J Huazhong Univ Sci Technol［Med Sci］ 34(6):861-868,2014 10.1007/s11596-014-1365-2 J DOI Huazhong Univ Sci Technol［Med Sci］ 34(6):2014
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An LC-MS/MS Method for the Simultaneous Determination of Lycorine and Galanthamine in Rat Plasma and Its Application to Pharmacokinetic Study of Lycoris Radiata Extract in Rats* Xin ZHOU (周 欣)†, Yue-bin LIU (刘月彬)† , Shan HUANG (黄 珊), Ying LIU (刘 莹)# Department of Pharmacy, Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of lycorine and galanthamine, two major constituents in Lycoris radiata extract, in rat plasma. Liquid-liquid extraction with ethyl ether was carried out using diphenhydramine as the internal standard. The two bioactive alkaloids were separated on a Zorbax SB-C18 reserved-phase column (150 mm × 4.6 mm, i.d., 5 μm) by gradient elution using a mobile phase consisting of methanol with 0.1% formic acid (A) and water with 0.1% formic acid (B) at a flow rate of 0.6 mL/min. All analytes showed good linearity over a wide concentration range (r2>0.99) and the lower limit of quantification was 3.00 ng/mL for each analyte. The average extraction recovery of the analytes from rat plasma was more than 82.15%, and the intra-day and inter-day accuracy and precision of the assay were less than 12.6%. The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of two Amaryllidaceous alkaloids in rat plasma after an oral administration of Lycoris radiata extract. Key words: liquid chromatography-tandem mass spectrometry; Lycoris radiata extract; pharmacokinetic; rat plasma
Lycoris radiata, commonly known as Shi-Suan, is used in China as a traditional folk medicine for treating laryngeal trouble, furuncles, carbuncles, and suppurative wounds[1]. This plant contains a large group of alkaloids, such as lycorine, galanthamine, homolycorine, pseudo-lycorine, and lycoramine[2, 3]. Among them, lycorine and galanthamine (fig. 1) are the most common alkaloids in Amaryllidaceae plants[4–7]. Galanthamine has already been used to treat mild to moderate Alzheimer’s disease and other memory disorders such as dementia[8]. It mainly acts by reversibly inhibiting acetylcholinesterase and it has been approved as a prescription drug by the US Food and Drug Administration (FDA) in 2001[9]. Lycorine is believed to possess antiviral, antifungal, anti-inflammatory, and anticancer properties[10, 11]. It has been clinically used in Russia as an expectorant for treating chronic and acute inflammations in the lungs and bronchial diseases[12]. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been reported for determining galanthamine alone or with other drugs in biological samples[13–15]. However, to our knowledge, there is no study that has reported the simultaneous determination of lycorine and galanthamine in biological Xin ZHOU, E-mail: [email protected]; Yue-bin LIU, E-mail: [email protected] † Both authors contributed equally to this work. # Corresponding author, E-mail: [email protected] * This project was supported by the Natural Science Foundation of Fujian Province of China (No. 2013J01382).
samples. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the two Amaryllidaceous alkaloids in rat plasma. The validated assay was successfully applied to a pharmacokinetic study in rats. 1 MATERIALS AND METHODS 1.1 Chemicals and Materials Lycorine, galanthamine and diphenhydramine (fig. 1) as internal standard (IS) were purchased from Sigma Chemical Co. (USA). HPLC grade methanol and formic acid were bought from Tedia Co. (USA). The deionized water was purified by a Milli-Q water purification system (USA). All other chemicals were of analytical grade. Lycoris radiata was procured from a traditional Chinese medicinal store in Anhui province in China (Bozhou, China). The LC system (Palo Alto, CA) was equipped with an isocratic pump (1100 series) and interfaced with an autosampler (Reliance, Holland). The analytical column was a YMC Hydrosphere C18 (50 × 2 mm i.d., 3 µm; YMC Co., Japan). MS analysis was performed using an API 2000 mass spectrometer system (Applied Biosystems, USA) equipped with an ESI interface and operated in the positive ionization mode. The ion source parameters were set as follows: curtain gas, 35 psi; GS1, 50 psi and GS2, 50 psi; ion spray voltage, 4500 V; ion source temperature, 250°C; collision-activated dissociation (CAD), 8.0. Data acquisition and analysis were per-
862 formed using the analyst software Peak Simple Chromatography Data system version 1.4.1. (Applied Biosys-
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tems, USA).
Fig. 1 Chemical structures of lycorine, galanthamine and diphenhydramine (IS)
1.2 Preparation of Lycoris radiata Extract Powdered Lycoris radiata (100 g) was extracted with 70% methanol (600 mL×3) under reflux on a water bath at 80°C for 4 h. The extraction solutions were combined for filtration and concentrated to 200 mL by using a rotary evaporator in vacuum. The concentrated solution was then added with aqueous ammonia for adjusting the pH to be 11.0, and extracted by CHCl3 (600 mL×3)[16]. The obtained CHCl3 extract was evaporated to dryness, yielding about 1.26 g of the extract powder. The contents of two alkaloids in the Lycoris radiata extract were quantitatively determined with an external standard LC coupled with diode array detection. The contents of lycorine and galanthamine in the extract were 6.21, and 5.10 mg/g, respectively. 1.3 Instrumentation and Analytical Conditions Analysis was performed with an Agilent 1200 series LC system equipped with an Agilent 6410 triple quadrupole mass spectrometric detector, a binary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column apartment (Agilent Technologies, USA). Chromatographic separations were performed with a Zorbax SB-C18 reserved-phase column (150 mm×4.6 mm, i.d., 5 μm) maintained at 40°C. The mobile phases were comprised of solvent A (methanol mixed with 0.1% formic acid), and solvent B (water mixed with 0.1% formic acid). The gradient elution program was set as follows: 0–5 min, A 20% → 100%; 5–6.5 min, A 100%; 6.5–6.6 min, A 100% → 20%; 6.6–13 min, A 20%. The flow rate was set at 0.6 mL/min and the injection volume was 20 µL. Agilent 6410 Triple Quad LC/MS equipped with electrospray ionization (ESI) was controlled by Agilent MassHunter Workstation B.01.03. The compounds were ionized in the positive ion polarity mode. The fragmentation energies of Q1 for the analytes were set at 135 V. The optimized collision energies of 23, 18 and 25 eV were used for lycorine, galanthamine and IS, respectively. Quantification was performed using selected reaction monitoring (SRM) mode at m/z 288.2→147.1 for lycorine, m/z 288.2→213.2 for galanthamine, and m/z 256.0→167.2 for IS. Nitrogen was employed as drying gas and nebulizer gas at a pressure of 35 psi, and high
purity nitrogen was as collision gas at a pressure of 0.1 MPa. Other parameters of the mass spectrometer were set to obtain the highest intensity of protonated molecules of the analytes as follows: drying gas flow, 10.0 L/min; drying gas temperature, 300°C; capillary voltage, 4000 V. During the data acquisition, the deltapotential of the electron multiplier (EMV) was set to 200 V. 1.4 Preparation of Standard and Quality Control (QC) Samples Standards and QCs stock solutions of lycorine and galanthamine were prepared separately by dissolving an accurately weighted amount of each reference standard in a mixture of water-methanol (1:1, v/v) to yield a concentration of 2.0 mg/mL, respectively. The combined working standard solutions were prepared by serial dilution of the stock solutions with methanol. The calibration plasma standards were prepared by spiking aliquots of 900 μL blank rat plasma with 100 μL of each of the combined working standard solution, covering the ranges from 3.00 to 1000 ng/mL for each analyte. QC samples were prepared at three concentrations of 10, 100, and 900 ng/mL for each analyte, with six replicates at each concentration level. The IS was prepared in methanol at a concentration of 10 μg/mL and further diluted to 50 ng/mL as a working solution by dissolving in methanol. All the stock and working standard solutions were stored under refrigeration at 4°C. 1.5 Sample Preparation A simple liquid-liquid extraction (LLE) method was performed for extraction of lycorine and galanthamine from rat plasma. IS working solution (50 µL, 50 ng/mL) was added to an aliquot of 50 µL plasma, and mixed for 20 s. After the addition of 3 mL of ethyl ether as extraction solvent, the samples were placed on a reciprocating shaker for 10 min at 300 r/min, followed by centrifugation for 5 min at 3500 r/min. The upper organic layer was then transferred to another clean glass tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 200 µL of methanol-water (50:50, v/v), and transferred to an autosampler vial. An aliquot of 20 µL was injected into the LC-MS/MS system for analysis.
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1.6 Method Validation A full validation according to the FDA guidelines was performed for the assay in rat plasma[17]. 1.6.1 Selectivity The specificity of the method was evaluated by analyzing blank plasma from six different blank plasma samples, blank plasma spiked with each analyte at 10 ng/mL and IS at 50 ng/mL, as well as plasma sample in preclinical study to ensure that there were no potential interferences at the LC peak region for analytes and IS. 1.6.2 Accuracy and Precision The accuracy and precision of the method were determined by QC samples at low, medium and high concentrations. The accuracy was defined as the relative error (RE), which was calculated using the following formula: RE% = [(found concentrations – added concentrations)/added concentrations]×100. The intra-day and inter-day precisions were defined as the relative standard deviation (RSD). 1.6.3 Linearity, Sensitivity and Matrix Effect The calibration curve was acquired by plotting the ratio of peak areas of lycorine and galanthamine to those of IS against the nominal plasma concentration using a 1/x2 weighted linear least-squares regression model in duplicate on three consecutive days. The LLOQ of the assay was defined as the lowest concentration on the calibration curve that could be quantitatively determined with an acceptable accuracy within ±20% and precision less than 20%. The matrix effect of the analytes and IS was assessed by comparing the peak areas of analytes and IS spiked in pre-extracted blank plasma samples to those of the corresponding standards at the equivalent concentrations. 1.6.4 Recovery and Stability The recovery was determined at three concentration levels by comparing the peak areas in the post-extraction QC samples (n=5) to those of post-extraction blank matrix extracts (direct extract from blank plasma) spiked at equivalent concentrations. The storage stability was investigated by assaying QC plasma samples (n=5) stored at –80°C for two weeks, the freeze and thaw stability was performed by determining QC plasma samples undergoing three freeze (–80°C)-thaw (room temperature) cycles, the short-term stability was assessed by analyzing QC samples stored for 6 h at room temperatures, and the post-preparation stability was checked by assaying the extracted QC samples stored in the auto-sampler for 12 h. 1.7 Application to Pharmacokinetic Study The study (permission number 13-R351) was approved by the Animal Ethics Committee of Fuzhou General Hospital of Nanjing Military Command (Fuzhou, China). Six male Wistar rats (230±20 g) were purchased from the Experiment Animal Center of Fuzhou General
Hospital of Nanjing Military Command (Fuzhou, China). They were housed in an environmentally controlled breeding room at a temperature of 22±2°C and a relative humidity of 50±10%. The rats were fasted with free access to water overnight prior to the experiment. The Lycoris radiata extract was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) aqueous solution and was administered to the rats (628 mg extract/kg bodyweight, equivalent to 3.9 mg/kg of lycorine, 3.2 mg/kg of galanthamine) by oral gavage. Approximately 300 µL blood samples were collected from oculi chorioideae vein at 0, 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 5, 8, 12, 18 and 24 h following oral administration. Blood samples were transferred to heparinized eppendorf tubes and centrifuged at 5000× g for 10 min to separate the plasma. The harvested plasma samples were transferred to clean eppendorf tubes and frozen at –80°C until LC–MS/MS analysis. 2 RESULTS 2.1 Mass Spectrometry Electrospray MS/MS was used to analyze the analytes as it is beneficial in developing a selective and sensitive method. The positive ion [M+H]+ was the predominant ion in the Q1 spectrum and was used as the precursor ion to obtain product ion spectra. The product ion mass spectrum of lycorine, galanthamine and the IS are shown in fig. 2A–2C, respectively. The most sensitive mass transition was from m/z 288.2 to 147.1 for lycorine, m/z 288.2 to 213.2 for galanthamine, and from m/z 256.0 to 167.1 for the IS. 2.2 Method Validation 2.2.1 Selectivity Specificity was assessed by comparing the chromatograms of drug-free rat plasma with the corresponding spiked plasma for checking the endogenous interferences. Under the described LC-MS/MS conditions, the retention time of lycorine, galanthamine, and IS was 2.9, 3.1, and 6.3 min, respectively (fig. 3B and 3C). No interfering peak was observed in the extracted blank plasma or drug-free rat plasma samples. 2.2.2 Precision and Accuracy The intra-day precision was ≤7.3% for lycorine and ≤5.5% for galanthamine, respectively. The intra-day accuracy in terms of RE was within the range of 0.8% to 12.6% for lycorine, and –3.6% to 5.2% for galanthamine, respectively (table 1). The inter-day precision and accuracy were determined by pooling all individual assay results of six replicates of QC samples over the five separate batch runs. The inter-day precision was ≤9.1% for lycorine and ≤8.3% for galanthamine, respectively. The inter-day accuracy was within the range of 3.1% to 8.9% for lycorine, and –3.1% to 0.6% for galanthamine, respectively (table 1).
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Fig. 2 Full-scan production spectra of [M+H]+ for lycorine (A), galanthamine (B) and IS (C) at their optimized collision energies Table 1 Precision and accuracy for the determination of lycorine and galanthamine in rat plasma Conccentration Intra-day Inter-day (ng/mL) Concentration Accuracy Precision Concentration Accuracy Precision (ng/mL)
(RE, %)
(RSD, %)
(ng/mL)
(RE, %)
(RSD, %)
Lycorine 3 (LLOQ) 10 (LQC)
3.08±0.19
2.6
6.2
3.16±0.18
5.4
5.6
10.44±0.64
4.4
6.1
10.49±0.96
4.9
9.1
100 (MQC)
100.79±7.32
0.8
7.3
103.12±8.25
3.1
8.0
900 (HQC)
1013.78±38.13
12.6
3.8
980.45±74.54
8.9
7.6
Galanthamine 3 (LLOQ)
2.99±0.23
–0.3
7.8
3.04±0.23
1.2
7.4
10 (LQC)
9.81±0.42
–1.9
4.3
10.06±0.83
0.6
8.3
100 (MQC)
96.45±5.30
–3.6
5.5
98.62±7.05
–1.4
7.1
900 (HQC)
946.95±51.97
5.2
5.5
871.92±46.37
–3.1
5.3
RSD: relative standard deviation; RE%: [(found concentrations − added concentrations)/added concentrations] × 100
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Fig. 3 Representative SRM chromatograms of lycorine, galanthamine and IS in rat plasma A: blank plasma; B: blank plasma spiked with each analyte at 10 ng/mL and IS at 50 ng/mL; C: rat plasma samples at 1.5 h after an oral dose of Lycoris radiata extract
2.2.3 Linearity, LLOQ and Matrix Effect Linear responses were obtained in concentration range from 3.00 to 1000 ng/mL for both lycorine and galanthamine. Typical equations for the calibration curves were: y (ng/mL)= 6.8966×10–4x–4.2953×10–4, r2=0.9961 for lycorine, and y (ng/mL)=9.0936×10–4x–1.9394×10–3, r2=0.9960 for galanthamine. The LLOQ was found to be 3.00 ng/mL for lycorine and galanthamine with 20 µL of sample solution by injecting into the LC–MS/MS column. The precision (RSD%) and accuracy (RE%) of LLOQs for the analytes were less than 7.8% and within ±5.4%, respectively (table 1).
No obvious matrix effects were found for all the analytes as the test results ranged from 85% to 115% (data not shown), which were within the acceptable limit. The same matrix effect evaluation was performed on the IS and no significant matrix effects were observed as well. 2.2.4 Recovery and Stability The mean recoveries of lycorine and galanthamine ranged from 82.15% to 85.88%, and from 84.39% to 88.17%, respectively. The RSD of the mean recoveries of lycorine and galanthamine at three QC levels are shown in table 2. The recovery of the IS was 89.65%.
Table 2 Extraction recovery of lycorine, galantamin and IS in rat plasma (n=5) Extraction recovery ±s (%) RSD (%) Lycorine (ng/mL) 10 82.15±3.21 3.9 100 85.88±2.05 2.4 900 84.42±0.97 1.1 Analyte
Galanthamine (ng/mL) 10 100 900 IS (ng/mL) 50
88.17±7.03 86.38±3.48 84.39±6.52
8.0 4.0 7.7
89.65±5.32
5.9
Stability data are summarized in table 3. The analytes were stable in plasma under different temperature and timing conditions. 2.3 Pharmacokinetics The plasma concentration-time curve of lycorine and galanthamine is presented in fig. 4. The main pharmacokinetic parameters from a non-compartmental model analysis (Drug and Statistics, DAS version 2.0)
are listed in table 4. By applying the method, the drug concentration in plasma should be detected until 24 h after administration. The maximum plasma concentration (Cmax) and time at which the concentration reached the maximum (Tmax) for lycorine were found to be 205.47±24.47 ng/mL and 2.83±0.41 h, respectively. While Cmax and Tmax for galanthamine were 116.88±27.27 ng/mL and 1.37±0.98 h, respectively. The plasma con-
866 centration-time curve from 0 h to the last measurable concentration (AUC0–t) and area under plasma concentration-time curve from 0 h to infinity (AUC0–∞) for lycorine were 2048.13±198.49 and 2185.03±235.43
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ng·h/mL, and for galanthamine were 811.21±101.22 and 896.60± 123.04 ng·h/mL, respectively. The terminal half-life (t1/2) was found to be 5.72±1.00 h for lycorine and 7.23±3.20 h for galanthamine.
Fig. 4 Plasma concentration-time profiles of lycorine and galanthamine in rats after an oral dose of 628 mg/kg Lycoris radiata extract ( ±s, n=6) Table 3 Stability of lycorine and galanthamine in rat plasma at three QC levels (n=5) Analyte Concentration (ng/mL) Precision (RSD, %) Added Found Short-term stability Lycorine 10 8.75±0.42 4.8 (6 h, room temperature) 100 98.37±8.13 8.3 900 898.55±57.2 6.4 Galanthamine 10 9.25±0.79 8.6 100 97.79±6.62 6.8 900 925.36±48.1 5.2 Post-preparation stability Lycorine 10 10.03±0.78 7.8 (in the auto-sampler for 12 h) 100 103.12±6.94 6.7 900 937.53±50.7 5.4 Galanthamine 10 9.75±0.44 4.5 100 99.17±6.74 6.8 900 946.03±26.2 2.8 Freeze and thaw stability Lycorine 10 9.87±0.78 7.9 (–80°C to room temperature) 100 103.90±6.79 6.5 900 956.21±19.7 2.1 Galanthamine 10 9.41±0.54 5.7 100 100.84±6.88 0.8 900 873.71±29.4 3.4 Storage stability Lycorine 10 10.09±0.85 8.4 (–80°C for two weeks) 100 97.52±3.39 3.5 900 888.04±55.3 6.2 Galanthamine 10 10.53±0.68 6.4 100 94.12±6.26 6.7 900 908.04±43.3 4.8 Storage conditions
Accuracy (RE, %) –12.5 –1.6 –0.2 –7.5 –2.2 2.8 0.3 3.1 4.2 –2.5 –0.8 5.1 –1.4 3.9 6.3 –5.9 6.8 –2.9 0.9 –2.5 –1.3 5.3 –5.9 0.9
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Table 4 Pharmacokinetic parameters of lycorine and galanthamine in rats after an oral dose of 628 mg/kg Lycoris radiata extract ( ±s, n=6) Pharmacokinetic parameters Lycorine Galanthamine AUC0–t (ng·h/mL)
2048.13±198.49
811.21±101.22
AUC0–∞ (ng·h/mL)
2185.03±235.43
896.60±123.04
7.87±0.24
6.97±0.86
MRT0–t (h) t1/2 (h)
5.72±1.00
7.23±3.20
CLz/F (L/h/kg)
1.80±0.20
3.63±0.51
Vz/F (L/kg)
14.81±2.55
36.91±13.16
Cmax (ng/mL)
205.47±24.47
116.88±27.27
2.83±0.41
1.37±0.98
Tmax (h)
3 DISCUSSION Lycorine and galanthamine are two major alkaloids of Lycoris radiate, and attracting more and more attention recently due to their significant biological activities. In the present study, the oral pharmacokinetics of the two compounds were investigated after Lycoris radiata extract was given to rats, to explore their ADME properties in the body and better understand their pharmacological activities. The method validated in this study is selective, sensitive, and accurate for simultaneous quantification of lycorine and galanthamine in rat plasma by LC-MS/MS. The triple quadrupole mass spectrometer was operated in the positive ion mode, and the SRM chromatograms obtained were used for quantification. SRM scan mode was a powerful technique for pharmacokinetic studies since it met requirements of selectivity, specificity and sensitivity for analytical methods. The SRM state file parameters such as fragmentation energy and collision energy were optimized to maximize the response for the analytes. SRM transitions of m/z 288.2→147.1 and m/z 288.2→213.2 were optimized for lycorine and galanthamine, respectively. They were more specific and sensitive than those obtained using the previously reported method[18, 19], where the m/z values of the parent [M+H]+ ions of lycorine and galanthamine were both 288.2, with their product ions of m/z 147.1 and m/z 213.2, respectively. The chromatographic conditions were optimized by varying the mobile phase, gradient elution type, flow rate, and type of chromatographic columns. Methanol-water and acetonitrile-water systems were tested in various proportions as the mobile phase. The responses of the analytes were higher with the methanol-water mobile phase than with the acetonitrile-water mobile phase. Moreover, adding formic acid into the mobile phase improved the peak shape and enhanced the signal intensity of the analytes and IS. Considering the response, retention times, and peak shapes of both analytes and the IS, a methanol-water-formic acid system was finally used as the mobile phase. Sample preparation is a key factor for reliable and accurate LC–MS/MS assays. Currently, the most widely employed biological sample preparation methodologies included protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). Several analytical methods for determining galanthamine in bio-
logical fluids have been reported[13, 14, 20]. Nirogi et al extracted galanthamine from plasma samples using toluene as the extraction solvent in the LC-MS/MS method[13], and Verhaeghe et al reported that adding 0.1 mol/L sodium hydroxide and a saturated potassium chloride solution can extract galanthamine from plasma samples using toluene in the LC-MS/MS method[14]. Our study indicated that LLE could produce a relatively clean sample, which is helpful for enhancing the sensitivity, and avoiding the introduction of highly polar materials into the MS system. Several organic extraction solvents including chloroform, dichloromethane, ethyl acetate, ethyl ether, and n-hexane were investigated. It was found ethyl ether could yield the highest recovery (>82%). In our preliminary stage of the experiment, several alkaloids, structurally similar to the analytes, were tested to select a suitable IS. Among these alkaloids, diphenhydramine showed the highest extraction efficiency and the best response stability. Thus, diphenhydramine was chosen as the IS, because there was no endogenous interference, and its stable mass spectrometric response and extraction efficiency were similar to those of the analytes. To the best of our knowledge, there is no publication on pharmacokinetics of lycorine so far. The present study firstly reported its pharmacokinetics, and the AUC0–t of lycorine was 2048.13±198.49 ng·h/mL, higher than that of its analogue galanthamine (811.21±101.22 ng h/mL). The compounds lycorine and galanthamine have very similar chemical structure. However, their absorption and pharmacokinetic parameters were found to be different (table 4). Galanthamine was found more quickly absorbed and more slowly eliminated from the body than lycorine. The most possible reason for their different pharmacokinetic properties might be related with the absorption and/or metabolism difference when passing through the small intestine and/or liver, which deserves further investigation. In conclusion, a rapid, sensitive, and selective LC-MS/MS method has been developed, for the first time, for the simultaneous determination of lycorine and galanthamine in rat plasma after oral administration of 628 mg/kg Lycoris radiata extract. This method is selective and sensitive, with high accuracy, and meets all requirements in bioanalytical method. It was successfully applied to the pharmacokinetic studies of two Amaryllidaceous alkaloids of Lycoris radiata extract, which might be helpful for investigating the bioactivity mechanism and clinical application of Lycoris radiate.
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An LC-MS/MS Method for the Simultaneous Determination of Lycorine and Galanthamine in Rat Plasma and Its Application to Pharmacokinetic Study of Lycoris Radiata Extract in Rats* Xin ZHOU (周 欣)†, Yue-bin LIU (刘月彬)† , Shan HUANG (黄 珊), Ying LIU (刘 莹)# Department of Pharmacy, Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of lycorine and galanthamine, two major constituents in Lycoris radiata extract, in rat plasma. Liquid-liquid extraction with ethyl ether was carried out using diphenhydramine as the internal standard. The two bioactive alkaloids were separated on a Zorbax SB-C18 reserved-phase column (150 mm × 4.6 mm, i.d., 5 μm) by gradient elution using a mobile phase consisting of methanol with 0.1% formic acid (A) and water with 0.1% formic acid (B) at a flow rate of 0.6 mL/min. All analytes showed good linearity over a wide concentration range (r2>0.99) and the lower limit of quantification was 3.00 ng/mL for each analyte. The average extraction recovery of the analytes from rat plasma was more than 82.15%, and the intra-day and inter-day accuracy and precision of the assay were less than 12.6%. The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of two Amaryllidaceous alkaloids in rat plasma after an oral administration of Lycoris radiata extract. Key words: liquid chromatography-tandem mass spectrometry; Lycoris radiata extract; pharmacokinetic; rat plasma
Lycoris radiata, commonly known as Shi-Suan, is used in China as a traditional folk medicine for treating laryngeal trouble, furuncles, carbuncles, and suppurative wounds[1]. This plant contains a large group of alkaloids, such as lycorine, galanthamine, homolycorine, pseudo-lycorine, and lycoramine[2, 3]. Among them, lycorine and galanthamine (fig. 1) are the most common alkaloids in Amaryllidaceae plants[4–7]. Galanthamine has already been used to treat mild to moderate Alzheimer’s disease and other memory disorders such as dementia[8]. It mainly acts by reversibly inhibiting acetylcholinesterase and it has been approved as a prescription drug by the US Food and Drug Administration (FDA) in 2001[9]. Lycorine is believed to possess antiviral, antifungal, anti-inflammatory, and anticancer properties[10, 11]. It has been clinically used in Russia as an expectorant for treating chronic and acute inflammations in the lungs and bronchial diseases[12]. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been reported for determining galanthamine alone or with other drugs in biological samples[13–15]. However, to our knowledge, there is no study that has reported the simultaneous determination of lycorine and galanthamine in biological Xin ZHOU, E-mail: [email protected]; Yue-bin LIU, E-mail: [email protected] † Both authors contributed equally to this work. # Corresponding author, E-mail: [email protected] * This project was supported by the Natural Science Foundation of Fujian Province of China (No. 2013J01382).
samples. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the two Amaryllidaceous alkaloids in rat plasma. The validated assay was successfully applied to a pharmacokinetic study in rats. 1 MATERIALS AND METHODS 1.1 Chemicals and Materials Lycorine, galanthamine and diphenhydramine (fig. 1) as internal standard (IS) were purchased from Sigma Chemical Co. (USA). HPLC grade methanol and formic acid were bought from Tedia Co. (USA). The deionized water was purified by a Milli-Q water purification system (USA). All other chemicals were of analytical grade. Lycoris radiata was procured from a traditional Chinese medicinal store in Anhui province in China (Bozhou, China). The LC system (Palo Alto, CA) was equipped with an isocratic pump (1100 series) and interfaced with an autosampler (Reliance, Holland). The analytical column was a YMC Hydrosphere C18 (50 × 2 mm i.d., 3 µm; YMC Co., Japan). MS analysis was performed using an API 2000 mass spectrometer system (Applied Biosystems, USA) equipped with an ESI interface and operated in the positive ionization mode. The ion source parameters were set as follows: curtain gas, 35 psi; GS1, 50 psi and GS2, 50 psi; ion spray voltage, 4500 V; ion source temperature, 250°C; collision-activated dissociation (CAD), 8.0. Data acquisition and analysis were per-
862 formed using the analyst software Peak Simple Chromatography Data system version 1.4.1. (Applied Biosys-
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tems, USA).
Fig. 1 Chemical structures of lycorine, galanthamine and diphenhydramine (IS)
1.2 Preparation of Lycoris radiata Extract Powdered Lycoris radiata (100 g) was extracted with 70% methanol (600 mL×3) under reflux on a water bath at 80°C for 4 h. The extraction solutions were combined for filtration and concentrated to 200 mL by using a rotary evaporator in vacuum. The concentrated solution was then added with aqueous ammonia for adjusting the pH to be 11.0, and extracted by CHCl3 (600 mL×3)[16]. The obtained CHCl3 extract was evaporated to dryness, yielding about 1.26 g of the extract powder. The contents of two alkaloids in the Lycoris radiata extract were quantitatively determined with an external standard LC coupled with diode array detection. The contents of lycorine and galanthamine in the extract were 6.21, and 5.10 mg/g, respectively. 1.3 Instrumentation and Analytical Conditions Analysis was performed with an Agilent 1200 series LC system equipped with an Agilent 6410 triple quadrupole mass spectrometric detector, a binary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column apartment (Agilent Technologies, USA). Chromatographic separations were performed with a Zorbax SB-C18 reserved-phase column (150 mm×4.6 mm, i.d., 5 μm) maintained at 40°C. The mobile phases were comprised of solvent A (methanol mixed with 0.1% formic acid), and solvent B (water mixed with 0.1% formic acid). The gradient elution program was set as follows: 0–5 min, A 20% → 100%; 5–6.5 min, A 100%; 6.5–6.6 min, A 100% → 20%; 6.6–13 min, A 20%. The flow rate was set at 0.6 mL/min and the injection volume was 20 µL. Agilent 6410 Triple Quad LC/MS equipped with electrospray ionization (ESI) was controlled by Agilent MassHunter Workstation B.01.03. The compounds were ionized in the positive ion polarity mode. The fragmentation energies of Q1 for the analytes were set at 135 V. The optimized collision energies of 23, 18 and 25 eV were used for lycorine, galanthamine and IS, respectively. Quantification was performed using selected reaction monitoring (SRM) mode at m/z 288.2→147.1 for lycorine, m/z 288.2→213.2 for galanthamine, and m/z 256.0→167.2 for IS. Nitrogen was employed as drying gas and nebulizer gas at a pressure of 35 psi, and high
purity nitrogen was as collision gas at a pressure of 0.1 MPa. Other parameters of the mass spectrometer were set to obtain the highest intensity of protonated molecules of the analytes as follows: drying gas flow, 10.0 L/min; drying gas temperature, 300°C; capillary voltage, 4000 V. During the data acquisition, the deltapotential of the electron multiplier (EMV) was set to 200 V. 1.4 Preparation of Standard and Quality Control (QC) Samples Standards and QCs stock solutions of lycorine and galanthamine were prepared separately by dissolving an accurately weighted amount of each reference standard in a mixture of water-methanol (1:1, v/v) to yield a concentration of 2.0 mg/mL, respectively. The combined working standard solutions were prepared by serial dilution of the stock solutions with methanol. The calibration plasma standards were prepared by spiking aliquots of 900 μL blank rat plasma with 100 μL of each of the combined working standard solution, covering the ranges from 3.00 to 1000 ng/mL for each analyte. QC samples were prepared at three concentrations of 10, 100, and 900 ng/mL for each analyte, with six replicates at each concentration level. The IS was prepared in methanol at a concentration of 10 μg/mL and further diluted to 50 ng/mL as a working solution by dissolving in methanol. All the stock and working standard solutions were stored under refrigeration at 4°C. 1.5 Sample Preparation A simple liquid-liquid extraction (LLE) method was performed for extraction of lycorine and galanthamine from rat plasma. IS working solution (50 µL, 50 ng/mL) was added to an aliquot of 50 µL plasma, and mixed for 20 s. After the addition of 3 mL of ethyl ether as extraction solvent, the samples were placed on a reciprocating shaker for 10 min at 300 r/min, followed by centrifugation for 5 min at 3500 r/min. The upper organic layer was then transferred to another clean glass tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 200 µL of methanol-water (50:50, v/v), and transferred to an autosampler vial. An aliquot of 20 µL was injected into the LC-MS/MS system for analysis.
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1.6 Method Validation A full validation according to the FDA guidelines was performed for the assay in rat plasma[17]. 1.6.1 Selectivity The specificity of the method was evaluated by analyzing blank plasma from six different blank plasma samples, blank plasma spiked with each analyte at 10 ng/mL and IS at 50 ng/mL, as well as plasma sample in preclinical study to ensure that there were no potential interferences at the LC peak region for analytes and IS. 1.6.2 Accuracy and Precision The accuracy and precision of the method were determined by QC samples at low, medium and high concentrations. The accuracy was defined as the relative error (RE), which was calculated using the following formula: RE% = [(found concentrations – added concentrations)/added concentrations]×100. The intra-day and inter-day precisions were defined as the relative standard deviation (RSD). 1.6.3 Linearity, Sensitivity and Matrix Effect The calibration curve was acquired by plotting the ratio of peak areas of lycorine and galanthamine to those of IS against the nominal plasma concentration using a 1/x2 weighted linear least-squares regression model in duplicate on three consecutive days. The LLOQ of the assay was defined as the lowest concentration on the calibration curve that could be quantitatively determined with an acceptable accuracy within ±20% and precision less than 20%. The matrix effect of the analytes and IS was assessed by comparing the peak areas of analytes and IS spiked in pre-extracted blank plasma samples to those of the corresponding standards at the equivalent concentrations. 1.6.4 Recovery and Stability The recovery was determined at three concentration levels by comparing the peak areas in the post-extraction QC samples (n=5) to those of post-extraction blank matrix extracts (direct extract from blank plasma) spiked at equivalent concentrations. The storage stability was investigated by assaying QC plasma samples (n=5) stored at –80°C for two weeks, the freeze and thaw stability was performed by determining QC plasma samples undergoing three freeze (–80°C)-thaw (room temperature) cycles, the short-term stability was assessed by analyzing QC samples stored for 6 h at room temperatures, and the post-preparation stability was checked by assaying the extracted QC samples stored in the auto-sampler for 12 h. 1.7 Application to Pharmacokinetic Study The study (permission number 13-R351) was approved by the Animal Ethics Committee of Fuzhou General Hospital of Nanjing Military Command (Fuzhou, China). Six male Wistar rats (230±20 g) were purchased from the Experiment Animal Center of Fuzhou General
Hospital of Nanjing Military Command (Fuzhou, China). They were housed in an environmentally controlled breeding room at a temperature of 22±2°C and a relative humidity of 50±10%. The rats were fasted with free access to water overnight prior to the experiment. The Lycoris radiata extract was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) aqueous solution and was administered to the rats (628 mg extract/kg bodyweight, equivalent to 3.9 mg/kg of lycorine, 3.2 mg/kg of galanthamine) by oral gavage. Approximately 300 µL blood samples were collected from oculi chorioideae vein at 0, 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 5, 8, 12, 18 and 24 h following oral administration. Blood samples were transferred to heparinized eppendorf tubes and centrifuged at 5000× g for 10 min to separate the plasma. The harvested plasma samples were transferred to clean eppendorf tubes and frozen at –80°C until LC–MS/MS analysis. 2 RESULTS 2.1 Mass Spectrometry Electrospray MS/MS was used to analyze the analytes as it is beneficial in developing a selective and sensitive method. The positive ion [M+H]+ was the predominant ion in the Q1 spectrum and was used as the precursor ion to obtain product ion spectra. The product ion mass spectrum of lycorine, galanthamine and the IS are shown in fig. 2A–2C, respectively. The most sensitive mass transition was from m/z 288.2 to 147.1 for lycorine, m/z 288.2 to 213.2 for galanthamine, and from m/z 256.0 to 167.1 for the IS. 2.2 Method Validation 2.2.1 Selectivity Specificity was assessed by comparing the chromatograms of drug-free rat plasma with the corresponding spiked plasma for checking the endogenous interferences. Under the described LC-MS/MS conditions, the retention time of lycorine, galanthamine, and IS was 2.9, 3.1, and 6.3 min, respectively (fig. 3B and 3C). No interfering peak was observed in the extracted blank plasma or drug-free rat plasma samples. 2.2.2 Precision and Accuracy The intra-day precision was ≤7.3% for lycorine and ≤5.5% for galanthamine, respectively. The intra-day accuracy in terms of RE was within the range of 0.8% to 12.6% for lycorine, and –3.6% to 5.2% for galanthamine, respectively (table 1). The inter-day precision and accuracy were determined by pooling all individual assay results of six replicates of QC samples over the five separate batch runs. The inter-day precision was ≤9.1% for lycorine and ≤8.3% for galanthamine, respectively. The inter-day accuracy was within the range of 3.1% to 8.9% for lycorine, and –3.1% to 0.6% for galanthamine, respectively (table 1).
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Fig. 2 Full-scan production spectra of [M+H]+ for lycorine (A), galanthamine (B) and IS (C) at their optimized collision energies Table 1 Precision and accuracy for the determination of lycorine and galanthamine in rat plasma Conccentration Intra-day Inter-day (ng/mL) Concentration Accuracy Precision Concentration Accuracy Precision (ng/mL)
(RE, %)
(RSD, %)
(ng/mL)
(RE, %)
(RSD, %)
Lycorine 3 (LLOQ) 10 (LQC)
3.08±0.19
2.6
6.2
3.16±0.18
5.4
5.6
10.44±0.64
4.4
6.1
10.49±0.96
4.9
9.1
100 (MQC)
100.79±7.32
0.8
7.3
103.12±8.25
3.1
8.0
900 (HQC)
1013.78±38.13
12.6
3.8
980.45±74.54
8.9
7.6
Galanthamine 3 (LLOQ)
2.99±0.23
–0.3
7.8
3.04±0.23
1.2
7.4
10 (LQC)
9.81±0.42
–1.9
4.3
10.06±0.83
0.6
8.3
100 (MQC)
96.45±5.30
–3.6
5.5
98.62±7.05
–1.4
7.1
900 (HQC)
946.95±51.97
5.2
5.5
871.92±46.37
–3.1
5.3
RSD: relative standard deviation; RE%: [(found concentrations − added concentrations)/added concentrations] × 100
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Fig. 3 Representative SRM chromatograms of lycorine, galanthamine and IS in rat plasma A: blank plasma; B: blank plasma spiked with each analyte at 10 ng/mL and IS at 50 ng/mL; C: rat plasma samples at 1.5 h after an oral dose of Lycoris radiata extract
2.2.3 Linearity, LLOQ and Matrix Effect Linear responses were obtained in concentration range from 3.00 to 1000 ng/mL for both lycorine and galanthamine. Typical equations for the calibration curves were: y (ng/mL)= 6.8966×10–4x–4.2953×10–4, r2=0.9961 for lycorine, and y (ng/mL)=9.0936×10–4x–1.9394×10–3, r2=0.9960 for galanthamine. The LLOQ was found to be 3.00 ng/mL for lycorine and galanthamine with 20 µL of sample solution by injecting into the LC–MS/MS column. The precision (RSD%) and accuracy (RE%) of LLOQs for the analytes were less than 7.8% and within ±5.4%, respectively (table 1).
No obvious matrix effects were found for all the analytes as the test results ranged from 85% to 115% (data not shown), which were within the acceptable limit. The same matrix effect evaluation was performed on the IS and no significant matrix effects were observed as well. 2.2.4 Recovery and Stability The mean recoveries of lycorine and galanthamine ranged from 82.15% to 85.88%, and from 84.39% to 88.17%, respectively. The RSD of the mean recoveries of lycorine and galanthamine at three QC levels are shown in table 2. The recovery of the IS was 89.65%.
Table 2 Extraction recovery of lycorine, galantamin and IS in rat plasma (n=5) Extraction recovery ±s (%) RSD (%) Lycorine (ng/mL) 10 82.15±3.21 3.9 100 85.88±2.05 2.4 900 84.42±0.97 1.1 Analyte
Galanthamine (ng/mL) 10 100 900 IS (ng/mL) 50
88.17±7.03 86.38±3.48 84.39±6.52
8.0 4.0 7.7
89.65±5.32
5.9
Stability data are summarized in table 3. The analytes were stable in plasma under different temperature and timing conditions. 2.3 Pharmacokinetics The plasma concentration-time curve of lycorine and galanthamine is presented in fig. 4. The main pharmacokinetic parameters from a non-compartmental model analysis (Drug and Statistics, DAS version 2.0)
are listed in table 4. By applying the method, the drug concentration in plasma should be detected until 24 h after administration. The maximum plasma concentration (Cmax) and time at which the concentration reached the maximum (Tmax) for lycorine were found to be 205.47±24.47 ng/mL and 2.83±0.41 h, respectively. While Cmax and Tmax for galanthamine were 116.88±27.27 ng/mL and 1.37±0.98 h, respectively. The plasma con-
866 centration-time curve from 0 h to the last measurable concentration (AUC0–t) and area under plasma concentration-time curve from 0 h to infinity (AUC0–∞) for lycorine were 2048.13±198.49 and 2185.03±235.43
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ng·h/mL, and for galanthamine were 811.21±101.22 and 896.60± 123.04 ng·h/mL, respectively. The terminal half-life (t1/2) was found to be 5.72±1.00 h for lycorine and 7.23±3.20 h for galanthamine.
Fig. 4 Plasma concentration-time profiles of lycorine and galanthamine in rats after an oral dose of 628 mg/kg Lycoris radiata extract ( ±s, n=6) Table 3 Stability of lycorine and galanthamine in rat plasma at three QC levels (n=5) Analyte Concentration (ng/mL) Precision (RSD, %) Added Found Short-term stability Lycorine 10 8.75±0.42 4.8 (6 h, room temperature) 100 98.37±8.13 8.3 900 898.55±57.2 6.4 Galanthamine 10 9.25±0.79 8.6 100 97.79±6.62 6.8 900 925.36±48.1 5.2 Post-preparation stability Lycorine 10 10.03±0.78 7.8 (in the auto-sampler for 12 h) 100 103.12±6.94 6.7 900 937.53±50.7 5.4 Galanthamine 10 9.75±0.44 4.5 100 99.17±6.74 6.8 900 946.03±26.2 2.8 Freeze and thaw stability Lycorine 10 9.87±0.78 7.9 (–80°C to room temperature) 100 103.90±6.79 6.5 900 956.21±19.7 2.1 Galanthamine 10 9.41±0.54 5.7 100 100.84±6.88 0.8 900 873.71±29.4 3.4 Storage stability Lycorine 10 10.09±0.85 8.4 (–80°C for two weeks) 100 97.52±3.39 3.5 900 888.04±55.3 6.2 Galanthamine 10 10.53±0.68 6.4 100 94.12±6.26 6.7 900 908.04±43.3 4.8 Storage conditions
Accuracy (RE, %) –12.5 –1.6 –0.2 –7.5 –2.2 2.8 0.3 3.1 4.2 –2.5 –0.8 5.1 –1.4 3.9 6.3 –5.9 6.8 –2.9 0.9 –2.5 –1.3 5.3 –5.9 0.9
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Table 4 Pharmacokinetic parameters of lycorine and galanthamine in rats after an oral dose of 628 mg/kg Lycoris radiata extract ( ±s, n=6) Pharmacokinetic parameters Lycorine Galanthamine AUC0–t (ng·h/mL)
2048.13±198.49
811.21±101.22
AUC0–∞ (ng·h/mL)
2185.03±235.43
896.60±123.04
7.87±0.24
6.97±0.86
MRT0–t (h) t1/2 (h)
5.72±1.00
7.23±3.20
CLz/F (L/h/kg)
1.80±0.20
3.63±0.51
Vz/F (L/kg)
14.81±2.55
36.91±13.16
Cmax (ng/mL)
205.47±24.47
116.88±27.27
2.83±0.41
1.37±0.98
Tmax (h)
3 DISCUSSION Lycorine and galanthamine are two major alkaloids of Lycoris radiate, and attracting more and more attention recently due to their significant biological activities. In the present study, the oral pharmacokinetics of the two compounds were investigated after Lycoris radiata extract was given to rats, to explore their ADME properties in the body and better understand their pharmacological activities. The method validated in this study is selective, sensitive, and accurate for simultaneous quantification of lycorine and galanthamine in rat plasma by LC-MS/MS. The triple quadrupole mass spectrometer was operated in the positive ion mode, and the SRM chromatograms obtained were used for quantification. SRM scan mode was a powerful technique for pharmacokinetic studies since it met requirements of selectivity, specificity and sensitivity for analytical methods. The SRM state file parameters such as fragmentation energy and collision energy were optimized to maximize the response for the analytes. SRM transitions of m/z 288.2→147.1 and m/z 288.2→213.2 were optimized for lycorine and galanthamine, respectively. They were more specific and sensitive than those obtained using the previously reported method[18, 19], where the m/z values of the parent [M+H]+ ions of lycorine and galanthamine were both 288.2, with their product ions of m/z 147.1 and m/z 213.2, respectively. The chromatographic conditions were optimized by varying the mobile phase, gradient elution type, flow rate, and type of chromatographic columns. Methanol-water and acetonitrile-water systems were tested in various proportions as the mobile phase. The responses of the analytes were higher with the methanol-water mobile phase than with the acetonitrile-water mobile phase. Moreover, adding formic acid into the mobile phase improved the peak shape and enhanced the signal intensity of the analytes and IS. Considering the response, retention times, and peak shapes of both analytes and the IS, a methanol-water-formic acid system was finally used as the mobile phase. Sample preparation is a key factor for reliable and accurate LC–MS/MS assays. Currently, the most widely employed biological sample preparation methodologies included protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). Several analytical methods for determining galanthamine in bio-
logical fluids have been reported[13, 14, 20]. Nirogi et al extracted galanthamine from plasma samples using toluene as the extraction solvent in the LC-MS/MS method[13], and Verhaeghe et al reported that adding 0.1 mol/L sodium hydroxide and a saturated potassium chloride solution can extract galanthamine from plasma samples using toluene in the LC-MS/MS method[14]. Our study indicated that LLE could produce a relatively clean sample, which is helpful for enhancing the sensitivity, and avoiding the introduction of highly polar materials into the MS system. Several organic extraction solvents including chloroform, dichloromethane, ethyl acetate, ethyl ether, and n-hexane were investigated. It was found ethyl ether could yield the highest recovery (>82%). In our preliminary stage of the experiment, several alkaloids, structurally similar to the analytes, were tested to select a suitable IS. Among these alkaloids, diphenhydramine showed the highest extraction efficiency and the best response stability. Thus, diphenhydramine was chosen as the IS, because there was no endogenous interference, and its stable mass spectrometric response and extraction efficiency were similar to those of the analytes. To the best of our knowledge, there is no publication on pharmacokinetics of lycorine so far. The present study firstly reported its pharmacokinetics, and the AUC0–t of lycorine was 2048.13±198.49 ng·h/mL, higher than that of its analogue galanthamine (811.21±101.22 ng h/mL). The compounds lycorine and galanthamine have very similar chemical structure. However, their absorption and pharmacokinetic parameters were found to be different (table 4). Galanthamine was found more quickly absorbed and more slowly eliminated from the body than lycorine. The most possible reason for their different pharmacokinetic properties might be related with the absorption and/or metabolism difference when passing through the small intestine and/or liver, which deserves further investigation. In conclusion, a rapid, sensitive, and selective LC-MS/MS method has been developed, for the first time, for the simultaneous determination of lycorine and galanthamine in rat plasma after oral administration of 628 mg/kg Lycoris radiata extract. This method is selective and sensitive, with high accuracy, and meets all requirements in bioanalytical method. It was successfully applied to the pharmacokinetic studies of two Amaryllidaceous alkaloids of Lycoris radiata extract, which might be helpful for investigating the bioactivity mechanism and clinical application of Lycoris radiate.
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