Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 160–164
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A sensitive LC–MS/MS method for simultaneous determination of amygdalin and paeoniflorin in human plasma and its application Xiaobing Li a,b,c , Fuguo Shi a,b , Pan Gu a,b , Lingye Liu a,b , Hua He b,c,∗∗ , Li Ding a,b,∗ a
Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China c Department of Analytical Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 24 January 2014 Keywords: Amygdalin Paeoniflorin Huoxue-Tongluo lyophilized powder for injection LC–MS/MS Pharmacokinetics
a b s t r a c t A simple and sensitive HPLC–MS/MS method was developed and fully validated for the simultaneous determination of amygdalin (AD) and paeoniflorin (PF) in human plasma. For both analytes, the method exhibited high sensitivity (LLOQs of 0.6 ng/mL) by selecting the ammonium adduct ions ([M+NH4 ]+ ) as the precursor ions and good linearity over the concentration range of 0.6–2000 ng/mL with the correlation coefficients > 0.9972. The intra- and inter-day precision was lower than 10% in relation to relative standard deviation, while accuracy was within ±2.3% in terms of relative error for both analytes. The developed method was successfully applied to a pilot pharmacokinetic study of AD and PF in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huoxue-Tongluo lyophilized powder for injection (HTLPI), a simplified formulated Chinese medicine derived from the well-known traditional Chinese medicine prescription Tao-HongSi-Wu-Tang, is mainly made from the extracts of Persicae Semen and Paeoniae Radix Rubra and used for treating blood-stasis obstruction syndrome in the acute stage of cerebral ischemic stroke. HTLPI has passed the Phase I clinical trial (Chinese CFDA: 2004L01097) and its safety has been verified. The Phase II and III clinical trials are carrying out. Amygdalin (AD) and paeoniflorin (PF), the phytochemical markers for the quality control of Persicae Semen and Paeoniae Radix Rubra (Pharmacopeia of PR China, 2010), are regarded as the principal constituents responsible for
the pharmacological activities [1–6] and selected as the indicators in the pharmacokinetic study of HTLPI. Various chromatographic methods have been published for the determination of AD or PF in animal biological fluids using HPLCUV [7–9], LC–MS [10,11], LC–MS/MS [12–15], UPLC–MS/MS [16]. However, to the best of our knowledge, there is presently no report on the determination of AD or PF in human plasma for the clinical pharmacokinetic study. In the present investigation, a simple and highly sensitive (LLOQs of 0.6 ng/mL) LC–MS/MS method was developed and fully validated for simultaneous determination of AD and PF in human plasma for the first time. This method was successfully applied to a pilot clinical pharmacokinetic study of AD and PF after intravenous infusion administration of HTLPI to healthy Chinese volunteers. 2. Experimental
∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 025 8327 1485; fax: +86 025 8327 1485. ∗∗ Corresponding author at: Department of Analytical Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 025 8327 1505; fax: +86 025 8327 1505. E-mail addresses: [email protected] (H. He), [email protected], [email protected] (L. Ding). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.020
2.1. Chemicals and reagents Huoxue-Tongluo lyophilized powders for injection (6 g, containing 25.3 mg of amygdalin and 35.8 mg of paeoniflorin) were supplied by Jiangsu Kanion Pharmaceutical Co., Ltd. (Lianyungang, China). Amygdalin (AD, purity 93.6%), paeoniflorin (PF, purity 96.5%) and geniposide (IS, purity 99.7%) were purchased from
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National Institutes for Food and Drug Control (Beijing, China). Methanol and acetonitrile were HPLC grade and purchased from Merck KGaA (Darmstadt, Germany). Ammonium acetate and formic acid (analytical grade) were purchased from Nanjing Chemical Reagents Co., Ltd. (Nanjing, China). Water was prepared with double distillation. 2.2. LC–MS/MS instruments and conditions The liquid chromatography was performed on an Agilent 1260 Series liquid chromatography (Agilent Technologies, Palo Alto, CA, USA), which comprised an Agilent 1260 binary pump (model G1312B), a vacuum degasser (model G4225A), an autosampler (model G1367E) and an Agilent 1290 temperature controlled column compartment (model G1330B). Chromatographic separation was carried out on a 38 ◦ C Hedera ODS-2 analytical column (2.1 mm × 150 mm, 5 m; Hanbon Science and Technology, Huai’an, China) with a security Guard-C18 column (4 mm × 2.0 mm, 5 m; Phenomenex, Torrance, CA, USA). The mobile phase consisted of acetonitrile and 5 mM ammonium acetate buffer solution containing 0.05% formic acid (20:80, v/v) at a flow rate of 300 L/min. Autosampler temperature was maintained at 6 ◦ C and the injection volume was 10 L. A 10-port switching valve (Valco, Houston, TX, USA) was used to direct flow eluate to a waste container in the first 1.5 min of the chromatographic run and afterwards to the mass spectrometer. The total run time was 4.5 min. Mass spectrometric detection was performed on an API 4000 tandem mass spectrometer (Applied Biosystems, Toronto, Canada) equipped with a Turbo-V® ionspray source operation in the positive ESI mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 475.2 → 163.1 for AD, m/z 498.2 → 179.1 for PF and m/z 406.2 → 227.1 for IS, respectively (see Fig. 1). The ion spray temperature and ion spray voltage were maintained at 400 ◦ C and 5500 V, with nebulizer gas (GS1) and heater gas (GS2) set at 70 and 70 psi, respectively. The curtain gas (CUR) was kept at 30 psi and the collision gas (CAD) was 6 psi. The specific parameters for each analyte are displayed in Table 1. The system control and data analysis were performed by AB Sciex Analyst software (version 1.5.2). 2.3. Preparation of calibration standards and quality control samples The mixture standard stock solution of AD and PF was prepared in methanol at the concentration of 1 mg/mL. Standard working solutions ranged from 0.006 to 20 g/mL were prepared by diluting the stock solution with methanol for both analytes. Calibration standards were prepared using blank human plasma spiked with 20 L of mixture working solutions, to yield the concentrations of 0.6, 2, 6, 20, 60, 200, 600, 1200 and 2000 ng/mL for both analytes, respectively. QC samples were prepared in the same way as the calibration samples at three concentration levels: 1.5, 100 and 1500 ng/mL for both analytes. The stock solution (1 mg/mL) of the IS was prepared in methanol and was further diluted with methanol to 1 g/mL. All the solutions were kept at −20 ◦ C and brought to room temperature before use. 2.4. Sample preparation All samples were stored in a freezer at −20 ◦ C and allowed to thaw at room temperature by vortex before processing. A 200 L of plasma sample was mixed with 20 L IS solution (1 g/mL). The mixture was deproteinized with 600 L of methanol, vortexed for 5 min, and then centrifuged at 15,600 rpm for 10 min. An aliquot of 0.2 mL supernatant was diluted by adding 0.2 mL of water. After
Fig. 1. Positive product ion mass spectra of [M+NH4 ]+ of AD (A), PF (B) and IS (C) and their proposed fragmentation patterns.
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Table 1 Optimized mass parameters for AD, PF and the IS. Analytes
Precursor ion (m/z)
Product ion (m/z)
Dwell time (ms)
DP (V)
EP (V)
CE (V)
CXP (V)
AD PF IS
475.2 498.2 406.1
163.1 179.1 227.1
450 450 450
55 51 50
11 8 7
23 25 13
10 11 17
DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: collision cell exit potential.
being vortex-mixed for 5 s, the mixture was transferred into an autosampler vial and 10 L was injected into the LC–MS/MS system for analysis. 2.5. Bioanalytical method validation The method was validated for specificity, carry-over effect, linearity, LLOQ, accuracy and precision, recovery and matrix effect, stability during the sample storage and processing procedures according to guidelines set by the United States Food and Drug Administration (FDA) for bioanalytical method validation [17]. 2.6. Application A pilot pharmacokinetic study was performed in two healthy Chinese volunteers (1 male, 1 female) approved by the Ethical Committee of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. Both the volunteers were recruited after a thorough medical, biochemical and physical examination and were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. A dose of 6 g HTLPI was dissolved in 250 mL of sodium chloride injection and intravenously infused to volunteers at a constant rate over 4 h. Blood samples (4 mL) were collected into heparin tubes at 0, 0.5, 1, 1.5, 2, 3, 4, 4.08, 4.25, 4.5, 5, 5.5, 6, 7, 8, 10, 12, 14, 16 and 24 h after administration and centrifuged immediately. The plasma fractions were stored at −20 ◦ C until analysis. 3. Results and discussion 3.1. Method development 3.1.1. Mass spectrometry In order to optimize the mass spectrometric conditions, the standard solutions of AD, PF and the IS were respectively infused
into the mass spectrometer by a syringe pump. According to the Q1 full-scan mass spectra, the protonated molecule ion [M+H]+ was invisible for AD and also in low abundance for PF under the positive ESI mode. Sodiated adduct ions [M+Na]+ were the base peak (the highest abundance) for both analytes, and hence selected as the precursor ions initially. But as the collision energy increased, the signal intensity of [M+Na]+ decreased significantly and no product ions were observed, which meant that [M+Na]+ ions could not be used as the precursor ions for the MRM detection of AD and PF. Afterwards, we found that ammonium adduct ions [M+NH4 ]+ of the analytes and IS also produced high abundance by adding ammonium acetate into the infusion solution. Moreover, the precursor [M+NH4 ]+ ions were fragile so that they could be fragmented to generate abundant product ions with high yield [18]. Therefore, the [M+NH4 ]+ ions at m/z 475.2, 498.2 and 406.2 were selected as the precursor ions for AD, PF and the IS, respectively. On the basis of that, to generate a high yield of stable and dominating product ion from [M+NH4 ]+ , mild collision-induced dissociation (CID) conditions such as low collision energy (CE) and low CAD were applied. The final MRM transitions used for quantification were m/z 475.2 → 163.1 for AD, m/z 498.2 → 179.1 for PF and m/z 406.2 → 227.1 for IS (see Fig. 1). 3.1.2. Chromatography Chromatographic conditions, especially the composition of the mobile phase were optimized to enhance the sensitivity and achieve symmetric peak shape, as well as shorten the run time. In this study, acetonitrile, rather than methanol, was chosen as the organic portion of the mobile phase because it provided higher mass spectrometric response and lower background noise. To promote the formation of the precursor ions [M+NH4 ]+ and achieve stable and sensitive mass spectrometric response, ammonium acetate was added into the aqueous portion of the mobile phase.
Fig. 2. Representative MRM chromatograms of AD (I), PF (II) and IS (III): (A) blank plasma sample, (B) blank plasma sample spiked with the analytes at LLOQ and IS, and (C) a plasma sample from a healthy volunteer at 4 h after a single intravenous infusion administration of 6 g HTLPI.
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Table 2 Precision and accuracy data for the analysis of AD and PF in human plasma (n = 5). Concentration levels (mean ± SD, ng/mL)
RSD (%)
Added
Measured
Intra-day
Inter-day
Accuracy
AD
0.6240 1.560 104.0 1560
0.6319 1.587 103.5 1569
± ± ± ±
0.0626 0.0959 1.8 50
10.0 6.4 1.9 3.1
9.3 2.9 0.6 3.7
1.3 1.7 −0.5 0.5
PF
0.6060 1.515 101.0 1515
0.6161 1.549 101.1 1528
± ± ± ±
0.0488 0.069 2.1 51
7.4 4.6 2.1 2.9
9.4 3.4 2.0 5.0
1.7 2.3 0.1 0.9
Analytes
RE (%)
RSD: relative standard deviation; RE: relative error.
Different concentration levels of ammonium acetate (0, 2, 5 and 10 mM) were optimized by evaluating the signal intensity of the QC samples, which were injected into LC–MS/MS for 5 times at three concentration levels (1.5, 100 and 1500 ng/mL) of both analytes. It was found that the addition of 5 mM ammonium acetate in the aqueous portion of the mobile phase could produce best sensitivity and reproducibility. The peak shape and retention behavior of the analytes were significantly improved when 0.05% formic acid was added in the mobile phase. Finally, the mobile phase was optimized as acetonitrile: 5 mM ammonium acetate solution with 0.05% formic acid (20:80, v/v) using an isocratic elution. 3.1.3. Sample preparation Considering the high polarity of AD and PF, protein precipitation (PPT) was applied to prepare the plasma sample. In this study, methanol was adopted as the precipitant because it yielded consistent and high recovery rates without matrix interference. It was necessary to add water to the supernatant of the plasma sample after protein precipitation to avoid peak tailing. The volume ratio of supernatant to water of 2:1, 1:1 and 1:2 was optimized to acquire good performance in the aspects of peak shape and sensitivity. The ratio of 1:1 was finally selected as the optimal one. 3.2. Bioanalytical method validation As shown in Fig. 2, there is no significant endogenous interference observed at the retention times of AD, PF and the IS which were 2.14, 3.18 and 2.28 min, respectively.
No carry-over peaks were observed at the retention times of AD, PF and the IS in the chromatogram of blank plasma injected after the ULOQ sample. This indicated the former sample analysis would not affect the accuracy of the latter sample, regardless of the concentration of the former sample. The calibration curves were linear over the concentration range of 0.6–2000 ng/mL in plasma with correlation coefficients >0.9972 for AD and >0.9981 for PF. The typical equations of the calibration curves were: f = 0.005766C + 0.0004963 for AD, f = 0.007594C + 0.001307 for PF, where f represents the peak area ratio of the analytes to the IS and C represents the concentration of the analytes. The LLOQs of both analytes were 0.6 ng/mL, which were the lowest reported so far [7–16]. Table 2 summarizes the intra- and inter-day precision and accuracy for AD and PF evaluated by assaying the LLOQ and QC samples. The results demonstrated that the values on both the occasions (intra- and inter-day) were all within the acceptable variability limits and the method was accurate and precise. The recoveries of the analytes at three concentration levels (1.5, 100 and 1500 ng/mL) were 99.2 ± 6.9%, 100.2 ± 2.7% and 100.5 ± 0.9% for AD and 95.2 ± 7.6%, 100.9 ± 3.9% and 99.9 ± 1.9% for PF, respectively. Mean recovery for the IS (1 g/mL) was 99.9 ± 2.4%. The matrix effect of six different batches of human plasma at three concentration levels (1.5, 100 and 1500 ng/mL) were 101.9 ± 6.4%, 100.3 ± 2.8% and 100.2 ± 1.5% for AD and 98.9 ± 7.7%, 102.0 ± 3.7% and 102.2 ± 1.3% for PF, respectively. The matrix effect of IS (1 g/mL) was 100.3 ± 2.8%. No significant matrix effect was observed for AD, PF and IS, indicating that no co-eluting substance influenced the ionization of the analytes.
Table 3 Stability of AD and PF in human plasma under various storage conditions (n = 3). Storage conditions
Ambient temperature, 9 h
Analytes
AD PF
Freezing for 7 weeks (−20 ◦ C)
AD PF
Three freeze/thaw cycles
AD PF
Autosampler for 10 h (6 ◦ C)
AD PF
RSD: relative standard deviation; RE: relative error; n: number of replicates.
Concentration levels (mean ± SD, ng/mL)
RSD (%)
RE (%)
Added
Measured
1.560 1560 1.515 1515
1.693 1602 1.629 1589
± ± ± ±
0.047 27 0.086 13
2.8 1.7 5.3 0.8
8.5 2.7 7.5 4.9
1.560 1560 1.515 1515 1.560 1560 1.515 1515
1.449 1425 1.582 1466 1.633 1616 1.628 1560
± ± ± ± ± ± ± ±
0.046 7 0.088 10 0.056 18 0.088 32
3.2 0.5 5.6 0.7 3.4 1.1 5.4 2.0
−7.1 −8.7 4.4 −3.2 4.7 3.6 7.4 3.0
1.560 1560 1.515 1515
1.561 1615 1.558 1619
± ± ± ±
0.123 5 0.071 24
7.9 0.3 4.5 1.5
0.1 3.5 2.8 6.9
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X. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 160–164
Fig. 3. Mean plasma concentration–time profiles of AD and PF in human plasma after a single intravenous infusion administration of 6 g HTLPI (n = 2).
The stability results summarized in Table 3 show that AD and PF were stable in human plasma and processed samples under different storage conditions. 3.3. Application This validated LC–MS/MS method was successfully applied to a pilot pharmacokinetic study of AD and PF in two healthy Chinese volunteers after a single intravenous infusion administration of 6 g HTLPI. Mean plasma concentration-time profiles of AD and PF are presented in Fig. 3. The maximum plasma concentration (Cmax ) for AD and PF were 692.4 ± 173.4 and 919.1 ± 253.0 ng/mL, respectively. The area under the curve (AUC0–24 ) for AD and PF were 3294 ± 522 and 4023 ± 884 ng h/mL while the elimination half-life (t1/2 ) were 2.4 ± 0.1 and 2.4 ± 0.1 h, respectively. 4. Conclusion For the first time, a rapid, simple and highly sensitive LC–MS/MS method was developed and successfully applied to the pharmacokinetic study of AD and PF in healthy volunteers after intravenous infusion administration of HTLPI. The validated method was proven to be precise, accurate and reproducible for the analysis of AD and PF and the results of the pharmacokinetic study would be helpful to provide some references to clinical application of this medicine. Acknowledgments This study was supported by the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (No. 2011ZX09101-002-01) and the National Natural Science Foundation of China (No. 81273482). References [1] R. Tanaka, M. Yamazaki, K. Hasada, A. Nagatsu, Application of quantitative 1 H NMR method to determination of paeoniflorin in paeoniae radix, J. Nat. Med. 67 (2013) 657–661.
[2] L. Liu, J.A. Duan, Y. Tang, J. Guo, N. Yang, H. Ma, X. Shi, Taoren–Honghua herb pair and its main components promoting blood circulation through influencing on hemorheology, plasma coagulation and platelet aggregation, J. Ethnopharmacol. 139 (2012) 381–387. [3] S.L. Li, J.Z. Song, F.F. Choi, C.F. Qiao, Y. Zhou, Q.B. Han, H.X. Xu, Chemical profiling of radix paeoniae evaluated by ultraliquid chromatography/photo-diode-array/quadrupole performance time-of-flight mass spectrometry, J. Pharm. Biomed. Anal. 49 (2009) 253–266. [4] N.C. Luo, W. Ding, J. Wu, D.W. Qian, Z.H. Li, Y.F. Qian, J.M. Guo, J.A. Duan, UPLC-QTOF/MS coupled with multivariate statistical analysis as a powerful technique for rapidly exploring potential chemical markers to differentiate between radix paeoniae alba and radix paeoniae rubra, Nat. Prod. Commun. 8 (2013) 487–491. [5] R.B. Guo, G.F. Wang, A.P. Zhao, J. Gu, X.L. Sun, G. Hu, Paeoniflorin protects against ischemia-induced brain damages in rats via inhibiting MAPKs/NF-B-mediated inflammatory responses, PLoS One 7 (2012) e49701. [6] G. Chen, L. Zhang, P. Yang, Rapid determination of paeoniflorin and three sugars in radix paeoniae alba by capillary electrophoresis, Anal. Sci. 21 (2005) 247–251. [7] C.H. Wang, R. Wang, X.M. Cheng, Y.Q. He, Z.T. Wang, C. Wu, J. Cao, Comparative pharmacokinetic study of paeoniflorin after oral administration of decoction of radix paeoniae rubra and radix paeoniae alba in rats, J. Ethnopharmacol. 117 (2008) 467–472. [8] X.G. Yang, B. Peng, G.H. Zhang, L.L. Wei, S.F. Nie, W.S. Pan, Studies of the pharmacokinetics of paeoniflorin in two Jing-Zhi-Guan-Xin formulations after oral administration to beagle dogs, J. Pharm. Biomed. Anal. 41 (2006) 320–324. [9] A.G. Rauws, L.G. Gramberg, M. Olling, Determination of amygdalin and its major metabolite prunasin in plasma and urine by high pressure liquid chromatography, Pharm. Weekbl. Sci. 4 (1982) 172–175. [10] C. Feng, M. Liu, X. Shi, W. Yang, D. Kong, K. Duan, Q. Wang, Pharmacokinetic properties of paeoniflorin, albiflorin and oxypaeoniflorin after oral gavage of extracts of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats, J. Ethnopharmacol. 130 (2010) 407–413. [11] X. Zhao, W. Zang, X. Zhao, S. Wang, X. Zheng, J. Zheng, HPLC with column switching coupled to APCI–MS for pharmacokinetic study of amygdalin in rabbit plasma, Chromatographia 65 (2006) 149–153. [12] X.Y. Wang, X.H. Ma, W. Li, Y. Chu, J.H. Guo, S.M. Li, J.M. Wang, H.C. Zhang, S.P. Zhou, Y.H. Zhu, Simultaneous determination of five phenolic components and paeoniflorin in rat plasma by liquid chromatography–tandem mass spectrometry and pharmacokinetic study after oral administration of Cerebralcare granule, J. Pharm. Biomed. Anal. 86C (2013) 82–91. [13] X.B. Li, C.H. Liu, R. Zhang, X.T. Huang, Y.Y. Li, L. Han, M.L. Xu, S.Q. Mi, N.S. Wang, Determination and pharmacokinetics of amygdalin in rats by LC–MS-MS, J. Chromatogr. Sci. (2013) (Epub ahead of print). [14] S.M. Xia, R. Shen, X.Y. Sun, L.L. Shen, Y.M. Yang, Y. Ke, Y. Wang, D.Y. Chen, X.M. Han, Development and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the determination of paeoniflorin in rat brain and its application to pharmacokinetic study, J. Chromatogr. B 857 (2007) 32–39. [15] B.Y. Ge, H.X. Chen, F.M. Han, Y. Chen, Identification of amygdalin and its major metabolites in rat urine by LC–MS/MS, J. Chromatogr. B 857 (2007) 281–286. [16] J. Wen, Y. Qiao, J. Yang, X. Liu, Y. Song, Z. Liu, F. Li, UPLC–MS/MS determination of paeoniflorin, naringin, naringenin and glycyrrhetinic acid in rat plasma and its application to a pharmacokinetic study after oral administration of Si-Ni-San decoction, J. Pharm. Biomed. Anal. 66 (2012) 271–277. [17] US DHHS, FDA, CDER, Guidance for Industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CV), 2001, Available at: http://www/fda.gov/cder/guidance/index.htm [18] F. Guan, A. Ishii, H. Seno, K. Watanabe-Suzuki, T. Kumazawa, O. Suzuki, Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS, Anal. Chem. 71 (1999) 4034–4043.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A sensitive LC–MS/MS method for simultaneous determination of amygdalin and paeoniflorin in human plasma and its application Xiaobing Li a,b,c , Fuguo Shi a,b , Pan Gu a,b , Lingye Liu a,b , Hua He b,c,∗∗ , Li Ding a,b,∗ a
Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China c Department of Analytical Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 24 January 2014 Keywords: Amygdalin Paeoniflorin Huoxue-Tongluo lyophilized powder for injection LC–MS/MS Pharmacokinetics
a b s t r a c t A simple and sensitive HPLC–MS/MS method was developed and fully validated for the simultaneous determination of amygdalin (AD) and paeoniflorin (PF) in human plasma. For both analytes, the method exhibited high sensitivity (LLOQs of 0.6 ng/mL) by selecting the ammonium adduct ions ([M+NH4 ]+ ) as the precursor ions and good linearity over the concentration range of 0.6–2000 ng/mL with the correlation coefficients > 0.9972. The intra- and inter-day precision was lower than 10% in relation to relative standard deviation, while accuracy was within ±2.3% in terms of relative error for both analytes. The developed method was successfully applied to a pilot pharmacokinetic study of AD and PF in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huoxue-Tongluo lyophilized powder for injection (HTLPI), a simplified formulated Chinese medicine derived from the well-known traditional Chinese medicine prescription Tao-HongSi-Wu-Tang, is mainly made from the extracts of Persicae Semen and Paeoniae Radix Rubra and used for treating blood-stasis obstruction syndrome in the acute stage of cerebral ischemic stroke. HTLPI has passed the Phase I clinical trial (Chinese CFDA: 2004L01097) and its safety has been verified. The Phase II and III clinical trials are carrying out. Amygdalin (AD) and paeoniflorin (PF), the phytochemical markers for the quality control of Persicae Semen and Paeoniae Radix Rubra (Pharmacopeia of PR China, 2010), are regarded as the principal constituents responsible for
the pharmacological activities [1–6] and selected as the indicators in the pharmacokinetic study of HTLPI. Various chromatographic methods have been published for the determination of AD or PF in animal biological fluids using HPLCUV [7–9], LC–MS [10,11], LC–MS/MS [12–15], UPLC–MS/MS [16]. However, to the best of our knowledge, there is presently no report on the determination of AD or PF in human plasma for the clinical pharmacokinetic study. In the present investigation, a simple and highly sensitive (LLOQs of 0.6 ng/mL) LC–MS/MS method was developed and fully validated for simultaneous determination of AD and PF in human plasma for the first time. This method was successfully applied to a pilot clinical pharmacokinetic study of AD and PF after intravenous infusion administration of HTLPI to healthy Chinese volunteers. 2. Experimental
∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 025 8327 1485; fax: +86 025 8327 1485. ∗∗ Corresponding author at: Department of Analytical Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 025 8327 1505; fax: +86 025 8327 1505. E-mail addresses: [email protected] (H. He), [email protected], [email protected] (L. Ding). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.020
2.1. Chemicals and reagents Huoxue-Tongluo lyophilized powders for injection (6 g, containing 25.3 mg of amygdalin and 35.8 mg of paeoniflorin) were supplied by Jiangsu Kanion Pharmaceutical Co., Ltd. (Lianyungang, China). Amygdalin (AD, purity 93.6%), paeoniflorin (PF, purity 96.5%) and geniposide (IS, purity 99.7%) were purchased from
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National Institutes for Food and Drug Control (Beijing, China). Methanol and acetonitrile were HPLC grade and purchased from Merck KGaA (Darmstadt, Germany). Ammonium acetate and formic acid (analytical grade) were purchased from Nanjing Chemical Reagents Co., Ltd. (Nanjing, China). Water was prepared with double distillation. 2.2. LC–MS/MS instruments and conditions The liquid chromatography was performed on an Agilent 1260 Series liquid chromatography (Agilent Technologies, Palo Alto, CA, USA), which comprised an Agilent 1260 binary pump (model G1312B), a vacuum degasser (model G4225A), an autosampler (model G1367E) and an Agilent 1290 temperature controlled column compartment (model G1330B). Chromatographic separation was carried out on a 38 ◦ C Hedera ODS-2 analytical column (2.1 mm × 150 mm, 5 m; Hanbon Science and Technology, Huai’an, China) with a security Guard-C18 column (4 mm × 2.0 mm, 5 m; Phenomenex, Torrance, CA, USA). The mobile phase consisted of acetonitrile and 5 mM ammonium acetate buffer solution containing 0.05% formic acid (20:80, v/v) at a flow rate of 300 L/min. Autosampler temperature was maintained at 6 ◦ C and the injection volume was 10 L. A 10-port switching valve (Valco, Houston, TX, USA) was used to direct flow eluate to a waste container in the first 1.5 min of the chromatographic run and afterwards to the mass spectrometer. The total run time was 4.5 min. Mass spectrometric detection was performed on an API 4000 tandem mass spectrometer (Applied Biosystems, Toronto, Canada) equipped with a Turbo-V® ionspray source operation in the positive ESI mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 475.2 → 163.1 for AD, m/z 498.2 → 179.1 for PF and m/z 406.2 → 227.1 for IS, respectively (see Fig. 1). The ion spray temperature and ion spray voltage were maintained at 400 ◦ C and 5500 V, with nebulizer gas (GS1) and heater gas (GS2) set at 70 and 70 psi, respectively. The curtain gas (CUR) was kept at 30 psi and the collision gas (CAD) was 6 psi. The specific parameters for each analyte are displayed in Table 1. The system control and data analysis were performed by AB Sciex Analyst software (version 1.5.2). 2.3. Preparation of calibration standards and quality control samples The mixture standard stock solution of AD and PF was prepared in methanol at the concentration of 1 mg/mL. Standard working solutions ranged from 0.006 to 20 g/mL were prepared by diluting the stock solution with methanol for both analytes. Calibration standards were prepared using blank human plasma spiked with 20 L of mixture working solutions, to yield the concentrations of 0.6, 2, 6, 20, 60, 200, 600, 1200 and 2000 ng/mL for both analytes, respectively. QC samples were prepared in the same way as the calibration samples at three concentration levels: 1.5, 100 and 1500 ng/mL for both analytes. The stock solution (1 mg/mL) of the IS was prepared in methanol and was further diluted with methanol to 1 g/mL. All the solutions were kept at −20 ◦ C and brought to room temperature before use. 2.4. Sample preparation All samples were stored in a freezer at −20 ◦ C and allowed to thaw at room temperature by vortex before processing. A 200 L of plasma sample was mixed with 20 L IS solution (1 g/mL). The mixture was deproteinized with 600 L of methanol, vortexed for 5 min, and then centrifuged at 15,600 rpm for 10 min. An aliquot of 0.2 mL supernatant was diluted by adding 0.2 mL of water. After
Fig. 1. Positive product ion mass spectra of [M+NH4 ]+ of AD (A), PF (B) and IS (C) and their proposed fragmentation patterns.
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Table 1 Optimized mass parameters for AD, PF and the IS. Analytes
Precursor ion (m/z)
Product ion (m/z)
Dwell time (ms)
DP (V)
EP (V)
CE (V)
CXP (V)
AD PF IS
475.2 498.2 406.1
163.1 179.1 227.1
450 450 450
55 51 50
11 8 7
23 25 13
10 11 17
DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: collision cell exit potential.
being vortex-mixed for 5 s, the mixture was transferred into an autosampler vial and 10 L was injected into the LC–MS/MS system for analysis. 2.5. Bioanalytical method validation The method was validated for specificity, carry-over effect, linearity, LLOQ, accuracy and precision, recovery and matrix effect, stability during the sample storage and processing procedures according to guidelines set by the United States Food and Drug Administration (FDA) for bioanalytical method validation [17]. 2.6. Application A pilot pharmacokinetic study was performed in two healthy Chinese volunteers (1 male, 1 female) approved by the Ethical Committee of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. Both the volunteers were recruited after a thorough medical, biochemical and physical examination and were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. A dose of 6 g HTLPI was dissolved in 250 mL of sodium chloride injection and intravenously infused to volunteers at a constant rate over 4 h. Blood samples (4 mL) were collected into heparin tubes at 0, 0.5, 1, 1.5, 2, 3, 4, 4.08, 4.25, 4.5, 5, 5.5, 6, 7, 8, 10, 12, 14, 16 and 24 h after administration and centrifuged immediately. The plasma fractions were stored at −20 ◦ C until analysis. 3. Results and discussion 3.1. Method development 3.1.1. Mass spectrometry In order to optimize the mass spectrometric conditions, the standard solutions of AD, PF and the IS were respectively infused
into the mass spectrometer by a syringe pump. According to the Q1 full-scan mass spectra, the protonated molecule ion [M+H]+ was invisible for AD and also in low abundance for PF under the positive ESI mode. Sodiated adduct ions [M+Na]+ were the base peak (the highest abundance) for both analytes, and hence selected as the precursor ions initially. But as the collision energy increased, the signal intensity of [M+Na]+ decreased significantly and no product ions were observed, which meant that [M+Na]+ ions could not be used as the precursor ions for the MRM detection of AD and PF. Afterwards, we found that ammonium adduct ions [M+NH4 ]+ of the analytes and IS also produced high abundance by adding ammonium acetate into the infusion solution. Moreover, the precursor [M+NH4 ]+ ions were fragile so that they could be fragmented to generate abundant product ions with high yield [18]. Therefore, the [M+NH4 ]+ ions at m/z 475.2, 498.2 and 406.2 were selected as the precursor ions for AD, PF and the IS, respectively. On the basis of that, to generate a high yield of stable and dominating product ion from [M+NH4 ]+ , mild collision-induced dissociation (CID) conditions such as low collision energy (CE) and low CAD were applied. The final MRM transitions used for quantification were m/z 475.2 → 163.1 for AD, m/z 498.2 → 179.1 for PF and m/z 406.2 → 227.1 for IS (see Fig. 1). 3.1.2. Chromatography Chromatographic conditions, especially the composition of the mobile phase were optimized to enhance the sensitivity and achieve symmetric peak shape, as well as shorten the run time. In this study, acetonitrile, rather than methanol, was chosen as the organic portion of the mobile phase because it provided higher mass spectrometric response and lower background noise. To promote the formation of the precursor ions [M+NH4 ]+ and achieve stable and sensitive mass spectrometric response, ammonium acetate was added into the aqueous portion of the mobile phase.
Fig. 2. Representative MRM chromatograms of AD (I), PF (II) and IS (III): (A) blank plasma sample, (B) blank plasma sample spiked with the analytes at LLOQ and IS, and (C) a plasma sample from a healthy volunteer at 4 h after a single intravenous infusion administration of 6 g HTLPI.
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Table 2 Precision and accuracy data for the analysis of AD and PF in human plasma (n = 5). Concentration levels (mean ± SD, ng/mL)
RSD (%)
Added
Measured
Intra-day
Inter-day
Accuracy
AD
0.6240 1.560 104.0 1560
0.6319 1.587 103.5 1569
± ± ± ±
0.0626 0.0959 1.8 50
10.0 6.4 1.9 3.1
9.3 2.9 0.6 3.7
1.3 1.7 −0.5 0.5
PF
0.6060 1.515 101.0 1515
0.6161 1.549 101.1 1528
± ± ± ±
0.0488 0.069 2.1 51
7.4 4.6 2.1 2.9
9.4 3.4 2.0 5.0
1.7 2.3 0.1 0.9
Analytes
RE (%)
RSD: relative standard deviation; RE: relative error.
Different concentration levels of ammonium acetate (0, 2, 5 and 10 mM) were optimized by evaluating the signal intensity of the QC samples, which were injected into LC–MS/MS for 5 times at three concentration levels (1.5, 100 and 1500 ng/mL) of both analytes. It was found that the addition of 5 mM ammonium acetate in the aqueous portion of the mobile phase could produce best sensitivity and reproducibility. The peak shape and retention behavior of the analytes were significantly improved when 0.05% formic acid was added in the mobile phase. Finally, the mobile phase was optimized as acetonitrile: 5 mM ammonium acetate solution with 0.05% formic acid (20:80, v/v) using an isocratic elution. 3.1.3. Sample preparation Considering the high polarity of AD and PF, protein precipitation (PPT) was applied to prepare the plasma sample. In this study, methanol was adopted as the precipitant because it yielded consistent and high recovery rates without matrix interference. It was necessary to add water to the supernatant of the plasma sample after protein precipitation to avoid peak tailing. The volume ratio of supernatant to water of 2:1, 1:1 and 1:2 was optimized to acquire good performance in the aspects of peak shape and sensitivity. The ratio of 1:1 was finally selected as the optimal one. 3.2. Bioanalytical method validation As shown in Fig. 2, there is no significant endogenous interference observed at the retention times of AD, PF and the IS which were 2.14, 3.18 and 2.28 min, respectively.
No carry-over peaks were observed at the retention times of AD, PF and the IS in the chromatogram of blank plasma injected after the ULOQ sample. This indicated the former sample analysis would not affect the accuracy of the latter sample, regardless of the concentration of the former sample. The calibration curves were linear over the concentration range of 0.6–2000 ng/mL in plasma with correlation coefficients >0.9972 for AD and >0.9981 for PF. The typical equations of the calibration curves were: f = 0.005766C + 0.0004963 for AD, f = 0.007594C + 0.001307 for PF, where f represents the peak area ratio of the analytes to the IS and C represents the concentration of the analytes. The LLOQs of both analytes were 0.6 ng/mL, which were the lowest reported so far [7–16]. Table 2 summarizes the intra- and inter-day precision and accuracy for AD and PF evaluated by assaying the LLOQ and QC samples. The results demonstrated that the values on both the occasions (intra- and inter-day) were all within the acceptable variability limits and the method was accurate and precise. The recoveries of the analytes at three concentration levels (1.5, 100 and 1500 ng/mL) were 99.2 ± 6.9%, 100.2 ± 2.7% and 100.5 ± 0.9% for AD and 95.2 ± 7.6%, 100.9 ± 3.9% and 99.9 ± 1.9% for PF, respectively. Mean recovery for the IS (1 g/mL) was 99.9 ± 2.4%. The matrix effect of six different batches of human plasma at three concentration levels (1.5, 100 and 1500 ng/mL) were 101.9 ± 6.4%, 100.3 ± 2.8% and 100.2 ± 1.5% for AD and 98.9 ± 7.7%, 102.0 ± 3.7% and 102.2 ± 1.3% for PF, respectively. The matrix effect of IS (1 g/mL) was 100.3 ± 2.8%. No significant matrix effect was observed for AD, PF and IS, indicating that no co-eluting substance influenced the ionization of the analytes.
Table 3 Stability of AD and PF in human plasma under various storage conditions (n = 3). Storage conditions
Ambient temperature, 9 h
Analytes
AD PF
Freezing for 7 weeks (−20 ◦ C)
AD PF
Three freeze/thaw cycles
AD PF
Autosampler for 10 h (6 ◦ C)
AD PF
RSD: relative standard deviation; RE: relative error; n: number of replicates.
Concentration levels (mean ± SD, ng/mL)
RSD (%)
RE (%)
Added
Measured
1.560 1560 1.515 1515
1.693 1602 1.629 1589
± ± ± ±
0.047 27 0.086 13
2.8 1.7 5.3 0.8
8.5 2.7 7.5 4.9
1.560 1560 1.515 1515 1.560 1560 1.515 1515
1.449 1425 1.582 1466 1.633 1616 1.628 1560
± ± ± ± ± ± ± ±
0.046 7 0.088 10 0.056 18 0.088 32
3.2 0.5 5.6 0.7 3.4 1.1 5.4 2.0
−7.1 −8.7 4.4 −3.2 4.7 3.6 7.4 3.0
1.560 1560 1.515 1515
1.561 1615 1.558 1619
± ± ± ±
0.123 5 0.071 24
7.9 0.3 4.5 1.5
0.1 3.5 2.8 6.9
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Fig. 3. Mean plasma concentration–time profiles of AD and PF in human plasma after a single intravenous infusion administration of 6 g HTLPI (n = 2).
The stability results summarized in Table 3 show that AD and PF were stable in human plasma and processed samples under different storage conditions. 3.3. Application This validated LC–MS/MS method was successfully applied to a pilot pharmacokinetic study of AD and PF in two healthy Chinese volunteers after a single intravenous infusion administration of 6 g HTLPI. Mean plasma concentration-time profiles of AD and PF are presented in Fig. 3. The maximum plasma concentration (Cmax ) for AD and PF were 692.4 ± 173.4 and 919.1 ± 253.0 ng/mL, respectively. The area under the curve (AUC0–24 ) for AD and PF were 3294 ± 522 and 4023 ± 884 ng h/mL while the elimination half-life (t1/2 ) were 2.4 ± 0.1 and 2.4 ± 0.1 h, respectively. 4. Conclusion For the first time, a rapid, simple and highly sensitive LC–MS/MS method was developed and successfully applied to the pharmacokinetic study of AD and PF in healthy volunteers after intravenous infusion administration of HTLPI. The validated method was proven to be precise, accurate and reproducible for the analysis of AD and PF and the results of the pharmacokinetic study would be helpful to provide some references to clinical application of this medicine. Acknowledgments This study was supported by the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (No. 2011ZX09101-002-01) and the National Natural Science Foundation of China (No. 81273482). References [1] R. Tanaka, M. Yamazaki, K. Hasada, A. Nagatsu, Application of quantitative 1 H NMR method to determination of paeoniflorin in paeoniae radix, J. Nat. Med. 67 (2013) 657–661.
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