Journal of Chromatography B, 988 (2015) 187–194
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of the antitubercular drug PA-824 in rat plasma, lung and brain tissues by liquid chromatography tandem mass spectrometry: Application to a pharmacokinetic study Dominika Bratkowska a , Adeola Shobo a , Sanil Singh b , Linda A. Bester b , Hendrik G. Kruger a , Glenn E.M. Maguire a , Thavendran Govender a,∗ a b
Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa Biomedical Resource Unit, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa
a r t i c l e
i n f o
Article history: Received 16 October 2014 Accepted 26 February 2015 Available online 9 March 2015 Keywords: LC–(ESI)MS/MS PA-824 Tuberculosis Rat plasma Rat tissues Pharmacokinetics
a b s t r a c t A selective, sensitive and high performance liquid chromatography-tandem mass spectrometry (LC–(ESI)MS/MS) method has been developed and validated for the quantification of the potent antitubercular drug candidate, PA-824, in rat plasma, lung and brain tissues. Sample clean-up involved protein precipitation and solid-phase extraction. Chromatographic separation was performed on YMC Triart C18 column (150 mm × 3.0 mm, 3.0 m). The method was validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Evaluation of the pharmacokinetic properties of PA-824 utilized Sprague Dawley rats with a dosage of 20 mg/kg at various time points. The new method was applied successfully for the determination of PA-824 with liquid desorption followed by liquid chromatography with ultra-high resolution quadrupole time-of-flight mass spectrometry in the different biological samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The HIV pandemic has created circumstances where coinfection, the emergence of multidrug resistant and extensively drug resistant tuberculosis (MDR-TB and XDR-TB, respectively), and thus mortality attributed to tuberculosis worldwide is increasing [1–4]. Therefore, there is a need for novel drugs with activity against MDR, XDR and latent TB as well as shorter treatment periods. In particular these new agents should be safe and effective for use in HIV-infected TB patients. Nitroimidazoles belong to a class of antimycobacterial agents that are active against drugsusceptible and drug-resistant organisms [4–7]. Nitroimidazoles present similar activity against replicating and non-replicating organisms, which may indicate their potential to shorten therapy timelines [8]. PA-824 is a nitroimidazo-oxazine and a metronidazole derivative from the nitroimidazopyran class [9], developed by the TB Alliance. PA-824 is an antitubercular agent, whose mode of action affects protein and lipid synthesis of M. tuberculosis. In addition, it has demonstrated potential bactericidal activity comparable
∗ Corresponding author. Tel.: +27 31 2601845; fax: +27 31 2603091. E-mail address: [email protected] (T. Govender). http://dx.doi.org/10.1016/j.jchromb.2015.02.041 1570-0232/© 2015 Elsevier B.V. All rights reserved.
to that of isoniazid [10]. In the past several years, significant technological improvements in mass spectrometry have had a great impact on drug discovery and development [11–14]. At present, LC–MS is a well-established analytical method for the identification and quantification of analytes in sample mixtures and has been widely used in bioanalytical studies [15,16]. LC–MS/MS methods [17–21] frequently provide specific, selective and sensitive quantitative results, often with reduced sample preparation and analysis times compared with other commonly used techniques. Up to now, several methods had been developed for the pharmacokinetics and therapeutic drug monitoring of a number of antitubercular drugs [22–24]. Recently, Wang et al. [25] reported an analytical method for simultaneous determination of three antitubercular drugs, including PA-824 after an oral administration. To examine the preclinical plasma pharmacokinetics and tissue distribution of PA-824 in a reproducible and precise manner, a validated assay is necessary. To the best of our knowledge, there is no published report literature that demonstrates validation of a sensitive assay for the determination of PA-824 in lungs and brain. The objective of the present study was to develop simple and sensitive method based on SPE/LC–(ESI)MS/MS for the determination of the PA-824 in plasma, lung and brain tissues to be applied in a pharmacokinetic study after oral and intraperitoneal administrations to rats.
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2. Materials and methods 2.1. Reagents and standards PA-824 was purchased from DLD Scientific (Durban, South Africa). The chemical structure of the analyte is presented in Fig. 1. Acetonitrile (ACN), formic acid, methanol (MeOH) were of analytical grade all from Sigma Aldrich. Ultra-pure water was obtained using a Milli-Q purification system from Millipore Corporation (Bedford, MA, USA). Hybrid-SPE cartridges (30 mg, 1.0 mL) were supplied from Supelco-Sigma (St. Louis, MO). An internal standard (IS), carnidazole, was obtained from Sigma-Aldrich (Steinheim, Germany). Deuterated IS is recommended whenever possible; however, this was not commercially available for PA-824, therefore we decided to use a structurally similar nitroimidazole. 2.2. Instrumentation The liquid chromatography tandem mass spectrometry (LC–MS) system consisted of a Shimadzu LC-20 AD series HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a maXis 4G electrospray ionization (ESI) time-of-flight-mass spectrometry (TOF-MS) instrument (Bruker Daltonics, Bremen, Germany). All results were stored and analyzed with Data Analysis 4.0 SP 5 (Bruker Daltonics). 2.3. Preparation of standards and calibration curves Separate stock solutions of PA-824 and IS were prepared by dissolving 10 mg of each substance in 10 mL of methanol, and the solutions were stored at refrigerated temperature (0–4 ◦ C). A series of PA-824 working standard solutions and an IS solution, were prepared by appropriate dilutions of their stock solutions with ACN:deionized water (1:1, v/v). Calibration standards were prepared by spiking working standard solutions and IS into 100 L of blank rat plasma or different tissue homogenates of untreated rats to yield PA-824 concentrations of 75, 150, 250, 500, 750, 1000 and 1500 ng/mL (in plasma), and IS concentration of 250 ng/mL. Quality control (QC) samples at lower limit of quantification (LLQC), low (LQC), middle (MQC) and high (HQC) concentrations (75; 100, 800 and 1400 ng/mL) were prepared separately in the same fashion. More detailed information can be found in Section 2.8. 2.4. Chromatographic conditions For HPLC separation, a YMC Triart C18 column (YMC Europe Gmbh, Dislanken, Germany), with spherical hybrid silica particles
(150 mm × 3.0 mm; particle size 3 m) equipped with the corresponding guard column (4 mm × 3.0 mm) was used. The mobile phase was Milli-Q water (0.1% v/v formic acid) and ACN (0.1% v/v formic acid). The flow rate was 0.2 mL min−1 and the temperature of the column oven was set at 25 ◦ C. The gradient profile was initially from 30 to 90% ACN in 10 min (held 2 min), after which time the mobile phase was returned to the initial conditions (30% ACN) in 3 min. The sample injection volume was 5 L. Column reequilibration was set at 5 min. 2.5. Mass spectrometric analysis To obtain accurate mass spectra of target analyte a maXis 4G ESI time-of-flight-mass spectrometry (TOF-MS) instrument (Bruker Daltonics) was used. The MS conditions were optimized. Acquisition parameters were: source type, ESI; ion polarity, positive; nebulizer, 1.5 bar; capillary, 3500 V; dry heater, 200 ◦ C; scan range, m/z 100–1500; end plate offset, 500 V; dry gas, 8.0 L/min; collision cell radiofrequency, 600 Vpp; collision energy, 21 eV. 2.6. Plasma and tissue samples Drug free plasma samples were purchased from Life Technologies (Burlington, ON, Canada) and stored at −20 ◦ C prior to analysis. Plasma samples were thawed at room temperature before analysis. Rat brains and other tissues were separated and weighed, then placed into liquid nitrogen immediately. The organs were weighed, cut it into small pieces using scissors and mixed with ultra-pure water (3 L/mg tissue). The tissues were then homogenized. All homogenate was stored at −80 ◦ C till analysis. 2.7. Sample preparation To an aliquot of 100 L of spiked plasma sample, IS was added at a concentration level of 250 ng/mL and vortexed briefly. Subsequently, 800 L of acetonitrile or methanol was added to induce the precipitation of plasma proteins. The mixture was vigorously mixed for 1 min, followed by centrifugation at 13,000 rpm for 15 min. The supernatants were filtered through a Hybrid-SPE-Phospholipid cartridge (30 mg/1 mL). The filtered samples (500 L) were evaporated until dryness under a gentle stream of nitrogen, and reconstituted in 500 L of mobile phase. All the vials containing samples were vortexed briefly and transferred into autosampler vials for injection into the chromatographic system. All thawing of frozen plasma samples and tissues were completed at room temperature. For the brain and lung tissue samples, the IS was added to 100 L brain homogenates samples and followed by addition of ultra-pure water. The IS was added to the homogenized tissue sample and thoroughly mixed. Subsequent extraction procedure of the drug from the tissue samples was the same as described for the plasma. 2.8. Method validation
Fig. 1. Chemical structure of the target analyte.
During the process of method validation, specificity, linearity, lower limit of quantification (LLOQ), limit of detection, precision, accuracy, extraction recovery, and stability were evaluated. The present method was also validated using total error approach [26–28]. For each level, the limit of the -expectation tolerance interval (-TI) was calculated. -TI is the interval within which the average proportion of future  results will fall. Calculations are detailed in the work of Rozet et al. [28]. -TI should not exceed the threshold of 30% selected for the acceptance limits, in agreement with the recommendations [29].
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2.8.1. Specificity and selectivity The specificity and selectivity of the method were performed by examining the presence or absence of interference, comparing chromatograms of six lots of blank plasma and tissue samples from different sources, blank samples spiked with standard and rat samples after oral and intraperitoneal administration of PA-824. In accordance with the validation guideline [29], absence of interfering components is accepted where the response is less than 20% of the LLOQ for the analyte and 5% for the IS. 2.8.2. Linearity and carry-over effect Each calibration curve should consist of a blank sample, a zero sample and six to eight calibration concentration levels, among which blank and zero samples were only analyzed to confirm the absence of interferences. Calibration curves were plotted by the peak area ratio versus analyte concentrations using a 1/x2 weighted linear least-squares regression model. The LLOQ was evaluated by analyzing six replicates of spiked samples at the relevant concentration. Carry-over effects were assessed, with relevant criteria, by injecting blank samples following the calibration standard at the highest concentration. Carry over in the blank should not exceed 20% of the LLOQ and 5% for the IS [29]. 2.8.3. Accuracy and precision Accuracy was determined as the ratio between the calculated concentration of the target compound and its theoretical concentration. Intra- and inter-day precisions were assessed by assay of six replicates of QC different samples at low, medium, and high concentrations on the same day and on three different days. The acceptance criteria were within 15% RSD and 85–115% (except LLOQ QC) of nominal concentration for accuracy and precision, respectively. QC samples concentrations were as following: 75, 100, 800 and 1400 ng/mL for plasma samples; 50, 75, 400 and 1100 ng/g for lung tissue; and 100, 150, 800 and 1400 ng/g for brain tissue. 2.8.4. Matrix effect and extraction recovery Matrix effects were investigated using the matrix factor by calculating the ratio of the peak area in the presence of matrix (measured by analyzing blank matrix spiked after extraction with analyte), to the peak area in absence of matrix (pure solution of the analyte). Matrix factor should not be greater than 15% [29]. This was determined at a low, medium and at a high level of concentration (maximum of three times the LLOQ and close to the upper limit of quantification, ULOQ). The extraction recovery of PA-824 was performed at the three QC levels (low, medium and high) in five replicates. 2.8.5. Stability Stabilities of PA-824 in plasma were estimated by assay of three replicates of QC samples at low, medium, and high concentrations under the following conditions: short-term stability after storage at room temperature (25 ◦ C) for 6 h; freeze-thaw stability through three freeze-thaw cycles (−80–25 ◦ C). The post-preparative stability was examined after 24 h in the autosampler maintained at 25 ◦ C. In agreement with guideline [29], the QC samples are analyzed against a calibration curve, obtained from freshly spiked calibration standards, and the obtained concentrations are compared to the nominal concentrations. The mean concentration at each level should be within ±15% of the nominal concentration. The stock solution stabilities of PA-824 and IS were demonstrated following storage at −20 ◦ C. The measured concentrations of stabilities are compared to the nominal concentrations. The acceptable RSD was within ±15%.
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3. Animals All animal experiments were performed with approval from the Animal Ethics Research Committee of University of KwaZulu-Natal (069/14/Animal). The study was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) which was approved by the Lund/Malmö, Sweden Ethical Committee on Animal Experiments (1988). Sprague Dawley female rats (weight 130 ± 10 g, 6 weeks) were purchased from the Biomedical Resource Unit (UKZN, Durban, South Africa). The animals were allowed a 7 days acclimatization period prior to experimentation. They were kept in a climate controlled facility (25 ◦ C, 60% relative humidity) with 12 h light-dark cycle. Food and water were given ad libitum. 3.1. Application to pharmacokinetic study 3.1.1. Drug administration and tissue collection The PA-824 was dissolved in a 40% (v/v) aqueous solution of DMSO. Two groups of Sprague Dawley rats were administered an oral dose or intraperitoneal injection of the PA-824 solution, respectively. The animals were anesthetized with halothane and sacrificed after the required time frames. Blood samples were collected at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 h post-dosing. Blood samples were collected in K3 -EDTA-coated tubes and centrifuged at 12,000 rpm for 10 min, and plasma was separated and stored at −80 ◦ C before bioanalysis. Tissues (brain and lungs) were collected after cervical dislocation of the anaesthetized rat. Organs were separated and placed into liquid nitrogen immediately. Tissues were homogenized and all homogenates were stored at −80 ◦ C until analysis. 3.1.2. Application to pharmacokinetic study Maximum concentration (Cmax ) of PA-824 and time to Cmax (Tmax ) were determined directly from the data. Other pharmacokinetic parameters were determined by analyzing the individual rat plasma profiles using non-compartmental equations in Microsoft Excel [30]. The area under the curve (AUC0→∞ ) was calculated by the linear trapezoidal rule. Apparent oral clearance (Cl/F) was calculated using the equation: Cl = dose/AUC0→∞ . Mean residence time (MRT) was calculated using the equation: MRT = AUMC0→∞/ AUC0→∞ . 4. Results and discussion 4.1. Method development During method development, different options were evaluated to optimize mass spectrometry detection parameters, chromatography and sample extraction. 4.1.1. LC–MS/MS analysis The instrument was optimized to obtain sensitivity and signal stability during infusion of the analyte. That was performed in the continuous flow of mobile phase to electrospray ion source operated at both polarities at a flow rate of 180 L/min. Target analytes gave good response in positive ion mode. For polar analytes, ESI is the preferred ionization technique. The addition of 0.1% formic acid improves the formation of protonated adducts, which can be detected in positive mode. Under these conditions the single charged quasi-molecular ion at 360.1 m/z is the base peak but the dimeric ion at 719.2 m/z was also detected. Using the m/z of the base peak, chromatograms of selected ions of interest were extracted. Fig. 1S a shows a typical mass spectrum of the product ion of m/z 360.1 for PA-824 (see Supporting Information).
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Table 1 Recoveries and matrix effects of PA-824 in different biological samples. Concentration level Plasma LQC MQC HQC Lungs LQC MQC HQC Brain LQC MQC HQC
Mean recovery (%)
%RSD
Matrix effect (%)
%RSD
90.2 91.0 90.1
1.6 1.1 2.1
−8.4 −1.9 −6.3
4.8 2.1 1.7
95.6 94.5 94.2
2.3 1.3 1.1
−1.6 −6.6 −6.8
3.0 3.5 3.8
90.1 91.5 90.2
2.0 1.2 1.8
−2.3 −1.3 −4.9
4.5 2.9 2.4
The mass spectrometer was operated in positive ion mode using MRM mode to monitor the mass transitions. The optimum MSMS conditions were optimized. Two fragmentations were acquired for the target analyte (m/z 360.1 → m/z 175.0 and m/z 360.1 → m/z 201.1). To quantify the analyte, its most intense transition was chosen. The transition m/z 360.1 → m/z 175.0 was selected for PA-824. Fig. 1S(b) presents a typical MS/MS spectrum obtained from collision activated dissociation of m/z 360.1 of PA-824 (see Supporting Information). 4.1.2. Optimization of extraction procedure Prior to loading the sample for LC injection, co-extracted proteins and phospholipids should be removed from the prepared solution. For this purpose, we tested two different techniques, protein precipitation (PP) and solid phase extraction (SPE). First for PP we used MeOH. The recoveries found employing this approach for PA-824 were 62–70%. Higher recoveries (more than 92%) were observed using ACN for PP. Although, this PP procedure removed gross levels of proteins providing good recoveries, in order to remove phospholipids that remained in the extract, an additional step was required. An appropriate pre-treatment protocol for plasma samples used before chromatographic determination was achieved using SPE. Plasma was first subjected to protein precipitation via the addition and mixing of acetonitrile. Precipitated proteins were then removed by centrifugation, and the resulting supernatant was loaded onto the HybridSPE-Phospholipid cartridge, which acts as a chemical filter that specifically targets the removal of endogenous sample phospholipids. The manufacturer’s specifications of HybridSPE-Phospholipid cartridges indicated the direct injection of the eluate. However, in the developed clean-up step, the eluate was evaporated to dryness and reconstituted with the mobile phase to obtain better separation. This simple methodology provided cleaner extracts of plasma samples containing the target analyte. Table 1 shows the recoveries of PA-824 in plasma at three concentration levels. The mean extraction recovery of IS (250 ng/mL) was more than 92.7% (%RSD 2.5%). This extraction procedure resulted in accurate and precise detection of PA-824 in plasma. 4.1.3. Evaluation of matrix effects Matrix effects may be defined as a composite of some undesirable effects that originate from a biological matrix [31]. Due to the influence of co-eluting compounds in the ionization process of the target analyte, these components may result in ion suppression/enhancement as well as decrease/increase in sensitivity of analytes over a period of time, increased baseline, imprecision of data, drift in retention time and distortion or tailing of a chromatographic output. The results of the post-extracted experiment (see Table 1) indicate that there is no significant ion suppression/enhancement observed at retention time of the analyte.
4.1.4. Method validation The method was validated according to the most recent European Medicine Agency (EMA) guideline on bioanalytical method validation [29]. Linearity, LLOQ, inter-day and intra-day precision and accuracy as well as recovery and stability of the PA-824 were evaluated. After appropriate adjustment of the chromatographic separation conditions, a reproducible separation of PA-824 from plasma was achieved. The detection of PA-824 and IS was highly selective with no interference from the endogenous substances. Fig. 2S(a) represents typical chromatograms of blank plasma, Fig. 2S(b) blank plasma spiked with IS at 250 ng/mL and PA-824 at 75 ng/mL (LLQC level) (see Supporting Information). Typical retention time for PA-824 was 8.8 min. Based on these findings, and the fact that the peaks for PA-824 and IS were symmetrical, they were judged to be adequate for subsequent analyses. Typical linear regression equations of the calibration curves for different matrices were equal to: y = 0.0065x − 0.1025; y = 1.536518x + 0.097110 and y = 1.774116x + 0.103510 (for plasma, lungs and brain, respectively), where y was the peak area ratio of the analyte to the internal standard and x the concentration of the analyte. The mean regression coefficients of the calibration curves were 0.9982, 0.9989 and 0.9974 (in plasma, lungs and brain, respectively). Calibration curves of PA-824 in plasma tissue samples were established over a concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Batches, consisting of duplicate calibration standards at each concentration, were analyzed on three different days to complete the method validation. In each batch, QC samples at 75, 100, 800, 1400 ng/mL in plasma, 50, 75, 400, 1100 ng/g and 100, 150, 800, 1400 ng/g in lungs and brain, respectively, were assayed in sets of six replicates. The calibration curves were linear over the concentration range of 75–1500 ng/mL (plasma), 50–1200 ng/g (lungs) and 100–1500 ng/g (brain) with the correlation coefficient greater than 0.997. The LLOQ was evaluated by analyzing six replicates of spiked samples at the concentration of 75 ng/mL. The limit of detection (LOD), calculated using a signal to noise ratio of ≥3, was 25 ng/mL for plasma, 25 ng/g for lungs and 50 ng/g for brain. The accuracy observed for the standards ranged from 95.5 to 104.5% for PA-824 (data not shown). Meanwhile, in complex plasma and tissue samples, the observed accuracy ranged from 85.5 to 98.5% (except LLQC level >82%). As can be seen, Table 2 summarizes the intra- and inter-day precision and relative error for the PA-824, evaluated by assaying the QC samples. The inter-day and intra-day precision (%RSD) of the spiked quality control samples for the analyte in plasma was lower than 12.7. The obtained results were within the acceptance criteria. Moreover, the accuracy of the method takes into consideration the total error related to the test result [26,27]. As shown in Table 2, the ˇ expectations were around 30% for each concentration level with a probability of 95%. Carryover evaluation was performed in each analytical run to ensure that the accuracy and the precision of the proposed method were not affected. There was a negligible carryover observed during the autosampler carryover experiment. No enhancement in the response was observed in the double blank after subsequent injection of the highest calibration standard (aqueous and extracted) at the retention time of analyte. 4.2. Stability 4.2.1. Stability in plasma Bench top and autosampler stability for PA-824 were investigated at LQC, MQC and HQC levels. The results revealed that PA-824 was stable in plasma for at least 6 h at room temperature, and 24 h in an autosampler. All the values of accuracy and precision were
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Table 2 Accuracy and precision for PA-824 in different biological matrices. Matrix
Concentration level
LLQC
LQC
MQC
HQC
Plasma
Theoretical concentration (ng/mL) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
75.0
100.0
800.0
1400.0
62.9 83.9 11.5
87.9 88.0 11.3
707.1 88.4 10.7
1260 90.0 12.3
61.5 82.0 8.9 [−2.3; 29.8]
85.6 85.5 12.4 [−5.5; 29.5]
704.6 88.1 11.3 [−4.6; 27.8]
1256.3 89.7 12.7 [−5.4; 26.6]
Theoretical concentration (ng/g) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
50
75
400
1100
44.4 88.7 9.1
73.9 98.5 5.1
380.0 95.0 4.8
952.4 86.6 1.7
44.8 89.6 2.8 [−3.6; 25.5]
73.4 97.9 1.1 [−12.7; 16.3]
376.4 94.2 1.1 [−8.4; 19.3]
946.9 86.1 1.8 [−4.9; 22.3]
Theoretical concentration (ng/g) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
100
150
800
1400
90.4 90.4 5.5
128.9 86.0 3.6
720.0 90.0 3.5
1261.6 90.1 2.1
89.8 89.8 2.1 [−8.2; 28.1]
128.1 85.4 1.0 [−3.4; 25.1]
718.5 89.8 3.8 [−8.1; 28.1]
1255.5 89.7 2.6 [−1.6; 21.8]
Lungs
Brain
-TI: -expectation tolerance limits.
in the range from 85.2 to 100.5% and 1.0 to 3.6%, respectively. PA824 in plasma and tissue homogenate samples was stable in the processed samples after three freeze/thaw circles. It was confirmed that repeated freezing and thawing of different samples spiked
with PA-824 at three concentration levels, which indicated that PA-824 had no significant degradation under the conditions previously described. The stability results obtained from the study are shown in Table 3.
Table 3 Stability of the plasma and tissue samples. Sample
Storage condition
Plasma
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Lungs
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Brain
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Concentration (ng/mL)
Accuracy (%)
%RSD
Added
Found
100 800 1400 100 800 1400 100 800 1400
90.2 747.6 1262.3 90.4 712.7 1260.4 85.2 696.8 1211.2
90.2 93.4 90.2 90.4 89.1 90.0 85.2 87.1 86.5
2.2 3.2 2.4 3.5 1.3 1.2 1.3 1.9 2.5
75 400 1100 75 400 1100 75 400 1100
74.1 375.6 1058.3 72.3 377.6 1033.9 69.5 369.9 1001.7
98.9 93.9 96.2 96.4 94.4 94.0 92.7 92.5 91.1
1.4 1.9 1.9 2.0 1.8 1.9 2.4 1.4 1.1
150 800 1400 150 800 1400 150 800 1400
138.2 750.9 1272.2 137.2 762.9 1280.7 128.1 740.3 1224.2
92.1 93.9 90.9 91.4 95.4 91.5 85.4 92.5 87.4
2.4 1.5 1.1 3.6 1.5 1.0 1.8 1.9 1.6
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Fig. 2. Mean plasma concentration–time profiles for PA-824 after oral (a) and intraperitoneal (b) administration of a single dose of 20 mg/kg to the rats.
4.3. Stock solution stability Stock solution stability was performed to check stability of PA824 and in stock solutions prepared in ACN and stored at 2–8 ◦ C in a refrigerator. The freshly prepared stock solutions were compared with stock solutions prepared before 30 days. The percentage change for PA-824 were less than 10% respectively indicating that stock solutions were stable for at least 30 days. 4.4. Application of method To investigate the suitability of this analytical method for pharmacokinetic studies, it was used for determination of plasma concentrations of PA-824 after oral and intraperitoneal administration of a single dose (20 mg/kg) to rats (n = 8). Pharmacokinetic parameters following both routes of administration of drug are shown in Table 4. Pharmacokinetic data after oral administration obtained by analysis of the individual profiles enabled estimation of Cmax of 626.7 ng/mL reached with a Tmax of 6 h. The area under the curve (AUC0→∞ ) using the trapezoidal rule was 3724.8 ng × h/mL, consistent with previous report [25]. The temporal profile for PA-824 in plasma after an oral administration is shown in Fig. 2a. In this Table 4 Pharmacokinetic data following oral and intraperitoneal administration of PA-824 (20 mg/kg) in a rat. Pharmacokinetic variable
Oral administration
Intraperitoneal administration
Cmax (ng/mL) (%RSD) Tmax (h) AUC0→∞ (ng × h/mL) Cl/F (mL/h/kg) MRT0→∞ (h)
626.7 (4.1%) 6.0 3724.8 5.37 12.17
1146.6 (1.5%) 0.25 3988.5 5.01 9.27
paper, the pharmacokinetic parameters of PA-824 were in accordance with previous report [25]. In the next step of this study, we investigated pharmacokinetic data after intraperitoneal administration of PA-824. As reported by Manley et al. [32] peritonitis may increase peritoneal permeability, and result in faster drug absorption as well as increase of peritoneal drug clearance during non-drug-containing dwells. When single dose of PA-824 was administered via the intraperitoneal route, the AUC0→∞ was 3988.5 ng × h/mL. The mean plasma concentration–time profile for PA-824 in plasma after an intraperitoneal administration is illustrated in Fig. 2b. After intraperitoneal administration, PA-824 was absorbed quickly. The concentration of the drug was readily measurable in the plasma samples collected up to 24 h post dose. As can be seen on Fig. 2b, after intraperitoneal administration, the plasma concentration of PA-824 first decreased rapidly and then more slowly, that is to say, the plasma concentration of PA-824 after intraperitoneal administration decreased polyexponentially and the terminal elimination half-life was relatively long (about 8 h). In addition, a similar tendency was reported by Xu et al. [33] for intraperitoneal administration of oridonin, one of the promising anti-cancer compounds. Moreover, in both cases, after 24 h traces of PA-824 were detected, but the concentration of the drug was just below the limit of quantification. This method was also successfully applied to determine the concentration of PA-824 in rat lungs and brain following oral and intraperitoneal administration. As an example, typical MRM chromatograms of rat plasma, lungs and brain samples, respectively, are presented in Fig. 3 (see attached ppt). The tendency graphs of PA-824 in rat lungs and brain after oral (Fig. 3S, Supporting Information) and intraperitoneal administration routes can be seen in Fig. 4. After oral administration due to first-pass metabolism the concentration PA-824 was determined at 6 and 8 h, concentrations of
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Fig. 3. Representative MRM chromatograms of PA-824 in rat plasma, lungs and brain homogenates. Blank plasma and blank tissue homogenates (a); blank plasma and tissue homogenates spiked with the analyte at LLOQ (b); and plasma and homogenates sample obtained 15 min after intraperitoneal injection (c).
the drug at the rest of post dosing points was below the limit of quantification. PA-824 reached a peak concentration of 216.3 ng/g (RSD = 11.7%) and 194.9 ng/g (RSD = 3.0%) at Tmax = 8 h, in lungs and brain respectively. Lungs and brain pharmacokinetics of PA-824 were also investigated following a single intraperitoneal dose to demonstrate the applicability of this assay. According to the results after intraperitoneal administration in rat lungs and brain, PA-824 features concentration–time profile, as shown in Fig. 4. Maximum concentrations of PA-824 for lungs were seen at 4 h after administration, whereas the highest concentration in brain was found at 2 h. The highest concentration levels of PA824 detected for tissues examined were 457.3 ng/g (RSD = 7.7%) in lungs. In brain PA-824 reached peak concentration of 236.8 ng/g (RSD = 2.6%) at 2 h post dosing. The results indicated that PA-824 was mostly eliminated from tissues after 24 h. The main pharmacokinetic parameters for PA-824 in rat lungs and brain after intraperitoneal administration are presented in
Table 5. Non-compartmental analysis of PA-824 concentration time profiles after intraperitoneal administration yielded Cmax of 4 and 2 h in lungs and brain tissue, respectively. Clearance was about 19.8 mL/h for lungs and 18.7 mL/h for brain. Tissue concentrations of PA-824 were considerably lower than the corresponding plasma concentrations. For PA-824, the plasma concentration was 2.5-fold higher than the tissue concentration of the lungs, whereas in comparison to brain plasma concentration was 5-fold higher. In addition, the plasma area under the curve from time zero to the last time point (AUC0→∞ ) was compared to those calculated for examined tissues. The mean ratio of PA-824 in the tissue to plasma was 0.25 for lungs and 0.27 for brain. By comparing the Cmax , Tmax and AUC0→∞ of rat plasma and brain, it is possible that PA-824 penetrates the blood–brain barrier. These results seem to indicate that PA-824 is absorbed from the gastrointestinal tract in rat. However, further investigations with other administration routes are necessary to confirm those findings. Generally, the observations indicate that the analytical method is suitable for measurement of concentrations of PA-824 in biological matrices to aid in more detailed pharmacokinetic and tissue distribution studies. In future studies this method can be used to study the effect of controlled release formulations on the delivery of the target drug to lungs and brain. Intranasal delivery is an efficient route to transport drugs across the blood–brain barrier. Since formulation and their
Table 5 Pharmacokinetic data following intraperitoneal administration of PA-824 in a rat lungs and brain.
Fig. 4. Concentration of PA-824 in rat lungs and brain after intraperitoneal administration at 20 mg/kg. Data are means ± SD.
Pharmacokinetic variable
Lungs
Brain
Cmax (ng/g) (%RSD) Tmax (h) AUC0→∞ (ng × h/mL) Cl (mL/h)
457.3 (7.7%) 4.0 1008.6 19.8
236.8 (2.6%) 2.0 1069.9 18.7
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components are vital factors for effective drug delivery to the brain, encapsulating the drug into nasal spray or nanoparticles [34,35] can also be considered in the development of PA-824 formulation. 5. Conclusion A sensitive and simple SPE/LC–(ESI)MS/MS method for the determination of PA-824 in rat plasma was developed and validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. The applicability of the established assay to the pharmacokinetic characterization of PA-824 in rats was investigated. The LC–MS/MS method was successfully applied to a pharmacokinetic study of PA-824 after oral administration of single dosage 20 mg/kg to rats. Moreover, based on the data reported, the assay appears to be applicable for the determination of pharmacokinetic characteristics of PA-824 involving the administration of single dose of the drug. Therefore, the analytical method may be useful in terms of characterizing the pharmacokinetic profiles in preclinical studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2015.02.041. References [1] [2] [3] [4] [5]
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of the antitubercular drug PA-824 in rat plasma, lung and brain tissues by liquid chromatography tandem mass spectrometry: Application to a pharmacokinetic study Dominika Bratkowska a , Adeola Shobo a , Sanil Singh b , Linda A. Bester b , Hendrik G. Kruger a , Glenn E.M. Maguire a , Thavendran Govender a,∗ a b
Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa Biomedical Resource Unit, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa
a r t i c l e
i n f o
Article history: Received 16 October 2014 Accepted 26 February 2015 Available online 9 March 2015 Keywords: LC–(ESI)MS/MS PA-824 Tuberculosis Rat plasma Rat tissues Pharmacokinetics
a b s t r a c t A selective, sensitive and high performance liquid chromatography-tandem mass spectrometry (LC–(ESI)MS/MS) method has been developed and validated for the quantification of the potent antitubercular drug candidate, PA-824, in rat plasma, lung and brain tissues. Sample clean-up involved protein precipitation and solid-phase extraction. Chromatographic separation was performed on YMC Triart C18 column (150 mm × 3.0 mm, 3.0 m). The method was validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Evaluation of the pharmacokinetic properties of PA-824 utilized Sprague Dawley rats with a dosage of 20 mg/kg at various time points. The new method was applied successfully for the determination of PA-824 with liquid desorption followed by liquid chromatography with ultra-high resolution quadrupole time-of-flight mass spectrometry in the different biological samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The HIV pandemic has created circumstances where coinfection, the emergence of multidrug resistant and extensively drug resistant tuberculosis (MDR-TB and XDR-TB, respectively), and thus mortality attributed to tuberculosis worldwide is increasing [1–4]. Therefore, there is a need for novel drugs with activity against MDR, XDR and latent TB as well as shorter treatment periods. In particular these new agents should be safe and effective for use in HIV-infected TB patients. Nitroimidazoles belong to a class of antimycobacterial agents that are active against drugsusceptible and drug-resistant organisms [4–7]. Nitroimidazoles present similar activity against replicating and non-replicating organisms, which may indicate their potential to shorten therapy timelines [8]. PA-824 is a nitroimidazo-oxazine and a metronidazole derivative from the nitroimidazopyran class [9], developed by the TB Alliance. PA-824 is an antitubercular agent, whose mode of action affects protein and lipid synthesis of M. tuberculosis. In addition, it has demonstrated potential bactericidal activity comparable
∗ Corresponding author. Tel.: +27 31 2601845; fax: +27 31 2603091. E-mail address: [email protected] (T. Govender). http://dx.doi.org/10.1016/j.jchromb.2015.02.041 1570-0232/© 2015 Elsevier B.V. All rights reserved.
to that of isoniazid [10]. In the past several years, significant technological improvements in mass spectrometry have had a great impact on drug discovery and development [11–14]. At present, LC–MS is a well-established analytical method for the identification and quantification of analytes in sample mixtures and has been widely used in bioanalytical studies [15,16]. LC–MS/MS methods [17–21] frequently provide specific, selective and sensitive quantitative results, often with reduced sample preparation and analysis times compared with other commonly used techniques. Up to now, several methods had been developed for the pharmacokinetics and therapeutic drug monitoring of a number of antitubercular drugs [22–24]. Recently, Wang et al. [25] reported an analytical method for simultaneous determination of three antitubercular drugs, including PA-824 after an oral administration. To examine the preclinical plasma pharmacokinetics and tissue distribution of PA-824 in a reproducible and precise manner, a validated assay is necessary. To the best of our knowledge, there is no published report literature that demonstrates validation of a sensitive assay for the determination of PA-824 in lungs and brain. The objective of the present study was to develop simple and sensitive method based on SPE/LC–(ESI)MS/MS for the determination of the PA-824 in plasma, lung and brain tissues to be applied in a pharmacokinetic study after oral and intraperitoneal administrations to rats.
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2. Materials and methods 2.1. Reagents and standards PA-824 was purchased from DLD Scientific (Durban, South Africa). The chemical structure of the analyte is presented in Fig. 1. Acetonitrile (ACN), formic acid, methanol (MeOH) were of analytical grade all from Sigma Aldrich. Ultra-pure water was obtained using a Milli-Q purification system from Millipore Corporation (Bedford, MA, USA). Hybrid-SPE cartridges (30 mg, 1.0 mL) were supplied from Supelco-Sigma (St. Louis, MO). An internal standard (IS), carnidazole, was obtained from Sigma-Aldrich (Steinheim, Germany). Deuterated IS is recommended whenever possible; however, this was not commercially available for PA-824, therefore we decided to use a structurally similar nitroimidazole. 2.2. Instrumentation The liquid chromatography tandem mass spectrometry (LC–MS) system consisted of a Shimadzu LC-20 AD series HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a maXis 4G electrospray ionization (ESI) time-of-flight-mass spectrometry (TOF-MS) instrument (Bruker Daltonics, Bremen, Germany). All results were stored and analyzed with Data Analysis 4.0 SP 5 (Bruker Daltonics). 2.3. Preparation of standards and calibration curves Separate stock solutions of PA-824 and IS were prepared by dissolving 10 mg of each substance in 10 mL of methanol, and the solutions were stored at refrigerated temperature (0–4 ◦ C). A series of PA-824 working standard solutions and an IS solution, were prepared by appropriate dilutions of their stock solutions with ACN:deionized water (1:1, v/v). Calibration standards were prepared by spiking working standard solutions and IS into 100 L of blank rat plasma or different tissue homogenates of untreated rats to yield PA-824 concentrations of 75, 150, 250, 500, 750, 1000 and 1500 ng/mL (in plasma), and IS concentration of 250 ng/mL. Quality control (QC) samples at lower limit of quantification (LLQC), low (LQC), middle (MQC) and high (HQC) concentrations (75; 100, 800 and 1400 ng/mL) were prepared separately in the same fashion. More detailed information can be found in Section 2.8. 2.4. Chromatographic conditions For HPLC separation, a YMC Triart C18 column (YMC Europe Gmbh, Dislanken, Germany), with spherical hybrid silica particles
(150 mm × 3.0 mm; particle size 3 m) equipped with the corresponding guard column (4 mm × 3.0 mm) was used. The mobile phase was Milli-Q water (0.1% v/v formic acid) and ACN (0.1% v/v formic acid). The flow rate was 0.2 mL min−1 and the temperature of the column oven was set at 25 ◦ C. The gradient profile was initially from 30 to 90% ACN in 10 min (held 2 min), after which time the mobile phase was returned to the initial conditions (30% ACN) in 3 min. The sample injection volume was 5 L. Column reequilibration was set at 5 min. 2.5. Mass spectrometric analysis To obtain accurate mass spectra of target analyte a maXis 4G ESI time-of-flight-mass spectrometry (TOF-MS) instrument (Bruker Daltonics) was used. The MS conditions were optimized. Acquisition parameters were: source type, ESI; ion polarity, positive; nebulizer, 1.5 bar; capillary, 3500 V; dry heater, 200 ◦ C; scan range, m/z 100–1500; end plate offset, 500 V; dry gas, 8.0 L/min; collision cell radiofrequency, 600 Vpp; collision energy, 21 eV. 2.6. Plasma and tissue samples Drug free plasma samples were purchased from Life Technologies (Burlington, ON, Canada) and stored at −20 ◦ C prior to analysis. Plasma samples were thawed at room temperature before analysis. Rat brains and other tissues were separated and weighed, then placed into liquid nitrogen immediately. The organs were weighed, cut it into small pieces using scissors and mixed with ultra-pure water (3 L/mg tissue). The tissues were then homogenized. All homogenate was stored at −80 ◦ C till analysis. 2.7. Sample preparation To an aliquot of 100 L of spiked plasma sample, IS was added at a concentration level of 250 ng/mL and vortexed briefly. Subsequently, 800 L of acetonitrile or methanol was added to induce the precipitation of plasma proteins. The mixture was vigorously mixed for 1 min, followed by centrifugation at 13,000 rpm for 15 min. The supernatants were filtered through a Hybrid-SPE-Phospholipid cartridge (30 mg/1 mL). The filtered samples (500 L) were evaporated until dryness under a gentle stream of nitrogen, and reconstituted in 500 L of mobile phase. All the vials containing samples were vortexed briefly and transferred into autosampler vials for injection into the chromatographic system. All thawing of frozen plasma samples and tissues were completed at room temperature. For the brain and lung tissue samples, the IS was added to 100 L brain homogenates samples and followed by addition of ultra-pure water. The IS was added to the homogenized tissue sample and thoroughly mixed. Subsequent extraction procedure of the drug from the tissue samples was the same as described for the plasma. 2.8. Method validation
Fig. 1. Chemical structure of the target analyte.
During the process of method validation, specificity, linearity, lower limit of quantification (LLOQ), limit of detection, precision, accuracy, extraction recovery, and stability were evaluated. The present method was also validated using total error approach [26–28]. For each level, the limit of the -expectation tolerance interval (-TI) was calculated. -TI is the interval within which the average proportion of future  results will fall. Calculations are detailed in the work of Rozet et al. [28]. -TI should not exceed the threshold of 30% selected for the acceptance limits, in agreement with the recommendations [29].
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2.8.1. Specificity and selectivity The specificity and selectivity of the method were performed by examining the presence or absence of interference, comparing chromatograms of six lots of blank plasma and tissue samples from different sources, blank samples spiked with standard and rat samples after oral and intraperitoneal administration of PA-824. In accordance with the validation guideline [29], absence of interfering components is accepted where the response is less than 20% of the LLOQ for the analyte and 5% for the IS. 2.8.2. Linearity and carry-over effect Each calibration curve should consist of a blank sample, a zero sample and six to eight calibration concentration levels, among which blank and zero samples were only analyzed to confirm the absence of interferences. Calibration curves were plotted by the peak area ratio versus analyte concentrations using a 1/x2 weighted linear least-squares regression model. The LLOQ was evaluated by analyzing six replicates of spiked samples at the relevant concentration. Carry-over effects were assessed, with relevant criteria, by injecting blank samples following the calibration standard at the highest concentration. Carry over in the blank should not exceed 20% of the LLOQ and 5% for the IS [29]. 2.8.3. Accuracy and precision Accuracy was determined as the ratio between the calculated concentration of the target compound and its theoretical concentration. Intra- and inter-day precisions were assessed by assay of six replicates of QC different samples at low, medium, and high concentrations on the same day and on three different days. The acceptance criteria were within 15% RSD and 85–115% (except LLOQ QC) of nominal concentration for accuracy and precision, respectively. QC samples concentrations were as following: 75, 100, 800 and 1400 ng/mL for plasma samples; 50, 75, 400 and 1100 ng/g for lung tissue; and 100, 150, 800 and 1400 ng/g for brain tissue. 2.8.4. Matrix effect and extraction recovery Matrix effects were investigated using the matrix factor by calculating the ratio of the peak area in the presence of matrix (measured by analyzing blank matrix spiked after extraction with analyte), to the peak area in absence of matrix (pure solution of the analyte). Matrix factor should not be greater than 15% [29]. This was determined at a low, medium and at a high level of concentration (maximum of three times the LLOQ and close to the upper limit of quantification, ULOQ). The extraction recovery of PA-824 was performed at the three QC levels (low, medium and high) in five replicates. 2.8.5. Stability Stabilities of PA-824 in plasma were estimated by assay of three replicates of QC samples at low, medium, and high concentrations under the following conditions: short-term stability after storage at room temperature (25 ◦ C) for 6 h; freeze-thaw stability through three freeze-thaw cycles (−80–25 ◦ C). The post-preparative stability was examined after 24 h in the autosampler maintained at 25 ◦ C. In agreement with guideline [29], the QC samples are analyzed against a calibration curve, obtained from freshly spiked calibration standards, and the obtained concentrations are compared to the nominal concentrations. The mean concentration at each level should be within ±15% of the nominal concentration. The stock solution stabilities of PA-824 and IS were demonstrated following storage at −20 ◦ C. The measured concentrations of stabilities are compared to the nominal concentrations. The acceptable RSD was within ±15%.
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3. Animals All animal experiments were performed with approval from the Animal Ethics Research Committee of University of KwaZulu-Natal (069/14/Animal). The study was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) which was approved by the Lund/Malmö, Sweden Ethical Committee on Animal Experiments (1988). Sprague Dawley female rats (weight 130 ± 10 g, 6 weeks) were purchased from the Biomedical Resource Unit (UKZN, Durban, South Africa). The animals were allowed a 7 days acclimatization period prior to experimentation. They were kept in a climate controlled facility (25 ◦ C, 60% relative humidity) with 12 h light-dark cycle. Food and water were given ad libitum. 3.1. Application to pharmacokinetic study 3.1.1. Drug administration and tissue collection The PA-824 was dissolved in a 40% (v/v) aqueous solution of DMSO. Two groups of Sprague Dawley rats were administered an oral dose or intraperitoneal injection of the PA-824 solution, respectively. The animals were anesthetized with halothane and sacrificed after the required time frames. Blood samples were collected at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 h post-dosing. Blood samples were collected in K3 -EDTA-coated tubes and centrifuged at 12,000 rpm for 10 min, and plasma was separated and stored at −80 ◦ C before bioanalysis. Tissues (brain and lungs) were collected after cervical dislocation of the anaesthetized rat. Organs were separated and placed into liquid nitrogen immediately. Tissues were homogenized and all homogenates were stored at −80 ◦ C until analysis. 3.1.2. Application to pharmacokinetic study Maximum concentration (Cmax ) of PA-824 and time to Cmax (Tmax ) were determined directly from the data. Other pharmacokinetic parameters were determined by analyzing the individual rat plasma profiles using non-compartmental equations in Microsoft Excel [30]. The area under the curve (AUC0→∞ ) was calculated by the linear trapezoidal rule. Apparent oral clearance (Cl/F) was calculated using the equation: Cl = dose/AUC0→∞ . Mean residence time (MRT) was calculated using the equation: MRT = AUMC0→∞/ AUC0→∞ . 4. Results and discussion 4.1. Method development During method development, different options were evaluated to optimize mass spectrometry detection parameters, chromatography and sample extraction. 4.1.1. LC–MS/MS analysis The instrument was optimized to obtain sensitivity and signal stability during infusion of the analyte. That was performed in the continuous flow of mobile phase to electrospray ion source operated at both polarities at a flow rate of 180 L/min. Target analytes gave good response in positive ion mode. For polar analytes, ESI is the preferred ionization technique. The addition of 0.1% formic acid improves the formation of protonated adducts, which can be detected in positive mode. Under these conditions the single charged quasi-molecular ion at 360.1 m/z is the base peak but the dimeric ion at 719.2 m/z was also detected. Using the m/z of the base peak, chromatograms of selected ions of interest were extracted. Fig. 1S a shows a typical mass spectrum of the product ion of m/z 360.1 for PA-824 (see Supporting Information).
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Table 1 Recoveries and matrix effects of PA-824 in different biological samples. Concentration level Plasma LQC MQC HQC Lungs LQC MQC HQC Brain LQC MQC HQC
Mean recovery (%)
%RSD
Matrix effect (%)
%RSD
90.2 91.0 90.1
1.6 1.1 2.1
−8.4 −1.9 −6.3
4.8 2.1 1.7
95.6 94.5 94.2
2.3 1.3 1.1
−1.6 −6.6 −6.8
3.0 3.5 3.8
90.1 91.5 90.2
2.0 1.2 1.8
−2.3 −1.3 −4.9
4.5 2.9 2.4
The mass spectrometer was operated in positive ion mode using MRM mode to monitor the mass transitions. The optimum MSMS conditions were optimized. Two fragmentations were acquired for the target analyte (m/z 360.1 → m/z 175.0 and m/z 360.1 → m/z 201.1). To quantify the analyte, its most intense transition was chosen. The transition m/z 360.1 → m/z 175.0 was selected for PA-824. Fig. 1S(b) presents a typical MS/MS spectrum obtained from collision activated dissociation of m/z 360.1 of PA-824 (see Supporting Information). 4.1.2. Optimization of extraction procedure Prior to loading the sample for LC injection, co-extracted proteins and phospholipids should be removed from the prepared solution. For this purpose, we tested two different techniques, protein precipitation (PP) and solid phase extraction (SPE). First for PP we used MeOH. The recoveries found employing this approach for PA-824 were 62–70%. Higher recoveries (more than 92%) were observed using ACN for PP. Although, this PP procedure removed gross levels of proteins providing good recoveries, in order to remove phospholipids that remained in the extract, an additional step was required. An appropriate pre-treatment protocol for plasma samples used before chromatographic determination was achieved using SPE. Plasma was first subjected to protein precipitation via the addition and mixing of acetonitrile. Precipitated proteins were then removed by centrifugation, and the resulting supernatant was loaded onto the HybridSPE-Phospholipid cartridge, which acts as a chemical filter that specifically targets the removal of endogenous sample phospholipids. The manufacturer’s specifications of HybridSPE-Phospholipid cartridges indicated the direct injection of the eluate. However, in the developed clean-up step, the eluate was evaporated to dryness and reconstituted with the mobile phase to obtain better separation. This simple methodology provided cleaner extracts of plasma samples containing the target analyte. Table 1 shows the recoveries of PA-824 in plasma at three concentration levels. The mean extraction recovery of IS (250 ng/mL) was more than 92.7% (%RSD 2.5%). This extraction procedure resulted in accurate and precise detection of PA-824 in plasma. 4.1.3. Evaluation of matrix effects Matrix effects may be defined as a composite of some undesirable effects that originate from a biological matrix [31]. Due to the influence of co-eluting compounds in the ionization process of the target analyte, these components may result in ion suppression/enhancement as well as decrease/increase in sensitivity of analytes over a period of time, increased baseline, imprecision of data, drift in retention time and distortion or tailing of a chromatographic output. The results of the post-extracted experiment (see Table 1) indicate that there is no significant ion suppression/enhancement observed at retention time of the analyte.
4.1.4. Method validation The method was validated according to the most recent European Medicine Agency (EMA) guideline on bioanalytical method validation [29]. Linearity, LLOQ, inter-day and intra-day precision and accuracy as well as recovery and stability of the PA-824 were evaluated. After appropriate adjustment of the chromatographic separation conditions, a reproducible separation of PA-824 from plasma was achieved. The detection of PA-824 and IS was highly selective with no interference from the endogenous substances. Fig. 2S(a) represents typical chromatograms of blank plasma, Fig. 2S(b) blank plasma spiked with IS at 250 ng/mL and PA-824 at 75 ng/mL (LLQC level) (see Supporting Information). Typical retention time for PA-824 was 8.8 min. Based on these findings, and the fact that the peaks for PA-824 and IS were symmetrical, they were judged to be adequate for subsequent analyses. Typical linear regression equations of the calibration curves for different matrices were equal to: y = 0.0065x − 0.1025; y = 1.536518x + 0.097110 and y = 1.774116x + 0.103510 (for plasma, lungs and brain, respectively), where y was the peak area ratio of the analyte to the internal standard and x the concentration of the analyte. The mean regression coefficients of the calibration curves were 0.9982, 0.9989 and 0.9974 (in plasma, lungs and brain, respectively). Calibration curves of PA-824 in plasma tissue samples were established over a concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. Batches, consisting of duplicate calibration standards at each concentration, were analyzed on three different days to complete the method validation. In each batch, QC samples at 75, 100, 800, 1400 ng/mL in plasma, 50, 75, 400, 1100 ng/g and 100, 150, 800, 1400 ng/g in lungs and brain, respectively, were assayed in sets of six replicates. The calibration curves were linear over the concentration range of 75–1500 ng/mL (plasma), 50–1200 ng/g (lungs) and 100–1500 ng/g (brain) with the correlation coefficient greater than 0.997. The LLOQ was evaluated by analyzing six replicates of spiked samples at the concentration of 75 ng/mL. The limit of detection (LOD), calculated using a signal to noise ratio of ≥3, was 25 ng/mL for plasma, 25 ng/g for lungs and 50 ng/g for brain. The accuracy observed for the standards ranged from 95.5 to 104.5% for PA-824 (data not shown). Meanwhile, in complex plasma and tissue samples, the observed accuracy ranged from 85.5 to 98.5% (except LLQC level >82%). As can be seen, Table 2 summarizes the intra- and inter-day precision and relative error for the PA-824, evaluated by assaying the QC samples. The inter-day and intra-day precision (%RSD) of the spiked quality control samples for the analyte in plasma was lower than 12.7. The obtained results were within the acceptance criteria. Moreover, the accuracy of the method takes into consideration the total error related to the test result [26,27]. As shown in Table 2, the ˇ expectations were around 30% for each concentration level with a probability of 95%. Carryover evaluation was performed in each analytical run to ensure that the accuracy and the precision of the proposed method were not affected. There was a negligible carryover observed during the autosampler carryover experiment. No enhancement in the response was observed in the double blank after subsequent injection of the highest calibration standard (aqueous and extracted) at the retention time of analyte. 4.2. Stability 4.2.1. Stability in plasma Bench top and autosampler stability for PA-824 were investigated at LQC, MQC and HQC levels. The results revealed that PA-824 was stable in plasma for at least 6 h at room temperature, and 24 h in an autosampler. All the values of accuracy and precision were
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191
Table 2 Accuracy and precision for PA-824 in different biological matrices. Matrix
Concentration level
LLQC
LQC
MQC
HQC
Plasma
Theoretical concentration (ng/mL) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
75.0
100.0
800.0
1400.0
62.9 83.9 11.5
87.9 88.0 11.3
707.1 88.4 10.7
1260 90.0 12.3
61.5 82.0 8.9 [−2.3; 29.8]
85.6 85.5 12.4 [−5.5; 29.5]
704.6 88.1 11.3 [−4.6; 27.8]
1256.3 89.7 12.7 [−5.4; 26.6]
Theoretical concentration (ng/g) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
50
75
400
1100
44.4 88.7 9.1
73.9 98.5 5.1
380.0 95.0 4.8
952.4 86.6 1.7
44.8 89.6 2.8 [−3.6; 25.5]
73.4 97.9 1.1 [−12.7; 16.3]
376.4 94.2 1.1 [−8.4; 19.3]
946.9 86.1 1.8 [−4.9; 22.3]
Theoretical concentration (ng/g) Intra-day Mean concentration Accuracy (%) %RSD Inter-day Mean concentration Accuracy (%) %RSD -TI (%)
100
150
800
1400
90.4 90.4 5.5
128.9 86.0 3.6
720.0 90.0 3.5
1261.6 90.1 2.1
89.8 89.8 2.1 [−8.2; 28.1]
128.1 85.4 1.0 [−3.4; 25.1]
718.5 89.8 3.8 [−8.1; 28.1]
1255.5 89.7 2.6 [−1.6; 21.8]
Lungs
Brain
-TI: -expectation tolerance limits.
in the range from 85.2 to 100.5% and 1.0 to 3.6%, respectively. PA824 in plasma and tissue homogenate samples was stable in the processed samples after three freeze/thaw circles. It was confirmed that repeated freezing and thawing of different samples spiked
with PA-824 at three concentration levels, which indicated that PA-824 had no significant degradation under the conditions previously described. The stability results obtained from the study are shown in Table 3.
Table 3 Stability of the plasma and tissue samples. Sample
Storage condition
Plasma
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Lungs
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Brain
Bench top, 6 h, RT
Autosampler, 24 h, RT
Three freeze/thaw cycles
Concentration (ng/mL)
Accuracy (%)
%RSD
Added
Found
100 800 1400 100 800 1400 100 800 1400
90.2 747.6 1262.3 90.4 712.7 1260.4 85.2 696.8 1211.2
90.2 93.4 90.2 90.4 89.1 90.0 85.2 87.1 86.5
2.2 3.2 2.4 3.5 1.3 1.2 1.3 1.9 2.5
75 400 1100 75 400 1100 75 400 1100
74.1 375.6 1058.3 72.3 377.6 1033.9 69.5 369.9 1001.7
98.9 93.9 96.2 96.4 94.4 94.0 92.7 92.5 91.1
1.4 1.9 1.9 2.0 1.8 1.9 2.4 1.4 1.1
150 800 1400 150 800 1400 150 800 1400
138.2 750.9 1272.2 137.2 762.9 1280.7 128.1 740.3 1224.2
92.1 93.9 90.9 91.4 95.4 91.5 85.4 92.5 87.4
2.4 1.5 1.1 3.6 1.5 1.0 1.8 1.9 1.6
192
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Fig. 2. Mean plasma concentration–time profiles for PA-824 after oral (a) and intraperitoneal (b) administration of a single dose of 20 mg/kg to the rats.
4.3. Stock solution stability Stock solution stability was performed to check stability of PA824 and in stock solutions prepared in ACN and stored at 2–8 ◦ C in a refrigerator. The freshly prepared stock solutions were compared with stock solutions prepared before 30 days. The percentage change for PA-824 were less than 10% respectively indicating that stock solutions were stable for at least 30 days. 4.4. Application of method To investigate the suitability of this analytical method for pharmacokinetic studies, it was used for determination of plasma concentrations of PA-824 after oral and intraperitoneal administration of a single dose (20 mg/kg) to rats (n = 8). Pharmacokinetic parameters following both routes of administration of drug are shown in Table 4. Pharmacokinetic data after oral administration obtained by analysis of the individual profiles enabled estimation of Cmax of 626.7 ng/mL reached with a Tmax of 6 h. The area under the curve (AUC0→∞ ) using the trapezoidal rule was 3724.8 ng × h/mL, consistent with previous report [25]. The temporal profile for PA-824 in plasma after an oral administration is shown in Fig. 2a. In this Table 4 Pharmacokinetic data following oral and intraperitoneal administration of PA-824 (20 mg/kg) in a rat. Pharmacokinetic variable
Oral administration
Intraperitoneal administration
Cmax (ng/mL) (%RSD) Tmax (h) AUC0→∞ (ng × h/mL) Cl/F (mL/h/kg) MRT0→∞ (h)
626.7 (4.1%) 6.0 3724.8 5.37 12.17
1146.6 (1.5%) 0.25 3988.5 5.01 9.27
paper, the pharmacokinetic parameters of PA-824 were in accordance with previous report [25]. In the next step of this study, we investigated pharmacokinetic data after intraperitoneal administration of PA-824. As reported by Manley et al. [32] peritonitis may increase peritoneal permeability, and result in faster drug absorption as well as increase of peritoneal drug clearance during non-drug-containing dwells. When single dose of PA-824 was administered via the intraperitoneal route, the AUC0→∞ was 3988.5 ng × h/mL. The mean plasma concentration–time profile for PA-824 in plasma after an intraperitoneal administration is illustrated in Fig. 2b. After intraperitoneal administration, PA-824 was absorbed quickly. The concentration of the drug was readily measurable in the plasma samples collected up to 24 h post dose. As can be seen on Fig. 2b, after intraperitoneal administration, the plasma concentration of PA-824 first decreased rapidly and then more slowly, that is to say, the plasma concentration of PA-824 after intraperitoneal administration decreased polyexponentially and the terminal elimination half-life was relatively long (about 8 h). In addition, a similar tendency was reported by Xu et al. [33] for intraperitoneal administration of oridonin, one of the promising anti-cancer compounds. Moreover, in both cases, after 24 h traces of PA-824 were detected, but the concentration of the drug was just below the limit of quantification. This method was also successfully applied to determine the concentration of PA-824 in rat lungs and brain following oral and intraperitoneal administration. As an example, typical MRM chromatograms of rat plasma, lungs and brain samples, respectively, are presented in Fig. 3 (see attached ppt). The tendency graphs of PA-824 in rat lungs and brain after oral (Fig. 3S, Supporting Information) and intraperitoneal administration routes can be seen in Fig. 4. After oral administration due to first-pass metabolism the concentration PA-824 was determined at 6 and 8 h, concentrations of
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Fig. 3. Representative MRM chromatograms of PA-824 in rat plasma, lungs and brain homogenates. Blank plasma and blank tissue homogenates (a); blank plasma and tissue homogenates spiked with the analyte at LLOQ (b); and plasma and homogenates sample obtained 15 min after intraperitoneal injection (c).
the drug at the rest of post dosing points was below the limit of quantification. PA-824 reached a peak concentration of 216.3 ng/g (RSD = 11.7%) and 194.9 ng/g (RSD = 3.0%) at Tmax = 8 h, in lungs and brain respectively. Lungs and brain pharmacokinetics of PA-824 were also investigated following a single intraperitoneal dose to demonstrate the applicability of this assay. According to the results after intraperitoneal administration in rat lungs and brain, PA-824 features concentration–time profile, as shown in Fig. 4. Maximum concentrations of PA-824 for lungs were seen at 4 h after administration, whereas the highest concentration in brain was found at 2 h. The highest concentration levels of PA824 detected for tissues examined were 457.3 ng/g (RSD = 7.7%) in lungs. In brain PA-824 reached peak concentration of 236.8 ng/g (RSD = 2.6%) at 2 h post dosing. The results indicated that PA-824 was mostly eliminated from tissues after 24 h. The main pharmacokinetic parameters for PA-824 in rat lungs and brain after intraperitoneal administration are presented in
Table 5. Non-compartmental analysis of PA-824 concentration time profiles after intraperitoneal administration yielded Cmax of 4 and 2 h in lungs and brain tissue, respectively. Clearance was about 19.8 mL/h for lungs and 18.7 mL/h for brain. Tissue concentrations of PA-824 were considerably lower than the corresponding plasma concentrations. For PA-824, the plasma concentration was 2.5-fold higher than the tissue concentration of the lungs, whereas in comparison to brain plasma concentration was 5-fold higher. In addition, the plasma area under the curve from time zero to the last time point (AUC0→∞ ) was compared to those calculated for examined tissues. The mean ratio of PA-824 in the tissue to plasma was 0.25 for lungs and 0.27 for brain. By comparing the Cmax , Tmax and AUC0→∞ of rat plasma and brain, it is possible that PA-824 penetrates the blood–brain barrier. These results seem to indicate that PA-824 is absorbed from the gastrointestinal tract in rat. However, further investigations with other administration routes are necessary to confirm those findings. Generally, the observations indicate that the analytical method is suitable for measurement of concentrations of PA-824 in biological matrices to aid in more detailed pharmacokinetic and tissue distribution studies. In future studies this method can be used to study the effect of controlled release formulations on the delivery of the target drug to lungs and brain. Intranasal delivery is an efficient route to transport drugs across the blood–brain barrier. Since formulation and their
Table 5 Pharmacokinetic data following intraperitoneal administration of PA-824 in a rat lungs and brain.
Fig. 4. Concentration of PA-824 in rat lungs and brain after intraperitoneal administration at 20 mg/kg. Data are means ± SD.
Pharmacokinetic variable
Lungs
Brain
Cmax (ng/g) (%RSD) Tmax (h) AUC0→∞ (ng × h/mL) Cl (mL/h)
457.3 (7.7%) 4.0 1008.6 19.8
236.8 (2.6%) 2.0 1069.9 18.7
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components are vital factors for effective drug delivery to the brain, encapsulating the drug into nasal spray or nanoparticles [34,35] can also be considered in the development of PA-824 formulation. 5. Conclusion A sensitive and simple SPE/LC–(ESI)MS/MS method for the determination of PA-824 in rat plasma was developed and validated over the concentration range of 75–1500 ng/mL for plasma, 50–1200 ng/g for lungs and 100–1500 ng/g for brain tissue. The applicability of the established assay to the pharmacokinetic characterization of PA-824 in rats was investigated. The LC–MS/MS method was successfully applied to a pharmacokinetic study of PA-824 after oral administration of single dosage 20 mg/kg to rats. Moreover, based on the data reported, the assay appears to be applicable for the determination of pharmacokinetic characteristics of PA-824 involving the administration of single dose of the drug. Therefore, the analytical method may be useful in terms of characterizing the pharmacokinetic profiles in preclinical studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2015.02.041. References [1] [2] [3] [4] [5]
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