Accepted Manuscript Title: Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry Author: Ming Liu Hongyun Wang Hongzhong Liu Ao Peng Fen Yang Wenjie Wang Liya Zhu Haihong Huang Ji Jiang Pei Hu PII: DOI: Reference:
S1570-0232(14)00129-9 http://dx.doi.org/doi:10.1016/j.jchromb.2014.02.033 CHROMB 18803
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
12-9-2013 13-2-2014 17-2-2014
Please cite this article as: M. Liu, H. Wang, H. Liu, A. Peng, F. Yang, W. Wang, L. Zhu, H. Huang, J. Jiang, P. Hu, Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.02.033 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.
*Manuscript
Title: Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry
Affiliations:
a
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Wenjie Wang b, Liya Zhu b, Haihong Huang b, Ji Jiang a, Pei Hu a
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Authors: Ming Liu a, Hongyun Wang a, Hongzhong Liu a, Ao Peng a, Fen Yang a,
Clinical Pharmacology Research Center, Peking Union Medical
us
College Hospital and Chinese Academy of Medical Sciences,
No. 1 Shuai Fu Yuan, Dong Cheng District, Beijing 100730, P.R.China Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
an
b
Union Medical College,
Corresponding Author: Pei Hu
M
No. 1 Xiannongtan Street, Xi Cheng District, Beijing 100050, P.R.China
100730, P.R.China.
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Present/permanent address: No.1, Shuaifuyuan. Dongcheng District, Beijing
Contact information: Tel: +8610-69158366; fax: +8610-69158364;
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E-mail: [email protected]
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1 2 3 4
Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry
Ming Liu a, Hongyun Wang a, Hongzhong Liu a, Ao Peng a, Fen Yang a, Wenjie Wang b
, Liya Zhu b, Haihong Huang b, Ji Jiang a, Pei Hu a, *
5
a
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Academy of Medical Sciences, No. 1 Shuai Fu Yuan, Dong Cheng District, Beijing 100730,
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P.R.China;
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b
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College, No. 1 Xiannongtan Street, Xi Cheng District, Beijing 100050, P.R.China
Clinical Pharmacology Research Center, Peking Union Medical College Hospital and Chinese
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Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
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Abstract
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A rapid and selective HPLC-MS/MS method was developed for the determination of
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LBPT in human plasma. The analyte was extracted from plasma samples by
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solid-phase extraction and then chromatographed on a C18 analytical column. The
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mobile phase consisted of acetonitrile-10mM ammonium formate in 0.1% formic acid
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(30:70, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a
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triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM)
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mode using positive electrospray ionization (ESI). The method was validated over the
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concentration range of 0.2-100 ng/mL. Inter- and intra-day precision (RSD %) were
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less than 9.2% and the accuracy (RE %) ranged from 0 to 11.0%. The lower limit of
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quantitation (LLOQ) was 0.2 ng/mL. The extraction recovery was on average 75 %
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and the detection was not affected by the matrix. The method was successfully
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applied to the pharmacokinetic study of LBPT in healthy Chinese subjects.
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Keywords: LBPT; HPLC-MS/MS; Chinese; pharmacokinetics
* Correspondence to Pei Hu. Email: [email protected]
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1. Introduction LBPT, (E)-ethyl 1-(5-(4-chlorophenyl)-3-oxopent-4-enyl) piperidine-4-carboxylate,
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is a platelet activated factor (PAF) receptor antagonist with the potential of
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anti-inflammation and gastric mucosa protecting property. Compared to the
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nonsteroidal anti-inflammatory drugs (NSAIDs) which are known as to induce
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ulceration in the gastrointestinal tract by chronic ingestion [1], the PAF receptor
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antagonists have anti-inflammation effect through a different mechanism [2-5]. LBPT
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is developed for the treatment of rheumatoid arthritis and is currently being evaluated
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in phase I trials.
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To date, few references are available about the determination of LBPT in biological
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samples. In this paper an HPLC-MS/MS method was developed for the determination
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of LBPT in human plasma. The method was validated for specificity, linearity, lower
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limit of quantification (LLOQ), accuracy, precision, stability, and extraction recovery
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and matrix effect. The HPLC-MS/MS method was successfully applied to the
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measurement of human plasma samples from healthy Chinese adults for the
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evaluation of the pharmacokinetics after single oral administration of LBPT tablet.
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2. Materials and methods
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2.1. Drug, chemical standard and reagents
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LBPT hydrochloride (Fig. 1) and its internal standard diphenhydramine
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hydrochloride were provided by the Institute of Material Medica, Chinese Academy
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of Medical Sciences & Peking Union medical college (Beijing, China). Ammonium
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formate was purchased from Sigma–Adrich chemicals (St. Louis, MO USA).
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Acetonitrile and methanol of HPLC grade was purchased from Honeywell Burdick &
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Jackson (Muskegon, MI, USA). HPLC grade water was prepared using a Milli Q 2
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system. LBPT tablets (25 and 100 mg/tablet) were provided by the Institute of
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Material Medica, Chinese Academy of Medical Sciences & Peking Union medical
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college. (Beijing, China).
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2.2. Chromatographic Conditions
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The samples were separated on an XTerra MS C18 analytical column (2.1 mm×50
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mm, 2.5µm) from Waters Corporation (Milford, MA, USA). The mobile phase
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consisted of acetonitrile and 10mM ammonium formate in 0.1% formic acid (30:70,
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v/v). The Chromatography was carried out via a binary system at a flow rate of 0.2
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mL/min at a preset column temperature of 40◦C. The temperature of the sample room
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was set at 15 ◦C and the injection volume was 10 µL.
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2.3. Mass Spectrometry Conditions
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An API 4000 triple quadrapole mass spectrometer (AB Sciex, Forster City, CA,
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USA) was employed for detection. An operation of multiple reaction monitoring
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(MRM) under unit mass resolution (0.7 amu) in both the Q1 and Q3 mass analyzers
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was applied after ionization in the positive mode of an electro-spray ionization (ESI)
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source. After optimization, the source parameters were set as follows: curtain gas, 10
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units; nebulizer gas, 70 units; turbo gas, 80 units; ion spray voltage, 5 kV; and
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temperature, 300 ◦C. The MRM transition was m/z 350→170 for LBPT and m/z
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256→167 for IS, respectively. The optimal collision energy was 25 eV for LBPT and
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17 eV for IS, respectively. The product ion spectra of LBPT and IS were shown in Fig
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2. Data acquiring and processing were performed using the Sciex Analyst software
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version 1.4.1.
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2.4. Stock solutions, calibration and quality control samples The stock solutions of LBPT (1 mg/mL) were prepared in duplicate, one for
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calibration curve and the other for quality control (QC) samples. Calibration standard
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samples and QC samples were prepared by spiking drug free human plasma (mixture
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from 6 individuals) with the stock solutions. The final concentrations in calibration
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standards were 0.2, 0.5, 1, 2, 5, 20, 50 and 100 ng/mL; and the concentrations in QC
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samples were 0.4, 8 and 80 ng/mL.
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The internal standard (IS) was weighed and dissolved in methanol to achieve the
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stock solution at the concentration of 1 mg/mL. The IS working solution (20 ng/mL)
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was prepared by the appropriate dilution of the stock IS solution in water. All stock
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solutions, working solutions, calibration standards and quality control samples were
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immediately stored at -30 ◦C.
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2.5. Sample preparation
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200µL of plasma sample, 20µL of working IS solution (20 ng/mL) and 500µL of
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10mM ammonium formate in 1% formic acid were mixed (30 seconds Vortex) and
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loaded on an OASIS HLB 96 well-plate for SPE (Waters Corporation, Milford, MA,
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USA) that was conditioned with 800µL of methanol and 800µL of water. The solid
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phase was then washed with 800µL of methanol/water (30:70, v/v), and eluted with
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800µL of methanol. The eluate was evaporated to dryness under a stream of nitrogen
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at 40°C and then reconstituted with 220µL of 10mM ammonium formate in 1%
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formic acid. Finally, 10µL of the reconstituted solution was injected to the
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HPLC-MS/MS system.
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2.6. Method validation
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Validation of the method included the assessment of selectivity, linearity, accuracy
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and precision, LLOQ, stability of the analyte at various test conditions, matrix effect
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and recovery. All terms of assay validation were performed according to FDA
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guideline [6]. The selectivity was assessed by analyzing six lots of drug-free human plasma from
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different sources. Endogenous interference at the retention time of LBPT and IS was
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evaluated. The area of interference peak should be within 20% of that of LLOQ and
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5% of that of the internal standard if interference was eluted with LBPT and IS at the
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same retention time. The intra and inter-run precision and accuracy for the method
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were determined by analyzing QC samples at low, medium and high concentration
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levels with 5 replicates per concentration on 3 independent runs. LLOQ was defined
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as the lowest non-zero calibration standard and five replicates of LLOQ samples were
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analyzed. For stability testing, QC samples at low, medium and high concentration
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levels (n=5) were evaluated under the following conditions: (1) 3 freeze and thaw
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cycles(from -30oC to 25oC); (2) in autosampler for 24 h after sample treatment; (3) at
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room temperature for 24 h; (4) at -30oC for 4 months. The extraction recovery was
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evaluated by comparing the peak area of extracted low, medium and high QC samples
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(n=5) with that of the analytes spiked to the blank plasma post-extraction at the same
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concentration.
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Matrix effect was evaluated based on the method described by Matuszewski et al
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[7-9]. Matrix effect was assessed by comparing the peak area of LBPT in different
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lots of plasma, and by comparing peak areas of LBPT spiked into plasma after
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extraction to that of neat solution at the same concentration. The extraction recovery
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was determined by comparing the peak area of the extracted plasma samples spiked
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before and after extraction.
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126 127
2.7. Data acquisition and analysis Data acquiring and processing were performed using the Sciex Analyst (version
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1.4.1, AB Sciex, Forster City, CA, USA). Standard regression and statistical analysis
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were performed with a Watson LIMS software system (version 7.3, Thermo Scientific,
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MA, USA). Calculated concentration data was rounded to the 3 decimal places.
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3.1. Method validation
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3.1.1. Selectivity
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Representative chromatograms of double blank, blank, LLOQ and a plasma sample
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from subject were shown in Fig. 3. It was clearly indicated that no endogenous
138
interference was observed at the retention time of LBPT and IS in extracted plasma
139
samples.
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3.1.2. Linearity
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In this paper, a 1/x2 weighted linear regression of the type Y = aX + b was fitted to
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the calibration curves of LBPT. Three batches of calibration curves were assayed to
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assess the linearity (each batch included two sets of calibration curves), and the
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back-calculated values for each concentration level were shown in Table 1. The
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RSD % for all runs was less than 12.7%, and the RE % from the nominal value varied
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from -12.0% to 13.0%. The mean of R-squared was 0.9866 for all the runs.
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3.1.3. Precision and accuracy
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The intra and inter-run precision and accuracy of the method were determined using
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QC samples at three different concentrations with 5 replicates (i.e., low QC, medium
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QC and high QC). The precision and accuracy of LLOQ were determined using 5 6
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replicates. The results of LLOQ, intra and inter-run precision and accuracy are
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summarized in Table 2. For the assessment of LLOQ, intra and inter-run precision, the
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RSD % of all runs were less than 9.2%, and for accuracy, the RE % ranged from 0 to
154
11.0%.
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3.1.4. Stability
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Plasma QC samples (at three concentration levels) were proved to be stable under
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the following storage conditions: -30oC for 112 days, at room temperature for 24 , 3
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freeze and thaw cycles(from -30oC to 25oC), and storage in the autosampler at 15 oC
159
for 24 h The stability results are summarized in Table 3. The RE % to nominal
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concentration values for the test QC samples was between -8.4% and 6.0%.
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3.1.5. Matrix effect and extraction recovery
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For the assessment of matrix effect, 6 lots of human plasma were extracted as
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described in “sample preparation” and spiked with three concentration levels of LBPT,
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peak areas were compared with relevant levels of sample in neat solution. Results
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were shown in Table 4.
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Matrix effect among different lots of plasma was assessed based on the modified
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[8]
. The “relative” matrix effect, possibility
167
approach described by Matuszewski et al
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of matrix differences between the various lots, was assessed by comparing the analyte
169
peak area corresponding to the different lots at each concentration level, and the
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results were shown in Table 4, the RSD % of the mean peak areas of LBPT at any
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given concentration in six different plasma lots were less than 3.8%, indicating little
172
or no difference in ionization efficiency of LBPT from different plasma lots.
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Furthermore, the “absolute” matrix effect was estimated by comparing mean peak
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area ratios of LBPT for samples spiked after extraction from plasma with the similar
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peak area ratios obtained by injecting neat samples at same concentration directly. As
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showed in Table 4, the matrix effects in three levels of LBPT were 117.8%, 104.4%
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and 102.7%, respectively, showing very little difference of matrix effect in three
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concentration levels. The extraction recovery was calculated by comparing the mean peak areas of LBPT
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spiked before extraction divided by the areas of LBPT of samples spiked after
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extraction and multiplied by 100. Results summarized in Table 5, recoveries for three
182
levels of LBPT were 77.5%, 76.4% and 71.9%, indicating that very little difference in
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extraction recovery was found from low, medium and high concentrations of LBPT.
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3.2. Application in human pharmacokinetic study
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The validated HPLC-MS/MS method was used to determine the plasma samples
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from a pharmacokinetic study of LBPT in healthy Chinese subjects. This clinical
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study was approved by the Ethics Committee of the Peking Union medical
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colleague
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placebo-controlled, parallel group, dose-ranging study to assess the safety and
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pharmacokinetics of LBPT in healthy Chinese subjects. Forty subjects were
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randomized to 4 treatment groups in which they received a single oral dose of 225 mg,
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300 mg, 400mg or 500 mg LBPT on day 1. Serial blood samples were collected at
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specific time points and assayed by the validated method. The mean plasma
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concentration time profiles of LBPT in Chinese subjects after a single dose of 225,
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300,400 or 500 mg were shown in Fig.4.
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This
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4. Discussion
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An HPLC–MS/MS method was developed for the determination of LBPT in human
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plasma. ESI was chosen as the ionization source and the voltage was optimized to
200
acquire the protonated molecular ion [M+H]+ for analyte and IS. The MRM 8
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transitions of m/z 350→170 and m/z 256→167 were selected for LBPT and IS. The
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ionspray voltage was optimized as 5 kV, and collision energy as 25 and 17 eV for
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LBPT and IS. The chromatographic conditions of LBPT were tested on 3 different kinds of
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analytical column products (Symmetry C18, XTerra MS C18 and XTerra RP-18) with
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different microns from Waters corporation (Milford, MA, USA). The results showed that
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the XTerra MS C18, 2.5 micron column would give a most narrow and symmetric
208
peak shape of LBPT. A satisfactory signal-to-noise (S/N) ratio of 75 could be
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achieved using the column, which may provide good accuracy and precision of LLOQ
210
to this study and also allows for determinations of decreased level of LLOQ in the
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future for more extensive pharmacokinetic studies of LBPT.
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The mobile phase system was optimized by trying different kinds of solvents. In
213
our extensive preliminary experiments, methanol, acetonitrile and buffer solution
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(ammonium formate and ammonium acetate with different pH value) had been
215
investigated. Acetonitrile was proved better than methanol to obtain a better
216
sensitivity. The use of 0.1% formic acid solution could also obtain better sensitivity
217
than water solution. Ammonium formate was much better than ammonium acetate for
218
obtaining a good peak shape. Good results in terms of peak shape, retention time and
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separation from interference were obtained using the simple isocratic elution of
220
acetonitrile/10mM ammonium acetate in 0.1% formic acid (30:70, v/v).
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Furthermore, the solid-phase extraction (SPE) conditions were optimized by
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trying different wash solvents and eluting solvents. A methanol/water (30/70, v/v) was
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chosen as the wash solvent which could wash out most of the interference in the
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samples while not eluting any of the analyte or IS. A 100% methanol was proved to
225
obtain a good recovery of both analyte and IS. A reconstitution solution with the same
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constituents of the mobile phase was chosen for obtaining good peak shape and
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sensitivity.
228 229
5. Conclusion An HPLC-MS/MS method for the determination of LBPT in human plasma was
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developed and validated. The limit of reliable quantification was 0.2 ng/mL. The
232
assay afforded the specificity, sensitivity, accuracy and precision needed for
233
quantitative measurements of LBPT. The method had been fully validated and was
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successfully applied to access pharmacokinetic studies of LBPT in Chinese healthy
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subjects.
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Acknowledgements
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The authors would like to thank Institute of Materia Medica, Chinese Academy
239
of Medical Sciences & Peking Union medical college for funding this study. We also
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thank the subjects enrolled in the study and thank the staff of Phase I Unit in Clinical
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Pharmacology Research Center of Peking Union Medical College Hospital.
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References
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[1] J.L. Wallace, Physiol. Rev. 88 (2008) 1547-1565.
247
[2] J. Casals-Stenzel, G. Muacevic, K.H. Weber, J. Pharmacol. Exp. Ther. 24 (1987)
248 249
974-981. [3] M. Merlos, M. Giral, R. Ferrando, M. Quetalt, A. Puigdemont, J. García-Rafanell,
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J. Forn, J. Pharmacol. Exp. Ther. 280 (1997) 114-121.
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255
Herrero-Beaumont, J. Rheumatol. 26 (1999) 1080-1086. [5] H. Oztürk, H. Oztürk, A.I. Dokucu, S. Otcu, Acta. Gastroenterol. Belg. 69 (2006)
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[4] I. Palacios, R. Miqué lez, O. Sá nchez-Pernaute, S. Gutierrez, J. Eqido, G.
197-202.
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[6] US Department of Health and Human Services, Food and Drug Administration
257
Center for Drug Evaluation and Research Center for Veterinary Medicine,
258
Guidance for Industry Bioanalytical Method Validation 2001.
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[7] C.T. Viswanathan, S. Bansal, B. Booth, A.J. DeStefano, M.J. Rose, J. Sailstad,
260
V.P. Shah, J.P. Skelly, P.G. Swann, R. Weiner, Pharm. Res. 24 (2007)
261
1962-1973.
264 265 266
882-889.
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[8] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 70 (1998)
[9] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 75 (2003)
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M
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3019-3030.
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Figure Captions
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Fig. 1. Chemical structure of: a) LBPT hydrochloride; b) Diphenhydramine
269
hydrochloride (IS).
270
Fig. 2. Product ion spectra of: a) LBPT (at CE 25 eV); b) IS (at CE 17 eV).
271
Fig. 3. Representative chromatograms of: a) blank plasma sample without addition of
272
LBPT (left panel) and internal standatd (right panel); b) blank sample with addition of
273
internal standard, c) LLOQ; d) sample from healthy Chinese volunteers.
274
Fig. 4. Mean plasma concentration-time profiles of LBPT in Chinese male and female
275
subjects after a single oral dose of 225, 300, 400 and 500 mg (mean±SD, n=8).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Tables
Table 1 Back-calculated calibration standards of LBPT (n=3) Conc. (ng/mL)
0.500
0.205 0.499 0.00839 0.0635 4.1 12.7 2.5 -0.2 0.9866
1.00
2.00
5.00
20.0
50.0
100
0.910 0.0184 2.0 -9.0
1.76 0.0495 2.8 -12.0
4.52 0.208 4.6 -9.6
19.7 0.643 3.3 -1.5
55.1 1.80 3.3 10.2
113 2.52 2.2 13.0
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Mean SD Precision (RSD %) Accuracy (RE %) R-squared (Mean of 3)
0.200
1
Page 18 of 23
Table 2 LLOQ, intra and inter-run precision and accuracy for LBPT LLOQ
Low
Medium
High
Nominal concentration (ng/mL) Mean SD Precision (RSD %) Accuracy (RE %)
0.200 0.200 0.00832 4.2 0.0
0.400 0.444 0.0356 8.0 11.0
8.00 8.61 0.325 3.8 7.6
80.0 88.1 6.36 7.2 10.1
0.400 0.435 0.0278 6.4 8.8
8.00 8.33 0.415 5.0 4.1
80.0 83.0 7.62 9.2 3.8
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Inter-run (n=15) Nominal concentration (ng/mL) Mean SD Precision (RSD %) Accuracy (RE %)
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Intra-run (n=5)
2
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Table 3 Stability results of LBPT in different test conditions Test conditions 0.400 0.405 1.2
8.00 7.34 -8.2
80.0 77.4 -3.3
Freeze and thaw (3 cycles)
Nominal conc.(ng/mL) Mean RE %
0.400 0.390 -2.6
8.00 7.89 -1.4
80.0 79.4 -0.7
Short term (room temp., 12 h)
Nominal conc.(ng/mL) Mean RE %
0.400 0.408 1.9
8.00 8.06 0.7
Nominal conc.(ng/mL) Mean RE %
0.400 0.401 0.2
(-30℃, 120 days)
80.0 84.8 6.0
cr
Long term
8.00 7.33 -8.4
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(15℃, 24 h)
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Nominal conc.(ng/mL) a Mean RE %
Auto sampler
80.0 78.9 -1.4
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a Nominal concentrations were 0.400, 8.00, 80.0 ng/mL for LBPT in all test conditions.
3
Page 20 of 23
Table 4 Matrix effect results of LBPT Nominal conc. (ng/mL)
Peak area mean (n=6)
0.4 8 80
31833 782833 7871667
Mean peak area of neat sample (n=6) 27033 749500 7666667
SD
RSD %
Matrix effect a (n=6)
1221 18137 199341
3.8 2.3 2.5
117.8 104.4 102.7
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a Matrix effect (%) expressed as the ratio of the mean peak area ratio of LBPT spiked into six different lots of blank plasma samples after extraction to the mean peak area ratio of the LBPT in neat samples and multiplied by 100.
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Table 5 Recovery results of LBPT Mean peak area of extracted (n=5)
Mean peak area of unextracted (n=5)
Recovery a (n=5)
0.4 8 80
24620 600400 5622000
31760 785600 7820000
77.5 76.4 71.9
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Nominal conc. (ng/mL)
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a Recovry (%) expressed as the ratio of the mean peak area of LBPT spiked into plasma before extraction to the mean peak areas of LBPT spiked into plasma after extraction and multiplied by100.
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Page 22 of 23
Highlights
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A rapid and selective HPLC-MS/MS method was developed for the determination of LBPT in human plasma. LBPT was detected on a triple quadrupole tandem mass spectrometer in MRM mode using positive electrospray ionization. Reversed phase chromatographic separation was used to provide good retention and selectivity. The method was validated for specificity, linearity, accuracy and precision, stability, recovery, and matrix effect. The method was applied to the pharmacokinetic studies of LBPT in Chinese subjects.
Page 23 of 23
S1570-0232(14)00129-9 http://dx.doi.org/doi:10.1016/j.jchromb.2014.02.033 CHROMB 18803
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
12-9-2013 13-2-2014 17-2-2014
Please cite this article as: M. Liu, H. Wang, H. Liu, A. Peng, F. Yang, W. Wang, L. Zhu, H. Huang, J. Jiang, P. Hu, Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.02.033 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.
*Manuscript
Title: Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry
Affiliations:
a
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Wenjie Wang b, Liya Zhu b, Haihong Huang b, Ji Jiang a, Pei Hu a
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Authors: Ming Liu a, Hongyun Wang a, Hongzhong Liu a, Ao Peng a, Fen Yang a,
Clinical Pharmacology Research Center, Peking Union Medical
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College Hospital and Chinese Academy of Medical Sciences,
No. 1 Shuai Fu Yuan, Dong Cheng District, Beijing 100730, P.R.China Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
an
b
Union Medical College,
Corresponding Author: Pei Hu
M
No. 1 Xiannongtan Street, Xi Cheng District, Beijing 100050, P.R.China
100730, P.R.China.
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Present/permanent address: No.1, Shuaifuyuan. Dongcheng District, Beijing
Contact information: Tel: +8610-69158366; fax: +8610-69158364;
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E-mail: [email protected]
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Page 1 of 23
1 2 3 4
Determination of LBPT in human plasma by high performance liquid chromatography-tandem mass spectrometry
Ming Liu a, Hongyun Wang a, Hongzhong Liu a, Ao Peng a, Fen Yang a, Wenjie Wang b
, Liya Zhu b, Haihong Huang b, Ji Jiang a, Pei Hu a, *
5
a
6
Academy of Medical Sciences, No. 1 Shuai Fu Yuan, Dong Cheng District, Beijing 100730,
7
P.R.China;
8
b
9
College, No. 1 Xiannongtan Street, Xi Cheng District, Beijing 100050, P.R.China
Clinical Pharmacology Research Center, Peking Union Medical College Hospital and Chinese
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Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
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Abstract
12
A rapid and selective HPLC-MS/MS method was developed for the determination of
13
LBPT in human plasma. The analyte was extracted from plasma samples by
14
solid-phase extraction and then chromatographed on a C18 analytical column. The
15
mobile phase consisted of acetonitrile-10mM ammonium formate in 0.1% formic acid
16
(30:70, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a
17
triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM)
18
mode using positive electrospray ionization (ESI). The method was validated over the
19
concentration range of 0.2-100 ng/mL. Inter- and intra-day precision (RSD %) were
20
less than 9.2% and the accuracy (RE %) ranged from 0 to 11.0%. The lower limit of
21
quantitation (LLOQ) was 0.2 ng/mL. The extraction recovery was on average 75 %
22
and the detection was not affected by the matrix. The method was successfully
23
applied to the pharmacokinetic study of LBPT in healthy Chinese subjects.
24 25
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Keywords: LBPT; HPLC-MS/MS; Chinese; pharmacokinetics
* Correspondence to Pei Hu. Email: [email protected]
1 Page 2 of 23
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1. Introduction LBPT, (E)-ethyl 1-(5-(4-chlorophenyl)-3-oxopent-4-enyl) piperidine-4-carboxylate,
28
is a platelet activated factor (PAF) receptor antagonist with the potential of
29
anti-inflammation and gastric mucosa protecting property. Compared to the
30
nonsteroidal anti-inflammatory drugs (NSAIDs) which are known as to induce
31
ulceration in the gastrointestinal tract by chronic ingestion [1], the PAF receptor
32
antagonists have anti-inflammation effect through a different mechanism [2-5]. LBPT
33
is developed for the treatment of rheumatoid arthritis and is currently being evaluated
34
in phase I trials.
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To date, few references are available about the determination of LBPT in biological
36
samples. In this paper an HPLC-MS/MS method was developed for the determination
37
of LBPT in human plasma. The method was validated for specificity, linearity, lower
38
limit of quantification (LLOQ), accuracy, precision, stability, and extraction recovery
39
and matrix effect. The HPLC-MS/MS method was successfully applied to the
40
measurement of human plasma samples from healthy Chinese adults for the
41
evaluation of the pharmacokinetics after single oral administration of LBPT tablet.
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2. Materials and methods
44
2.1. Drug, chemical standard and reagents
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LBPT hydrochloride (Fig. 1) and its internal standard diphenhydramine
46
hydrochloride were provided by the Institute of Material Medica, Chinese Academy
47
of Medical Sciences & Peking Union medical college (Beijing, China). Ammonium
48
formate was purchased from Sigma–Adrich chemicals (St. Louis, MO USA).
49
Acetonitrile and methanol of HPLC grade was purchased from Honeywell Burdick &
50
Jackson (Muskegon, MI, USA). HPLC grade water was prepared using a Milli Q 2
Page 3 of 23
51
system. LBPT tablets (25 and 100 mg/tablet) were provided by the Institute of
52
Material Medica, Chinese Academy of Medical Sciences & Peking Union medical
53
college. (Beijing, China).
54
2.2. Chromatographic Conditions
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The samples were separated on an XTerra MS C18 analytical column (2.1 mm×50
57
mm, 2.5µm) from Waters Corporation (Milford, MA, USA). The mobile phase
58
consisted of acetonitrile and 10mM ammonium formate in 0.1% formic acid (30:70,
59
v/v). The Chromatography was carried out via a binary system at a flow rate of 0.2
60
mL/min at a preset column temperature of 40◦C. The temperature of the sample room
61
was set at 15 ◦C and the injection volume was 10 µL.
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2.3. Mass Spectrometry Conditions
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An API 4000 triple quadrapole mass spectrometer (AB Sciex, Forster City, CA,
65
USA) was employed for detection. An operation of multiple reaction monitoring
66
(MRM) under unit mass resolution (0.7 amu) in both the Q1 and Q3 mass analyzers
67
was applied after ionization in the positive mode of an electro-spray ionization (ESI)
68
source. After optimization, the source parameters were set as follows: curtain gas, 10
69
units; nebulizer gas, 70 units; turbo gas, 80 units; ion spray voltage, 5 kV; and
70
temperature, 300 ◦C. The MRM transition was m/z 350→170 for LBPT and m/z
71
256→167 for IS, respectively. The optimal collision energy was 25 eV for LBPT and
72
17 eV for IS, respectively. The product ion spectra of LBPT and IS were shown in Fig
73
2. Data acquiring and processing were performed using the Sciex Analyst software
74
version 1.4.1.
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2.4. Stock solutions, calibration and quality control samples The stock solutions of LBPT (1 mg/mL) were prepared in duplicate, one for
78
calibration curve and the other for quality control (QC) samples. Calibration standard
79
samples and QC samples were prepared by spiking drug free human plasma (mixture
80
from 6 individuals) with the stock solutions. The final concentrations in calibration
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standards were 0.2, 0.5, 1, 2, 5, 20, 50 and 100 ng/mL; and the concentrations in QC
82
samples were 0.4, 8 and 80 ng/mL.
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The internal standard (IS) was weighed and dissolved in methanol to achieve the
84
stock solution at the concentration of 1 mg/mL. The IS working solution (20 ng/mL)
85
was prepared by the appropriate dilution of the stock IS solution in water. All stock
86
solutions, working solutions, calibration standards and quality control samples were
87
immediately stored at -30 ◦C.
89
2.5. Sample preparation
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200µL of plasma sample, 20µL of working IS solution (20 ng/mL) and 500µL of
91
10mM ammonium formate in 1% formic acid were mixed (30 seconds Vortex) and
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loaded on an OASIS HLB 96 well-plate for SPE (Waters Corporation, Milford, MA,
93
USA) that was conditioned with 800µL of methanol and 800µL of water. The solid
94
phase was then washed with 800µL of methanol/water (30:70, v/v), and eluted with
95
800µL of methanol. The eluate was evaporated to dryness under a stream of nitrogen
96
at 40°C and then reconstituted with 220µL of 10mM ammonium formate in 1%
97
formic acid. Finally, 10µL of the reconstituted solution was injected to the
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HPLC-MS/MS system.
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99 100
2.6. Method validation
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Validation of the method included the assessment of selectivity, linearity, accuracy
102
and precision, LLOQ, stability of the analyte at various test conditions, matrix effect
103
and recovery. All terms of assay validation were performed according to FDA
104
guideline [6]. The selectivity was assessed by analyzing six lots of drug-free human plasma from
106
different sources. Endogenous interference at the retention time of LBPT and IS was
107
evaluated. The area of interference peak should be within 20% of that of LLOQ and
108
5% of that of the internal standard if interference was eluted with LBPT and IS at the
109
same retention time. The intra and inter-run precision and accuracy for the method
110
were determined by analyzing QC samples at low, medium and high concentration
111
levels with 5 replicates per concentration on 3 independent runs. LLOQ was defined
112
as the lowest non-zero calibration standard and five replicates of LLOQ samples were
113
analyzed. For stability testing, QC samples at low, medium and high concentration
114
levels (n=5) were evaluated under the following conditions: (1) 3 freeze and thaw
115
cycles(from -30oC to 25oC); (2) in autosampler for 24 h after sample treatment; (3) at
116
room temperature for 24 h; (4) at -30oC for 4 months. The extraction recovery was
117
evaluated by comparing the peak area of extracted low, medium and high QC samples
118
(n=5) with that of the analytes spiked to the blank plasma post-extraction at the same
119
concentration.
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120
Matrix effect was evaluated based on the method described by Matuszewski et al
121
[7-9]. Matrix effect was assessed by comparing the peak area of LBPT in different
122
lots of plasma, and by comparing peak areas of LBPT spiked into plasma after
123
extraction to that of neat solution at the same concentration. The extraction recovery
124
was determined by comparing the peak area of the extracted plasma samples spiked
125
before and after extraction.
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126 127
2.7. Data acquisition and analysis Data acquiring and processing were performed using the Sciex Analyst (version
129
1.4.1, AB Sciex, Forster City, CA, USA). Standard regression and statistical analysis
130
were performed with a Watson LIMS software system (version 7.3, Thermo Scientific,
131
MA, USA). Calculated concentration data was rounded to the 3 decimal places.
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134
3.1. Method validation
135
3.1.1. Selectivity
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Representative chromatograms of double blank, blank, LLOQ and a plasma sample
137
from subject were shown in Fig. 3. It was clearly indicated that no endogenous
138
interference was observed at the retention time of LBPT and IS in extracted plasma
139
samples.
140
3.1.2. Linearity
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In this paper, a 1/x2 weighted linear regression of the type Y = aX + b was fitted to
142
the calibration curves of LBPT. Three batches of calibration curves were assayed to
143
assess the linearity (each batch included two sets of calibration curves), and the
144
back-calculated values for each concentration level were shown in Table 1. The
145
RSD % for all runs was less than 12.7%, and the RE % from the nominal value varied
146
from -12.0% to 13.0%. The mean of R-squared was 0.9866 for all the runs.
147
3.1.3. Precision and accuracy
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148
The intra and inter-run precision and accuracy of the method were determined using
149
QC samples at three different concentrations with 5 replicates (i.e., low QC, medium
150
QC and high QC). The precision and accuracy of LLOQ were determined using 5 6
Page 7 of 23
replicates. The results of LLOQ, intra and inter-run precision and accuracy are
152
summarized in Table 2. For the assessment of LLOQ, intra and inter-run precision, the
153
RSD % of all runs were less than 9.2%, and for accuracy, the RE % ranged from 0 to
154
11.0%.
155
3.1.4. Stability
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Plasma QC samples (at three concentration levels) were proved to be stable under
157
the following storage conditions: -30oC for 112 days, at room temperature for 24 , 3
158
freeze and thaw cycles(from -30oC to 25oC), and storage in the autosampler at 15 oC
159
for 24 h The stability results are summarized in Table 3. The RE % to nominal
160
concentration values for the test QC samples was between -8.4% and 6.0%.
161
3.1.5. Matrix effect and extraction recovery
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For the assessment of matrix effect, 6 lots of human plasma were extracted as
163
described in “sample preparation” and spiked with three concentration levels of LBPT,
164
peak areas were compared with relevant levels of sample in neat solution. Results
165
were shown in Table 4.
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Matrix effect among different lots of plasma was assessed based on the modified
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166
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[8]
. The “relative” matrix effect, possibility
167
approach described by Matuszewski et al
168
of matrix differences between the various lots, was assessed by comparing the analyte
169
peak area corresponding to the different lots at each concentration level, and the
170
results were shown in Table 4, the RSD % of the mean peak areas of LBPT at any
171
given concentration in six different plasma lots were less than 3.8%, indicating little
172
or no difference in ionization efficiency of LBPT from different plasma lots.
173
Furthermore, the “absolute” matrix effect was estimated by comparing mean peak
174
area ratios of LBPT for samples spiked after extraction from plasma with the similar
175
peak area ratios obtained by injecting neat samples at same concentration directly. As
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Page 8 of 23
176
showed in Table 4, the matrix effects in three levels of LBPT were 117.8%, 104.4%
177
and 102.7%, respectively, showing very little difference of matrix effect in three
178
concentration levels. The extraction recovery was calculated by comparing the mean peak areas of LBPT
180
spiked before extraction divided by the areas of LBPT of samples spiked after
181
extraction and multiplied by 100. Results summarized in Table 5, recoveries for three
182
levels of LBPT were 77.5%, 76.4% and 71.9%, indicating that very little difference in
183
extraction recovery was found from low, medium and high concentrations of LBPT.
184
3.2. Application in human pharmacokinetic study
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The validated HPLC-MS/MS method was used to determine the plasma samples
186
from a pharmacokinetic study of LBPT in healthy Chinese subjects. This clinical
187
study was approved by the Ethics Committee of the Peking Union medical
188
colleague
189
placebo-controlled, parallel group, dose-ranging study to assess the safety and
190
pharmacokinetics of LBPT in healthy Chinese subjects. Forty subjects were
191
randomized to 4 treatment groups in which they received a single oral dose of 225 mg,
192
300 mg, 400mg or 500 mg LBPT on day 1. Serial blood samples were collected at
193
specific time points and assayed by the validated method. The mean plasma
194
concentration time profiles of LBPT in Chinese subjects after a single dose of 225,
195
300,400 or 500 mg were shown in Fig.4.
196 197
This
was
a
phase
I,
double-blinded,
randomized,
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4. Discussion
198
An HPLC–MS/MS method was developed for the determination of LBPT in human
199
plasma. ESI was chosen as the ionization source and the voltage was optimized to
200
acquire the protonated molecular ion [M+H]+ for analyte and IS. The MRM 8
Page 9 of 23
201
transitions of m/z 350→170 and m/z 256→167 were selected for LBPT and IS. The
202
ionspray voltage was optimized as 5 kV, and collision energy as 25 and 17 eV for
203
LBPT and IS. The chromatographic conditions of LBPT were tested on 3 different kinds of
205
analytical column products (Symmetry C18, XTerra MS C18 and XTerra RP-18) with
206
different microns from Waters corporation (Milford, MA, USA). The results showed that
207
the XTerra MS C18, 2.5 micron column would give a most narrow and symmetric
208
peak shape of LBPT. A satisfactory signal-to-noise (S/N) ratio of 75 could be
209
achieved using the column, which may provide good accuracy and precision of LLOQ
210
to this study and also allows for determinations of decreased level of LLOQ in the
211
future for more extensive pharmacokinetic studies of LBPT.
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The mobile phase system was optimized by trying different kinds of solvents. In
213
our extensive preliminary experiments, methanol, acetonitrile and buffer solution
214
(ammonium formate and ammonium acetate with different pH value) had been
215
investigated. Acetonitrile was proved better than methanol to obtain a better
216
sensitivity. The use of 0.1% formic acid solution could also obtain better sensitivity
217
than water solution. Ammonium formate was much better than ammonium acetate for
218
obtaining a good peak shape. Good results in terms of peak shape, retention time and
219
separation from interference were obtained using the simple isocratic elution of
220
acetonitrile/10mM ammonium acetate in 0.1% formic acid (30:70, v/v).
221
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Furthermore, the solid-phase extraction (SPE) conditions were optimized by
222
trying different wash solvents and eluting solvents. A methanol/water (30/70, v/v) was
223
chosen as the wash solvent which could wash out most of the interference in the
224
samples while not eluting any of the analyte or IS. A 100% methanol was proved to
225
obtain a good recovery of both analyte and IS. A reconstitution solution with the same
9
Page 10 of 23
226
constituents of the mobile phase was chosen for obtaining good peak shape and
227
sensitivity.
228 229
5. Conclusion An HPLC-MS/MS method for the determination of LBPT in human plasma was
231
developed and validated. The limit of reliable quantification was 0.2 ng/mL. The
232
assay afforded the specificity, sensitivity, accuracy and precision needed for
233
quantitative measurements of LBPT. The method had been fully validated and was
234
successfully applied to access pharmacokinetic studies of LBPT in Chinese healthy
235
subjects.
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236
Acknowledgements
M
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The authors would like to thank Institute of Materia Medica, Chinese Academy
239
of Medical Sciences & Peking Union medical college for funding this study. We also
240
thank the subjects enrolled in the study and thank the staff of Phase I Unit in Clinical
241
Pharmacology Research Center of Peking Union Medical College Hospital.
243 244
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References
246
[1] J.L. Wallace, Physiol. Rev. 88 (2008) 1547-1565.
247
[2] J. Casals-Stenzel, G. Muacevic, K.H. Weber, J. Pharmacol. Exp. Ther. 24 (1987)
248 249
974-981. [3] M. Merlos, M. Giral, R. Ferrando, M. Quetalt, A. Puigdemont, J. García-Rafanell,
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J. Forn, J. Pharmacol. Exp. Ther. 280 (1997) 114-121.
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255
Herrero-Beaumont, J. Rheumatol. 26 (1999) 1080-1086. [5] H. Oztürk, H. Oztürk, A.I. Dokucu, S. Otcu, Acta. Gastroenterol. Belg. 69 (2006)
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[4] I. Palacios, R. Miqué lez, O. Sá nchez-Pernaute, S. Gutierrez, J. Eqido, G.
197-202.
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[6] US Department of Health and Human Services, Food and Drug Administration
257
Center for Drug Evaluation and Research Center for Veterinary Medicine,
258
Guidance for Industry Bioanalytical Method Validation 2001.
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[7] C.T. Viswanathan, S. Bansal, B. Booth, A.J. DeStefano, M.J. Rose, J. Sailstad,
260
V.P. Shah, J.P. Skelly, P.G. Swann, R. Weiner, Pharm. Res. 24 (2007)
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1962-1973.
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882-889.
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263
[8] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 70 (1998)
[9] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 75 (2003)
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M
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3019-3030.
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Figure Captions
268
Fig. 1. Chemical structure of: a) LBPT hydrochloride; b) Diphenhydramine
269
hydrochloride (IS).
270
Fig. 2. Product ion spectra of: a) LBPT (at CE 25 eV); b) IS (at CE 17 eV).
271
Fig. 3. Representative chromatograms of: a) blank plasma sample without addition of
272
LBPT (left panel) and internal standatd (right panel); b) blank sample with addition of
273
internal standard, c) LLOQ; d) sample from healthy Chinese volunteers.
274
Fig. 4. Mean plasma concentration-time profiles of LBPT in Chinese male and female
275
subjects after a single oral dose of 225, 300, 400 and 500 mg (mean±SD, n=8).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Tables
Table 1 Back-calculated calibration standards of LBPT (n=3) Conc. (ng/mL)
0.500
0.205 0.499 0.00839 0.0635 4.1 12.7 2.5 -0.2 0.9866
1.00
2.00
5.00
20.0
50.0
100
0.910 0.0184 2.0 -9.0
1.76 0.0495 2.8 -12.0
4.52 0.208 4.6 -9.6
19.7 0.643 3.3 -1.5
55.1 1.80 3.3 10.2
113 2.52 2.2 13.0
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Mean SD Precision (RSD %) Accuracy (RE %) R-squared (Mean of 3)
0.200
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Table 2 LLOQ, intra and inter-run precision and accuracy for LBPT LLOQ
Low
Medium
High
Nominal concentration (ng/mL) Mean SD Precision (RSD %) Accuracy (RE %)
0.200 0.200 0.00832 4.2 0.0
0.400 0.444 0.0356 8.0 11.0
8.00 8.61 0.325 3.8 7.6
80.0 88.1 6.36 7.2 10.1
0.400 0.435 0.0278 6.4 8.8
8.00 8.33 0.415 5.0 4.1
80.0 83.0 7.62 9.2 3.8
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Inter-run (n=15) Nominal concentration (ng/mL) Mean SD Precision (RSD %) Accuracy (RE %)
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Intra-run (n=5)
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Table 3 Stability results of LBPT in different test conditions Test conditions 0.400 0.405 1.2
8.00 7.34 -8.2
80.0 77.4 -3.3
Freeze and thaw (3 cycles)
Nominal conc.(ng/mL) Mean RE %
0.400 0.390 -2.6
8.00 7.89 -1.4
80.0 79.4 -0.7
Short term (room temp., 12 h)
Nominal conc.(ng/mL) Mean RE %
0.400 0.408 1.9
8.00 8.06 0.7
Nominal conc.(ng/mL) Mean RE %
0.400 0.401 0.2
(-30℃, 120 days)
80.0 84.8 6.0
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Long term
8.00 7.33 -8.4
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Nominal conc.(ng/mL) a Mean RE %
Auto sampler
80.0 78.9 -1.4
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Table 4 Matrix effect results of LBPT Nominal conc. (ng/mL)
Peak area mean (n=6)
0.4 8 80
31833 782833 7871667
Mean peak area of neat sample (n=6) 27033 749500 7666667
SD
RSD %
Matrix effect a (n=6)
1221 18137 199341
3.8 2.3 2.5
117.8 104.4 102.7
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a Matrix effect (%) expressed as the ratio of the mean peak area ratio of LBPT spiked into six different lots of blank plasma samples after extraction to the mean peak area ratio of the LBPT in neat samples and multiplied by 100.
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Table 5 Recovery results of LBPT Mean peak area of extracted (n=5)
Mean peak area of unextracted (n=5)
Recovery a (n=5)
0.4 8 80
24620 600400 5622000
31760 785600 7820000
77.5 76.4 71.9
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Nominal conc. (ng/mL)
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a Recovry (%) expressed as the ratio of the mean peak area of LBPT spiked into plasma before extraction to the mean peak areas of LBPT spiked into plasma after extraction and multiplied by100.
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Highlights
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A rapid and selective HPLC-MS/MS method was developed for the determination of LBPT in human plasma. LBPT was detected on a triple quadrupole tandem mass spectrometer in MRM mode using positive electrospray ionization. Reversed phase chromatographic separation was used to provide good retention and selectivity. The method was validated for specificity, linearity, accuracy and precision, stability, recovery, and matrix effect. The method was applied to the pharmacokinetic studies of LBPT in Chinese subjects.
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