Development of a nsitive and rapid UPLC-MS/MS method for the determination of koumine in rat plasma:application to a pharmacokinetic study
Jian-Zhong Chen a *,Yu Li a ,Jian-Ping Xiao b ,Shui-Sheng Wu a and Heng-Wen Song a
ABSTRACT:A rapid,lective and nsitive method using UPLC-MS/MS was first developed and validated for quantitative analysis of koumine in rat plasma.A one-step protein precipitation with methanol was employed as a sample preparation technique.Plasma samples were parated on an Acquity UPLC BEH C 18column (50Â2.1mm,i.d.1.7m m)with a gradient mobile pha consisting of methanol with 0.1%(v/v)formic acid and water containing 0.1%(v/v)formic acid at a flow rate of 0.3mL/min.Detection and quanti fication were performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization.Good linearity (r >0.9997)was achieved using weighted (1/x 2)least squares linear regression over a concentration range of 0.025–15m g/mL with a lower limit of quanti fication of 0.025m g/mL for koumine.The intra-and inter-precisions (relative standard deviation)of the assay at all three quality control samples were 5.6–14.1%with an accuracy (relative error)of 5.0–14.0%,which meets the requirements of the US Food and Drug Administration guidance.This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous do of 20mg/kg koumine.Copyright ©2012Joh
n Wiley &Sons,Ltd.Keywords:koumine;determination;pharmacokinetics;UPLC-MS/MS
Introduction
Gelmium elegans Benth.(Loganiaceae)is a well-known toxic plant widely distributed in China,South East Asia and Indo-Malaysia (Dutt et al .,2010).Many cas of poisoning by inadver-tent ingestion of this plant have been reported in China.In vere poisoning,it even can cau death (Xie et al .,2006).Although it is toxic,it has commonly been ud in Chine folk medicine for treatment of malignant tumors,skin dias (e.g.psoriasis),rheumatic arthritis and pain (Xu et al .,1991;Editorial Committee of Chine Materia Medica,the Administra-tion Bureau of Traditional Chine Medicine,2000;Lu et al .,1990).So far,a ries of indole/oxindole alkaloids with diver chemical structures,biological activities and toxicity,such as koumine (Fig.1),gelmine,gelnicine and gelvirine,have been isolated from G .elegans Benth.(Yamada et al .,2011;Xu et al .,2012b).Koumine is one of the major components among the alkaloids.Recent studies have found that koumine posss a variety of interesting pharmacological effects,including anti-tumor (Cai et al .,2009;Chi et al .,2007;Wang et al .,2009),anti-stress (Cai et al .,2007),anti-psoriasis and analgesia (Xu et al .,2012a).Compared with other alkaloids from G .elegans Benth.,koumine showed relatively mild toxicity,indicating that it might be a promising lead drug.To better understand the pharmacologic
al activities of the component,an investigation of drug metabolism and pharmacokinetics is required.However,there has not been any report on the determination of koumine in plasma or investigation of its pharmacokinetics.
This paper describes a simple,rapid and speci fic ultra-performance liquid chromatography –mass spectrometry (UPLC-MS/MS)method
for the quanti fication of koumine in rat plasma.The method referred in this paper could meet high-throughput analysis needs,and it facilitated pharmacokinetic studies of koumine after intrave-nous administration.
Experimental
Chemicals and reagents
Reference standards of koumine (99.0%purity)and gelmine (internal standard,IS,99.0%purity)were purchad from Shanghai R&D Centre for Standardization of Chine Medicines (Shanghai,China).Methanol of HPLC grade was purchad from Fisher Scienti fic (Fair Lawn,NJ,USA).Formic acid of HPLC grade was obtained from Tedia (Fair field,OH,USA).HPLC-grad
e water (>18m Ω)was obtained from a Milli-Q water puri fication system (Bedford,MA,USA).Other chemicals ud were of analytical grade.Blank plasma samples were obtained from six different Sprague –Dawley rats,which were provided by the Experimental Animal Centre,Fujian University of Traditional Chine Medicine,China.
*Correspondence to:Jian-Zhong Chen,School of Pharmacy,Fujian University of Traditional Chine Medicine,Fuzhou 350108,China.E-mail:
a
School of Pharmacy,Fujian University of Traditional Chine Medicine,Fuzhou,350108,China
b
Department of Pharmacy,The Second People ’s Hospital of Fujian Province,Fuzhou,350003,China
Abbreviations ud:ESI,electrospray ionization;MRM,multiple reaction monitoring;PPT,protein precipitation;UPLC,ultra performance liquid chromatography.
Biomed.Chromatogr.2013;27:736–740Copyright ©2012John Wiley &Sons,Ltd.
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Rearch article
Received:1September 2012,
Revid:5November 2012,
eventful
Accepted:5November 2012
Published online in Wiley Online Library:7December 2012
()DOI 10.1002/bmc.2852
西班牙语在线学习736
Apparatus and operation conditions
Liquid chromatography.The paration was performed on an Acquity UPLC with Quattro micro Mass Spectrometer system (Waters Corp.,Milford,MA,USA)using an Acquity UPLC BEH C 18column (50Â2.1mm,i.d.1.7m m,Waters,USA)maintained at 40 C.The mobile pha was compod of methanol with 0.1%(v/v)formic acid (A)and aqueous 0.1%(v/v)formic acid (B)at a flow rate of 0.3mL/
min.A gradient program was ud as follows (time,min/%A):0/20,1/35,3.5/35,4/20,6/20.The injection volume was 5m L.After each injection,the sample manager underwent a needle wash process,including strong wash (methanol –water,80:20)and weak wash (methanol –water,20:80).
Mass spectrometric conditions.A triple-quadrupole Quattro micro mass spectrometer (Waters Corp.,Milford,MA,USA)equipped with an electrospray ionization (ESI)interface was ud for analytical detection.ESI/MS/MS was operated in positive mode under the following operating parameters:capillary voltage,2.8kV;cone voltage,35V;source tempera-ture,110 C;desolvation temperature,350 C;desolvation gas (nitrogen),550L/h;cone gas (nitrogen),50L/h.Quantitative analysis was per-formed using multiple-reaction monitoring (MRM)of the transitions of m/z 307.1!69.76for koumine and m/z 323.1!69.76for IS,with a scan time of 0.20s per transition.All data were acquired in centroid mode and procesd using Masslynx 4.1software (Waters Corp.,Milford,MA,USA).
Stock and working solutions
Standard stock solution of koumine (1.1mg/mL)was prepared by dissol-ving the correct amount of accurately weighed reference substance in methanol.The standard stock solutions were then rially
diluted with methanol to provide standard working solutions in the concentration range of 0.050–30m g/mL for koumine.An IS working solution (1.0m g/mL)was also prepared by dissolving the correct amount of gelmine in methanol.All of the solutions were stored at 4 C and brought to room temperature before u.
Calibration standards and quality control samples
Calibration standards were prepared daily by spiking appropriate standard working solutions (50m L of koumine)into 100m L of blank plasma.Effective concentrations in plasma samples were 0.025,0.050,0.25,0.50,1.0,2.5,5.0,10and 15m g/mL for koumine.The low,medium,and high quality control (QC)samples were prepared in a similar way to the calibration standards to give three final concentrations of 0.050,0.50and 10m g/mL for koumine.The spiked plasma samples (calibration standards and quality control samples)were then treated following the protein precipitation (PPT)procedure as described below.
Plasma sample preparation
To an aliquot 100m L of plasma samples,50m L of IS solution (1.0m g/mL)and 450m L of methanol were added in a 1.5mL polypropylene micro-centrifuge tube.The sample mixture was vortex-mixed fo
r approximate 1min,and centrifuged at 17,000rpm for 15min.Then the supernatant (550m L)was carefully removed,and transferred to another clean test tube and evaporated to dryness under nitrogen at 45 C.The dried residue was reconstituted in 100m L of methanol –water solution
(10:90,v/v)followed by vortex-mixing for 1min.After centrifuging at 17,000rpm for 15min,a 5m L aliquot of the supernatant was injected into the chromatographic systems for analysis.
Method validation
Assay validation was performed according to the currently accepted US Food and Drug Administration bioanalytical method validation guide-line (Food and Drug Administration,Center for Drug Evaluation and Rearch,2001).The lectivity was evaluated by comparing the MRM ion chromatograms of six different batches of blank plasma obtained from six subjects with tho of corresponding spiked plasma containing koumine and IS,and plasma sample at 0.5h after intravenous do of koumine.The calibration curves of koumine were constructed using spiked plasma samples at nine concentrations ranging from 0.025to 15m g/mL with weighted (1/x 2)least square linear regression method through measurement of the peak area ratio of koumine to IS.The lower limit of quanti fication (LLOQ),de fined as the lowest concentration on the calibration curve at
which both precision and accuracy were less than or equal to a deviation of Æ20%,were evaluated by analyzing spiked plasma samples which were prepared in six replicates.
To evaluate the precision and accuracy of the method,QC samples at four concentration levels (LLOQ,Low QC,Medium QC and High QC)were analyzed in six replicates on the same day and on three different days,respectively.Each run consisted of two calibration curves and six replicates of each concentration level.The assay accuracy was expresd by relative errors (RE,%)and precision by relative standard deviation (RSD,%).
The recoveries of koumine at three QC levels were determined by comparing peak area ratios of koumine to IS in samples that were spiked with koumine prior to extraction with tho in samples to which koumine had been added post-extraction.The matrix effect at three QC levels was measured by comparing the peak respon of samples spiked post-extraction (A)with that of pure standard solution containing an equivalent amount of koumine (B).The ratio (A/B Â100%)was ud to evaluate the matrix effect.The extraction recovery and matrix effect of IS were also simultaneously evaluated using the same method.
The stability of koumine in rat plasma was assd by analyzing repli-cates (n =6)of low and high Q
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liliaC samples during the sample storage and processing procedures.The freeze –thaw stability was determined after three freeze –thaw cycles.Post-preparation stability was estimated by analyzing QC samples at 0and 12h in the refrigerator at 4 C.Six aliquots of QC samples were stored at –20 C for 30days and at ambient temper-ature for 4h to determine long-and short-term stability,respectively.All stability testing QC samples were determined by using calibration curves of freshly prepared standards.
Pharmacokinetic study
The pharmacokinetic study was approved by the local Ethics Committee of Fujian University of Traditional Chine Medicine.The Sprague –Dawley rats (200–230g,three males and three females)were provided by the Experimental Animal Centre,Fujian University of Traditional Chine Medicine,China.The rats were fasted for 12h with free access to water prior to a single injection of 20mg/kg koumine via the tail vein (in saline solution).Blood samples (each 0.25mL)were collected before and at 1,5,10,20,30,60,90,120and 240min post-dosing.The plasma was immediately parated by centrifugation at 3500rpm for 10min and stored at –20 C until analysis.The main pharmacokinetic parameters such as elimination rate constant (K e ),half-life (t 1/2),apparent volume of distribution (V d ),clearance rate (Cl )and mean residence time (MRT)were analyzed using the non
compartmental pharmacokinetics data analysis software of PK solution 2™(Summit Rearch Service,USA).The area under the plasma concentration –time curve from zero to in fin-ity (AUC 0–1)was calculated by means of the linear trapezoidal rule with extrapolation to in finity with terminal elimination rate constant (K e
).
Figure 1.Structures of koumine (A)and gelmine (B).
老师用英语怎么读Quantitative determination of koumine in rat plasma
Biomed.Chromatogr.2013;27:736–740Copyright ©2012John Wiley &Sons,/journal/bmc
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Results and discussiondie in your arms
Mass conditions and chromatographic conditions optimization
MS analytical parameters were carefully optimized for the determi-nation of koumine in rat plasma.The mass spectrometer was tuned in both positive and negative ionization modes.Koumine produced much stronger signal in positive ion mode than in neg-ative ion mode owing to the prence of nitrogen atom in its struc-ture.In the precursor ion full-scan spectra,the most abundant ions were protonated molecules [M +H]+m/z 307.1and 323.1for kou-mine and IS,respectively.And other parameters in positive ion mode,such as capillary and cone voltage,desolvation tempera-ture,ESI source temperature,flow rate of desolvation gas and cone gas were further tuned to obtain the optimal intensity of proton-ated molecules [M +H]+of each compound.Both of them showed high abundance fragment ions at m/z 69.76in their product ion scan spectrum.The collision energy of collision-induced-decom-position was optimized for the optimal respon of the fragmenta-tion of koumine.Under the optimal conditions,multiple reaction monitoring with the precursor !product ion transition of m/z 307.1!69.76and m/z 323.1!69.76,respectively,was ud for quanti fication of koumine and IS.
Various mobile phas were evaluated for chromatographic behavior,sample throughput and the ionization respons of koumine and IS:acetonitrile –water (1),methanol –water (2),ace-tonitrile –water (0.1%formic acid)(3),methanol –water (0.1%formic acid)(4),acetonitrile (0.1%formic acid)–water (0.1%for-mic acid)(5)and methanol (0.1%formic acid)–water (0.1%
formic acid)(6).It was found that better peaks shape and ioniza-tion respons of them could be obtained when the mobile phas 5and 6were adopted.However,the best respon and peak shape were obtained from a gradient delivery of a mixture of water containing 0.1%formic acid and methanol with 0.1%formic acid.The overall chromatographic run time was completed within 6min.Sample preparation
In terms of sample preparation,solid-pha extraction and liquid –liquid extraction are techniques widely ud in the preparation of biological samples for their ability to improve the nsitivity and robustness of assay.However,a one-step PPT method was adopted that provided high recovery for both koumine and IS in the prent study.Compared with solid-pha extraction and liquid –liquid extraction,the sample preparation procedure of a PPT method was relatively simple and low-cost.Methanol was chon as the protein precipitant for its satisfactory ef ficiency in precipitating and its better compatibility with mobile pha.Method validation
Selectivity.There were no interfering peaks at the elution times for koumine and IS (gelmine).The typical MRM chroma-tograms of blank plasma (A),spiked plasma containing koumine (0.050m g/mL)and IS (0.50m g/mL)(B),and plasma collected at 0.5h after a single injection of 20mg/kg koumine via tail vein (C)are shown in Fig.
2.
Figure 2.Typical MRM chromatograms of koumine (m/z 307.1>69.76)and gelmine (IS,m/z 323.1>69.76)in rat plasma.(A)A blank plasma sample;(B)a blank plasma sample spiked with koumine (0.050m g/mL)and IS (0.50m g/mL);(C)a plasma sample from a rat at 0.5h after intravenous administration of koumine.
J.-Z.Chen et al .
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Linearity and the lower limit of quanti fication.The peak area ratios of koumine to IS in rat plasma varied linearly over the concentration range tested (0.025–15m g/mL).The best linear fit and least-square residual for the calibration curve was achieved with a 1/x 2weighting factor.A typical equation for the calibration curve was:y =1.053Â10À1x +1.03Â10À2,r =0.9997for koumine,where y reprents the peak area ratios of koumine to IS and x reprents the plasma concentration of koumine in micrograms per milliliter.
For koumine,the prent UPLC-ESI/MS/MS method offered an LLOQ of 25ng/mL,with the accuracy of À16.0%in terms of RE and the intra-and inter-day precision were <10.0%in terms of RSD (3days,n =6).
Precision and accuracy.The data of inter-and intra-day precision and accuracy for koumine are listed in Table 1,and conformed to the criteria for the analysis of biological samples according to the guidance of the US Food and Drug Administra-tion.The accuracy was required to be within Æ15%(20%for LLOQ),and the intra-and inter-day precisions were not to exceed Æ15%(20%for LLOQ).The results demonstrate that the values are within the acceptable range and the method is accurate and preci.
Extraction recovery and matrix effect.The extraction recov-eries of koumine from QC samples were 70.0,78.9and 76.5%at low,medium and high concentrations,respectively,whereas the recovery was 72.7%for IS.In terms of matrix effect,all the ratios de fined above were within the acceptable limits (85–115%).No signi ficant matrix effect for koumine was obrved,indicating that ion suppression and enhancement from plasma were negli-gible for this method.
Stability.The results from all stability tests are prented in Table 2,and indicated that koumine was st
able under routine laboratory conditions.The method is therefore proved to be applicable for routine analysis.
Pharmacokinetic study
The validated UPLC-MS/MS method was successfully applied to a pharmacokinetic study following a single intravenous administra-tion of 20mg/kg koumine to rats.The mean plasma concentra-tion –time curve of koumine is shown in Fig.3.After intravenous administration of koumine,the t 1/2and K e were 42.9Æ18.9min and 0.018Æ0.0061/min,respectively.The AUC 0–t and AUC 0–1values obtained were 346.992Æ41.048and 352.071Æ36.887m g min/mL,respectively.The Cl and V d were 57.298Æ5.655mL/min/kg and 3.646Æ1.950L/kg,respectively.
Conclusion
A rapid,reliable and nsitive UPLC-MS/MS method for the determination of koumine in rat plasma is described for the first time.One-step protein precipitation of the plasma sample with methanol simpli fied the sample preparation and required a smaller plasma volume of 100m L.The short retention time of koumine of 2.65min promis high sample throughout.This validated method has successfully been applied to a pharmacoki-netic study of koumine in rats.
Table 1.The accuracy and precision for the analysis of koumine in rat plasma (intra-day,n =6;inter-day,n =6ries per day,3days)
Added concentration (m g/mL)Found concentration (m g/mL)
Accuracy,RE (%)
Intra-day RSD (%)
Inter-day RSD (%)
0.025(LLOQ)0.021Æ0.001À16.0 4.810.00.050(LQC)0.054Æ0.0038.0 5.614.10.50(MQC)0.57Æ0.0514.08.7 6.710(HQC)
10.50Æ0.69
5.0
6.6
8.9
LLOQ,Lower limit of quanti fication;LQC,low quality control;MQC,medium quality control;HQC,high quality
control.
Figure 3.Mean plasma concentration –time pro files of koumine in rats following intravenous administration of 20mg/kg koumine (n =6,each point reprents mean ÆSD).
Table 2.The stability of koumine in rat plasma at low and high QC levels (n =6)(mean ÆSD)
Stability
Koumine
0.050(m g/mL)10(m g/mL)Freeze –thaw stability 0.056Æ0.0089.5Æ0.2Long-term stability 0.053Æ0.0029.6Æ0.3Short-term stability
0.055Æ0.0059.9Æ0.3Post-preparative stability
0.057Æ0.008
9.9Æ0.2
Quantitative determination of koumine in rat plasma
nianBiomed.Chromatogr.2013;27:736–740Copyright ©2012John Wiley &Sons,/journal/bmc
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Acknowledgments
This work was supported by a grant from the Natural Science Foundation of Fujian Province(no.2011J05072),People’s Republic of China.
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