Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202

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Development and validation of a highly sensitive LC–MS/MS method for quantification of IC87114 in mice plasma, bronchoalveolar lavage and lung samples: Application to pharmacokinetic study Anu Marahatta a , Bidur Bhandary a , Yong-Chul Lee b , So-Ri Kim b , Han-Jung Chae a,∗ a b

Department of Pharmacology, School of Medicine, Chonbuk National University, Jeonju 560-182, Republic of Korea Department of Internal Medicine, School of Medicine, Chonbuk National University, Jeonju 560-182, Republic of Korea

a r t i c l e

i n f o

Article history: Received 12 September 2013 Received in revised form 2 November 2013 Accepted 5 November 2013 Available online 14 November 2013 Keywords: IC87114 Pharmacokinetic LC–MS/MS Intratracheal PI3K

a b s t r a c t IC87114 is a selective PI3K␦ inhibitor. A simple, sensitive and reliable LC–MS/MS method with rapid sample preparation was developed and validated for the determination of IC87114 in mouse plasma, bronchoalveolar lavage, and lung. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C18 column (150 mm × 2.1 mm internal diameter, 3.5 ␮m particle size). Mass spectrometric detection was conducted by electrospray ionization in positive ion multiple reaction monitoring modes. The calibration curve was linear over a concentration range of 0.01–1000 ng/mL for plasma/BAL and 0.1–250 ng/mL for lung tissue. Recoveries were as high as 97.29%, 102.81% and 89.70% for plasma, BAL fluid and lung sample, respectively. The lower limit of quantification was 0.01 ng/mL. Intra-day and interday accuracy and precision were within the acceptable limits of ±15% at all concentrations. Finally, the method was successfully used in a pharmacokinetic study that measured IC87114 in mouse plasma, BAL fluid and lung tissue after administration of a single 1 mg/kg intratracheal dose of IC87114. The percentage change for incurred sample reanalysis (ISR) was within ±15.0% and met the acceptance criteria for ISR. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Phosphatidylinositide 3-kinases (PI3K) family proteins catalyze the phosphorylation of phosphoinositides at the 3-hydroxyl position and generate lipids that control a wide variety of intracellular signaling pathways. PI3Ks can be classified into class I, class II, and class III subfamilies according to their structure and substrate specificity [1,2]. Class I PI3Ks are responsible for the production of phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2), and phosphatidylinositol (3,4,5)trisphosphate (PI(3,4,5)P3). PI3Ks are involved in different cellular functions such as cell growth, proliferation, motility, differentiation, survival and intracellular trafficking [3]. PI3Ks affect key signaling pathways via several downstream molecules such as protein kinase B (PKB/AKT), 3 -phosphoinositide dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and glycogen synthase kinase 3 (GSK3), thus modulating different biological functions such as cell proliferation, cell survival, cell differentiation, migration, metabolism and chemotaxis [4]. There are four isoforms of PI3K class I: ␣, ␤, ␥, and ␦. Among these, the ␣, ␤ and ␦ subunits are grouped together as class IA due to their association with the

∗ Corresponding author. Tel.: +82 63 270 3092; fax: +82 63 275 2855. E-mail address: [email protected] (H.-J. Chae). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.

adapter subunit, p85. PI3K␦ isoforms are predominantly expressed in hematopoietic cells [5]. PI3K␦ inhibitors are also novel therapeutic targets to treat rheumatoid arthritis and other inflammatory diseases. Use of PI3K␦ inhibitor has even been shown to enhance the sensitivity of beta(2)-agonist formoterol against oxidative stressinduced corticosteroid resistant chronic obstructive pulmonary disease (COPD) [6]. Asthma is one of the most common chronic diseases in the world and is characterized by reversible airway obstruction and airway hyperresponsiveness [7]. Corticosteroids are the most potent of the anti-inflammatory drugs used to treat asthma. Some reports have linked poor response to corticosteroids in patients with COPD or asthma to oxidative stress. Corticosteroids are also well known to have other side-effects. Hence, there the need exists to develop new drugs to treat pulmonary diseases such as asthma and COPD. Recently a selective inhibitor of PI3K␦, IC87114 (Fig. 1), has been widely studied for the treatment of pulmonary diseases including asthma and COPD [6,8,9]. This inhibitor was found to reduce the polarized morphology of neutrophils, formyl-methionyl-leucylphenylalanine (fMLP)-stimulated PIP3 production and chemotaxis. It has also been used to explore the importance of PI3K␦ in neutrophil migration [10]. The objective of this study was to develop and validate a highly sensitive LC–MS/MS method for the quantitative analysis of IC87114 in mouse plasma, BAL fluid and lung samples.


A. Marahatta et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202 Table 1 Multiple reaction monitoring (MRM) transition and condition for IC87114 and IS. Compound

Precursor ion

Product ion

Frag (V)

CE (V)


IC87114 Acetaminophen (IS)

398 151.900

146 110

130 130

35 20

Positive Positive

a capillary. The chest cavity was exposed to allow expansion, after which the trachea was carefully intubated and the catheter secured with ligatures. The lungs were lavaged with 1 mL of 0.9% NaCl. The bronchoalveolar lavage (BAL) fluid was centrifuged at 400 × g for 10 min at 4 ◦ C to pellet the cell fraction, and the supernatant was stored at −80 ◦ C until the measurements were carried out. Fig. 1. Chemical structure of IC87114.

2.6. Chromatography 2. Experimental 2.1. Chemicals and reagents IC87114 was purchased from Calbiochem (San Diego, CA). Acetaminophen was purchased from Sigma (St. Louis, MO, USA). Water was purified by a Milli-Q system from Millipore (Bedford, MA, USA). LC–MS grade acetonitrile, methanol and formic acid were from Fischer Scientific (Fair Lawn, NJ, USA). 2.2. Preparation of calibration standards and quality control (QC)

HPLC separation was performed on an Agilent 1100 system (Agilent, Palo Alto, CA, USA). Chromatographic separation was achieved using a Zorbax Eclipse XDB-C18 column (150 mm × 2.1 mm internal diameter, 3.5 ␮m particle size). Separation of IC87114 and internal standard was carried out by gradient elution. The mobile phase was composed of (A) 10 mM ammonium formate, and (B) acetonitrile. The gradient run started from 10% solvent B for 1 min, increasing up to 100% over 6 min and remaining there for another 1 min, followed by a return to 10% for 15 min. Chromatography was performed at 30 ◦ C with a flow rate of 0.3 mL/min, and the run time was 15 min. The injection volume was 2 ␮L of each sample.

Stock solutions of IC87114 were prepared by dissolving 5 mg of the drug in 1 mL of dimethyl sulfoxide (DMSO) and were stored at −20 ◦ C. Internal standard (IS; acetaminophen) was dissolved in methanol. Calibration standards were prepared by spiking working standards and IS into 50 ␮L of blank plasma, BAL fluid or lung tissue homogenates from an untreated mouse. The final concentrations of the standard curve samples were 0.01–1000 ng/mL for plasma and BAL, and 0.1–250 ng/mL for lung tissue. The IS concentration was 1 ng/mL in each sample. QC samples were prepared at six concentrations (1000, 500, 100, 10, 0.1, 0.01 ng/mL for plasma/BAL fluid, or 250, 100, 50, 10, 0.1 ng/g for lung tissue). The calibration standard samples and QC samples were stored at −20 ◦ C until analysis.

An Agilent Technologies 6410 triple quadruple mass spectrometer equipped with electrospray ionization (ESI) in the positive ionization mode was used. The following conditions were found to be optimal for analysis: capillary voltage 4 kV, gas temperature 300 ◦ C, dwell time 200 ms and gas flow 10 L/min. Samples were analyzed by multiple reactions monitoring (MRM). Precursor ions, product ions, and MS/MS parameters are reported in Table 1. Mass hunter software was used to control the LC–MS/MS system and for data analysis.

2.3. Animals

2.8. Sample preparation procedure

The animal ethics committee at Chonbuk National University of Korea approved all experiments. C57B/L6 mice were obtained from Damul Science (Deajeon, South Korea). Eight-week-old male mice were kept in a fully acclimatized room at constant temperature and humidity on a 24-h light/dark cycle. The animals had free access to food and water.

2.8.1. Plasma and BAL fluid Proteins were precipitated by the addition of 5 ␮L of IS (100 ng/mL) and 445 ␮L of methanol to 50 ␮L plasma/BAL. The resulting solution was vortexed for 30 s and centrifuged at 13,000 × g for 20 min. The supernatant was transferred to a HPLC vial and injected.

2.4. Nonsurgical intratracheal administration method

2.8.2. Lung Lung tissues were cut into small pieces with scissors and were homogenized in a phosphate buffer saline solution (PBS) using a Polytron PT 1200C homogenizer (Kinematica INC., Switzerland). Proteins were precipitated by the addition of 5 ␮L of IS (100 ng/mL) and 445 ␮L of methanol to 50 ␮L lung solution. The resulting solution was vortexed for 30 s, centrifuged at 13,000 × g for 20 min and an aliquot of the supernatant was used for analysis.

Mice were given a single dose of IC87114 (1 mg/kg) intratracheally (IT) by a nonsurgical method [11]. We choose a dose of 1 mg/kg because of its proven efficacy for treatment of pulmonary disease [9,12,13]. The tongue was pulled out with forceps and held to the side. Using the other hand, the bent gavage needle of a 1 mL syringe containing the drug was inserted into the trachea and the plunger was pushed down to deliver the drug. Blood samples were collected at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 18, 24, 48 and 72 h. Six animals were scarified at each time point. 2.5. Bronchoalveolar lavage Mice were anesthetized using intraperitoneal pentobarbital sodium. Blood was collected from the eyes of euthanized mice using

2.7. Mass spectrometry

2.9. Specificity Specificity is the ability of an analytical method to differentiate and quantify the analytes in presence of other components in the sample. The specificity of the method was evaluated by screening six different lots of blank plasma, BAL fluid and lung. Selectivity was evaluated by analyzing different blank plasma, BAL fluid and

A. Marahatta et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202


Fig. 2. The representative chromatograms of IC87114: (A) std 10 ng/mL, (B) blank lung sample, (C) 12 h BAL fluid, (D) 48 h lung sample, (E) 15 min plasma sample, (F) spiked 50 ng/mL lung sample, (G) IS 1 ng/mL, and (H) blank plasma spiked with IS.

lung samples to investigate the potential interfaces at the retention times of IC87114 and IS. 2.10. Linearity and limit of quantification (LOQ) Calibration curves of IC87114 in plasma/BAL and tissue samples were established over a concentration range of 1000–0.01 ng/mL for plasma/BAL and 250–0.1 ng/g for tissue. Calibration curves were

Table 2 Recovery and matrix effect of IC87114 in plasma, BAL fluid and lung tissue (n = 5). Recovery (%)


Concentration (ng/mL or ng/g)


0.010 0.100 10 100 500 1000

97.296 95.594 85.449 78.408 90.944 92.011

± ± ± ± ± ±

7.802 9.805 9.563 11.349 5.321 5.510

90.532 102.198 94.957 89.148 102.669 92.598

± ± ± ± ± ±

12.949 5.013 3.343 7.319 1.899 3.824

0.010 0.100 10 100 500 1000

100.435 88.685 97.001 93.620 85.950 102.811

± ± ± ± ± ±

1.013 9.769 10.825 5.962 5.429 10.284

95.562 95.583 90.320 97.094 100.873 90.117

± ± ± ± ± ±

9.781 4.253 2.880 4.584 8.433 3.519

0.100 10 50 100 250

84.436 85.622 89.703 78.079 85.497

± ± ± ± ±

9.542 10.705 1.786 7.466 7.445

87.615 91.156 87.652 93.166 94.527

± ± ± ± ±

9.059 6.356 6.344 4.082 4.401

BAL fluid


Matrix effect (%)

obtained by determining the best fit of the peak-area ratios (peak area analyte/peak area IS) versus concentration, and were fitted to a linear model y = mx + c using a weighting factor (1/X2 ). The LOQ was defined as the lowest concentration that provided a peak area with signal to noise ratio >10. 2.11. Recovery and matrix effect Extraction recovery tests were performed by comparing the peak areas of the sample prepared by plasma/BAL and lung tissue extraction to those from directly injected standards. Recoveries were evaluated at five concentrations for plasma/BAL and tissue samples. The matrix effects may cause ionization suppression or enhancement, generally due to the influence of co-eluting compounds on the ionization process of the actual analyte [14]. 2.12. Precision and accuracy Accuracy and precision were evaluated at the QC concentrations of 0.01, 0.1, 10, 100, 500, and 1000 ng/mL for plasma, BAL fluid and lung samples. Intra-day and inter-day accuracy (relative error) and precision (% relative standard deviation or RSD) were assessed by analyzing sample concentrations at 1000, 500, 100, 10, 0.1, and 0.01 ng/mL for IC87114. To determine the intra-day precision of the method, five different concentrations of plasma/BAL and lung tissue were analyzed five times on the same day. The inter-day precision and accuracy were evaluated five times in other independent samples extracted on three separate days. Accuracy was calculated as the percent deviation from the nominal concentration.


A. Marahatta et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202

Table 3 Intra-day precision and accuracy. Sample

Concentration (ng/mL or g)

Intraday (n = 3) IC87114 ± SD

Precision (%RSD)

Accuracy (%bias)


0.010 0.100 10 100 500 1000

0.009 0.095 8.744 88.310 469.810 905.961

8.019 10.257 11.192 1.126 11.427 6.398

10.000 5.000 12.560 11.690 6.038 9.404

BAL fluid

0.010 0.100 10 100 500 1000

0.010 0.097 9.021 95.699 443.154 970.858

0.000 0.471 7.323 5.429 1.986 2.239

0.000 3.000 9.790 4.301 11.369 2.914

0.100 10 50 100 250

0.087 9.315 43.256 89.984 218.925

9.043 5.417 9.811 2.331 7.108

13.000 6.850 13.488 10.016 12.430


Table 4 Inter-day precision and accuracy. Sample

Concentration (ng/mL or g)

2.14. Incurred sample reanalysis Reproducibility of the analytical method was further evaluated by re-analysis of ISR. An incurred sample re-analysis was also conducted by selection of 7 samples from plasma, BAL fluid and lung samples, which were near Cmax and in elimination phase of the pharmacokinetic profile of drug. The re-analysis data for IC87114 were compared with data from the original study.

3. Results and discussions Interday (n = 3)

3.1. Mass spectrometry IC87114 ± SD

Precision (%RSD)

Accuracy (%bias)


0.010 0.100 10 100 500 1000

0.009 0.095 8.954 92.354 444.920 947.190

8.019 10.374 4.901 4.608 10.324 3.732

10.000 5.000 10.460 7.646 11.016 5.281

BAL fluid

0.010 0.100 10 100 500 1000

0.009 0.094 9.159 99.584 446.273 898.921

11.818 7.174 3.469 2.638 13.765 8.578

10.000 6.000 8.410 0.416 10.745 10.107

0.100 10 50 100 250

0.092 9.039 43.778 97.092 228.221

0.105 6.590 8.892 5.035 3.246

8.000 9.610 12.444 2.908 8.712


(Pharsight, Mountain View, CA, USA). The area under the plasma concentration–time curve (AUC) was calculated using the linear trapezoidal method. The maximum plasma concentration (Cmax ) and the time to reach the maximum plasma concentration (tmax ) were observed values from the experimental data. The elimination rate constant (Kel) was estimated by regression analysis from the slope of the line of best fit, and the half-life (t1/2 ) of the drug was obtained by 0.693/Kel. Total plasma clearance was calculated by Dose/AUC.

The accuracy and precision of the assay were determined by analyzing samples at the lower limit of quantification (LLOQ). Accuracy was calculated at each test concentration as: (mean measured concentration/nominal concentration) × 100%. 2.13. Pharmacokinetic analysis Pharmacokinetic analysis of IC87114 was carried out by noncompartmental analysis using WinNolin version 5.2.1

The tandem mass ESI conditions for IC87114 and IS were optimized in both positive and negative mode, but more stable and intense ions were observed in positive ion mode. Protonated molecular ions of [M+H]+, m/z 398 and 151.9 were obtained for IC87114 and IS respectively. The most abundant ions in the product ion mass spectrum were 146 and 110 for IC87114 and IS, respectively. The MRM transitions of m/z 398 → 146 and 151.9 → 110 were found for IC87114 and IS, respectively. Chromatographic separation of IC87114 and IS was attempted using various combinations of acetonitrile, methanol and buffers (formic acid, ammonium bicarbonate, ammonium acetate and ammonium formate) over a range of pH values with either isocratic or gradient elution in order to develop a simple, rapid and robust LC–MS/MS method. Peak intensity was increased by adjusting the pH. Resolution and peak intensity were increased with 10 mM ammonium formate pH 3.3 ± 0.02 and acetonitrile with a flow rate of 0.3 mL/min on a Zorbax C18 column. These conditions were found to be suitable for the determination of electrospray response for IC87114 and IS. The choice of internal standard was troublesome since no deuterated candidate was commercially available. Similarly, IC87048 and PIK-39 analog of IC87114 were also not commercially available. Acetaminophen did not have any significant impact on matrix effect. Reproducibility study of IS in spiked plasma, BAL fluid and lung sample was in acceptable range and also not present in biological samples, so we choose acetaminophen as internal standard. The retention times of IC87114 and IS were 8.7, and 3.5 min, respectively (Fig. 2).

Table 5 Pharmacokinetics of IC87114 in mouse following IT administration at a dose 1 mg/kg (n = 6). PK parameter


Cmax (ng/mL or g) Tmax (h) AUClast (h*ng/mL or g) T1/2 (h) V (mL/kg) CL (mL/h/kg) AUCtissue/BAL :AUCplasma

538.630 0.083 2322.946 12.365 7236.714 408.971

BAL fluid ± ± ± ± ± ±

122.274 0 111.959 0.767 314.515 20.297

172.329 ± 0.083 ± 1427.688 ± 14.388 ± 11,491.291 ± 569.817 ± 0.614

Lung 13.598 0 78.117 2.062 1123.076 33.717

75.101 ± 0.083 ± 443.011 ± 11.930 ± 33,052.903 ± 1971.885 ± 0.190

19.324 0 23.591 1.220 1979.763 129.185

A. Marahatta et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202

IC87114 concentration in plasma (ng/mL)



Plasma 600






0 0









IC87114 concentration in BAL/Lung (ng/mL or g)

Time (hr)

BAL fluid Lung

180 160 140 120 100 80 60 40 20 0 0








Time (hr) Fig. 3. (A) Concentration versus time profile of IC87114 in plasma of C57B/L mouse following i.t. administration of single dose 1 mg/kg (n = 6) in lung and BAL fluid (B) plasma.

3.2. Sample preparation Sample preparation by an efficient extraction step is crucial in determining IC87114 in biological matrices to ensure high recovery by easier and less time consuming separation methods. Protein precipitation has been found to be economical, convenient and fast, making it suitable for pharmacokinetic studies involving a large number of samples. In this study we chose to precipitate proteins with methanol due to excellent performance, a more symmetrical peak shape and less matrix effects compared to acetonitrile. 3.3. Validation of the method The assay was validated according to the Food and Drug Administration (FDA) Guidelines on bioanalytical method validation. The plasma, BAL fluid and lung assays were fully validated for calibration model, specificity, carry-over, linearity, precision and accuracy. 3.3.1. Selectivity and specificity Different biological matrices were tested with the aim of observing any possible interference between IC87114 and other compounds already present in the sample. The chromatographic condition was developed for both drug and IS peaks at high specificity and selectivity. The specificity of the method was evaluated

using blank mouse plasma, BAL and lung tissue samples from five different mice. As shown in Fig. 2 the chromatograms did not exhibit any peak interference at retention times of 3.5 min and 8.7 min for IS and IC87114, respectively. 3.3.2. Linearity and limit of quantification (LOQ) The calibration curves were linear over the following concentration ranges: 0.01–1000 ng/mL for plasma/BAL fluid and 0.1–250 ng/mL for lung tissue samples. Typical equations of the calibration curves were as follows: y = 0.8684x + 2.0626, r = 0.9978 for BAL fluid; y = 1.0405x − 11.353, r = 0.9977 for plasma; and y = 2.4142x − 6.7569, r = 0.9928 for lung. The LOQ was 0.01 ng/mL for plasma/BAL and 0.1 ng/g for lung tissue. This method was found to be about 600 times more sensitive than the previous method [15]. 3.3.3. Extraction recovery and matrix effect The mean recoveries of all analytes ranged from 78.40–97.29% for plasma, 85.95–102.81% for BAL fluid and 78.07–89.70% for lung tissue (Table 2). The extraction recovery of IS (1 ng/mL) was 90.64%, and the matrix effect was 97.31%. The simple protein precipitation procedure yielded satisfactory recovery of IC87114 from mouse plasma, BAL fluid and lung tissue. The detailed results of matrix effects are shown in Table 2. The matrix effect ranged from 87.61


A. Marahatta et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 197–202

Table 6 The incurred sample reanalysis of IC87114 in plasma, BAL fluid and lung sample. Original results (ng/mL or ng/g)

Repeated results (ng/mL or ng/g)

Percentage difference (%)

Plasma 475.358 381.180 228.671 18.456 8.914 9.162 6.330

411.261 392.432 227.358 17.239 8.811 9.543 5.628

14.459 −2.909 0.576 6.819 1.162 −4.074 11.741

Bal fluid 217.020 207.090 170.916 62.997 34.186 11.300 13.930

213.510 200.380 173.890 67.280 31.098 10.100 13.260

1.631 3.293 −1.725 −6.575 9.460 11.215 4.928

Lung 96.234 51.443 49.319 42.986 42.588 2.684 2.875

90.848 55.681 56.219 43.557 38.016 2.867 2.656

5.758 −7.912 −13.076 −1.320 11.344 −6.593 7.919

to 102.66%. The observed variation did not exceed the range of 85–115%. Thus, ion suppression or enhancement was not significant with the HPLC–MS/MS method. 3.3.4. Accuracy and precision The intra-day and inter-day precision and accuracy results are presented in Tables 3 and 4. Precision is presented in terms of % CV and the accuracy as % error. The intra-day precision was 6.39–11.42% for plasma, 0.46–7.32% for BAL fluid and 2.33–9.81% for lung tissue. The intra-day accuracy was −1.26 to −12.6 for plasma, 0.43 to −11.37 for BAL fluid and −6.9 to −13.5 for lung tissue. Inter-day precision was 3.73–10.37% for plasma, 2.63–13.76% for BAL fluid and 0.10–8.89% for lung tissue. The inter-day accuracy was −3.0 to −11.01 for plasma, −0.42 to −10.74 for BAL fluid and −2.91 to −12.46 for lung tissue. The intra-day and inter-day variation, as well as the accuracy, were within the acceptable range. 3.4. Application of the method The validated method was applied to a pharmacokinetic study in mice. This is the first report on the pharmacokinetics of nonsurgical intratracheally (IT) administered IC87114 in any mammalian species. The previously reported pharmacokinetic study (Thappali et al.) described the plasma concentration of IC87114 in wistar rats following oral gavage. In our study, C57B/L mice were given a single dose of IC87114 (1 mg/kg) IT and nonsurgically. Blood, BAL fluid and lung tissue samples were collected at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48 and 72 h. Pharmacokinetic analysis of IC87114 in mouse plasma, BAL fluid and lung tissue are summarized in Table 5 and Fig. 3. IC87114 was absorbed quickly with tmax at 0.083 h after IT administration. The previous study by Thappali et al. reported a Cmax of 1374 ng/mL and tmax of 4 h in plasma. In their study rats were orally administered with 5 mg/kg [15]. 3.5. Incurred sample reanalysis A total of 21 samples were analyzed (Table 6). The % differences between the re-assay concentrations and the original

concentrations were all less than 15% of their mean value and met the acceptance criteria for incurred sample analysis. 4. Conclusion A bioanalytical method was successfully developed and validated to determine IC87114 in mice plasma, BAL fluid and lung tissues. A quick and simple protein precipitation procedure was used. The advantage of this method is very sensitivity, efficiency and low cost. The major improvement over the previously reported LC–MS/MS method for plasma is a lower limit of quantification of about 600-fold lower. Thus the method is ideally suited to pharmacokinetic studies with IC87114 administration in small mice at low dosage. The applicability of the method was demonstrated in a pilot pharmacokinetic study by using nonsurgical method which provided new information about BAL fluid and lung tissue. Acknowledgments This work was supported by the Korea Healthcare Technology R&D Project (A121931), Ministry for Health and Welfare, Republic of Korea. We would like to thank Dr. Yun-Jo Jung from Korea Basic Science Institute, Jeonju, Korea for his technical assistance. References [1] B. Vanhaesebroeck, S.J. Leevers, K. Ahmadi, J. Timms, R. Katso, P.C. Driscoll, R. Woscholski, P.J. Parker, M.D. Waterfield, Synthesis and function of 3-phosphorylated inositol lipids, Annu. Rev. Biochem. 70 (2001) 535–602. [2] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002) 1655–1657. [3] S. Koyasu, The role of PI3K in immune cells, Nat. Immunol. 4 (2003) 313–319. [4] X. Ma, Y. Hu, Targeting PI3K/Akt/mTOR cascade: the medicinal potential, updated research highlights and challenges ahead, Curr. Med. Chem. 20 (2013) 2991–3010. [5] L. Liu, K.D. Puri, J.M. Penninger, P. Kubes, Leukocyte PI3Kgamma and PI3Kdelta have temporally distinct roles for leukocyte recruitment in vivo, Blood 110 (2007) 1191–1198. [6] C. Rossios, Y. To, G. Osoata, M. Ito, P.J. Barnes, K. Ito, Corticosteroid insensitivity is reversed by formoterol via phosphoinositide-3-kinase inhibition, Br. J. Pharmacol. 167 (2012) 775–786. [7] J. Bousquet, P.K. Jeffery, W.W. Busse, M. Johnson, A.M. Vignola, Asthma From bronchoconstriction to airways inflammation and remodeling, Am. J. Respir. Crit. Care Med. 161 (2000) 1720–1745. [8] B.N. Kang, S.G. Ha, X.N. Ge, M. Reza Hosseinkhani, N.S. Bahaie, Y. Greenberg, M.N. Blumenthal, K.D. Puri, S.P. Rao, P. Sriramarao, The p110delta subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice, Am. J. Physiol. Lung Cell. Mol. Physiol. 302 (2012) L1179–L1191. [9] K.S. Lee, H.K. Lee, J.S. Hayflick, Y.C. Lee, K.D. Puri, Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model, FASEB J. 20 (2006) 455–465. [10] E. Collmann, T. Bohnacker, R. Marone, J. Dawson, M. Rehberg, R. Stringer, F. Krombach, C. Burkhart, E. Hirsch, G.J. Hollingworth, M. Thomas, M.P. Wymann, Transient targeting of phosphoinositide 3-kinase acts as a roadblock in mast cells’ route to allergy, J. Allergy Clin. Immunol. 132 (2013) 959–968. [11] M. Rayamajhi, E.F. Redente, T.V. Condon, M. Gonzalez-Juarrero, D.W. Riches, L.L. Lenz, Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry, J. Vis. Exp. (2011) e2702, [12] S.R. Kim, K.S. Lee, H.S. Park, S.J. Park, K.H. Min, H. Moon, K.D. Puri, Y.C. Lee, HIF1alpha inhibition ameliorates an allergic airway disease via VEGF suppression in bronchial epithelium, Eur. J. Immunol. 40 (2010) 2858–2869. [13] S.J. Park, K.S. Lee, S.R. Kim, K.H. Min, H. Moon, M.H. Lee, C.R. Chung, H.J. Han, K.D. Puri, Y.C. Lee, Phosphoinositide 3-kinase delta inhibitor suppresses interleukin-17 expression in a murine asthma model, Eur. Respir. J. 36 (2010 1448–1459. [14] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS, Anal. Chem. 75 (2003) 3019–3030. [15] S.R. Thappali, K.V. Varanasi, S. Veeraraghavan, S.K. Vakkalanka, K. Mukkanti, Simultaneous quantitation of IC87114, roflumilast and its active metabolite roflumilast N-oxide in plasma by LC–MS/MS: application for a pharmacokinetic study, J. Mass Spectrom. 47 (2012) 1612–1619.

MS method for quantification of IC87114 in mice plasma, bronchoalveolar lavage and lung samples: Application to pharmacokinetic study.

IC87114 is a selective PI3Kδ inhibitor. A simple, sensitive and reliable LC-MS/MS method with rapid sample preparation was developed and validated for...
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