Rapid and sensitive analysis of cyclobenzaprine by LC-MS/MS:Application to a pharmacokinetic study of cyclobenzaprine in dog

Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang 110016,China

Short Communication

Rapid and sensitive analysis of cyclobenzaprine by LC-MS/MS:Application to a pharmacokinetic study of cyclobenzaprine in dog

Wenhong Yu,Xiaojing Yang,Wenwen Sui,Haiyan Xu,Xinyi Luan, Xiangjun Wang,Yi Jin,Bo Yuan*

Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang 110016,China

A R T I C L E I N F O

Article history:

Received 11 December 2013

Received in revised form

8 January 2014

Accepted 13 January 2014

Available online 29 January 2014

Cyclobenzaprine

A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantif i cation of cyclobenzaprine in dog plasma.After extracted with organic solvent,post-treatment samples were separated on an Agela C18 column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode.Multiple reaction monitoring was performed using the transitions of m/z 276.2 → 216.1 and m/z 325.1 → 109.0 to quantify cyclobenzaprine and escitalopram(internal standard),respectively.The mobile phase consisted of acetonitrile: 5 mM ammonium acetate:formic acid(90:10:0.01,v/v/v)at a f l ow rate of 0.3 ml/min.The total analysis time was 2.4 min.The method was linear over the concentration range of 0.0200-10.0 ng/ml.The intra-and inter-day precision was within 12.8%in terms of relative standard deviation(RSD%)and the accuracy within 5.6%in terms of relative error.This method was successfully applied in a pharmacokinetic study of extended-release cyclobenzaprine in dogs.

© 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

1. Introduction

Cyclobenzaprine(Fig.1),3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-1-propanamine,is a centrally acting skeletal muscle relaxant shown to be effective for relief of musculoskeletal pain Refs.[1-3].The exact mechanism of action is unknown,but it is presumed to work at the brain stem,reducing tonic somatic motor activity at both γ-and αmotor neuron systems[4,5].Cyclobenzaprine has been usedfor the treatment of back,neck and myofascial pain for more than 30 years in clinic[6,7].

Fig.1-Structures of cyclobenzaprine(A)and escitalopram(IS,B).

In previous studies,different methods,including thin layer chromatography(TLC)[8],gas chromatography(GC)equipped with nitrogen selective detector or f l ame ionization detector [9,10],high performance liquid chromatography(HPLC)with ultraviolet absorbance detection(UV)[11,12],and liquid chromatography-tandem mass spectrometry(LC-MS/MS) with atmospheric pressure chemical ionization(APCI)[12]or eletrospray ionization source(ESI)[13,14],were developed for quantitative analysis of cyclobenzaprine in biological samples.The TLC,GC and HPLC-UV methods were not suitable for the quantif i cation of cyclobenzaprine following a low oral dosage because of long analysis time and poor sensitivity.The reported LC-MS/MS methods were rapid and sensitive with a run-time of 3-5 min and the lowest limit of quantif i cation (LLOQ)of 0.1-0.5 ng/ml;however,they suffered from tedious sample preparation[13]or used a bulk plasma volume of 1 ml [12,14].Xiang et al.established an LC-ESI-MS/MS method to determine cyclobenzaprine using a plasma volume of 0.5 ml with an LLOQ of 0.049 ng/ml[15].They separated cyclobenzaprine on an Ultimate XB-CN column.Cyano column is not used as commonly as C18 column in routine bioanalysis and its service life is easily decreased in reversed phase chromatography.Therefore,in this paper,we developed a rapid and sensitive LC-MS/MS method to determine cyclobenzaprine in dog plasma with a C18 column in a run-time of 2.4 min.A 0.4 ml aliquot of plasma was used in the present method and the LLOQ was 0.0200 ng/ml.The method was successfully applied to a pharmacokinetic study of cyclobenzaprine in beagle dogs.

2. Materials and methods

2.1. Chemicals and materials

Cyclobenzaprine(99.6%purity)and escitalopram(99.7%purity)were obtained from Suizhou Jiake Biomedicine Co. (Hubie,China)and National Institute for the Control of Pharmaceutical and Biological Products(Beijing,China),respectively.Cyclobenzaprine extended-release capsules(AMRIX®) were supplied by Cephalon Inc(PA,USA).HPLC-grade acetonitrile was purchased from Dikma Technology(CA,USA). Distilled water,prepared from demineralized water,was used throughout the study.All other chemicals and solvents were of analytical grade or better and used without further purif i cation.

2.2. Instrument and LC-MS/MS conditions

2.2.1. Chromatographic conditions

Chromatographic separation was performed on an Agela C18 column(50 × 2.1 mm i.d.,3.0 μm)using an Agilent 1290 LC system(Agilent,CA,USA).The column temperature was kept at 30°C.The mobile phase consisted of acetonitrile:5 mM ammonium acetate:formic acid(90:10:0.01,v/v/v)at a f l ow rate of 0.3 ml/min.The injection volume was 5 μl.

2.2.2. Mass spectrometric conditions

An API 4000 triple quadrupole tandem mass spectrometer (Applied Biosystem/MDS SCIEX,CA,USA)with electrospray source(ESI)was operated in positive ion mode.The quantif ication was performed using multiple reaction monitoring (MRM)method with the transitions of m/z 276.2 → 216.0 for cyclobenzaprine and m/z 325.1 → 109.0 for escitalopram (internal standard;IS).The main working parameters were set as follows:ionspray voltage,4.0 kV;ion source temperature,400°C;gas1,40 psi;gas2,40 psi;curtain gas,20 psi. Analyte concentrations were determined using the software Analyst 1.5.

2.3. Preparation of calibration standards and quality control samplesStock solutions of cyclobenzaprine and escitalopram(IS)were prepared in acetonitrile at a concentration of 500 μg/ml, respectively.A series of cyclobenzaprine working standard solutions with concentrations in the range of 0.200-100 ng/ml were obtained by further dilution of the stock solution with acetonitrile.The IS working solution was diluted with acetonitrile to a f i nal concentration of 50.0 ng/ml.All solutions were stored at-20°C until analysis and they were found to be stable for at least 30 d(data not shown).

Plasma calibration standards of cyclobenzaprine at concentrations of 0.0200,0.0400,0.200,1.00,5.00 and 10.0 ng/ml were prepared as follows:a 40 μl aliquot of cyclobenzaprine working standard solutions was spiked into an 8-ml glasstubes and the solvent was evaporated to dryness under air stream.A 400 μl aliquot of blank plasma was then added to each tube.Quality control(QC)samples of 0.0400,0.800 and 8.00 ng/ml were prepared in the same way as calibration standards.Additionalvalidation QCswerepreparedat 0.0200 ng/ml to test LLOQ.The spiked plasma(calibrators and QCs)were treated according to the sample preparation described below.

2.4. Sample preparation

To a 400 μl aliquot of each plasma sample,40 μl of IS solution (50.0ng/mlescitalopraminacetonitrile)and40 μlof1Msodium carbonate were added and vortex-mixed for 10 s.After the addition of 3 ml of n-hexane:dichloromethane:isopropanol (2:1:0.1,v/v/v),thesamplewasplacedonareciprocatingshaker for 10 min at 120 rpm,followed by centrifugation at 2110 g for 5 min.The organic layer was separated and evaporated to dryness at 40°C under an air stream.The residue was reconstituted in 100 μl of the mobile phase and 5 μl was injected for LC-MS/MS analysis.

2.5. Method validation

The validation process was conducted according to Guidance for Industry Bioanalytical Method Validation,recommended by FDA[16].

Specif i city of the method was evaluated by analyzing six different blank plasma samples to investigate the potential interferences at the retention times for the analyte and IS.

The linearity of the method was assessed by processing(in duplicate)a six-point calibration curve over the concentration range of 0.0200-10.0 ng/ml.The calibration curves were constructed by plotting the peak area ratios of the analyte to IS versus the nominal concentrations of the analyte by weighted (1/x2)least-squares linearregression.TheLLOQwas def i nedas the lowest plasma concentration in the calibration curve yielding a signal-to-noise ratio of at least 10.

The precision and accuracy were evaluated by analyzing QCs at plasma concentrations of 0.0200(LLOQ),0.0400,0.800 and 8.00 ng/ml in six replicates on three separated days.The criteria for acceptability of the data included accuracy within ±15%relative error(%RE)of the nominal values and a precision of within 15%relative standard deviation(%RSD)except for LLOQ at which both precision and accuracy were within 20%.

The matrix effect and extraction recovery for cyclobenzaprine and IS were evaluated by assaying three groups of samples:neat standard solutions of cyclobenzaprine and IS (group 1),blank plasma extracts from six different dogs spiked with cyclobenzaprine and IS after liquid-liquid extraction (group 2),and plasma extracts spiked with cyclobenzaprine and IS before extraction(group 3).Samples of each group were prepared at three cyclobenzaprine levels of 0.0400,0.800 and 8.00 ng/ml.The matrix effects were calculated as the ratio of peak area of an analyte spiked post-extraction(group 2)to its mean peak area from neat solution(group 1).The variability (RSD)of matrix effect at each concentration level should be less than 15%[17].The recovery was calculated as the ratio of the peak area of an analyte spiked prior to extraction(group 3) to its mean peak area after extraction(group 2).

The stability of cyclobenzaprine in dog plasma was assessed by analyzing triplicates of QCs at 0.0400 and 8.00 ng/ ml,which wereexposedto differenttemperatures and storage conditions.These QCs were analyzed after storage at room temperature for 4.0 h(bench-top),at-70°C for 24 d,after three freeze-thaw cycles at-70°C and after transportation with dry ice for2.0 h.The stabilityof cyclobenzaprine and IS in the injection solvent was determined periodically by reinjecting the processed QCs for up to 24 h(at room temperature)after the initial injection.Samples were considered stable if assay values were within the acceptable limits of accuracy(±15%RE)and precision(15%RSD).

The dilution integrity experiment was performed with an aim to validate the dilution test to be carried out on higher analyte concentrations above upper limit of quantif i cation (ULOQ),which maybe encountered duringreal subjectsample analysis.Dilution integrity experiment was carried out at 4 times ULOQ concentrations in six replicates.Samples at 4 times ULOQconcentration were5 times diluted with drug-free plasma.Their concentrations were calculated by applying the dilution factor 5 against the freshly prepared calibration curve.The criteria for acceptability of the data included accuracy within ±15%RE and precision within 15%RSD.

2.6. Pharmacokinetic study

Animal experiments were performed in accordance with the guidelines of the Experimental Animal Care and Use Committee of Shenyang Pharmaceutical University(Shenyang, China).Six beagle dogs(9.73 ± 0.31 kg,half male and half female)were supplied by Shenyang Kangping Laboratory Animal Institute(Shenyang,China).They were housed under standard conditions and had ad libitum access to water.After fasted for 12 h,dogs were given an oral dose of 15 mg extended-release cyclobenzaprine hydrochloride.Blood samples were collected into heparinized tubes before(0 h)and at 0.5,1.0,2.0,3.0,4.0,6.0,8.0,10,12,14,16,18,20,22,24,36 and 48 h after administration.Plasma was separated by centrifugation at 2110 g for 5 min and stored at-70°C until analysis.

Pharmacokineticparametersofcyclobenzaprinewere calculated by non-compartmental method using DAS 3.2 pharmacokinetic program(Chinese Pharmacology Society).

3. Results and discussion

3.1. Method development

An LC-MS/MS method with APCI ionization source was reported for the quantif i cation of cyclobenzaprine using 1 ml of plasma with an LLOQ of 0.1 ng/ml[12].ESI interface is used more widely than APCI interface in bioanalysis because most ofpolarandnon-polaranalytescanbeionizedinESIsource.In the present study,we found that cyclobenzaprine was easily ionized in ESI interface to form protonated ion of[M+H]+at m/z 276.2 and its signal intensity in ESI source was higher than that in APCI source.Therefore,ESI was chosen as the ionization source in this method.After the instrument parameterswere optimized,the ion transitions m/z 276.2 → 216.0 and m/z 325.1 → 109.0 were used to quantify cyclobenzaprine and IS, respectively(Fig.2).

Fig.2-Full scan MS/MS spectra of[M+H]+of cyclobenzaprine(A)and escitalopram(IS,B).

To improve the sensitivity of the method,various chromatographic conditions were tested.Xiang et al.separated cyclobenzaprine in human plasma on a cyano column in reversed phase chromatography[15].Because cyano bonds hydrolyze in water,the quality of cyano column becomes poor easily when it is used in reversed phase chromatographic system.An Agela C18 column packed with 3.0 μm particles was used in the present method.Compared with columns with bigger particle size(5.0 μm),the peak width of cyclobenzaprine was thinner on this column and signal-to-noise ratio was increased about 3 times.Additionally,the use of ammonium acetate as mobile phase decreased the tailing of the peak obviously and made the peak sharper.After careful comparison of the composition of mobile phase,eventually,a mixture of acetonitrile:5 mM ammonium acetate:formic acid (90:10:0.01,v/v/v)was adopted to achieve good sensitivity and shorten running time.In this chromatographic condition,the retention times of cyclobenzaprine and escitalopram(IS)were 1.9 and 1.7 min,respectively.The total run-time was 2.4 min. Compared with previously reported LC-ESI-MS/MS methods [13-15],this method was more sensitive with an LLOQ at 0.0200 ng/ml by using smaller sample volume(0.4 ml)and the analysis time was shorter.

3.2. Method validation

3.2.1. Specif i city

Potential interference from endogenous substanceswas investigated.Representative chromatograms obtained from blank plasma,blank plasma spiked with cyclobenzaprine at the LLOQ,and a dog plasma sample taken at 10 h after an oral administration of 15 mg extended-release cyclobenzaprine hydrochloride are shown in Fig.3.No interfering peaks with cyclobenzaprine or IS were observed which indicated that the developed method was specif i c for the analysis of cyclobenzaprine in dog plasma(Fig.4).

3.2.2. Linearity and LLOQ

Linearity of the method was observed in cyclobenzaprine concentration range of 0.0200-10.0 ng/ml.The representative calibration curve was y=1.38 × 10-1x+2.08 × 10-4,and the correlation coeff i cient was higher than 0.995.The LLOQ was conf i rmed to be 0.0200 ng/ml,at which the relative error wasnot more than 8.8%and the precision were below6.7% (Table 1).

Fig.3-Representative MRM chromatograms for cyclobenzaprine(peak I)and escitalopram(IS,peak II)in dog plasma samples.Panel A:a blank plasma sample;panel B:a blank plasma sample spiked with cyclobenzaprine at the LLOQ;panel C:a dog plasma sample collected at 10 h after drug administration.

Fig.4-Mean plasma concentration-time curve of cyclobenzaprine in beagle dogs after an oral administration of 15 mg extended-release cyclobenzaprine hydrochloride(n=6 per time point).Values are represented as mean ± SD.

3.2.3. Precision and accuracy

Table 1 summarizes the results for intra-and inter-day precision and accuracy for cyclobenzaprine measured by QCs. The intra-and inter-day precisions were all below 15%with a maximum RSD of 12.8%,and a maximum RE of 5.6%for accuracy was calculated.

3.2.4. Recovery and matrix effect

The recoveries of cyclobenzaprine extracted from plasma were 90.0 ± 4.5,101.1 ± 3.1 and 93.0 ± 2.7%at the concentrations of 0.0400,0.800 and 8.00 ng/ml,respectively.The recovery of IS at 5.0 ng/ml was 92.3 ± 4.2%.The matrix effects of cyclobenzaprine were in the range of 86.5-89.7%with RSD values below 8.5%at concentrations of 0.0400,0.800 and 8.00 ng/ml.The matrix effect of IS was 96.3%and the RSD value was 3.6%.These results indicated that the effect of matrix on the determination of cyclobenzaprine could be ignored.

Table 1-Precision and accuracy of the LC-MS/MSmethod to determine cyclobenzaprine in dog plasma(in 3 consecutive days,six replicates for each day).

3.2.5. Stability

Table 2 contains the results of investigations into the stability of cyclobenzaprine under the various conditions tested throughout the validation process.The tests were designed to cover anticipated conditions which might be encountered during sample handling and processing.The results indicated the stability of cyclobenzaprine in plasma after storage at room temperature for 4.0 h(bench-top),at-70°C for 24 d, after three freeze-thaw cycles at-70°C,during transportation for 2.0 h(dry ice)and in processed samples at room temperature for 24 h.

3.2.6. Dilution integrity

During the method development,saturated signal response of cyclobenzaprine was observed above the plasma concentration of 15 ng/ml.Preliminary pharmacokinetic study of cyclobenzaprine indicated that the concentrations of some plasma samples collected after administration were higher than 20 ng/ml.Basing on these results,four-fold dilution integrity was hence tested in the present study as described in Experimental.The mean back-calculated concentrations for 1/5 dilution samples were within ±4.1%RE of the nominal concentration with an RSD of 6.9%.

3.3. Pharmacokinetic study The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of cyclobenzaprine in beagle dogs after an oral administration of 15 mg extended-release cyclobenzaprine hydrochloride.The mean plasma concentration versus time prof i le of cyclobenzaprine is shown in Fig.4 and the main pharmacokinetic parameters are presented in Table 3.

Cyclobenzaprine reached the peak plasma concentration (Cmax)at approximately 3.50 h.It eliminated slowly from the plasma with a mean elimination half-time(T1/2)of 9.70 h. Marked interanimal variability was found in the pharmacokinetic prof i le of cyclobenzaprine in beagle dogs after drug administration.The values of Cmaxvaried from 2.19 to 32.9 ng/ ml.The area under curves(AUC0-t)in plasma was in the range from 24.4 to 33.9 ng·h/ml.Signif i cant individual deference was also observed in the pharmacokinetics ofcyclobenzaprine in humanbeings after administration of both common tablet and extended-release formulation[13-15], although it was not as obviously as that in dogs.

Table 2-Summary of stability of cyclobenzaprine under various storage in plasma(n=3).

Table 3-Pharmacokinetic parameters ofcyclobenzaprine after an oral administration of 15 mg extended-release cyclobenzaprinehydrochloride.

4. Conclusion

A rapid and sensitive LC-ESI-MS/MS method has been developed and validated for the analysis of cyclobenzaprine in dog plasma.The separation of cyclobenzaprine was carried out on a C18 column.This method showed high throughput (2.4 min each sample)and good sensitivity with an LLOQ of 0.0200 ng/ml.This method was successfully applied to characterize the pharmacokinetics of extended-release cyclobenzaprine hydrochloride in beagle dogs.

R E F E R E N C E S

[1]Nibbelink DW,Strickland SC,McLean LF,et al. Cyclobenzaprine,diazepam and placebo in the treatment of skeletal muscle spasm of local origin.Clin Ther 1978;1:409-424.

[2]Browning R,Jackson JL,O’Malley PG.Cyclobenzaprine and back pain:a meta-analysis.Arch Intern Med 2001;161:1613-1620.

[3]Toth PP,Urtis J.Commonly used muscle relaxant therapies for acute low back pain:a review of carisoprodol, cyclobenzaprine hydrochloride,and metaxalone.Clin Ther 2004;26:1355-1367.

[4]Chou R,Peterson K,Helfand M.Comparative eff i cacy and safety of skeletal muscle relaxants for spasticity and musculoskeletal conditions:a systematic review.J Pain Symptom Manage 2004;28:140-175.

[5]Share NN,McFarlane CS.Cyclobenzaprine:novel centrally acting skeletal muscle relaxant.Neuropharmacology 1975;14:675-684.

[6]Katz WA,Dube J.Cyclobenzaprine in the treatment of acute muscle spasm:review of a decade of clinical experience.Clin Ther 1988;10:216-228.

[7]Malanga GA,Ruoff GE,Weil AJ,et al.Cyclobenzaprine ER for muscle spasm associated with low back and neck pain:two randomized,double-blind,placebo-controlled studies of identical design.Curr Med Res Opin 2009;25:1179-1196.

[8]Faber DB,Man in’t Veld WA.A thin-layer chromatographic method for determining carbamazepine in blood.J Chromatogr 1974;93:238-242.

[9]Hucker HB,Stauffer SC.Gas-liquid chromatographic determination of nanogram amounts of cyclobenzaprine in plasma using a nitrogen detector.J Chromatogr 1976;124:164-168.

[10]Constanzer ML,Vincek WC,Bayne WF.Determination of cyclobenzaprine in plasma and urine using capillary gas chromatography with nitrogen-selective detection.J Chromatogr 1985;339:414-418.

[11]Hwang PTR,Young DA,Straughn AB,et al.Quantitative determination of cyclobenzaprine in human plasma by high pressure liquid chromatography.J Liq Chromatogr 1993;16:1163-1171.

[12]Constanzer M,Chavez C,Matuszewski B.Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine.J Chromatogr B 1995;666:117-126.

[13]Darwish M,Hellriegel ET,Xie F.Single-dose pharmacokinetics of once-daily cyclobenzaprine extended release 30 mg versus cyclobenzaprine immediate release 10 mg three times daily in healthy young adults:a randomized,open-label,two-period crossover,single-centre study.Clin Drug Investig 2008;28:793-801.

[14]Brioschi TM,Schramm SG,Kano EK,et al.Pharmacokinetics and bioequivalence evaluation of cyclobenzaprine tablets. Biomed Res Int 2013;2013:281392.

[15]Xiang Y,Zhou L,Qian Z,et al.Determination of cyclobenzaprine in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry and its application in a pharmacokinetic study. Biomed Chromatogr 2012;26:1083-1088.

[16]U.S.Department of Health and Human Services,Food and Drug Administration,Center for Drug Evaluation and Research.Guidance for industry,bioanalytical method validation;2001.

[17]Bansal S,DeStefano A.Key elements of bioanalytical method validation for small molecules.AAPS J 2007;9:E109-14.

*Corresponding author.Tel.:+86 24 23986468;fax:+86 24 23986250.

E-mail address:boyuan1962@163.com(B.Yuan).

Peer review under responsibility of Shenyang Pharmaceutical University

Production and hosting by Elsevier

1818-0876/$-see front matter © 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

http://dx.doi.org/10.1016/j.ajps.2014.01.002

LC-MS/MS

Pharmacokinetic

Plasma