HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究

李相鸿，孙华，贾元威，周理想，王伟佳，戴敏，赵亚男，谢海棠，*

1皖南医学院弋矶山医院临床药学部;2皖南医学院弋矶山医院安徽省药物临床评价中心，芜湖241001

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究

李相鸿1，孙华1，贾元威2，周理想1，王伟佳1，戴敏1，赵亚男1，谢海棠1，2*

1皖南医学院弋矶山医院临床药学部;2皖南医学院弋矶山医院安徽省药物临床评价中心，芜湖241001

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取，汉邦C18色谱柱(150 mm×4．6 mm，5 μm)进行分离，流动相为甲醇∶水(85∶15，v/ v)，水相含10 mmol/L的醋酸铵和0．1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测，草乌甲素及内标的反应离子对分别为644．4/584．4和237．2/194．2，草乌甲素血药浓度在0．010～1．0 ng/mL范围内线性关系良好，最低定量限为0．010 ng/mL可以满足口服0．4 mg草乌甲素后血药浓度的检测，日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中，20名志愿者口服0．4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0．325±0．110)，(0．323± 0．115)ng/mL;AUC0-16(1．627±0．489)，(1．732±0．556)ng·h/mL;AUC0-∞(1．730±0．498)，(1．831±0．562) ng·h/mL;t1/2(4．26±0．95)，(3．80±0．90)h;Tmax(1．34±0．54)，(1．83±0．99)h。

HPLC-MS/MS;草乌甲素;血药浓度;生物等效性

Introduction

Bulleyaconitine A(BLA，see Fig．1(A))was an anal-gesic and anti-inflammatory drug isolated from Aconitum plants，which can be used for the treatment of rheumatoid arthritis，osteoarthritis，periarthritis humeroscapularis，lumbar muscle strain，sprain，etc．It was reported that the analgesic effect of BLA might be related to the 5-HT level in the brain，while no physical dependence has been observed．Furthermore，the relative analgesic effect of BLA was found to be as much as 3，65，and 7000 times as potent as 3-acetylaconitine，morphine and aspirin，respectively．BLA has been approved for the treatment of chronic pain and rheumatoid arthritis by SFDA(Sino Food and Drug Administration)as intramuscular injections，tablets and soft gel capsules for clinical use in China．So far，no article reported the pharmacokinetics of oral administration of BLA，only three articles have reported the determination of BLA in human plasma．Two of articles is to use LC-MS method with a lowest limit of quantification(LLOQ)of only 0.12 ng/mL．［1，2］Daria etc．reported a modified-HPLC-MS/MS，but the lower limit of quantification only reached 0．0587 ng/mL．［3］All these methods need complex process of sample preparation and not sensitive enough for its oral administration pharmacokinetics study．

With respect to its extensively clinical application，it was urgent to develop a sensitive and practical BLA quantitation method．Here we developed a validated HLC-MS/MS method with the lower limit of quantification 0．010 ng/mL，which was sensitive enough and successfully applied to bioequivalence evaluation in Chinese healthy volunteers．

Fig.1 chemical structures for BLA

Experimental

Material

The standard of BLA(NO．201005，purity＞99．70%) and carbamazepinum were kindly provided by Haichen Medicine Co．，Ltd，Nanjing，PR China．BLA dispersible tablets with a batch No．20100301 and reference tablets (Yunnan Haobang Medicine Co．，LTD)with a batch No．100104，were also provided by this company．Methanol(Merck Company Inc．，Germany)was of chromatographic grade．All other reagents were of analytical grade．Milli-Q water(Millipore，Bedford，MA)was used throughout the study．

HPLC-MS/MS instrumentation and conditions

The HPLC system consisted of a Shimadzu LC-20AD HPLC series liquid chromatograph with a Kromasil C18column(150 mm×4．6 mm，5 μm，Hanbon Science＆Technology Co．，Ltd)．Mass spectrometric detection was performed on an API 3200 triple quadruple instrument(Applied Biosystem/MDS Sciex，Toronto，Canada)．

The mobile phase was composed of methonal–10 mmol/L ammonium acetate solution containing 0.1% formic acid(85∶15，v/v)at a flow rate of 0．5 mL/ min．The effluent from the HPLC column was directed into the ESI probe．Mass spectrometer conditions were optimized to obtain maximal sensitivity using MRM mode．A turbo ion spray interface was operated in positive ionization mode．Source conditions were as follows: the turbo-gas temperature was set at 600℃and the ion spray needle voltage was adjusted to 5500 V．The common parameters:gas 1，gas 2，curtain gas and collision gas，were set at 50，50，20 and 5 psi，respectively．The mass spectrometer was operated at unit resolution for both Q1 and Q3 in the MRM mode，with a dwell time of 100 ms per MRM channel．The［M+H］+precursor/ product ion pairs were m/z 644．4/584．4 for BLA and 237．2/194．2 for IS(Fig．2)．The declustering potential，entrance potential，collision energy，collision cell exit potential were set at 80，5，35，20 and 40，10，26，6 V for BLA and IS，respectively．Data acquisition was performed with analyst software(Version1．4．2)．

Preparation of the stock and standard solutions

The stock solutions containing 84．25 μg/mL of BLA and the internal standard(carbamazepinum 92．31 ng/ mL)were prepared in 50%methanol and stored at 4℃．The standard solutions were prepared by serial dilution with 50%methanol．

Fig.2 Mass spectra of the positive ion of the BLA (A)and the IS(B)

Sample preparation

A volume of 1 mL of drug-free human plasma was added to a disposable Eppendorf tube，followed by spiking 20 μL IS working solution(4 ng/mL)．After 1 min vortex，the well-vortexed plasma sample was loaded to Waters Oasis HLB solid-phase cartridge for elution，which was pre-conditioned by passing through 1 mL of methanol followed by 1 mL of water．Then，the solidphase extraction(SPE)cartridge was washed with 1 mL of water and 1mL 30%(v/v)methanol aqueous solution，and finally the SPE cartridge was slowly eluted by 1mL pure methanol．The eluted sample was transferred to a new Eppendorf tube for evaporation．The evaporated residue was reconstituted with 120 μL mobile phase，prior to transferral to HPLC vials and injection of 10 μL in the HPLC–MS/MS system．

Preparation of calibration curves and quality control samples

The samples for calibration curves were prepared by spiking 20 μL of the appropriate standard solutions in 1 mL blank human plasma to yield the following concentrations:0．010，0．020，0．050，0．10，0．20，0．50，1．0 ng/mL．The QC samples were prepared in 1 mL blank plasma at concentrations of 0．020，0．10 and 0．50 ng/ mL．

Method validation

The method validation assays were carried out according to the currently accepted U．S．Food and Drug Administration(FDA)and Chinese Food and Drug Administration(SFDA)bioanalytical method validation guidance．

The specificity of the method was evaluated by analyzing blank plasma samples from six independent humans．The LLOQ was defined as the lowest concentration on the calibration curve at which precision and accuracy were within 20%，respectively，and it was established by using six independent samples．

Calibration standards of seven BLA concentration levels at 0．01，0．020，0．050，0．10，0．20，0．50 ng/mL and 1．0 ng/mL were analyzed．To evaluate the linearity，the calibration standards were prepared and assayed on five separate runs．The calibration curve was constructed using the peak area ratios of BLA to IS vs the concentration of BLA，using weighed(1/C2)least squares linear regression．The precision and accuracy of the assay were determined from the QC samples by analyzing five replicates at 0．02，0．1 and 0．5 ng/mL．Intra-batch precision and accuracy were determined on one batch．Inter-batch precision and accuracy were determined on three consecutive days．The concentration of each sample was determined using the calibration curve prepared and analyzed on the same batch．Assay precision was calculated using the relative standard deviation (RSD%)．The accuracy is the degree of closeness of the determined value to the nominal true value under prescribed conditions．The accuracy is defined as a percentage of the calculated value(C)from its true value (T)．It was calculated using the formula:(C-T)/T× 100%．

The extraction recoveries of BLA at three QC levels were determined by comparison of the peak areas of BLA obtained from precipitated plasma samples with those from the standard solution at the same concentration．

The matrix effect was defined as the direct or indirect alteration or interference in response due to the presence of unintended analytes or other interfering substances in the samples．Three different concentration levels of BLA(0．020，0．10 ng/mL and 0．50 ng/mL) were evaluated by analyzing five samples at each level．The blank plasmas used in this study were from three different volunteers．If the peak area ratio is less than 85%or greater than 115%，a matrix effect is implied．The stability of BLA was investigated under a variety ofstorage and handling conditions at three QC levels．The Freeze and thaw stability was checked after three freeze–thaw cycles．The QC samples were stored at-70℃for 24 h and thawed unassisted at room temperature for 3 times，and then analyzed after the third cycle．The short-term stability was assessed by analyzing the samples that kept at room temperature for a period that exceeded the routine preparation time of the samples(4 h)．The Long-term stability was performed at-70℃for 5 d and 20 d．The autosampler stability was conducted by reanalyzing extracted QC samples kept under auto sampler conditions(4℃)for 24 h．

Bioequivalence study

A total of 20 healthy male volunteers from the local population participated in the study after signing an informed consent at the Organization for State Drug Clinical Trial of Yijishan Hospital，Wuhu，China．The subjects were aged from 19 to 24(21．7±1．9)years，with body weight from 53 to 76(64．9±6．2)kg and height from 1．64 to 1．81 m．Before the beginning of the study，they underwent physical examination，electrocardiography and clinical laboratory tests，including biochemistry and hematological profile．Subjects with history of drug allergies or idiosyncrasies，renal or hepatic impairment，any illness of the cardiovascular or hematological system，or alcohol and drug abuse were excluded．All the subjects were instructed to abstain from taking any medication for 2 weeks before and during the overall study period．They also abstained from beverages containing alcohol or caffeine and smoking for 1 week prior to each dose and until the collection of the last blood sample．The study protocols were approved by the relevant Ethical Review Committee in accordance with the principles of the Declaration of Helsinki，and the recommendations of State Food and Drug Administration of China．

The 20 volunteers participated in a single dose crossover bioequivalence study with a 1-week interval between each administration．A single dose of either formulation(reference or test)of 0．4 mg BLA according to the randomization plan was given to volunteers with 250 mL of water．In addition，water，lunch and dinner were given to all the volunteers according to the specially designed schedule．About 4 mL blood samples from the cubital vein were collected into clear tubes pre-dose(0 h)and at 0．25，0．5，0．75，1，1．5，2，3，4，6，8，10，12 h，and 16 h after drug administration and centrifuged at 6000 rpm for 5 min．The plasma samples were pipetted into labeled tubes and stored at-70°C until analysis．

Pharmacokinetic study and statistics analysis

To determine the pharmacokinetic parameters of BLA，the concentration-time data were analyzed by non-compartmental methods．All results were expressed as arithmetic mean±standard deviation(S．D．)．Bioavailability is calculated according to the following equation:

Bioavailability(F)=AUC0-t(test)/AUC0-t(reference)

AUC0-t，AUC0-∞and Cmaxwere considered as important variables for the bioequivalence analysis．Bioequivalence was evaluated by means of an analysis of variance (ANOVA)and a two-sided t-test(P＞0．05)for cross-over design and calculating standard 90%confidence intervals(CI)of the ratio of test/reference using log-transformed data．The products were considered bioequivalent if the difference between the compared parameters fell into the 80%-125%range(FDA，2001b)．Wilcoxonsigned-ranknonparametrictests were utilized to compare Tmax．

Results and Discussions

HPLC-MS/MS conditions

BLA responded higher in positive ionization mode and molecular ion［M+H］+were present as major peak．The parent ions of BLA and IS were 644．4 and 237．2，respectively，while the product ions for BLA and IS were 584．4 and 194．2，respectively．Carbamazepinum was chosen as the internal standard because its polarity and dissolubility is similar to BLA．To improve the chromatographic peak shapes and intensity of BLA and IS，a 10 mmol/L ammonium acetate buffer solution was adopted in the mobile phase．Under the present chromatographic conditions，the retention time was 3．5 min for BLA and 4．5 min for IS．

Assay validationSelectivity and calibration．The selectivity was assessed by comparing the chromatograms of six independent blank plasmas with the corresponding spiked plasmas．Fig．3 shows the typical chromatograms of blank plasma，LLOQ，spiked plasma sample with BLA and IS，and the plasma sample from a volunteer after oral administration．There was no significant interference from endogenous substances at the retention times of the analytes．

Fig.3 representative LC-MS/MS chromatograms(A)blank plasma;(B)LLOQ 0．010ng/mL;(C)the standard sample spiked with BLA(0．20 ng/mL)and IS;(D)a plasma sample from a volunteer 0．75 h after an oral administration of 0．4 mg BLA．

The calibration curve was obtained over the BLA concentration linearity ranges from 0．010 to 1．0 ng/mL．The mean regression equation from five replicate calibration curves on different days was:y=2．863C+ 0.0068(r=0．9993，1/C2)，y=peak area ratio of BLA to I．S．，C=BLA concentration in ng/mL．The lowest limit of quantification for BLA was 0．010 ng/ mL．The calibration curves exhibited good linearity within the chosen range．Based on the standard data presented in Table 1，it was concluded that the calibration curves used in this method were precise and accurate for the determination of BLA．

Precision and accuracy．Data for intra-batch and interbatch precision and accuracy are presented in Table 2．The accuracy deviation values are within 15%of the actual values．The precision(RSD%)determined at each concentration level does not exceed 15%．The results revealed good precision and accuracy．

Table 1 Precision and accuracy of calibration samples for BLA in human plasma

Table 2 Intra-and Inter-batch accuracy and precision of BLA in human plasma(n=5 runs，five replicates per run)

Extraction recovery and matrix effect．The extraction recoveries determined for BLA are shown to be consistent，precise and reproducible．Data are shown in Table 3．The ratios of matrix effect assay were 89．62%± 8.23%，102．25%±2．17%and 99．85%±1．14% (n=3)for the low，medium and high concentration levels，respectively．The results showed there was no matrix effect of the analytes observed in this study．

Stability．Table 4 summarizes the post-extraction stability，room temp tability，freeze and thaw stability，aut osampler stability，short-term stability and long-term stability data of BLA．All the results showed good stability during these tests and there were no stability related problems during the routine analysis．

Table 3 Recovery of BLA from human plasma(n=5)

Table 4 Stability of BLA in human plasma under various conditions(n=5)

Bioequivalence evaluation

The highly sensitive HPLC-MS/MS method has been successfully used in a bioequivalence study of 0．4 mg BLA tablets in humans following oral administration．The mean plasma concentration-time curves for two kinds of BLA dispersible tablets(test and reference)are shown in fig．4．The main pharmacokinetic parameters of BLA in human plasma are summarized in Table 5．Both the mean values and S．D．of the main pharmacokinetic parameters such as Cmax，Tmax，t1/2，AUC0-16and AUC0-∞were found to be very close between test and reference preparations，indicating that plasma profiles generated by test preparations were bioequivalent to reference preparations．There was no statistically significant difference in the data of Cmax，AUC0-16and AUC0-∞between the two preparations and two periods except for that between individuals(see table 6)．There was also a lack of statistically significant difference in the values of Tmaxfor both preparations by a Wilcoxon signed-ranks nonparametric test．The results demonstrated that the ratio of these pharmacokinetic parameters of two drugs lies within the SFDA’s accepted range of 80%-125%．Therefore，it can be concluded that the two BLA preparations(test and reference)are bioequivalent in healthy Chinese volunteers．

Conclusions

Fig.4 Mean plasma concentration-time profile of BLA after an oral administration of 0．4 mg test and reference BLA tablets(n=20)．

Table 5 Pharmacokinetic parameters of BLA in human plasma after oral administration of 0．4 mg test and reference tablets(n=20)

Table 6 ANOVA results of pharmacokinetic parameters between product，period and individual and 90% confidence intervals(CI)after a single oral dose of 0．4 mg test and reference BLA tablets

Its concentration in human plasma was extremely low after oral administration of 0．4 mg BLA(Cmaxabout 0.3 ng/mL)．So far，no article reported the pharmacokinetics of oral administration of BLA．Now，a sensitive and practical HPLC-MS/MS method with solid-phase extraction for determination of BLA in human plasma was developed and fully validated．The developed method showed acceptable specificity，precision，accuracy and linearity over a therapeutically relevant concentration range．In particular，the high sensitivity of our solid-phase extraction method allowed the detection of BLA at very low levels in human plasma，The method has been successfully applied to a bioequivalence study in 20 Chinese healthy volunteers who were administered the dose of 0．4 mg BLA in a clinical study．

1Weng WY，Xua HN，Huang JM，et al．Determination of BLA in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry．J Chromatogr B，2005，816:315-320．

2Weng WY，Xua HN，Huang JM，et al．A pharmacokinetic study of intramuscular administration of BLA in healthy volunteers．Biol Pharm Bull，2005，28:747-749

3Xiong X，Zhai SD，Yao ZQ．Sensitive quantification of bulleyaconitine A in human serum by Liquid Chromatography-Tandem Mass Spectrometry．Biosci Biotechnol Biochem，2009，73:1572-1577

4Wang CF，Gerner P，Schmidt B，et al．Use of bulleyaconitine A as an adjuvant for prolonged cutaneous analgesia in the rat．Anesth Anal，2008，107:1397-1405

5Wang CF，Gerner P，Wang SY，et al．Bulleyaconitine A isolated from aconitum plant displays long-acting local anesthetic properties in vitro and in vivo．Anesthesiology，2007，107:82-90．

Determination of Bulleyaconitine A in Human Plasma by HPLC-MS/MS and Its Bioequivalence Study

LI Xiang-hong1，SUN Hua1，JIA Yuan-wei2，ZHOU Li-xiang1，WANG Wei-jia1，DAI Min1，ZHAO Ya-nan1，XIE Hai-tang1，2*1Department of Clinical Pharmacy，Yijishan Hospital of Wanna Medical College;2Anhui Provincial Centre for Drug Clinical Evaluation，Yijishan Hospital of Wannan Medical College，Wuhu 241001，China

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry(HPLC-ESIMS/MS)method was developed and validated for the determination of Bulleyaconitine A(BLA)in human plasma using carbamazepinum as internal standard(IS)．Plasma samples were extracted with Waters Oasis HLB solid-phase cartridges，separated on a reversed C18column(150 mm×4．6 mm，5 μm)with a mobile phase of methonal–10 mmol/L ammonium acetate solution containing 0．1%formic acid(85∶15，v/v)．Bulleyaconitine A and IS were detected in the multiple reaction monitoring mode(MRM)with precursor to product ion transitions of m/z 644．4/584．4 and 237．2/ 194．2，respectively．The method exhibited a linear range of 0．010-1．0 ng/mL for BLA in human plasma．The lowest limit of quantification(LLOQ)was 0．010 ng/mL，which was sensitive enough for the pharmacokinetic study of BLA．Acceptable precision and accuracy were obtained for concentrations of the calibration standard and the quality control (QC)．The validated method was successfully applied for evaluation of a bioequivalence study in Chinese healthy volunteers．The main pharmacokinetics parameters after oral administration of 0．4 mg BLA test or reference formulation were as follows∶Cmax(0．325±0．110)，(0．323±0．115)ng/mL;AUC0-16(1．627±0．489)，(1．732±0．556)ng·h/mL; AUC0-∞(1．730±0．498)，(1．831±0．562)ng·h/mL;t1/2(4．26±0．95)，(3．80±0．90)h;Tmax(1．34±0．54)，(1．83±0．99)h．

HPLC-MS/MS;bulleyaconitine A;plasma drug concentration;bioequivalence

July 15，2011;Accepted September 9，2011

This research project was supported by the Youth Science Foundation of Wannan Medical College(No．WK200918F)．

book=2011,ebook=11

R969．1

A

1001-6880(2011)06-1031-07

*Corresponding author Tel:86-553-5739728;E-mail:xiehaitang@sina．

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