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MS method for determination of hordenine in rat plasma and its application to pharmacokinetic study
Journal of Pharmaceutical and Biomedical Analysis 111 (2015) 131–137
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Validated UPLC–MS/MS method for determination of hordenine in rat plasma and its application to pharmacokinetic study Jianshe Ma a,1 , Shuanghu Wang b,1 , Xueli Huang c , Peiwu Geng b , Congcong Wen c , Yunfang Zhou b , Linsheng Yu a,d,∗ , Xianqin Wang c,d,∗∗ a
School of Basic Medicine, Wenzhou Medical University, 325035 Wenzhou, China The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, 323000 Lishui, China c Analytical and Testing Center, Wenzhou Medical University, 325035 Wenzhou, China d Forensic Center, Wenzhou Medical University, 325000 Wenzhou, China b
a r t i c l e
i n f o
Article history: Received 22 December 2014 Received in revised form 20 March 2015 Accepted 26 March 2015 Available online 3 April 2015 Keywords: Hordenine UPLC–MS/MS Pharmacokinetics Rat plasma HILIC
a b s t r a c t Hordenine is an active compound found in several foods, herbs and beer. In this work, a sensitive and selective UPLC–MS/MS method for determination of hordenine in rat plasma was developed. After addition of caulophylline as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 166.1 → 121.0 for hordenine and m/z 205.1 → 58.0 for IS. Calibration plots were linear over the range of 2–2000 ng/mL for hordenine in rat plasma. Mean recoveries of hordenine in rat plasma were in the range of 80.4–87.3%. RSD of intraday and inter-day precision were both <8%. The accuracy of the method ranged from 97.0% to 107.7%. The method was successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hordenine (4-(2-dimethylaminoethyl)phenol, Fig. 1) is an alkaloid found particularly in malting barley; it is also found in several plants and fruits [1,2], such as Aconitum tanguticum (Maxim.) Stapf, seaweeds, Senecio scandens, Coryphantha ramillosa and orange. It has been shown that hordenine is biosynthesized by the stepwise N-methylation of tyramine, which is first converted to N-methyltyramine, and further methylated to hordenine [3]. Hordenine has been reported to possess various effects [4–6], such as stimulation of gastrin release, inhibition of monoamine oxidase B and antibacterial properties. Recent research also showed that hordenine could inhibit melanogenesis in human melanocytes [7]. As
∗ Corresponding author at: School of Basic Medicine, Wenzhou Medical University, University-town, Wenzhou 325035, China. Tel.: +86 577 86689961. ∗∗ Corresponding author at: Analytical and Testing Center, Wenzhou Medical University, University-town, Wenzhou 325035, China. Tel.: +86 577 86699156. E-mail addresses: [email protected] (L. Yu), [email protected] (X. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.03.032 0731-7085/© 2015 Elsevier B.V. All rights reserved.
hordenine is usually taken into the human body from edible plants, fruits and some herbs, therefore, it is important to develop analytical methods for the determination of hordenine in biological fluids for its further pharmacological and pharmacokinetic studies [8]. To date, all analytical methods described in the literature for the determination of hordenine in biological and other matrices involve high performance liquid chromatography with ultraviolet detection (HPLC–UV) [8–12], liquid chromatography mass spectrometry (LC–MS) [13] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [14,15]. Until now, to the best of our knowledge, there is no UPLC–MS/MS method for determination of hordenine to characterize the pharmacokinetic properties. In this study, we developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for determination of hordenine in rat plasma and it successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration. The column was packed with C18 particles of 1.7 mm, which contribute to higher column performance, efficient separation and a short analysis time [16,17]. In this study, the total run time for each injection was 3 min.
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and cone gas (50 L/h). The selected ion monitoring conditions were defined as follows: capillary voltage 2.5 kV; source temperature 150 ◦ C; desolvation temperature 500 ◦ C. The multiple reaction monitoring (MRM) mode of m/z 166.1 → 121.0 for hordenine and m/z 205.1 → 58.0 for IS was used as quantitative analysis. 2.3. Calibration standards and quality control samples
Fig. 1. The chemical structure of hordenine (a) and caulophylline (IS, (b)).
2. Experimental 2.1. Chemicals and reagents Hordenine (purity > 98%) was a gift from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Caulophylline (IS, purity > 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA, USA). The rat blank plasma samples were supplied from drug-free rats (Laboratory Animal Center of Wenzhou Medical University).
The stock solutions of hordenine (1.0 mg/mL) and caulophylline (IS) (100 g/mL) were prepared in methanol–water (50:50). Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 0.5 g/mL working standard solution of IS was prepared from the IS stock solution by dilution using methanol. All of the solutions were stored at 4 ◦ C and were brought to room temperature before use. The calibration curves and QC samples used to estimate precision and accuracy of the method prepared from same stock solutions. Hordenine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 2–2000 ng/mL for hordenine in rat plasma (2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL). Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations (4, 800 and 1600 ng/mL). The analytical standards and QC samples were stored at −20 ◦ C. 2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (0.5 g/mL) was added to 100 L of collected plasma sample followed by the addition of 200 L acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min at 4 ◦ C, the supernatant (2 L) was injected into the UPLC–MS/MS system for analysis. 2.5. Method validation
2.2. Instrumentation and conditions UPLC–MS/MS with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) interface were used to analyze the compounds. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). The Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control. Hordenine and caulophylline (IS) were separated using a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) maintained at 40 ◦ C. The initial mobile phase consisted of acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 L. Elution was in a linear gradient, with the acetonitrile (containing 10 mM ammonium formate) content changing from 90 to 60% between 0 and 1.0 min. The acetonitrile (containing 10 mM ammonium formate) content was maintained at 60% for 1.0 min and then increased to 90% within 0.5 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including a strong wash (methanol–water, 50/50, v/v) and a weak wash (methanol–water, 10/90, v/v). The mass spectrometric detection was performed on a triplequadrupole mass spectrometer equipped with an ESI interface in a positive mode. Nitrogen was used as the desolvation gas (1000 L/h)
The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA) [18], European Medicines Agency (EMA) [19] and literatures [20–23] for validation of bioanalytical method. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. The selectivity of the method was evaluated by analyzing six different lots of blank rat plasma, blank plasma spiked hordenine and IS and a rat plasma sample. The “cross-talk” between MRM transitions was evaluated by analyzing the different blank samples. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of hordenine to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 2–2000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, six different lots of blank rat plasma were extracted and then spiked with the analyte at 4, 800 and 1600 ng/mL. The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect. The matrix effect of IS was evaluated at the concentration (50 ng/mL) in the same manner.
J. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 111 (2015) 131–137
Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800 and 1600 ng/mL) in three validation days. The precision was expressed by RSD. The recovery of hordenine was evaluated by comparing peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in a similar way. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ) and the response was checked [24]. The stabilities of hordenine in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 4 and 1600 ng/mL, which were exposed to different conditions. These results were compared with those freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The freeze/thaw stability was evaluated after three complete freeze/thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 20 days. The stability of the IS (50 ng/mL) was evaluated in a similar way. Reproducibility of the analytical method was further evaluated by re-analysis of incurred samples [25]. A total of 11 samples (9.6% of the total number of samples) were re-analyzed, and included samples from samples collected from six rats after oral administration. The re-analysis data for hordenine were compared with data from the original assay.
2.6. Pharmacokinetic study Male Sprague-Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of hordenine. All twelve rats were housed at Laboratory Animal Center of Wenzhou Medical University. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available, the rats housed on wire bottom cages. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 h after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine. The samples were immediately centrifuged at 3000 × g for 10 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma hordenine concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
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The liquid chromatographic conditions were developed to separate as many interfering compounds as possible from the hordenine and IS. Different columns, such as UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) and UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 m) were compared for chromatographic separation. The UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) column demonstrated proper retention time and better peak shape for hordenine and IS than C18 column. An alternative for separation of hydrophilic compounds is hydrophilic interaction chromatography (HILIC). HILIC is a type of liquid chromatography that allows high-resolution separation of highly polar compounds [26,27]. The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization [28–30]. Various combinations of water, 0.1% formic acid, 10 mM ammonium formate in water, methanol and acetonitrile changed content of each component were investigated. Acetonitrile was chosen as the organic phase because it could provide sharper peak shape and lower pump pressure compared with methanol. The addition of 10 mM ammonium formate into mobile phase could improve the peak shape. Acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) was chosen as mobile phase because it could provide proper retention time and peak shape. An efficient clean-up for bio-samples to remove protein and potential interferences prior to LC–MS analysis was an important point in the method development [31–33]. The effective and simple protein precipitation was employed in our work. Acetonitrile–methanol (9:1, v/v) was chosen as the protein precipitation solvent because it exhibited acceptable recovery (80.4–87.3%), and it is fast and simple. We tried diazepam, carbamazepine, midazolam at first, however caulophylline was more suitable for IS in the present study. Select caulophylline as IS, because its chromatographic performance was similar with hordenine, and they had similar retention time; they both were polar substances, both suitable for detection in positive ion electrospray ionization interface and on a HILIC column.
3.2. Selectivity and matrix effect Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with hordenine and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The matrix effect for hordenine at concentrations of 4, 800 and 1600 ng/mL were measured to be 102.6, 104.5 and 106.2% with the RSD of 6.0, 6.6 and 3.5% (n = 6). The matrix effect for IS (50 ng/mL) was 104.2% with the RSD of 4.0% (n = 6). As a result, matrix effect from plasma was negligible in this method.
3.3. Calibration curve and sensitivity 3. Results and discussion 3.1. Method development The MS detector parameters were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS–MS conditions, the daughter ion spectrum of the [M+H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most abundant fragment was detected at m/z 121.1 with the capillary voltage of 2500 V and the collision energy of 15 V. Therefore, the m/z 166.1 → 121.1 transition was selected for further UPLC–MS/MS analysis in MRM mode.
The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2–2000 ng/mL for hordenine in rat plasma (Table 2). Typical equation of the calibration curve was: y = 0.00226532 × x − 0.000409021, r = 0.9990, the RSD for slope and coefficient of correlation were 6.5% and 5.3%, where y represents the ratios of hordenine peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of hordenine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 10.1% and 87.5%, respectively. The LOD, defined as a signal/noise ratio of 3, was 0.5 ng/mL for hordenine in rat plasma.
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Fig. 2. Representative UPLC–MS/MS chromatograms of hordenine and caulophylline (IS), (a) blank plasma; (b) blank plasma spiked with hordenine (2 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 2 h after oral administration of single dosage 15 mg/kg hordenine (105.6 ng/mL).
3.4. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over three validation days. Intra-day precision was 5% or less and the inter-day
precision was 8% or less at each QC level. The accuracy of the method ranged from 97.0% to 107.7% at each QC level. Mean recoveries of hordenine were better than 80.4%. The recovery of the IS (50 ng/mL) was 87.9%. Assay performance data was presented in Table 1.
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Table 1 Precision, accuracy and recovery for hordenine of QC sample in rat plasma (n = 6). Concentration (ng/mL)
4 800 1600
RSD (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
2.2 4.9 3.6
7.8 6.0 4.0
97.0 100.3 102.3
102.6 107.7 98.8
Recovery (%)
RSD (%)
87.3 80.4 82.0
6.7 7.3 3.5
Table 2 Precision, accuracy of calibrator standards in the calibration curve of hordenine (n = 6). Concentration (ng/mL)
Measured concentration (ng/mL)
Accuracy (%)
RSD (%)
2 5 10 20 50 100 200 500 1000 2000
1.8 4.9 10.8 21.6 53.8 107.7 205.3 511.7 1003.0 1941.0
87.5 98.8 108.2 108.2 107.7 107.7 102.6 102.3 100.3 97.0
10.1 8.4 2.8 5.5 7.8 4.0 6.0 3.6 4.9 2.2
300
None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding 0.5 extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over [24].
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3.6. Stability The auto-sampler, room temperature, freeze–thaw and longterm (20 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentrations were within ±9% of their nominal values, and the established method was suitable for the pharmacokinetic study. 3.7. Cross-talk The phenomenon of cross-talk may have occurred if the analytes had similar retention times, which will lead to greater deviation of the results. Therefore, it is essential to monitor two or more channels for MRM analysis so as to avoid cross-talk. Two methods were usually used to avoid cross-talk: selecting different quantitated ions to improve the selectivity; optimizing the mobile phase to completely separate the analytes [34]. In the present study, hordenine and caulophylline (IS) had different quantitated ions and retention time (tR = 1.30 min for hordenine, and tR = 1.88 min for caulophylline). The results indicated that there were no interference between the four monitoring channels, and no relevant cross-talk was observed. 3.8. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine was shown in Fig. 3. The main pharmacokinetic parameters from noncompartment model analysis were summarized in Table 3. A sudden dip in pharmacokinetic profile of oral administration, it may due to the tissue distribution is faster and the overall elimination is slow. The pharmacokinetic profile of hordenine in rat was characterized for the first time. It helps to better understand pharmacology features of hordenine. Furthermore, the bioavailability of hordenine was reported to be 66.2% for first time.
Concentration (ng/mL)
3.5. Carry-over
800
Oral administration Intravenous administration
600 200 400
150 100
200
50 0
0 0
5
10
15
20
25
Time (h) Fig. 3. Mean plasma concentration time profile after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine in rats.
Previously, there several LC–MS [13] and LC–MS/MS [14,15] developed for determination of hordenine in harding grass or bitter orange, however, these matrix were not biological samples; only Chen et al. developed a selective extraction based on poly(MAA-VBEGMDA) monolith followed by HPLC for determination of hordenine in plasma and urine samples [8], a novel sorbent was fabricated for selective solid-phase extraction (SPE) of hordenine in biological samples. The conditions for preparation were optimized to generate a poly(MAA-VB-EGMDA) monolith with good permeability. The proposed SPE-HPLC method presented good linearity (r = 0.9992) within 10–2000 ng/mL and the detection limits was 3 ng/mL with run-time of 10 min. In present study, our UPLC–MS/MS method with LOD of 0.5 ng/mL for hordenine in rat plasma was validated for selectivity, linearity, accuracy, precision, recovery and stability, and it only took 3 min for total run time, it is much faster and more sensitive than literature [8].
3.9. Incurred sample reanalysis A total of 11 samples were re-analyzed. The % differences between the reassay concentrations and the original concentrations were all less than 10% of their mean values and met the acceptance criteria for incurred sample reanalysis (Table 4).
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Table 3 The main pharmacokinetic parameters after oral and intravenous administration of hordenine in rats (n = 6). Parameters
Unit
Mean SD Oral administration 15 mg/kg
Mean SD Intravenous administration 5 mg/kg
AUC(0−t) AUC(0−∞) t1/2 Cmax /C5min Bioavailability, F
ng/mL × h ng/mL × h h ng/mL
565.8 585.3 4.6 184.2
285.0 287.6 1.0 599.1
105.3 106.3 1.6 85.7 66.2%
67.0 67.2 0.3 124.7
AUC, the area under the plasma concentration–time curve; t1/2 , the half-life; Cmax , the maximum plasma concentration (oral administration); C5min , the plasma concentration at 5 min (intravenous administration).
Table 4 Incurred sample reassay data of hordenine from rats of oral administration. Time (min)
Original result (ng/mL)
Repeat result (ng/mL)
Percentage differencea
0.08333333 0.25 0.5 1 1.5 2 4 6 8 12 24
10.8 168.7 180.0 170.7 138.3 102.7 33.9 33.1 22.6 11.2 2.9
9.8 175.6 185.7 165.8 140.8 95.8 31.6 34.8 21.3 10.6 3.1
9.7 −4.0 −3.1 2.9 −1.8 7.0 7.0 −5.0 5.9 5.5 −6.7
a
(Repeat-original)/average expressed as a percentage.
4. Conclusion The UPLC–MS/MS method described here allowed accurate and reproducible for determination of hordenine in rat plasma utilizing 100 L of plasma with an LLOQ of 2 ng/mL. The UPLC–MS/MS method successfully applied to a pharmacokinetic study of hordenine after oral and intravenous administration. The pharmacokinetic profile of hordenine in rat was characterized and the bioavailability of hordenine was reported to be 66.2% for first time. Acknowledgements This work was supported by fund of the National Natural Science Foundation of China, No. 81401558. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jpba.2015.03.032. References [1] M. Frank, T.J. Weckman, T. Wood, W.E. Woods, C.L. Tai, S.L. Chang, A. Ewing, J.W. Blake, T. Tobin, Hordenine: pharmacology, pharmacokinetics and behavioural effects in the horse, Equine Vet. J. 22 (1990) 437–441. [2] L.C. Sander, K. Putzbach, B.C. Nelson, C.A. Rimmer, M. Bedner, J.B. Thomas, B.J. Porter, L.J. Wood, M.M. Schantz, K.E. Murphy, K.E. Sharpless, S.A. Wise, J.H. Yen, P.H. Siitonen, R.L. Evans, A. Nguyen Pho, M.C. Roman, J.M. Betz, Certification of standard reference materials containing bitter orange, Anal. Bioanal. Chem. 391 (2008) 2023–2034. [3] J.V. Lovett, A.H. Hoult, O. Christen, Biologically active secondary metabolites of barley IV. Hordenine production by different barley lines, J. Chem. Ecol. 20 (1994) 1945–1954. [4] G.S. Rao, Identity of peyocactin, an antibiotic from peyote (Lophophora williamsii), and hordenine, J. Pharm. Pharmacol. 22 (1970) 544–545. [5] C.J. Barwell, A.N. Basma, M.A. Lafi, L.D. Leake, Deamination of hordenine by monoamine oxidase and its action on vasa deferentia of the rat, J. Pharm. Pharmacol. 41 (1989) 421–423. [6] Y. Yokoo, H. Kohda, A. Kusumoto, H. Naoki, N. Matsumoto, T. Amachi, Y. Suwa, H. Fukazawa, H. Ishida, K. Tsuji, H. Nukaya, Isolation from beer and structural determination of a potent stimulant of gastrin release, Alcohol Alcohol. 34 (1999) 161–168.
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Validated UPLC–MS/MS method for determination of hordenine in rat plasma and its application to pharmacokinetic study Jianshe Ma a,1 , Shuanghu Wang b,1 , Xueli Huang c , Peiwu Geng b , Congcong Wen c , Yunfang Zhou b , Linsheng Yu a,d,∗ , Xianqin Wang c,d,∗∗ a
School of Basic Medicine, Wenzhou Medical University, 325035 Wenzhou, China The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, 323000 Lishui, China c Analytical and Testing Center, Wenzhou Medical University, 325035 Wenzhou, China d Forensic Center, Wenzhou Medical University, 325000 Wenzhou, China b
a r t i c l e
i n f o
Article history: Received 22 December 2014 Received in revised form 20 March 2015 Accepted 26 March 2015 Available online 3 April 2015 Keywords: Hordenine UPLC–MS/MS Pharmacokinetics Rat plasma HILIC
a b s t r a c t Hordenine is an active compound found in several foods, herbs and beer. In this work, a sensitive and selective UPLC–MS/MS method for determination of hordenine in rat plasma was developed. After addition of caulophylline as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 166.1 → 121.0 for hordenine and m/z 205.1 → 58.0 for IS. Calibration plots were linear over the range of 2–2000 ng/mL for hordenine in rat plasma. Mean recoveries of hordenine in rat plasma were in the range of 80.4–87.3%. RSD of intraday and inter-day precision were both <8%. The accuracy of the method ranged from 97.0% to 107.7%. The method was successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hordenine (4-(2-dimethylaminoethyl)phenol, Fig. 1) is an alkaloid found particularly in malting barley; it is also found in several plants and fruits [1,2], such as Aconitum tanguticum (Maxim.) Stapf, seaweeds, Senecio scandens, Coryphantha ramillosa and orange. It has been shown that hordenine is biosynthesized by the stepwise N-methylation of tyramine, which is first converted to N-methyltyramine, and further methylated to hordenine [3]. Hordenine has been reported to possess various effects [4–6], such as stimulation of gastrin release, inhibition of monoamine oxidase B and antibacterial properties. Recent research also showed that hordenine could inhibit melanogenesis in human melanocytes [7]. As
∗ Corresponding author at: School of Basic Medicine, Wenzhou Medical University, University-town, Wenzhou 325035, China. Tel.: +86 577 86689961. ∗∗ Corresponding author at: Analytical and Testing Center, Wenzhou Medical University, University-town, Wenzhou 325035, China. Tel.: +86 577 86699156. E-mail addresses: [email protected] (L. Yu), [email protected] (X. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.03.032 0731-7085/© 2015 Elsevier B.V. All rights reserved.
hordenine is usually taken into the human body from edible plants, fruits and some herbs, therefore, it is important to develop analytical methods for the determination of hordenine in biological fluids for its further pharmacological and pharmacokinetic studies [8]. To date, all analytical methods described in the literature for the determination of hordenine in biological and other matrices involve high performance liquid chromatography with ultraviolet detection (HPLC–UV) [8–12], liquid chromatography mass spectrometry (LC–MS) [13] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [14,15]. Until now, to the best of our knowledge, there is no UPLC–MS/MS method for determination of hordenine to characterize the pharmacokinetic properties. In this study, we developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for determination of hordenine in rat plasma and it successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration. The column was packed with C18 particles of 1.7 mm, which contribute to higher column performance, efficient separation and a short analysis time [16,17]. In this study, the total run time for each injection was 3 min.
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and cone gas (50 L/h). The selected ion monitoring conditions were defined as follows: capillary voltage 2.5 kV; source temperature 150 ◦ C; desolvation temperature 500 ◦ C. The multiple reaction monitoring (MRM) mode of m/z 166.1 → 121.0 for hordenine and m/z 205.1 → 58.0 for IS was used as quantitative analysis. 2.3. Calibration standards and quality control samples
Fig. 1. The chemical structure of hordenine (a) and caulophylline (IS, (b)).
2. Experimental 2.1. Chemicals and reagents Hordenine (purity > 98%) was a gift from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Caulophylline (IS, purity > 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA, USA). The rat blank plasma samples were supplied from drug-free rats (Laboratory Animal Center of Wenzhou Medical University).
The stock solutions of hordenine (1.0 mg/mL) and caulophylline (IS) (100 g/mL) were prepared in methanol–water (50:50). Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 0.5 g/mL working standard solution of IS was prepared from the IS stock solution by dilution using methanol. All of the solutions were stored at 4 ◦ C and were brought to room temperature before use. The calibration curves and QC samples used to estimate precision and accuracy of the method prepared from same stock solutions. Hordenine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 2–2000 ng/mL for hordenine in rat plasma (2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL). Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations (4, 800 and 1600 ng/mL). The analytical standards and QC samples were stored at −20 ◦ C. 2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (0.5 g/mL) was added to 100 L of collected plasma sample followed by the addition of 200 L acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min at 4 ◦ C, the supernatant (2 L) was injected into the UPLC–MS/MS system for analysis. 2.5. Method validation
2.2. Instrumentation and conditions UPLC–MS/MS with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) interface were used to analyze the compounds. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). The Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control. Hordenine and caulophylline (IS) were separated using a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) maintained at 40 ◦ C. The initial mobile phase consisted of acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 L. Elution was in a linear gradient, with the acetonitrile (containing 10 mM ammonium formate) content changing from 90 to 60% between 0 and 1.0 min. The acetonitrile (containing 10 mM ammonium formate) content was maintained at 60% for 1.0 min and then increased to 90% within 0.5 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including a strong wash (methanol–water, 50/50, v/v) and a weak wash (methanol–water, 10/90, v/v). The mass spectrometric detection was performed on a triplequadrupole mass spectrometer equipped with an ESI interface in a positive mode. Nitrogen was used as the desolvation gas (1000 L/h)
The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA) [18], European Medicines Agency (EMA) [19] and literatures [20–23] for validation of bioanalytical method. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. The selectivity of the method was evaluated by analyzing six different lots of blank rat plasma, blank plasma spiked hordenine and IS and a rat plasma sample. The “cross-talk” between MRM transitions was evaluated by analyzing the different blank samples. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of hordenine to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 2–2000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, six different lots of blank rat plasma were extracted and then spiked with the analyte at 4, 800 and 1600 ng/mL. The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect. The matrix effect of IS was evaluated at the concentration (50 ng/mL) in the same manner.
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Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800 and 1600 ng/mL) in three validation days. The precision was expressed by RSD. The recovery of hordenine was evaluated by comparing peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in a similar way. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ) and the response was checked [24]. The stabilities of hordenine in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 4 and 1600 ng/mL, which were exposed to different conditions. These results were compared with those freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The freeze/thaw stability was evaluated after three complete freeze/thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 20 days. The stability of the IS (50 ng/mL) was evaluated in a similar way. Reproducibility of the analytical method was further evaluated by re-analysis of incurred samples [25]. A total of 11 samples (9.6% of the total number of samples) were re-analyzed, and included samples from samples collected from six rats after oral administration. The re-analysis data for hordenine were compared with data from the original assay.
2.6. Pharmacokinetic study Male Sprague-Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of hordenine. All twelve rats were housed at Laboratory Animal Center of Wenzhou Medical University. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available, the rats housed on wire bottom cages. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 h after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine. The samples were immediately centrifuged at 3000 × g for 10 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma hordenine concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
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The liquid chromatographic conditions were developed to separate as many interfering compounds as possible from the hordenine and IS. Different columns, such as UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) and UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 m) were compared for chromatographic separation. The UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) column demonstrated proper retention time and better peak shape for hordenine and IS than C18 column. An alternative for separation of hydrophilic compounds is hydrophilic interaction chromatography (HILIC). HILIC is a type of liquid chromatography that allows high-resolution separation of highly polar compounds [26,27]. The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization [28–30]. Various combinations of water, 0.1% formic acid, 10 mM ammonium formate in water, methanol and acetonitrile changed content of each component were investigated. Acetonitrile was chosen as the organic phase because it could provide sharper peak shape and lower pump pressure compared with methanol. The addition of 10 mM ammonium formate into mobile phase could improve the peak shape. Acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) was chosen as mobile phase because it could provide proper retention time and peak shape. An efficient clean-up for bio-samples to remove protein and potential interferences prior to LC–MS analysis was an important point in the method development [31–33]. The effective and simple protein precipitation was employed in our work. Acetonitrile–methanol (9:1, v/v) was chosen as the protein precipitation solvent because it exhibited acceptable recovery (80.4–87.3%), and it is fast and simple. We tried diazepam, carbamazepine, midazolam at first, however caulophylline was more suitable for IS in the present study. Select caulophylline as IS, because its chromatographic performance was similar with hordenine, and they had similar retention time; they both were polar substances, both suitable for detection in positive ion electrospray ionization interface and on a HILIC column.
3.2. Selectivity and matrix effect Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with hordenine and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The matrix effect for hordenine at concentrations of 4, 800 and 1600 ng/mL were measured to be 102.6, 104.5 and 106.2% with the RSD of 6.0, 6.6 and 3.5% (n = 6). The matrix effect for IS (50 ng/mL) was 104.2% with the RSD of 4.0% (n = 6). As a result, matrix effect from plasma was negligible in this method.
3.3. Calibration curve and sensitivity 3. Results and discussion 3.1. Method development The MS detector parameters were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS–MS conditions, the daughter ion spectrum of the [M+H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most abundant fragment was detected at m/z 121.1 with the capillary voltage of 2500 V and the collision energy of 15 V. Therefore, the m/z 166.1 → 121.1 transition was selected for further UPLC–MS/MS analysis in MRM mode.
The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2–2000 ng/mL for hordenine in rat plasma (Table 2). Typical equation of the calibration curve was: y = 0.00226532 × x − 0.000409021, r = 0.9990, the RSD for slope and coefficient of correlation were 6.5% and 5.3%, where y represents the ratios of hordenine peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of hordenine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 10.1% and 87.5%, respectively. The LOD, defined as a signal/noise ratio of 3, was 0.5 ng/mL for hordenine in rat plasma.
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Fig. 2. Representative UPLC–MS/MS chromatograms of hordenine and caulophylline (IS), (a) blank plasma; (b) blank plasma spiked with hordenine (2 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 2 h after oral administration of single dosage 15 mg/kg hordenine (105.6 ng/mL).
3.4. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over three validation days. Intra-day precision was 5% or less and the inter-day
precision was 8% or less at each QC level. The accuracy of the method ranged from 97.0% to 107.7% at each QC level. Mean recoveries of hordenine were better than 80.4%. The recovery of the IS (50 ng/mL) was 87.9%. Assay performance data was presented in Table 1.
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Table 1 Precision, accuracy and recovery for hordenine of QC sample in rat plasma (n = 6). Concentration (ng/mL)
4 800 1600
RSD (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
2.2 4.9 3.6
7.8 6.0 4.0
97.0 100.3 102.3
102.6 107.7 98.8
Recovery (%)
RSD (%)
87.3 80.4 82.0
6.7 7.3 3.5
Table 2 Precision, accuracy of calibrator standards in the calibration curve of hordenine (n = 6). Concentration (ng/mL)
Measured concentration (ng/mL)
Accuracy (%)
RSD (%)
2 5 10 20 50 100 200 500 1000 2000
1.8 4.9 10.8 21.6 53.8 107.7 205.3 511.7 1003.0 1941.0
87.5 98.8 108.2 108.2 107.7 107.7 102.6 102.3 100.3 97.0
10.1 8.4 2.8 5.5 7.8 4.0 6.0 3.6 4.9 2.2
300
None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding 0.5 extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over [24].
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3.6. Stability The auto-sampler, room temperature, freeze–thaw and longterm (20 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentrations were within ±9% of their nominal values, and the established method was suitable for the pharmacokinetic study. 3.7. Cross-talk The phenomenon of cross-talk may have occurred if the analytes had similar retention times, which will lead to greater deviation of the results. Therefore, it is essential to monitor two or more channels for MRM analysis so as to avoid cross-talk. Two methods were usually used to avoid cross-talk: selecting different quantitated ions to improve the selectivity; optimizing the mobile phase to completely separate the analytes [34]. In the present study, hordenine and caulophylline (IS) had different quantitated ions and retention time (tR = 1.30 min for hordenine, and tR = 1.88 min for caulophylline). The results indicated that there were no interference between the four monitoring channels, and no relevant cross-talk was observed. 3.8. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine was shown in Fig. 3. The main pharmacokinetic parameters from noncompartment model analysis were summarized in Table 3. A sudden dip in pharmacokinetic profile of oral administration, it may due to the tissue distribution is faster and the overall elimination is slow. The pharmacokinetic profile of hordenine in rat was characterized for the first time. It helps to better understand pharmacology features of hordenine. Furthermore, the bioavailability of hordenine was reported to be 66.2% for first time.
Concentration (ng/mL)
3.5. Carry-over
800
Oral administration Intravenous administration
600 200 400
150 100
200
50 0
0 0
5
10
15
20
25
Time (h) Fig. 3. Mean plasma concentration time profile after oral (15 mg/kg) and intravenous (5 mg/kg) administration of hordenine in rats.
Previously, there several LC–MS [13] and LC–MS/MS [14,15] developed for determination of hordenine in harding grass or bitter orange, however, these matrix were not biological samples; only Chen et al. developed a selective extraction based on poly(MAA-VBEGMDA) monolith followed by HPLC for determination of hordenine in plasma and urine samples [8], a novel sorbent was fabricated for selective solid-phase extraction (SPE) of hordenine in biological samples. The conditions for preparation were optimized to generate a poly(MAA-VB-EGMDA) monolith with good permeability. The proposed SPE-HPLC method presented good linearity (r = 0.9992) within 10–2000 ng/mL and the detection limits was 3 ng/mL with run-time of 10 min. In present study, our UPLC–MS/MS method with LOD of 0.5 ng/mL for hordenine in rat plasma was validated for selectivity, linearity, accuracy, precision, recovery and stability, and it only took 3 min for total run time, it is much faster and more sensitive than literature [8].
3.9. Incurred sample reanalysis A total of 11 samples were re-analyzed. The % differences between the reassay concentrations and the original concentrations were all less than 10% of their mean values and met the acceptance criteria for incurred sample reanalysis (Table 4).
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Table 3 The main pharmacokinetic parameters after oral and intravenous administration of hordenine in rats (n = 6). Parameters
Unit
Mean SD Oral administration 15 mg/kg
Mean SD Intravenous administration 5 mg/kg
AUC(0−t) AUC(0−∞) t1/2 Cmax /C5min Bioavailability, F
ng/mL × h ng/mL × h h ng/mL
565.8 585.3 4.6 184.2
285.0 287.6 1.0 599.1
105.3 106.3 1.6 85.7 66.2%
67.0 67.2 0.3 124.7
AUC, the area under the plasma concentration–time curve; t1/2 , the half-life; Cmax , the maximum plasma concentration (oral administration); C5min , the plasma concentration at 5 min (intravenous administration).
Table 4 Incurred sample reassay data of hordenine from rats of oral administration. Time (min)
Original result (ng/mL)
Repeat result (ng/mL)
Percentage differencea
0.08333333 0.25 0.5 1 1.5 2 4 6 8 12 24
10.8 168.7 180.0 170.7 138.3 102.7 33.9 33.1 22.6 11.2 2.9
9.8 175.6 185.7 165.8 140.8 95.8 31.6 34.8 21.3 10.6 3.1
9.7 −4.0 −3.1 2.9 −1.8 7.0 7.0 −5.0 5.9 5.5 −6.7
a
(Repeat-original)/average expressed as a percentage.
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