Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al h om epage: www.elsev ier .co Determ tyl [2′-(1H 3H (TM-25 ra Sung Heu k Je Eun Sook Sun a Drug Discove hemic b Center for Me Chem c College of Pha d College of Pha nd Dr South Korea a r t i c l e i n f o Article history: Received 20 October 2011 Received in revised form 2 January 2012 Accepted 28 January 2012 Available online 7 February 2012 Keywords: TM-25659 Method valida Pharmacokine LC–MS/MS Rat plasma a b s t r a c t TM-25659 compound, a novel TAZ modulator, is developed for the control of bone loss and obesity. TAZ is known to bind to a variety of transcription factors to control cell differentiation and organ development. A selective and sensitive method was developed for the determination of TM-25659 con- centrations in rat plasma. The drug was measured by liquid chromatography–tandem mass spectrometry 1. Introdu Transcri was origin also called 1 (WWTR1 stem cell d its regulati such as cor factor-2 (R receptor-�) progression actions wi 25659, 2-bu yl)-bipheny ∗ Correspon E-mail add 0731-7085/$ – doi:10.1016/j. tion tics after liquid–liquid extraction with ethyl acetate. TM-25659 and the internal standard imipramine were separated on a Hypersil GOLD C18 column with a mixture of acetonitrile–ammonium formate (10 mM) (90:10, v/v) as the mobile phase. The ions m/z 501.2 → 207.2 for TM-25659 and m/z 281.0 → 86.0 for imipramine in multiple reaction monitoring mode were used for the quantitation. The calibration range was 0.1–100 �g/ml with a correlation coefficient greater than 0.99. The lower limit of quantitation of TM-25659 in rat plasma was 0.1 �g/ml. The percent recoveries of TM-25659 and imipramine were 98.6% and 95.7% from rat plasma, respectively. The intra- and inter-batch precisions were 3.17–15.95% and the relative error was 0.38–10.82%. The developed assay was successfully applied to a pharmacokinetic study of TM-25659 administered intravenously (10 mg/kg) to rats. © 2012 Elsevier B.V. All rights reserved. ction ptional coactivator with PDZ-binding motif (TAZ) ally identified as a 14-3-3 – interacting protein, WW-domain-containing transcription co-regulator- ) [1,2]. TAZ is a key modulator for mesenchymal ifferentiation into osteoblasts and adipocytes through on of lineage-specific master transcription factors e-binding factor-1 (Cbfa1)/runt-related transcription unx2) and PPAR� (peroxisome proliferator-activated [3–7]. It is involved in the control of cell cycle , cell differentiation, and apoptosis through inter- th phosphorylated signaling molecules [8,9]. TM- tyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5- l-4-ylmethyl]-3H imidazo[4,5-b]pyridine], a newly ding author. Tel.: +82 42 860 7170. resses:
[email protected],
[email protected] (M.-S. Kim). synthesized small molecule, enhanced TAZ expression in the nucleus. The TAZ-dependent modulatory activity of TM-25659 in adipocyte and osteoblast differentiation was evidenced by loss of the anti-adipogenic and osteogenic activity of TM-25659 in TAZ- deficient cells. [10]. Thus, TM-25659 may play beneficial roles in the control of obesity and bone loss through activation of TAZ. Liquid chromatography coupled with tandem mass spec- trometry (LC–MS/MS) provides unique capabilities for preclinical biopharmaceutical and clinical pharmacology studies [11–13]. This method is applicable to a wide range of compounds of pharma- ceutical interest with sensitivity, selectivity, speed of analysis, and cost-effectiveness [14,12]. Multiple reaction monitoring (MRM) enables the detection of a specific precursor ion that is programmed to select certain ions chosen by the operator. The precursor ion is fragmented by certain collision energy and it is possible to detect a specific product ion following fragmentation [15,16]. The aim of this study was to develop a LC–MS/MS method for the determination of TM-25659 in rat plasma after simple liquid–liquid extraction (LLE). The method was validated for accuracy, precision, see front matter © 2012 Elsevier B.V. All rights reserved. jpba.2012.01.036 ination of a novel TAZ modulator, 2-bu -tetrazole-5-yl)-biphenyl-4-ylmethyl]- 659) in rat plasma by liquid chromatog m Choia, Kyeong-Ryoon Leea, Jae-Chun Wooa, Na Hwangd, Sung-Hoon Ahna, Myung Ae Baea, Min- ry Platform Technology Team, Medicinal Science Division, Korea Research Institute of C tabolic Syndrome Therapeutics, Medicinal Science Division, Korea Research Institute of rmacy, Chungbuk National University, Cheongju, South Korea rmacy and Division of Life and Pharmaceutical Sciences and Center for Cell Signaling a m/locate / jpba -5-methyl-6-(pyridine-3-yl)-3- imidazo[4,5-b]pyridine] phy–tandem mass spectrometry ong Kimb, Dong Cheul Moonc, Kima,∗ al Technology, Daejon 305-600, South Korea ical Technology, Daejon 305-600, South Korea ug Discovery Research, Ewha Womans University, Seoul 120-750, 48 S.H. Choi et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 selectivity, sensitivity, reproducibility, and stability. This is the first method for the biological quantitation of TM-25659 and its appli- cation for a preliminary pharmacokinetic study. 2. Experim 2.1. Chemic TM-256 at the Kore Korea). Imi MO, USA). O acetonitrile MI, USA). D lipore, Bedf the highest 2.2. Calibra Stock s methanol. W for preparin were made internal sta the stock IS (50:50, v/v) A calibra working sta and 100 �g for TM-256 They were p 2.3. Instrum Sample system (Ag trap Mass S equipped w mode for th MRM mode source tem 50 psi; heat 25659 and 2 the compou 25659, and (ver. 1.4; Ap data collect The LC c column (50 The mobile (10 mM; 90 flow rate w used isocra atures of th respectively 2.4. Sample A volum microfuge 1 �g/ml wa for 1 min. T ple. After vo (13,000 rpm to another room temp and vortexed for 1 min. Finally, 5 �l of the supernatant was injected onto the analytical column. 2.5. Validation anal ctivi f ana gate n cu tios ( . The nal-t d as t abov on an at th ucib four day a mean e err ion (R cura guide atrix mat ssess s (0.3 naly fere ring t 2. T rea o es. abilit stab in fiv abili 659 he s (b) e re to −80 nd 1 at ro y inc osur arm ee m 6.6 ls we t an 3 ◦C ligh intr 0:di (abou d 8 h uged ental als 59 was synthesized by the Medicinal Science Division a Research Institute of Chemical Technology (Daejeon, pramine was purchased from Sigma–Aldrich (St. Louis, rganic solvents of HPLC grade (ethyl acetate, methanol, ) were acquired from Burdick & Jackson Inc. (Muskegon, istilled water was obtained from a Milli-Q system (Mil- ord, MA, USA). All other chemicals and solvents were of analytical grade available. tion standard and quality control samples olution of TM-25659 (1 mg/ml) was prepared in orking standard solutions at the desired concentration g a calibration curve and quality control (QC) samples by serial dilution with methanol:water (50:50, v/v). An ndard (IS) working solution (1 �g/ml) was diluted from solution of imipramine (1 mg/ml) with methanol:water . All solutions were stored at −20 ◦C when not in use. tion curve for TM-25659 was prepared by spiking the ndard solution equivalent to levels of 0.1, 0.3, 5, 40, 80, /ml in blank plasma. QC samples were also prepared 59 concentrations of 0.3, 5, and 80 �g/ml in rat plasma. repared from independent weightings of the analytes. entation and chromatographic conditions analyses were carried out with a 1200 series HPLC ilent, Santa Clara, CA, USA) coupled to an API 4000 Q pectrometer (Applied Biosystems, Foster City, CA, USA) ith a turbo-electrospray interface in positive ionization e LC–MS/MS analysis. The spectrometer was used in . The optimized instrument conditions were as follows: perature, 400 ◦C; curtain gas, 20 psi; nebulizing (GS1), ing (GS2), 50 psi; collision energy (CE), 37 V for TM- 5 V for imipramine. The most abundant product ions of nds were at m/z 207.2 from the parent m/z 501.2 of TM- m/z 86.0 from the m/z 281.0 of the IS. Analyst software plied Biosystems) was used for instrument control and ion. hromatograph was equipped with a Hypersil GOLD C18 mm × 2.1 mm i.d., 3 �m; Thermo, Waltham, MA, USA). phase consisted of acetonitrile–ammonium formate :10, v/v), and was filtered and degassed before use. The as set at 0.3 ml/min for sample analysis. The method tic elution with a total run time of 2 min. The temper- e autosampler and column oven were 4 ◦C and 30 ◦C, . preparation e of 30 �l of rat plasma was aliquoted in a 1.5 ml tube. Then 20 �l of the IS solution of imipramine at s added to an individual sample tube and vortex mixed hen 1 ml of cold ethyl acetate was added to each sam- rtexing for another 10 min, the extract was centrifuged , 5 min, 4 ◦C). Next, the organic phase was transferred tube and evaporated to dryness under nitrogen gas at erature. The residue was dissolved in 1 ml mobile phase The to sele ence o investi ibratio area ra �g/ml the sig define titated Precisi of QCs reprod ples at single of the relativ deviat and ac (FDA) 2.6. M The were a tration of the a with re compa with se peak a five tim 2.7. St The levels tion st TM-25 tests. T bility, exposu sure to 1 day a phase stabilit (b) exp 2.8. Ph Thr 212.9 ± Anima rat die of 23 ± 12/12 h The PEG 40 Blood 2, 4, an centrif ytical method for TM-25659 was validated with regard ty, linearity, accuracy, and precision. For the pres- lytical interferences, the selectivity of the method was d by analyzing the extract from six different sources. Cal- rves were constructed by linear regression of the peak y) of TM-25659 to the IS versus the concentration (x) in lower limit of detection was calculated as three-times o-noise ratio. The lower limit of quantitation (LLOQ) was he lowest concentration that could be accurately quan- e the noise level with acceptable precision (within 20%). d accuracy of the method were evaluated by analyses ree levels in five replicates. The intra- and inter-assay ilities were investigated by analyzing the spiked sam- different concentrations (0.1, 0.3, 5 and 80 �g/ml) in a nd for 5 days, respectively. The percentage of deviation from calculated concentrations was expressed as the or (RE). Precision was expressed as the relative standard SD). All results were within the ranges of precision (%) cy (%) specified by US Food and Drug Administration lines [17]. effect and recovery rix effect, recovery, and process efficiency for TM-25659 ed by analyzing three sets of standards at three concen- , 5 and 80 �g/ml). To determine the matrix effect, those te-spiked post-extraction matrix (set 2) were compared nce standards (set 3). The recovery was determined by the peak areas of analyte spiked before extraction (set 1) he process efficiency was calculated by comparing the f set 3 with that of set 1. Each sample set was analyzed y ility of TM-25659 was assessed by analyzing three QC e replicates under different conditions. The stock solu- ty was estimated at −20 ◦C for 3 weeks. The study of stability in rat plasma included short- and long-term hort-term stability included (a) freeze–thaw cycle sta- xposure of samples to room temperature for 1 day, (c) 4 ◦C for 1 day, (d) exposure to −20 ◦C for 1 day, (e) expo- ◦C for 1 day, (f) exposure in the mobile phase at 4 ◦C for week after preparation, and (g) exposure in the mobile om temperature for 1 day after preparation. Long-term luded (a) exposure of samples to −80 ◦C for 30 days and e to −20 ◦C for 30 days. acokinetic study ale Sprague–Dawley rats, aged 8 weeks and weighing g, were used for the pharmacokinetic disposition study. re kept in plastic cages with free access to standard d water. The room was maintained at a temperature , relative humidity of 50 ± 10%, and an approximately t/dark cycle. avenous dose solutions (10 mg/kg) were prepared in stilled water:dimethyl sulfoxide at a ratio of 40:55:5. t 0.2 ml) was collected at pre-dose, 0.033, 0.167, 0.5, 1, after intravenous administration. Blood samples were immediately and stored at −80 ◦C until analysis. S.H. Choi et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 49 A non-compartmental method using the nonlinear least- squares regression program WinNonlin (Pharsight, Mountain View, CA, USA) was used to calculate the pharmacokinetic parameters. The area under the plasma concentration–time curve from time zero to the last measured concentration (AUC0 → last) and to infi- nite time (AUC0→ ∞) by adding extrapolated area were estimated. The terminal elimination half-life (t1/2), total body clearance (CL), volume of distribution at steady state (Vss), and mean residence time (MRT) for TM-25659 were determined by individual plasma concentration–time profiles. 3. Results and discussion 3.1. Mass spectra and chromatography The MRM mode produces very specific and sensitive responses for the selected analytes. TM-25659 has mass spectrometric response either in the positive ionization or in the negative ion- ization mode, although signal intensity in positive mode is higher than that in the negative mode. TM-25659 and the IS were inves- tigated for the abundant precursor ions [M+H]+ at m/z 501.2 and 281.0, respectively. The precursor ions of the analyte and IS were formed using declustering potentials of 111 and 75 V, respectively. The quantitation of analytes was performed using MRM mode for high selectivity and sensitivity of acquisition data. To confirm the correct identification and to prevent false positives, two or more different ions were selected for each analyte, and the peak area ratio of two selected ions (quantitative ion and confirmative ion) was compared with that of the standard compound. Fig. 1 shows the chemical structure and product ion mass spectra of TM-25659 and the IS (imipramine). The predominant ion at m/z 207.2 was chosen for the quantitation, and that at m/z 267.2 was used as the confir- mative ion for TM-25659. Imipramine was fragmented to produce intense product ion signals at m/z 281.0 → 86.0. The optimal values of the collision energy were 37 and 25 eV for TM-25659 and the IS, respectively. The chromatographic separation was performed on a Hypersil GOLD C18 column offering excellent peak shape, efficient separa- tion, desired linearity and reproducibility for TM-25659 and IS in plasma matrix. The optimization of the mobile phase was based on peak selec- tivity and retention time. Increasing the ratio of organic solvent, the peak shapes of TM-25659 and the IS were sharp, but the retention time decreased. Therefore, a mixture of acetonitrile–ammonium formate (10 mM) (90:10, v/v) was adopted as the isocratic mobile phase. TM-25659 and the IS were retained on the column with good peak shape at the retention times of 0.59 and 1.44 min, respectively. Fig. 2 shows the typical peak shapes and retention times of MRM chromatograms. 3.2. Sample preparation For the extraction, LLE was chosen because of its efficiency, specificity, and low cost. Ethyl acetate, methylene chloride, and methyl-t-butyl ether (MTBE) were tested as the LLE solvents to extract TM-25659 from spiked plasma. Methylene chloride and MTBE exhibited relatively low extraction efficiency (recoveries 63% and 14%, respectively). Ethyl acetate is the most effective solvent for the recovery of spiked TM-25659 (recovery 91%). Although the relatively polar solvent is able to extract polar impurities, no inter- ference peaks and a stable baseline appeared in MRM mode. The extraction time required no more than 10 min and 1 ml of solvent was enough to efficiently extract TM-25659 from a 30 �l plasma sample. 3.3. Validation and matrix effect Typical MRM chromatograms of six different lots of rat plasma, double blank plasma, blank plasma, and the LLOQ samples are TM-2 Fig. 1. The structures and product-ion scan spectra of (A) 5659 and (B) imipramine (IS). 50 S.H. Choi et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 Fig. 2. Repres TM-25659 and shown in F that directl IS. The cali ear least s area ratio v of 0.1–100 � quantificati Table 1 Reproducibilit Theoretical c 0.1 0.3 5 80 a RSD (%) = s b RE (%) = (ca entative MRM chromatograms of (a) double blank rat plasma, (b) blank rat plasma spik the IS, (d) a plasma sample obtained 1 h after intravenous administration of TM-25659 a ig. 2. No significant endogenous peaks were observed y interfered at the retention times of the analyte or bration curve for TM-25659 was generated by a lin- quares regression analysis of the TM-25659/IS peak ersus the amount of spiked TM-25659 in the range g/ml. The weighted regression (i.e., 1/x) was used for on of the analytes. The coefficient of determination (R2) for the mea 0.99, indica ducibilities precision. A 0.38 to 5.62 to 6.03%, an with RSD v accuracy. y and accuracy for TM-25659 in rat plasma (n = 5). oncentration (�g/ml) Intra-day Concentration found (�g/ml) RSDa (%) REb (% 0.11 6.03 5.62 0.29 3.73 1.95 5.18 3.97 3.63 80.31 3.17 0.38 tandard deviation of the concentration/mean concentration ×100. lculated concentration − theoretical concentration)/theoretical concentration × 100. ed with the IS, (c) blank rat plasma spiked with 0.1 �g/ml (LLOQ) of t 10 mg/kg to rats. n standard curve of five different lots of plasma was ting excellent linearity. The intra- and inter-assay repro- of the assay were investigated in terms of accuracy and s shown in Table 1, the intraday accuracy ranged from % (defined as RE), with RSD values ranging from 3.17 d the inter-day accuracy ranged from 3.85 to 10.82%, alues ranging from 8.17 to 15.95%, indicating excellent Inter-day ) Concentration found (�g/ml) RSD (%) RE (%) 0.11 15.95 10.82 0.27 8.17 9.21 4.81 13.15 3.85 85.95 14.04 7.44 S.H. Choi et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 51 Table 2 Recovery, matrix effect and process efficiency (n = 5). Concentration (�g/ml) Matrix effecta (%) Recoveryb (%) Process efficiencyc (%) TM-25659 0.3 74.1 99.5 72.2 5 79.8 98.9 78.5 80 77.9 97.5 76.3 Mean 77.3 98.6 75.7 IS (Imipramine) 1 94.6 95.7 90.6 a Matrix effect expressed as the ratio of the mean peak area of an analyte added post-extraction (set 2) to the mean peak area of the same analyte standards (set 3) multiplied by 100. b Recovery calculated as the ratio of the mean peak area of an analyte added before extraction (set 1) to the mean peak area of an analyte spiked post-extraction (set 2) multiplied by 100. c Process efficiency calculated as the ratio of the mean peak area of an analyte added before extraction (set 1) to the mean peak area of the same analyte standards (set 3) multiplied by 100. The matrix effects are considered attributable to co-eluting, reducing, or enhancing the ion intensity of the analytes. The mean matrix effects at TM-25659 concentrations of 0.3, 5, and 80 �g/ml were 74.1, 79.8, and 77.9%, respectively; the mean percentage recoveries at the three concentrations were 99.5, 98.9, and 97.5%, respectively (Table 2). These results indicate the presence of some matrix effect in terms of the TM-25659/imipramine response ratio, but the LLOQ signal intensity was sufficient. To evaluate the process efficiency, t with standa mined by a from the m TM-25659 w enough to a 3.4. Stabilit The stab ples under processed s (1 mg/ml) w 25659 was Table 3 TM-25659 i plasma we term, respe freeze–thaw on the quan room temp TM-25659 a ean plasma concentration–time plot of TM-25659 after intravenous admin- of TM-25659 at 10 mg/kg to rats (mean ± standard deviation, n = 3 rats). tively, and 82.5, 73.7, and 86.6% at 4 ◦C for 1 day. Therefore, sample storage conditions are required above −20 ◦C. plication to clinical testing method has been successfully applied to analyze plasma s obtained from male rats that received intravenous TM- at a dose of 10 mg/kg. Fig. 3 illustrates the mean plasma tration profiles of TM-25659 in rats. The concentration of 659 was readily measurable in plasma samples collected up Table 3 Stability of TM Condition te QCM (5 �g/ml) QCH (80 �g/ml) Mean RSD (%) RE (%) Mean RSD (%) RE (%) Short-term s Control sam – 1.2 – 7.6 – Freeze–thaw 4.98 0.6 −0.3 80.53 1.8 0.6 Bench (room Refrigerator Freezer (−20 Freezer (−80 Post-prepara Post-prepara Post-prepara Long-term s Freezer (−80 Freezer (−20 a RSD (%) = s b RE (%) = (ca he standards spiked before extraction were compared rds injected directly in the mobile phase. It is deter- combination of matrix effects and analyte recovery atrix by sample extraction. The process efficiency of as 72.2–78.5%. The sensitivity of this method was high nalyze samples during the pharmacokinetic study. y ility of TM-25659 was investigated in QC plasma sam- a variety of conditions used for sample handling and amples. The stock solution of TM-25659 in methanol as confirmed to be stable for 3 weeks at −20 ◦C; TM- stable, ranging from 90.0 to 98.8%. summarizes the short-term and long-term stability of n plasma. There was no significant change when fresh re kept at −20 ◦C and −80 ◦C for the short and long ctively, in QC samples (0.3, 5 and 80 �g/ml). Three cycles and post-preparative stability had little effect titation. However, the concentration of TM-25659 at erature and 4 ◦C for 1 day decreased. The stability of t room temperature for 1 day was 81.4, 70.0, and 73.6%, Fig. 3. M istration respec careful 3.5. Ap The sample 25659 concen TM-25 -25659 in rat plasma (n = 5). sted QCL (0.3 �g/ml) Mean RSDa (%) REb (%) tability ples (freshly prepared) – 10.0 – (−80 ◦C, 3 cycle) 0.28 1.6 −7.5 temperature, 1 day) 0.24 0.2 −18.6 3.5 (4 ◦C, 1 day) 0.25 0.9 −17.5 3.6 ◦C, 1 day) 0.27 0.6 −9.8 4.4 ◦C, 1 day) 0.33 0.8 9.8 4.7 tive stability (4 ◦C, 1 day) 0.30 1.0 −0.8 5.0 tive stability (4 ◦C, 1 week) 0.31 0.9 4.6 5.3 tive stability (room temperature, 1 day) 0.33 0.9 9.9 4.7 tability ◦C, 30 days) 0.32 0.8 7.3 4.8 ◦C, 30 days) 0.24 2.6 −19.6 4.8 tandard deviation of the concentration/mean concentration × 100. lculated concentration − theoretical concentration)/theoretical concentration × 100. 0 0.8 −30.0 58.81 1.9 −26.4 8 0.3 −26.3 69.25 1.5 −13.4 0 0.5 −12.0 78.34 1.5 −2.0 0 0.2 −6.0 80.12 0.8 0.1 0 0.3 −0.0 83.38 1.4 4.2 9 1.8 7.7 80.09 2.1 0.1 9 0.9 −4.2 73.72 1.1 −7.8 8 1.2 −2.4 71.08 1.5 −11.1 0 0.8 −3.9 69.04 2.8 −13.6 52 S.H. Choi et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 47– 52 to 8 h post-dose. The terminal half-life and AUC∞ values of TM- 25659 were 3.51 ± 0.62 h and 39.80 ± 3.75 �g h/ml, respectively. The AUC0 → last/AUC0 → ∞ ratio was higher than 75% for all sub- jects (mean values, 81.02 ± 5.53%). The CL, Vss, and MRT were 0.25 ± 0.02 l/h/kg, 1.09 ± 0.21 l/kg, and 2.20 ± 0.06 h, respectively. 4. Conclusions A sensitive and selective LC–MS/MS method was developed and validated for determining TM-25659 in rat plasma. Plasma sam- ples were treated with LLE followed by LC–MS/MS analysis. The present assay was demonstrated in terms of selectivity, linearity, accuracy, precision, and stability. The R2 was greater than 0.99, and the recovery of QC samples was 98.6%. The intraday and inter- day accuracy was ≤15.95% RSD and precision was less than 10.82% RE. In the stability test, some losses of TM-25659 were observed under room temperature and 4 ◦C, so the samples should be kept below −20 ◦C. The method was successfully applied to a pharma- cokinetic study of TM-25659 in rats. The successful application of this method to a pharmacokinetic study supports its applications in future preclinical studies on TM-25659. Acknowledgement This research was supported by the Ministry of Knowledge Economy (Grant NO. 2011-10033279). References [1] Z. Strakova, J. Reed, I. Ihnatovych, Human transcriptional coactivator with PDZ- binding motif (TAZ) is downregulated during decidualization, Biol. Reprod. 82 (2010) 1112–1118. [2] A. Mitani, T. Nagase, K. Fukuchi, H. Aburatani, R. Makita, H. Kurihara, Transcriptional coactivator with PDZ-binding motif is essential for normal alve- olarization in mice, Am. J. Respir. Crit. Care Med. 180 (2009) 326–338. [3] J.H. Hong, E.S. Hwang, M.T. McManus, A. Amsterdam, Y. Tian, R. Kalmukova, E. Mueller, T. Benjamin, B.M. 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Determination of a novel TAZ modulator, 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-... 1 Introduction 2 Experimental 2.1 Chemicals 2.2 Calibration standard and quality control samples 2.3 Instrumentation and chromatographic conditions 2.4 Sample preparation 2.5 Validation 2.6 Matrix effect and recovery 2.7 Stability 2.8 Pharmacokinetic study 3 Results and discussion 3.1 Mass spectra and chromatography 3.2 Sample preparation 3.3 Validation and matrix effect 3.4 Stability 3.5 Application to clinical testing 4 Conclusions Acknowledgement References