The inter-run R.S.D., calculated from QCs, was less than 10.87%. The accuracy as determined from the QCs was within 8.00% for bufadienolides. The clean-up of the rat plasma samples was achieved by a one-step LLE procedure with ethyl acetate:diethyl ether (4:1, v/v), which was much simpler than the reported method [12,17]. The mean extraction recoveries of bufalin, cinobufagin and resibufogenin at three concentrations were 86.3 ± 0.7%, 89.8 ± 0.5% and 92.8 ± 0.9%, while the recovery of the IS was 74.9 ± 0.3%. The recoveries of the present method conformed to the requirement for the analysis of biological samples.
3.4.4.
administration of 2.10 mg kg−1 bufadienolides to six male rats. The mean plasma concentration–time profiles of the bufadienolides are presented in Fig. 5. It can be seen that three bufadienolides were rapidly eliminated from rat plasma. The pharmacokinetic parameters were calculated using the drug and statistics (DAS) version 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). All the concentration–time curves of bufadienolides in rat plasma fitted a two-compartment model well with a weighting factor of 1c−2 . The main pharmacokinetic parameters of the bufadienolides are listed in Table 4.
Stability
The stability of the bufadienolides in rat plasma was investigated under a variety of storage and process conditions. The bufadienolides were found to be stable when stored at −25 ◦ C for 7 days and after three freeze–thaw cycles in rat plasma. The accuracies calculated from the QCs ranged from 88.2% to 104.3%. The bufadienolides were also shown to be stable in rat plasma at room temperature for 2.0 h (R.E. < 6.8%) and after the reconstitution at 4 ◦ C for 6.0 h (R.E. < 9.7%). Therefore, the method can be used for routine analysis.
3.5.
Fig. 5 – Mean plasma concentration–time curves of bufadienolides after a single intravenous administration of 2.10 mg kg−1 bufadienolides to Wistar rats (n = 6).
Pharmacokinetic application
The present method was successfully applied to a pharmacokinetic study of bufadienolides after a single intravenous
Table 4 – The main pharmacokinetics of bufadienolides after intravenous administration of 2.10 mg kg−1 bufadienolides. Parameter
Bufalin
Cinobufagin
Resibufogenin
t1/2␣ (h) t1/2 (h) V1 (L kg−1 ) CL (L h−1 kg−1 ) AUC0–2 (g L−1 h−1 ) AUC0–∞ (g L−1 h−1 ) K10 (L h−1 ) K12 (L h−1 ) K21 (L h−1 )
0.023 0.422 0.137 1.014 122.796 147.867 7.395 18.449 6.152
0.131 0.723 2.810 6.997 22.253 28.325 2.490 2.220 1.548
0.087 0.693 1.324 4.142 44.873 60.509 3.128 4.008 1.809
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4.
Conclusion
A novel UPLC-ESI-MS/MS method was developed and validated for the simultaneous determination of three bufadienolides (bufalin, cinobufagin and resibufogenin) in rat plasma. The method is sensitive, specific and rapid with an LLOQ of 1.0 ng mL−1 for bufadienolides using 200 L of rat plasma. With this method, simultaneous multi-compound quantification is possible in a short chromatographic run time (3.0 min). It also met the requirement for a high sample throughout. This method is suitable for preclinical pharmacokinetic studies of bufadienolides in rats following a single intravenous administration.
Acknowledgements Dr. David B. Jack is gratefully thanked for correcting the manuscript.
references
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Simultaneous determination of three bufadienolides in rat plasma after intravenous administration of bufadienolides extract by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry Yu Zhang, Xing Tang ∗ , Xiaoliang Liu, Fang Li, Xia Lin Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
A novel, rapid and specific ultra performance liquid chromatography electrospray ion-
Received 24 November 2007
ization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed for
Received in revised form
simultaneous determination and pharmacokinetic studies of three bufadienolides (bufalin,
26 December 2007
cinobufagin and resibufogenin) in rat plasma. The analytes, bufalin, cinobufagin, resibufo-
Accepted 10 January 2008
genin and the internal standard, diphenhydramine were extracted from rat plasma samples
Published on line 18 January 2008
by a one-step liquid–liquid extraction and separated on an ACQUITY UPLCTM BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water (containing
Keywords:
0.1% formic acid) at a flow rate of 0.20 mL min−1 . Detection was carried out on a triple-
Bufadienolides
quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode
Ultra performance liquid
via an electrospray ionization (ESI) interface. The three bufadienolides could be simul-
chromatography–tandem mass
taneously determined within 3.0 min. Linear calibration curves were obtained over the
spectrometry
concentration ranges of 1.0–200 ng mL−1 for all the analytes. The intra- and inter-day pre-
Pharmacokinetics
cisions (relative standard deviation (R.S.D.)) were less than 11.35 and 10.87%, respectively.
Rat plasma
The developed method was applied for the first time to the pharmacokinetic studies of bufadienolides in rats following a single intravenous administration of 2.10 mg kg−1 bufadienolides. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Toad venom, also called “Chan Su” in China, is obtained from the postauricular and skin glands of Bufo bufo gargarizans Cantor and the other species of the same genus, family bufonidae [1]. It is often found in traditional Chinese medicinal ingredients, such as Liu-Shen-Wan [2] and Niuhuangxiaoyan tablets [3]. These Chinese medicines have been widely used in China, Japan and other Asian countries for a long time, and
∗
Corresponding author. Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (X. Tang). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.01.029
over the last decade, they have gained considerable favor in the United States and elsewhere [4]. Toad venom in small doses is used for stimulation of myocardial contraction, to produce an anti-inflammatory effect and for pain relief. Due to its anesthetic and antibiotic actions, it is also used for a variety of other purposes including the treatment of tonsillitis, sore throat, and palpitation [5]. Recently, toad venom has been used for the treatment of cancer [6–9]. The principal biologically active components of toad
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venom are bufadienolides, a class of cardioactive C-24 steroids with a characteristic ␣-pyrone ring at C-17 [10–11]. However, cardioactive steroids, including bufadienolides, usually have a narrow therapeutic index, and intentional therapeutic intoxication is well documented [12]. Therefore, the detection and accurate quantitation of bufadienolides in bio-fluids play a very important role in therapeutic drug monitoring and pharmacokinetic investigations. Up to now, most investigations have been based on the pharmacodynamics or pharmacology of bufadienolides and very little attention has been devoted to the pharmacokinetic study of bufadienolides in vivo. Also, one essential difference between traditional medicines and synthetic drugs is that the therapeutic effects of the former are due to the joint contribution of multi-components, not only the major ones [13]. Therefore, it is necessary to develop a more comprehensive and global assay to fully evaluate the pharmacokinetics of bufadienolides. Several HPLC/UV methods have been developed to determine bufadienolides in rat serum [14–15], in rat plasma [16] and human liver [12]. However, these HPLC/UV methods were not sensitive enough to determine bufadienolides in biological matrix for pharmacokinetics study after intravenous administration. The lower limit of quantitation (LLOQ) was more than 6.8 ng mL−1 . Recently, two LC/MS methods have been established for determination of bufadienolides. One LC/TOF-MS method has been reported for the determination of bufalin, cinobufagin and resibufogenin in dog plasma after oral administration of Liu-Shen-Wan [17]. This method was sensitive enough for pharmacokinetic study, but the run time was about 30 min which did not meet acquirement of high-throughput determination of biosamples. Another LC/MS/MS method has been developed for determination of five bufadienolides in rat plasma after intragastric administration of 100 mg kg−1 Chan Su [18]. The purpose of this study was to develop a rapid, selective and specific UPLC-ESI-MS/MS method, which enables simultaneous determination of three bufadienolides (bufalin, cinobufagin and resibufogenin) at 1.0 ng mL−1 in rat plasma. The total run time of the method per sample was only 3.0 min which was almost 10 times shorter than the reported method [17]. To our knowledge, this is the first report of the development, validation and application of a UPLC-ESI-MS/MS method for simultaneous determination of three bufadienolides in rat plasma and a study of their pharmacokinetics after a single intravenous administration of 2.10 mg kg−1 bufadienolides.
2.
Experimental
2.1.
Materials and reagents
Bufadienolides were extracted from toad venom (Bufo bufo gargarizans Cantor) by the Department of Pharmaceutics, Shenyang Pharmaceutical University, China. The bufadienolides extract was mainly composed of bufalin, cinobufagin and resibufogenin, with a content of 7.09%:9.37%:11.85%, respectively. The reference standards: cinobufagin (Lot #803-9202) and resibufogenin (Lot #0718-9306) were purchased from the
Table 1 – Gradient condition of UPLC Time (min)
Flow rate (mL min−1 )
A (%)a
B (%)b
Curve
Initial 1.0 2.5 3.0
0.20 0.20 0.20 0.20
10.0 55.0 55.0 10.0
90.0 45.0 45.0 90.0
Initial 6c 6 1d
a b c d
Acetonitrile. 0.1% Fomic acid water. Linear. Pre-step.
National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and bufalin (Lot #1032050314) was obtained from National Engineering Research Center for Traditional Chinese Medicine (Nanchang, Jiangxi, China). Diphenhydramine (internal standard, IS) was a kind gift of the Department of Analytical Chemistry of Shenyang Pharmaceutical University (Shenyang, China). The structures of the three bufadienolides and IS are shown in Fig. 1. Acetonitrile and formic acid (HPLC grade) were supplied by Dikma (Richmond Hill, NY, USA). Water was purified in a Barnstead EASYpure® II RF/UV ultrapure water system (Dubuque, Lowa, USA) and passed through a 0.22 m filter prior to use in all the studies. Other chemicals were of analytical grade.
2.2.
Instrumentation and conditions
2.2.1.
Ultra performance liquid chromatography (UPLC)
Chromatography was performed on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a conditioned autosampler at 4 ◦ C. The separation was carried out on an ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm i.d., 1.7 m; Waters Corp., Milford, MA, USA). The column temperature was maintained at 35 ◦ C. The analysis was achieved with gradient elution using (A) acetonitrile and (B) water (containing 0.1% formic acid) as the mobile phase. The gradient conditions are shown in Table 1. The injection volume was 5 L and the partial loop mode was used for sample injection.
2.2.2.
Mass spectrometer
The Waters ACQUITYTM TQD triple-quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) was connected to the UPLC system via an electrospray ionization (ESI) interface. The ESI source was operated in positive ionization mode with the capillary voltage set at 3.8 kV. The extractor and RF voltages were 2.0 and 0.1 V, respectively. The temperature of the source and desolvation was set at 80 and 400 ◦ C, separately. Nitrogen was used as the desolvation gas (500 L h−1 ) and cone gas (50 L h−1 ). For collision-induced dissociation (CID), argon was used as the collision gas at a flow rate of 0.20 mL min−1 (approximately 2.81 × 10−3 mbar). The multiple reaction monitoring (MRM) mode was used for quantification. Transition reactions of the analytes and internal standards are given in Table 2. All data collected in centroid mode were acquired using MasslynxTM NT4.1 software (Waters Corp., Milford, MA,
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Fig. 1 – The structures of bufalin (a), cinobufagin (b), resibufogenin (c) and diphenhydramine (d).
USA). Post-acquisition quantitative analyses were performed using a QuanLynxTM program (Waters Corp., Milford, MA, USA).
2.3.
Animals and blood sampling
Male Wistar rats (8 weeks old, 200 ± 20 g) were obtained from the Laboratory Animal Center of Shenyang Pharmaceutical University. The experimental protocol was approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. Rats were housed in groups of two or three with 12-h light/12-h dark cycle at a temperature of 22 ± 3 ◦ C, relative humidity of 45–60%, for 1 week. Immediately before the day of administration, the rats were fasted for 12 h but were allowed water ad libitum. Then, 2.10 mg kg−1 aqueous solution of bufadienolide extract containing 10% propylene glycol was administered to the rats
(equivalent to 149.95 g kg−1 bufalin; 198.20 g kg−1 cinobufagin; 250.65 g kg−1 resibufogenin) intravenously via the femoral vein. Blood samples from each rat were collected into heparinized Eppendorf tubes (2.0 mL) by puncture of the retroorbital sinus. This was performed at 0 (predose), 5, 10, 15, 20, 30, 45, 60, 90 and 120 min after administration. As soon as possible, the heparinized blood was centrifuged for 10 min at 2000 × g, and the plasma obtained was stored frozen at −20 ◦ C until analysis.
2.4. Preparation of standard and quality control (QC) solutions Stock solutions of bufalin, cinobufagin and resibufogenin were prepared by dissolving the accurately weighed standard compounds in methanol to give final concentrations of 10 g mL−1 for each analyte in the same volumetric flask. The mixed solution was then further diluted with methanol
Table 2 – Transition reactions of the analytes and internal standards Molecule Bufalin Cinobufagin Resibufogenin I.S.
Transition 387.2 → 255.1 443.2 → 365.1 385.2 → 367.1 256.1 → 167.0
Dwell (s) 0.05 0.05 0.05 0.05
Cone voltage (V) 45.0 50.0 45.0 30.0
Collision energy (eV) 25.0 20.0 20.0 30.0
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to achieve standard working solutions of desired concentrations. The internal standard working solution (120 ng mL−1 ) was similarly prepared by diluting a stock standard solution of diphenhydramine with water. All the working solutions were kept refrigerated (4 ◦ C) and brought to room temperature before use.
2.5. Preparation of calibration standards and quality control samples (QCs) The standard solutions were used to spike 200 L of blank plasma samples either for calibration standards of bufadienolides or for QCs in a prestudy and during the pharmacokinetic studies. Calibration standards were prepared at plasma concentrations of 1.0, 2.0, 5.0, 10, 20, 50, 100 and 200 ng mL−1 for each analyte, while QCs were prepared with blank plasma at LLOQ, low, medium and high concentrations of 1.0, 2.0, 50 and 180 ng mL−1 .
2.6.
Plasma sample preparation
Frozen plasma samples were thawed at room temperature and thoroughly vortexed prior to extraction. To a 200 L aliquot of plasma, 50 L of the IS and 150 L of water were added, then vortexed for 30 s. The mixture samples were then extracted with 3.0 mL ethyl acetate–diethyl ether (4:1, v/v) by shaking for 10 min in a test-tube shaker. After centrifugation for 10 min at 3000 × g, the supernatant organic layer was transferred to a polyethylene tube (5.0 mL) and evaporated to dryness at 40 ◦ C in a centrifugal concentrator (Labconco Corp., Missouri, USA). The residue was reconstituted in 100 L acetonitrile–water (55:45, v/v) and a 5.0 L aliquot of the solution was injected into the UPLC-ESI-MS/MS system for analysis.
2.7.
Method validation
Typical method development and establishment for a bioanalytical method include determination of selectivity, accuracy, precision, recovery, construction of a calibration curve, and measurement of the analyte stability in spiked samples [19]. Validation runs were conducted on 3 consecutive days. Each validation run consisted of a minimum of one set of calibration standards and six sets of QC plasma samples at three concentrations [20].
2.7.1.
Selectivity and matrix effect
To investigate the selectivity, chromatograms of rat blank plasma from six different donors were compared with those of QC plasma samples and plasma samples after intravenous administration. Matrix effects on the ionization of analytes were evaluated by comparing the peak area of analytes in the samples spiked post-extraction (A) with that of bufadienolides standard solutions dried directly and reconstituted with the same volume of acetonitrile–water (55:45, v/v) (B). Three concentrations of bufadienolides, each in triplicate, were studied. The ratio (A/B × 100)% was used to evaluate the matrix effect. The same assay method was also applied to the internal standard.
2.7.2.
227
Linearity and LLOQ
To evaluate linearity, plasma calibration curves were prepared and assayed at plasma concentrations ranging from 1.0 to 200 ng mL−1 for bufadienolides on 3 successive days. The calibration curves were fitted by least-square regression using 1x−2 as the weighting factor of the peak area ratio of bufadienolides to IS versus individual bufadienolide plasma concentrations. The concentrations of bufadienolides in QCs or unknown samples were calculated by interpolation from the calibration curves. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve with acceptable precision (relative standard deviation (R.S.D.)) below 20% and accuracy (R.E.) within ±20%.
2.7.3.
Accuracy, precision and recovery
Accuracy and precision were assessed by determining QCs using six replicates at three concentration levels on 3 different validation days. Precision was calculated as the R.S.D. within a single run and between different runs. The accuracy was expressed as the relative error (R.E.), i.e. (calculated concentration − nominal concentration)/(nominal concentration) × 100%. The accuracy was required to be within ±15%, and the intra- and inter-run precisions should not exceed 15%. The extraction recoveries of bufadienolides at three QC levels were determined by comparing the mean peak areas of analytes obtained from plasma samples with bufadienolides spiked before extraction with those spiked after extraction, which represented 100% recovery. The extraction recovery of the IS was determined in a similar way using the medium QC as a reference.
2.7.4.
Stability
QC plasma samples at two concentrations (low and high) were subjected to the conditions below. Bench top stability was assessed by analyzing QC plasma samples left at room temperature for 2.0 h which was longer than the routine preparation time of the samples. Autosampler rack stability was determined by analyzing the extracted QC plasma samples kept in auotsampler at 4 ◦ C for 6.0 h. Freeze–thaw stability was investigated after three freeze (−20 ◦ C)–thaw (room temperature) cycles. Storage stability was investigated by analyzing QC plasma samples after storage at −20 ◦ C for 7 days.
3.
Results and discussion
3.1.
IS and extraction solvent
A stable isotope-labeled analyte is the ideal IS for LC–MS/MS assay. However, sometimes it is difficult to obtain such a reference standard. In fact, analogs of the analyte are usually used as the IS because they exhibit similar behavior to the analyte during the sample extraction, chromatographic elution and mass spectrometric detection. However, the components of Traditional Chinese Medicines (TCM) are extremely complex. The identities of some are even unknown. It is difficult to decide whether the analyte analogs to be present or not. Thus, it is more difficult to select a suitable
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IS for simultaneous determination of three bufadienolides. To solve the problem, a chemical reference standard may be more suitable as IS, if it has a high response and rapid elution under the same LC–MS/MS conditions as the analytes. Therefore, diphenhydramine was used as the IS throughout the investigations and in the final pharmaceutical study. As bufadienolides are lipophilic compounds, a liquid–liquid extraction (LLE) method was applied to extract the analytes. Several extraction solvents, such as Ter butyl methyl ether (TBME), ethyl acetate, diethyl ether–ethyl acetate (5:1, v/v), n-hexane–diethyl ether (3:2, v/v), and nhexane–dichlormethane–isopropanol (20:10:1, v/v/v), were considered. The mean extraction recovery of bufadienolides was in the order of ethyl acetate > diethyl ether–ethyl acetate (5:1, v/v) > TBME ≈ n-hexane–dichlormethane–isopropanol (20:10:1, v/v/v) > n-hexane–diethyl ether (3:2, v/v). A pilot experiment showed that ethyl acetate was the most efficient for the extraction of bufadienolides, but it had a lower efficiency for the IS (ca. 50%). A high extraction recovery for the IS was obtained with both diethyl ether–ethyl acetate (5:1, v/v) and n-hexane–diethyl ether (3:2, v/v). This showed that diethyl ether was a good choice for the extraction of the IS. Hence, another extraction solvent, ethyl acetate:diethyl ether (4:1, v/v), was investigated. Addition of a small quantity of diethyl ether could improve the recovery of IS. While, owing to the lower boiling point of diethyl ether, it could be evaporated to dryness more quickly.
3.2.
Fig. 2 – Product ion spectra of [M+H]+ of bufalin (a), cinobufagin (b), resibufogenin (c) and diphenhydramine (d).
Mass spectrometry
The UPLC-MS/MS method for simultaneous determination of the three bufadienolides in rat plasma was investigated. Initially, bufadienolide responses to electrospray ionization (ESI) were evaluated by acquiring direct inlet MS-scan mass spectra under positive ionization. In positive ESI mode, the bufadienolides and internal standard formed predominately protonated molecules [M+H]+ in MS-scan mass spectra. Fig. 2 shows the product ion spectra of [M+H]+ ions from bufadienolides and IS. Bufalin gave a higher signal at m/z 255.1, formed by the loss of two water molecules ([M+H-36]+ ) then this was followed by the elimination of a 1,2-pyrone group ([M+H-132]+ ). Meanwhile, the product ion at m/z 351.2 ([M+H-36]+ ) was also clearly apparent in the spectrum of bufalin, which was adopted in Xu et al. report [18]. Cinobufagin showed an intense product ion at m/z 365.1, sequentially lost an acetic acid plus a water molecule ([M+H-78]+ ). In the case of resibufogenin, one water molecule characteristically disappeared, leading to the main fragment ion at m/z 367.1. Diphenhydramine showed a major fragment ion at m/z 167.0 corresponding to a neutral loss of [HOCH2 CH2 N(CH3 )2 ]. During the development of the MRM method, it was found that the product spectra of [M+H]+ ions of bufadienolides were dependent on the collision energy (CE). As far as bufalin was concerned, at a low-collision energy (CE, below 25 eV), the response of the fragment ion at m/z 255.1 was enhanced with the increase in CE. The greatest response of the m/z 255.1 ion was obtained with a CE value of 25 eV, although the protonated molecule (m/z 387.2; [M+H]+ ) was still the base peak. On steadily increasing the CE value, both the abundance of precursor ion (m/z 387.2) and the product ion (m/z 255.1)
was reduced to zero. When the CE value reached to 60 eV, another unknown fragment ion at m/z 90.9 appeared, but this exhibited a low response and much noise. Similar results were found with cinobufagin and resibufogenin. In addition, they all had a common fragment ion at m/z 90.9 which is shown in Fig. 3. During the early stage of the method development, attempts were also made to use atmospheric pressure chemical ionization (APCI) as an alternative ionization method for bufadienolide analysis. However, no obvious improvement was obtained. The selected ion recording (SIR) mode of operation has a better sensitivity than the multiple reaction monitoring (MRM) mode, but it is not suitable for the simultaneous analysis of three bufadienolides by virtue of poor specificity. Therefore, the multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) was used as a quantitation mode for bufadienolides. The mass transitions chosen for quantification were m/z 387.2 → 255.1 for bufalin, m/z 443.2 → 365.1 for cinobufagin, m/z 385.2 → 367.1 for resibufogenin, and m/z 256.1 → 167.0 for IS.
Fig. 3 – The common product ion spectrum of bufadienolides.
a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 224–231
3.3.
229
Chromatography
The chromatographic conditions were optimized. It was found that gradient elution was important for UPLC. It not only increased the sensitivity but also improved the chromatographic peaks and shortened the analysis time significantly. A mobile phase composed of acetonitrile–water (containing 0.1% formic acid) was used for chromatographic separation by gradient elution. The presence of a small amount of formic acid in the mobile phase improved the ionization of the analytes in positive ion mode of the UPLC-ESI-MS/MS, and subsequently improved the sensitivity. A diverting valve, between the analytical column and the mass spectrometer, was used to reduce contamination of the mass spectrometer. It directed the UPLC flow to a waste container during the first 1.5 min of the chromatographic acquisition, and then allowed the eluate to pass through the mass spectrometer only during analyte elution (1.5–3.0 min). In addition, during sample preparation, it was usual practice to reconstitute the residues with the starting mobile phase to prevent deterioration in the chromatographic behavior of the analytes. However, bufadienolides have a very poor water-solubility. To reconstitute the residues completely and improve the response of bufadienolides indirectly, the acetonitrile–water (55:45, v/v) was used to reconstitute the residues without compromising the chromatographic peaks. Furthermore, better chromatographic peaks were obtained with an injection volume of 5 L compared with 10 L. As the sample was dissolved in 55% organic phase, an injection onto a gradient starting with mainly aqueous composition (90% water) would cause bandbroadening. The greater the injection volume, the worse the effect. Therefore, a 5.0 L aliquot of the sample was injected by partial loop mode for analysis. As shown in Fig. 4, four channels were used for recording the response, channel 1 for cinobufagin with a typical retention time of 2.40 min, channel 2 for bufalin with a typical retention time of 2.17 min, channel 3 for resibufogenin with a typical retention time of 2.44 min, and channel 4 for IS with a typical retention time of 1.83 min. Strangely, in Fig. 4c, a peak with a retention time of 2.68 was also detected in channel 3 of all the plasma samples from rats. In terms of peak area, the maximum was reached at 5.0 min after intravenous administration, while it was decreased with the passage of time. In this context, the dosing solution was analyzed to determine whether the peak observed in the rat plasma was dosed or a metabolite. However, no similar peak was found in channel 3, so it appeared that the peak (V) could not be an unknown constituent of the bufadienolides, but a metabolite.
3.4.
Method validation
3.4.1.
Selectivity and matrix effect
Fig. 4 shows the typical MRM chromatograms of a blank rat plasma sample (a), a blank rat plasma sample spiked with bufadienolides at the LLOQ (1.0 ng mL−1 ) and IS (30 ng mL−1 ) (b), and a plasma sample from a rat at 5.0 min after a single intravenous administration of 2.10 mg kg−1 bufadienolides (c). No significant interferences were observed at the retention times of the bufadienolides and IS. Typical retention times for
Fig. 4 – Representative MRM chromatograms for bufalin (peak II, channel 2), cinobufagin (peak III, channel 1), resibufogenin (peak IV, channel 3) and IS (peak I, channel 4) in rat plasma samples: (a) a blank plasma sample; (b) a blank plasma sample spiked with bufadienolides at the LLOQ (1.0 ng mL−1 ), and IS (30 ng mL−1 ); (c) a plasma sample from a rat at 5 min after a single intravenous administration of 2.10 mg kg−1 bufadienolides.
bufalin, cinobufagin, resibufogenin and IS are 2.17, 2.40, 2.44 and 1.83 min, respectively. While the corresponding retention factors (k) are 3.25, 3.70, 3.77 and 2.58, which are between 2 and 4. Both the analytes and IS seem to be robust in the region. With regard to a matrix effect, all the ratios (A/B × 100)% were between 85% and 115%, which means that no significant co-eluting “unseen” endogenous substances interfered with the ionization of the analytes and IS.
3.4.2.
Linearity and LLOQ
Linear calibration curves were obtained over the concentration ranges of 1.0–200 ng mL−1 for the three bufadienolides in rat plasma. Typical equations of calibration curves are as
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Table 3 – Summary of precision and accuracy from QC samples of rat plasma extracts (in pre-study, n = 18) Analyte
Added C
Found C
Intra-run R.S.D. (%)
Inter-run R.S.D. (%)
Relative error (%)
Bufalin
2.0 50.0 180.0
2.04 52.83 185.10
5.96 4.53 1.41
10.87 5.72 7.01
1.79 5.67 2.83
Cinobufagin
2.0 50.0 180.0
1.84 53.24 181.19
10.71 10.65 8.39
5.50 7.09 6.42
−8.00 6.47 0.66
Resibufogenin
2.0 50.0 180.0
2.10 53.58 178.59
7.65 11.35 7.55
8.34 4.11 7.81
4.90 7.17 −0.78
follows: Bufalin :
Y = 2.450 × 10−1 x + 3.002 × 10−1 ,
Cinobufagin :
r = 0.9953
Y = 2.062 × 10−1 x + 2.715 × 10−1 ,
Resibufogenin :
Y = 1.129 × 10−1 x + 3.998 × 10−1 ,
r = 0.9915 r = 0.9952
The lower limit of quantification (LLOQ) was 1.0 ng mL−1 for three bufadienolides, respectively. The precision and accuracy at this concentration level were acceptable, with 5.19% of the R.S.D. and 2.10% of the R.E. for bufalin, 6.02% of the R.S.D. and 3.47% of the R.E. for cinobufagin, and 2.65% of the R.S.D. and 1.17% of the R.E. for resibufogenin.
3.4.3.
Accuracy, precision and recovery
The method showed good precision and accuracy. Table 3 summarizes the intra- and inter-run precision and accuracy for bufadienolides from QCs. The intra-run R.S.D., calculated from QCs, was less than 11.35%. The inter-run R.S.D., calculated from QCs, was less than 10.87%. The accuracy as determined from the QCs was within 8.00% for bufadienolides. The clean-up of the rat plasma samples was achieved by a one-step LLE procedure with ethyl acetate:diethyl ether (4:1, v/v), which was much simpler than the reported method [12,17]. The mean extraction recoveries of bufalin, cinobufagin and resibufogenin at three concentrations were 86.3 ± 0.7%, 89.8 ± 0.5% and 92.8 ± 0.9%, while the recovery of the IS was 74.9 ± 0.3%. The recoveries of the present method conformed to the requirement for the analysis of biological samples.
3.4.4.
administration of 2.10 mg kg−1 bufadienolides to six male rats. The mean plasma concentration–time profiles of the bufadienolides are presented in Fig. 5. It can be seen that three bufadienolides were rapidly eliminated from rat plasma. The pharmacokinetic parameters were calculated using the drug and statistics (DAS) version 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). All the concentration–time curves of bufadienolides in rat plasma fitted a two-compartment model well with a weighting factor of 1c−2 . The main pharmacokinetic parameters of the bufadienolides are listed in Table 4.
Stability
The stability of the bufadienolides in rat plasma was investigated under a variety of storage and process conditions. The bufadienolides were found to be stable when stored at −25 ◦ C for 7 days and after three freeze–thaw cycles in rat plasma. The accuracies calculated from the QCs ranged from 88.2% to 104.3%. The bufadienolides were also shown to be stable in rat plasma at room temperature for 2.0 h (R.E. < 6.8%) and after the reconstitution at 4 ◦ C for 6.0 h (R.E. < 9.7%). Therefore, the method can be used for routine analysis.
3.5.
Fig. 5 – Mean plasma concentration–time curves of bufadienolides after a single intravenous administration of 2.10 mg kg−1 bufadienolides to Wistar rats (n = 6).
Pharmacokinetic application
The present method was successfully applied to a pharmacokinetic study of bufadienolides after a single intravenous
Table 4 – The main pharmacokinetics of bufadienolides after intravenous administration of 2.10 mg kg−1 bufadienolides. Parameter
Bufalin
Cinobufagin
Resibufogenin
t1/2␣ (h) t1/2 (h) V1 (L kg−1 ) CL (L h−1 kg−1 ) AUC0–2 (g L−1 h−1 ) AUC0–∞ (g L−1 h−1 ) K10 (L h−1 ) K12 (L h−1 ) K21 (L h−1 )
0.023 0.422 0.137 1.014 122.796 147.867 7.395 18.449 6.152
0.131 0.723 2.810 6.997 22.253 28.325 2.490 2.220 1.548
0.087 0.693 1.324 4.142 44.873 60.509 3.128 4.008 1.809
a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 224–231
4.
Conclusion
A novel UPLC-ESI-MS/MS method was developed and validated for the simultaneous determination of three bufadienolides (bufalin, cinobufagin and resibufogenin) in rat plasma. The method is sensitive, specific and rapid with an LLOQ of 1.0 ng mL−1 for bufadienolides using 200 L of rat plasma. With this method, simultaneous multi-compound quantification is possible in a short chromatographic run time (3.0 min). It also met the requirement for a high sample throughout. This method is suitable for preclinical pharmacokinetic studies of bufadienolides in rats following a single intravenous administration.
Acknowledgements Dr. David B. Jack is gratefully thanked for correcting the manuscript.
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