Accepted Manuscript Title: Liquid chromatography-tandem mass spectrometry for the quantification of flurbiprofen in human plasma and its application in a study of bioequivalence Author: Chenghan Mei Bin Li Qiangfeng Yin Jing Jin Ting Xiong Wenjuan He Xiujuan Gao Rong Xu Piqi Zhou Heng Zheng Hui Chen PII: DOI: Reference:
S1570-0232(15)00258-5 http://dx.doi.org/doi:10.1016/j.jchromb.2015.04.037 CHROMB 19428
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
18-1-2015 18-4-2015 26-4-2015
Please cite this article as: C. Mei, B. Li, Q. Yin, J. Jin, T. Xiong, W. He, X. Gao, R. Xu, P. Zhou, H. Zheng, H. Chen, Liquid chromatography-tandem mass spectrometry for the quantification of flurbiprofen in human plasma and its application in a study of bioequivalence, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.04.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Liquid chromatography-tandem mass spectrometry for the quantification of flurbiprofen in human plasma and its application in
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a study of bioequivalence
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Chenghan Meia, Bin Lia, Qiangfeng Yinb, Jing Jina, Ting Xionga, Wenjuan
Department of Pharmacology, Institute of Clinical Pharmacology, Tongji
an
a
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Hea, Xiujuan Gaoa, Rong Xua, Piqi Zhoub, Heng Zhenga, Hui Chenab.
Medical College, Huazhong University of Science & Technology, Wuhan
b
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430030, PR China.
Institute of Clinical Pharmacy, Union Hospital Affiliated to Tongji
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430022, PR China.
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Medical College, Huazhong University of Science & Technology, Wuhan
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Corresponding author: Hui Chen. E-mail:
[email protected] Tele: +86 02783692628
Address: 13 Hangkong Road, Qiaokou District, Wuhan, Hubei Province, China.
Keywords Flurbiprofen
LC-MS/MS
Human plasma
Pharmacokinetics
Bioequivalence
1/1 Page 1 of 26
Abstract A simple, quick and accurate LC-MS/MS method for the quantification of flurbiprofen in human plasma with indomethacin as internal standard (IS)
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was developed and validated. Samples were treated with methanol to
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precipitate proteins, then separated on a Ultimate C18 column (5 µm, 2.1×50 mm) with a gradient elusion process. Mobile phase A was
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comprised of water and formic acid, mobile phase B was comprised of
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acetonitrile and formic acid. Multi reaction monitoring (MRM) signals were saved on a negative ionization electrospray mass spectrometer. The
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calibration curve showed good linearity in the range of 40.00-10000.00 μg/L (r2=0.998). Intra-day RE was 0.2-2.2%. Inter-day RE was 0.5-3.4%.
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The samples showed good stability under the study conditions. No
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significant matrix effect was observed. The established method was then
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applied to a bioequivalence study of a flurbiprofen axetil formulation.
1. Introduction
Flurbiprofen axetil is a targeted nonsteroidal anti-inflammatory drug, mainly used in surgery and cancer pain management. Lipid microspheres as the carrier get preferentially delivered to lesion tissues and tumor cells after injecting into the systemic circulation. Flurbiprofen axetil, is then released from the lipid microspheres, and rapidly hydrolyzed to flurbiprofen by carboxyl esterase in less than 5 minutes [1]. The 2/2 Page 2 of 26
mechanism of flurbiprofen is to inhibit the synthesis of prostaglandin. It demonstrated comparable efficacy over other NSAIDs (e.g., aspirin,
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can be used as an in vivo probe for CYP2C9 activity [3].
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indomethacin, ibuprofen, naproxen, and diclofenac) [2]. Flurbiprofen
Several HPLC methods have been reported for the determination of
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flurbiprofen in human plasma. They vary in sample treatment, separation
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condition and detector use. Albert KS et al [4] reported a method of extraction with pentane-ether (80:20). Askholt et al [5] reported a
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method of detection of UV spectrometry. Hutzler et al [3] reported a method of fluorescence detection. With advancements in instrumentation,
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methods with no extraction procedure have become popular [3]. But
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such methods still suffer from long analytic time [6]. In the presented
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study we utilized LC-MS/MS, which has a better selectivity, thus the time necessary for separation was significantly reduced.
The objective of the present study was to develop and validate a rapid, selective, accurate and reliable LC-MS/MS method of flurbiprofen and apply this method to a bioequivalence study.
2. Materials and methods 2.1.
Chemicals and regents 3/3 Page 3 of 26
Flurbiprofen standard reference (chemical purity: 99.7%, lot No. 100725-200401), the IS (indomethacin) (chemical purity: 100.0%, lot No. 100258-200403) were purchased from National Institutes for Food and
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Drug Control (Beijing, China). Flurbiprofen axetil injection for test (5 mL:
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50 mg, lot No. 130802) was supplied by Wuhan Docan Pharmaceutical Co., Ltd. (Wuhan, China). And the reference formulation of the
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flurbiprofen axetil injection (5 mL: 50 mg, lot No. 5173K) was supplied
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by Beijing Tide Pharmaceutical Co., Ltd. (Beijing, China).Water was purified by a PURELAB classic (ELGA, England). Formic acid was MS
2.2.
Instrumentation
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grade (Fluka, Switzerland). Methanol and acetonitrile were HPLC grade.
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The LC-MS/MS system consisted of a Shimadzu UFLC LC-30 AD
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chromatography system (Shimadzu, Japan) and a QTRAR 4500 mass spectrometer (AB SCIEX, USA) equipped with electrospray ionization (ESI) source system. The analytes were separated on an Ultimate C18 column (5 μm, 2.1×50 mm) connected with a pre-column (4 3.0 mm I.D. Phenomenex). Mobile phase A was water-formic acid (99.9:0.1, v/v), and mobile phase B was acetonitrile-formic acid (99.9:0.1, v/v). Gradient elution was performed with 60:40 A-B for 0.1 min, 60:40 A-B to 15:85 A-B for 0.5 min, 85% B to 95% B for 0.4 min, 1 min hold at 95% B, return to 60:40 A-B for 0.1 min, 2.5 min run time, at a flow rate of 0.4 4/4 Page 4 of 26
mL/min. The column temperature was set 40 ◦C. A 5 μL sample was injected into the sampling system with the auto-sampler conditioned at 4
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◦C.
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The multiple reaction monitoring (MRM) transitions were performed at m/z 242.9→198.7 for flurbiprofen and m/z 355.9→312.0 for
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indomethacin (IS). No additional transitions were looked at. Optimized
an
values for collision energy (CE), declustering potential (DP), entrance potential (EP) and collision exit potential (CXP) were -12 V, -26 V, -8 V
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and -8 V for flurbiprofen, and -20 V, -30 V, -10 V and -10 V for indomethacin (IS), respectively. Curtain gas, Ion spray voltage, source
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40 psi and 40 psi.
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temperature, Ion source Gas1 and Gas 2 were 30 psi, -4500 V, 400 ℃,
2.3.
Preparation of calibration standards and quality control samples
Stock solutions were prepared by dissolving flurbiprofen and indomethacin (IS) in methanol to yield 1.0 mg/mL, 99400 μg/L solutions,
respectively. Flurbiprofen stock solution was diluted with methanol to get a series of standard working solutions, 400-100000 μg/L. A 49700 μg/L solution of IS was prepared by diluting the stock IS solution.
5/5 Page 5 of 26
Calibration standards of flurbiprofen at concentrations of 40.0, 100, 300, 900, 2500, 5000, 10000 μg/L were prepared by mixing 20 μL different standard working solutions, 20 μL IS solution (indomethacin 49700 μg/L)
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and 200 μL blank human plasma in a 1.5 mL plastic vial . Similarly,
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quality control (QC) samples (low QC, med QC and high QC) were
prepared by spiking blank human plasma at concentrations of 100, 1000
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2.4.
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and 8000 μg/L.
Sample preparation
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A human plasma sample (200 μL) was placed in a 1.5 mL plastic vial and then 20 μL of IS (49700 μg/L) was added and mixed with 780 μL
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methanol by vortexing for 30 sec. The mixture was centrifuged at 12000
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rpm for 10 min. Finally, the supernatant liquid (5 μL) was transferred to a
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sample vial for later separation with LC.
2.5.
Method validation
The validation of specificity, linearity, precision and accuracy, recovery and matrix effect, stability, dilution and carry-over of the presented method were carried out. Procedures were compliant with guidelines for bioanalytical method validation published by the US FDA [7] and other guidelines [8].
6/6 Page 6 of 26
Selectivity of flurbiprofen over interference from endogenous substances was assessed. Selectivity of flurbiprofen over interference from flurbiprofen axetil and flurbiprofen glucuronide was assessed. 6 blank
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human plasma samples were analyzed under the test condition. Absence
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of interfering components is accepted where the response is lower than 20%LLOQ for flurbiprofen and 5%for the IS. (CHANGE MADE IN
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RESPONSE TO REVIEWER COMMENTS) A selected subset of
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samples was subject to additional analysis. These samples were treated with the same preprocessing progress. These samples were separated
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under the same elution process. Selected Ion Monitoring (SIM) [9] of m/z 419.1 was used to see if flurbiprofen glucuronide should exist. SIM of
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m/z 329.1 was used to see if flurbiprofen axetil should exist. Neither
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should peak at the retention time of flurbiprofen. Neutral loss of 176 was
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used to see if flurbiprofen glucuronide undergo source induced fragmentation to flurbiprofen. Neutral loss of 86 was used to see if flurbiprofen axetil should undergo source induced fragmentation to flurbiprofen. No related peaks should be detected at the retention time of flurbiprofen. (ANSWER TO REVIEWER COMMENTS: To prove the presumption the ester prodrug and the acyl glucuroide metabolite should not coelute with flurbiprofen and undergo source induced fragmentation at the elution time of flurbiprofen, we conducted additional experiments on a selected subset of samples to by selected ions monitoring of 419.1 7/7 Page 7 of 26
and 329.1 and neutral loss of 86 and 176. No related peaks were observed.)
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A calibration curves was constructed by plotting the peak area ratios of
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flurbiprofen to the IS against the concentrations of flurbiprofen. A
replicate was conducted using the same standards after all samples were
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processed. And weighted least-squares linear regression with1/x2 as
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weighting factor was adopted to derive the overall equation from data
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points gathered both pre- and post-sample.
Accuracy and precision were assessed in five samples at three different
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QC levels on the same day and among three different days. RE should be
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te
within ±15% and the intra- and inter-day RSD should not exceed 15%.
The extraction recovery was evaluated at three QC levels and reckoned by comparing the peak areas received from plasma samples with the analytes spiked before extraction to those spiked after extraction. Matrix effect was determined by peak areas of flurbiprofen in two QC levels (100, 8000 μg/L), calculated as a percentage of the response of samples prepared by spiking flurbiprofen and the IS with drug-free human plasma (blank matrix) extracts from six different sources to those spiked with mobile phase. 8/8 Page 8 of 26
Stability was investigated at three QC levels under 4 different conditions: 24 h in the autosampler, 24 h at room temperature, three cycles of freeze
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thaw, and 32 days at −80 ◦C, with 5 replicates under each condition.
Carry-over was assessed by injecting a blank plasma sample following a
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sample of the LLOQ and a sample of the ULOQ. This measurement was
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repeated 3 times. All carry-over peaks should not exceed 15% LLOQ.
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The dilution integrity was demonstrated by diluting plasma samples with flurbiprofen 12500 μg/L 2.5- fold by blank plasma, five replicates. RE
Bioequivalence study
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2.6.
te
d
and RSD should be within ±15% and below 15% respectively.
The proposed method was applied to a Bioequivalence study of flurbiprofen axetil injection among healthy male Chinese volunteers. The study protocol was approved by the Ethics Committee of Huazhong University of Science & Technology and a written informed consent was obtained from all volunteers.
Subjects (n=24) were randomly assigned (1:1) into two groups, and received a single dose of either test or reference formulation of a 5mL 9/9 Page 9 of 26
(i.v.in not longer than 2min) flurbiprofen axetil, and a single dose of the alternate formulation after a 7-day washout period. Subjects were fasted for 12h prior to administration. Blood samples were collected in
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heparinized tubes immediately before (0 h) and 0.17, 0.25, 0.33, 0.5, 1,
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1.5, 2, 4, 6, 8, 12, and 24 h after administration. Samples were
centrifuged at 3000 rpm for 10 min and plasma was separated and stored
an
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at −70 ◦C until analysis.
A non-compartmental method was used to evaluate the pharmacokinetic
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parameters. Following guidelines [10] bioequivalence was assessed by means of an analysis of variance (ANOVA) and calculating the standard
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90% confidence intervals (90% CIs) of the ratio of average of
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log-transformed Cmax, AUC0-t and AUC0-∞. Bioequivalence was assumed
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when 90%CIs fit in the range of 80-125%.
3. Results and discussion 3.1.
Optimization of LC-MS/MS conditions
Parameters for ESI negative ion mode were optimized for the detection specificity of precursor and product ions of flurbiprofen and IS. Transition m/z 242.9→198.7 for flurbiprofen and m/z 355.9→312.0 for IS were used. Fig. 1 shows the mass spectra. Compared to method reported by Hutzler et al [3], which used acetonitrile as mobile phase, 10 / 10 Page 10 of 26
gradient elution described previously was found favorable regarding peak shape and separation time in our study. Sharper peaks were observed with
Method validation
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3.2.
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formic acid in the mobile phase.
3.2.1. Selectivity
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No significant peaks at the retention time of flurbiprofen or IS are found
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in blank plasma samples. (CHANGE MADE IN RESPONSE TO REVIEWER COMMENTS) Flurbiprofen axetil and flurbiprofen
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glucuronide did not peak at the retention time of flurbiprofen. No source induced fragmentation to flurbiprofen of flurbiprofen axetil or
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flurbiprofen glucuronide was observed at the retention time of
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flurbiprofen. Flurbiprofen axetil undergo a quick hydrolysis reaction in
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vivo, thus its plasma concentration should drop below significant level within a few minutes [1]. According to the chemical structure of flurbiprofen glucuronide, this species is a more polar molecule, thus should not coelute with flurbiprofen. Experimental evidence aligned with these hypotheses. (ANSER TO REVIEWER COMMENTS: To prove the presumption the ester prodrug and the acyl glucuroide metabolite should not coelute with flurbiprofen and undergo source induced fragmentation at the elution time of flurbiprofen, we conducted additional experiments on a selected subset of samples to by selected ions monitoring of 419.1 11 / 11 Page 11 of 26
and 329.1 and neutral loss of 86 and 176. No related peaks were observed.) Chromatograms obtained from the blank human plasma, bank human plasma spiked with the flurbiprofen at LLOQ level (40.0 μg/L)
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and IS, and plasma sample at concentration of 5760.22 μg/L after IV
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administration are shown in Fig. 2.
3.2.2. Calibration curves and lower limits of quantification
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y=0.000499x + 0.00121 (r2=0.998) was an equation derived from peak
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area ratio of flurbiprofen to the IS and the concentration of flurbiprofen. Linearity was established over the concentration 40.0-10000 μg/L of
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flurbiprofen. LLOQ was 40.0 μg/L (S/N > 10). The LLOQ of flurbiprofen in our study is smaller than other methods [3,12,13]. Accuracy and
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precision at LLOQ were acceptable. Systematic stasis in the LC-MS/MS
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system was confirmed by comparing pre- and post-sample calibration
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curves, thus we should assume that instrument condition remained still for all sample runs. Since two curves were measured symmetrically in time, minor (if any is present) drift should be reduced in the quantification results.
3.2.3. Precision and accuracy Intra- and inter-day precision and accuracy of the method proposed are presented in Table 1. Intra-day RE varied between 0.2% and 2.2%, RSD was 3.2-8.4%. The inter-day RE varied between -0.5 and 3.4%, RSD was 12 / 12 Page 12 of 26
5.4-8.7%. The precision and accuracy of the proposed assay method
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should be suitable for a pharmacokinetic study.
3.2.4. Recovery and matrix effect
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Recovery and matrix effect are presented in Table 2. The mean extraction recovery of flurbiprofen at three QC concentration levels were 92.5%
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95.3% and 105.4%, RSD were 2.6%, 4.5% and 2.1%. The mean recovery
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of IS was 95.6%, RSD was 9.6%. Mean matrix effect at 100 and 8000μg/L was 80.9% and 81.4%. Mean matrix effect on IS was 67.4%.
3.2.5. Stability
te
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matrix effect was observed.
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RSD of the IS-normalized MFs were 6.9% and 3.7%. No significant
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Stability of flurbiprofen in human plasma samples under various conditions are presented in Table 3. It can be seen that flurbiprofen maintained reasonable stability at room temperature for 24 h, in the auto-sampler for 24 h, after three freeze-thaw cycles, and at −80 ◦C for 32 day. No significant degradation of flurbiprofen was observed under the tested conditions.
3.2.6. Carry-over and dilution There was no carry-over detected in the proposed LC-MS/MS method. 13 / 13 Page 13 of 26
Resulted RE were within ±11.2%, and the RSD were less than 3.3%,
3.3.
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showed dilution of samples did not affect the accuracy.
Bioequivalence study
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As shown in Table 4 main pharmacokinetic parameters of the test and
reference formulation were: t1/2z, 4.94 ± 1.25 h, 4.81 ± 1.10 h; Tmax, 0.24
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± 0.09 h, 0.23 ± 0.09 h; Cmax, 8546.97 ± 1407.93 μg/L, 8611.48 ± 1810.00
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μg/L; AUC0–t, 33961.94 ± 7587.45 μg h/L, 34325.94 ± 10462.46 μg h/L, respectively. The mean plasma concentration-time curves are presented in
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Fig. 3, demonstrating a very similar pharmacokinetic profile.
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93.6-107.1%, 96.1-105.6% and 97.2 -105.7% were 90% CIs of the
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relative values (test: reference) of Cmax, AUC0-t and AUC0-∞. The test
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formulation of flurbiprofen should be bioequivalent to the reference formulation.
(CHANGE MADE IN RESPONSE TO REVIEWER COMMENTS)
3.4.
Comparison with previous studies
The present study used a LC-MS/MS method that provided improvements over legacy HPLC based methods, i.e. the LLOQ is lower without the need of complicated sample preprocessing [2,3,11], the analytical time is shorter [2,3,11], and by not allowing other ions reach 14 / 14 Page 14 of 26
the detector, the selectivity is better. Thus the present method is more suitable for analysis of big amount of samples. Compared with a previously published LC-MS/MS method [14], the present method used a
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simple preprocessing procedure, and achieved a close outcome with
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accuracy of similar level. We conducted additional experiments. The
results were not compromised with interference from source induced
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fragmentations of flurbiprofen axetil and flurbiprofen glucuronide.
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(ANSWER TO REVIEWER COMMENTS: At the end of the result and discussion section we appended comparison with published LC-MS based
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4. Conclusions
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methods.)
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(CHANGE MADE IN RESPONSE TO REVIEWER COMMENTS) We
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have developed and validated a rapid, selective, accurate and reliable LC-MS/MS method for the determination of flurbiprofen in human plasma samples. This method is suitable for measuring in vivo pharmacokinetic parameters of flurbiprofen. This method is suitable for evaluating bioequivalence of flurbiprofen axetil injection formulations. The scalability allows further adoption with large sample sizes. (ANSWER TO REVIEWER COMMENTS: we have rephrased the conclusion.)
15 / 15 Page 15 of 26
Acknowledgments The author would like to thank Chen Xi for technical assistance, Hangao Hu for experimental guidance and Wenming Cao for English writing
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recommendations.
References
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[1] Ohmukai O, Lipo-NSAID preparation[J]. Advanced drug delivery
an
reviews. 20 (1996) 203-207.
[2] A. Qayyum, M.H. Najmi, Determination of pharmacokinetics of
M
flurbiprofen in Pakistani population using modified HPLC method, J Chromatogr Sci. 49 (2011) 108-113.
d
[3] Hutzler, J. Matthew, Reginald F. Frye, and Timothy S. Tracy,
te
Sensitive and specific high-performance liquid chromatographic assay for
Ac ce p
4'-hydroxyflurbiprofen and flurbiprofen in human urine and plasma, J Chromatogr B Biomed Sci Appl. 749 (2000) 119-125. [4] K. Albert, W. Gillespie, A. Raabe, M. Garry, Determination of flurbiprofen in human serum by reverse-phase high-performance liquid chromatography with fluorescence detection, J Pharm Sci. 73 (1984) 1823-1825. [5] J. Askholt, F. Nielsen‐Kudsk, Rapid HPLC-determination of ibuprofen and flurbiprofen in plasma for therapeutic drug control and pharmacokinetic applications, Acta Pharmacol Toxicol (Copenh). 59 16 / 16 Page 16 of 26
(1986) 382-386. [6] M. Contin, S. Mohamed, C. Candela, F. Albani, R. Riva, A. Baruzzi, Simultaneous HPLC-UV analysis of rufinamide, zonisamide, lamotrigine,
ip t
oxcarbazepine monohydroxy derivative and felbamate in deproteinized
cr
plasma of patients with epilepsy, J Chromatogr B 878 (2010) 461-465.
Drug Administration, September 2013,
us
[7] Guidance for industry: bioanalytical method validation, US Food and
an
www.fda.gov/downloads/drugs/guidancecompliancereguLatoryinformatio n/guidances/ucm368107.pdf (accessed 6 May 2014).
M
[8] Guideline on bioanalytical method validation, European Medicines Agency, July 2011,
d
www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/
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2011/08/WC500109686.pdf (accessed 6 May 2014).
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[9] Murray K K, Boyd R K, Eberlin M N, et al, Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013), Pure and Applied Chemistry. 85 (2013) 1515-1609. [10] COMMITTEE FOR MEDICINAL PRODUCTS FOR HUMAN USE (CHMP), GUIDELINE ON THE INVESTIGATION OF BIOEQUIVALENCE, European Medicines Agency, January 2010, www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/ 2010/01/WC500070039.pdf (accessed 20 April 2014). [11] Jin, Yin-Xiu, Yi-Hong Tang, and Su Zeng, Analysis of flurbiprofen, 17 / 17 Page 17 of 26
ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug-protein binding in human plasma, J Pharm Biomed Anal. 46 (2008) 953-958.
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[12] L. Ozbay, D.O. Unal, I. Cakici, A. Fenercioglu, D. Erol, Clinical
Eur J Drug Metab Pharmacokinet. 34 (2009) 1-5.
cr
study on the bioequivalence of two tablet formulations of flurbiprofen,
us
[13] N. Charoo, A.A. Shamsher, K. Kohli, K. Pillai, Z. Rahman, Simple
an
and sensitive high-performance liquid chromatographic method for determination of transdermally applied flurbiprofen in rat plasma and
M
excised skin samples, Chromatographia. 62 (2005) 493-497. [14] Lee H I, Choi C I, Byeon J Y, et al, Simultaneous determination of
d
flurbiprofen and its hydroxy metabolite in human plasma by liquid
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chromatography-tandem mass spectrometry for clinical application, J
Ac ce p
Chromatogr B. 971 (2014) 58-63.
Figure Legends
Fig. 1. Full-scan product ion spectra of [M-H] - for flurbiprofen (A) and indomethacin (IS, B).
Fig. 2. Typical MRM chromatograms of (A) drug-free human plasma, (B) drug-free human plasma supplemented with IS (zero standard), (C) drug-free human plasma supplemented with IS and flurbiprofen at LLOQ of 40.00 μg/L, and (D) a plasma sample at concentration of 5760.22 μg/L. 18 / 18 Page 18 of 26
Peaks I and II represents flurbiprofen and IS, respectively. Fig. 3. Mean (±SD) concentration–time curves of the 2 formulations of flurbiprofen after a single dose (5mL flurbiprofen axetil injection) of the
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test formulation (Wuhan Docan Pharmaceutical Co., Ltd., Wuhan, China)
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and the reference formulation (Beijing Tide Pharmaceutical Co., Ltd.,
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te
d
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an
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Beijing, China) in healthy male Chinese volunteers (n = 24).
19 / 19 Page 19 of 26
Tables Table 1 Intra- and inter-run precision and accuracy for the quantification of
Precision
concentration
(mean ± SD)
RSD (%)
(µg/L)
(µg/L)
100.2 ± 8.4
1000
1022.0 ± 43.9
8000
8036.3 ± 259.8
4.3
2.2
3.2
0.5
103.4 ± 9.0
8.7
3.4
1025.3 ± 88.6
8.6
2.5
7956.3 ± 427.8
5.4
-0.5
d
Ac ce p
1000
0.2
te
100
8000
RE (%)
8.4
M
100
an
Intra-day
Inter-day
Accuracy
cr
Test
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Nominal
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flurbiprofen in human plasma by LC-MS/MS.
20 / 20 Page 20 of 26
Table 2 Extraction recovery and matrix effect of flurbiprofen and IS in human
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plasma (n =6). Nominal concentrations
Extraction recovery
RSD
Matrix effect
(µg/L)
(%)
(%)
100
92.5 ± 2.2
2.6 80 ± 5.1
1000
95.3 ± 3.8
4.5
8000
105.4 ± 2.0
2.1 81.4 ± 2.8
(%)
6.9
3.7
an
us
cr
(%)
RSD
M
Table 3
Stability of flurbiprofen in human plasma under various conditions.
d
cycles
◦C)
Room temperature
Long term (32 days,
(12h)
−80◦C)
1000
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Nominal
Three freeze/thaw
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Autosampler (24 h, 4
5.2
-1.6
3.5
-5.2
4.3
-0.3
2.9
0.1
8000
3.7
0.1
8.3
-3.9
1.1
-3.9
2.7
6.8
concentration (µg/L) 100
Precision
Accuracy
Precision
Accuracy
Precision
Accuracy
Precision
Accuracy
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
4.2
8.7
2.4
-5.9
7.1
3.6
5.9
5.8
21 / 21 Page 21 of 26
Table 4 Pharmacokinetic parameters of flurbiprofen after a single dose of
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50 mg (5 mL flurbiprofen axetil injection) of the test formulation and
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the reference formulation (5 mL flurbiprofen axetil injection) in
Testa
Parameters (unit)
Referenceb
4.94 ± 1.25
Tmax (h)
0.24 ± 0.0
0.23 ± 0.09
8546.97 ± 1407.93
8611.48 ± 1810.00
1.50 ± 0.36
1.53 ± 0.47
33961.94 ± 7587.45
34325.94 ± 10462.46
M
CLz (L/h)
te
d
AUC0–t (µg h/L) AUC0–∞ (µg h/L)
4.81 ± 1.10
an
t1/2z (h)
Cmax (µg/L)
35249.67 ± 8402.25
35447.94 ± 10723.76
flurbiprofen axetil injection (Wuhan Docan Pharmaceutical Co.,
Ac ce p
a
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healthy male Chinese volunteers (n = 24, mean±SD).
Ltd., Wuhan, China). b
flurbiprofen axetil injection (Beijing Tide Pharmaceutical Co., Ltd.,
Beijing, China).
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Highlights
We develop a simple and quick method for the quantification of flurbiprofen in human plasma by LC-MS/MS. Gradient elution in LC is applied.
Instrument conditions checked for stability prior to and after
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sample testing.
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A study of pharmacokinetics and bioequivalence is fulfilled.
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Figure 1
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Figure 2
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Figure 3
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