Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry

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Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry Aziza El Beqqali, Mazaher Ahmadi, Mohamed Abdel-Rehim ∗ Department of Environmental Sci. & Analytical Chemistry, Stockholm University, SE10691 Stockholm, Sweden

a r t i c l e

i n f o

Article history: Received 18 July 2016 Received in revised form 20 September 2016 Accepted 3 November 2016 Available online xxx Keywords: Microextraction by packed sorbent AZD6118 Dog plasma samples LC-ESI–MS/MS Validation

a b s t r a c t In this work, for the first time, a method has been developed for the determination of AZD6118, a candidate drug, in dog plasma samples. The method is based on microextraction by packed sorbent (MEPS) of the drug prior to liquid chromatography-electrospray ionization tandem mass spectrometry assay. Various important factors affecting MEPS performance were optimized, and under the optimized condition, a linear calibration curve in the concentration range of 20–25,000 nmol L−1 with a coefficient of determination over 0.99 was obtained. The back-calculated values of the calibration points showed good agreement with the theoretical concentrations (coefficients of variation percent between 0.3–3.8). The lower limit of quantification and limit of detection were 20.0 and 2.9 nmol L−1 , respectively. The repeatability and accuracy of the method was evaluated by determination of quality control samples at three concentration levels (low, medium and high) using the developed method, and the results (coefficients of variation values were between 1.9% and 3.2%, relative recoveries ranged between 93.5–102.1%) confirm that a powerful method has been developed for the extraction and determination of the investigated drug in dog plasma. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Introducing a new drug needs a valid, accurate, and sensitive method to study the drug pharmacokinetics, toxicity, adverse effects, interactions and therapeutic efficiency by monitoring the drug and its metabolites levels in body fluids such as urine and plasma [1]. Fortunately, nowadays there are some well-established methods such as chromatographic methods including liquid and gas chromatography (LC and GC) but unfortunately, these methods suffer from some limitations in facing to complex matrixes which contain various compounds [2]. Various co-existing compounds inside of the body fluids such as proteins, cells, carbohydrates, lipids, enzymes, amino acids, ions, etc. in addition to other xenobiotic compounds can affect the chromatography columns efficiency in terms of sensitivity, accuracy, durability, precision, etc. To this end, a sample preparation step is usually useful to increase the method total sensitivity and to decrease the effect of endogenous compounds in sample matrix [3–6].

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (M. Abdel-Rehim).

Sample preparation has many tasks such as sample cleanup, preconcentration, and subsequently signal enhancement and enhancement of method selectivity [7–12]. By far, various sample preparation techniques have been established including liquidliquid extraction (LLE), protein precipitation (PPT), and solid-phase extraction (SPE). However, nowadays these techniques have been regarded as conventional sample preparation techniques. Sample preparation has always been at the forefront of research and investigation for newer and more effective ways of extracting the analyte from the biological matrixes. Herein, an advanced sample preparation technique, i.e. microextraction by packed sorbent (MEPS), has been utilized for determination of a drug candidate (i.e. AZD6118) in plasma samples. MEPS is a miniaturized form of SPE technique with improved analytical, economical, and environmental friendship features [13–18]. In MEPS very low quantities of the sorbent (∼2 mg) is packed into a microsyringe barrel as a plug or between the needle and the barrel (BIN mode) or inside of the needle as a cartridge [9,13]. The packed syringe can also be used several times, more than 100 times with plasma or urine samples, and more than 400 times for water samples [13,16]. MEPS is specially invented for on-line fully-automated analysis coupled to GC or LC instruments and has four main steps including preconditioning, sampling, washing and elution [13].

http://dx.doi.org/10.1016/j.jchromb.2016.11.004 1570-0232/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: A. El Beqqali, et al., Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.11.004

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Fig. 1. The chemical structures of AZD6118 and the internal standard.

There is a great demand for novel drugs in the treatment of Alzheimer’s disease (AD) and other cognitive disorders. Neuronal nicotinic receptors (NNRs) play a key role in memory and cognitive processes, whereas NNR loss is involved in the cognitive deficits associated with aging and dementias [15]. AZD6118 is a candidate drug provided by AstraZeneca. AZD6118 is a selective agonist of the central ␣4␤2 and ␣2␤2 neuronal nicotinic cholinergic receptors (NNRs). Thus, AZD6118 might be a potential candidate for AD treatment and other cognitive disorders in the elderly. Herein, MEPS is utilized as an efficient sample preparation technique for extraction and enrichment of AZD6118 from plasma samples, and for matrix clean-up prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) assay. The bioanalytical method using LC-ESI–MS/MS has been set up, and the methodology has been validated using three different quality control samples. 2. Experimental 2.1. Chemicals AZD6118 was obtained from AstraZeneca (Södertälje, Sweden) as its succinate adducts salt (Fig. 1). [15 N1 ,13 C4 ]-AZD6118was used as the internal standard (I.S.). For LC-ESI–MS/MS measurements, isopropanol, methanol, formic acid, and ammonium formate with HPLC-grade from Merck (Darmstadt, Germany) were used. Liquid nitrogen from AGA (Stockholm, Sweden) was used as both the drying and nebulizing gasses. High-purity argon (99.9999%) also from AGA (Stockholm, Sweden) was used as the collision gas. The investigated drug and the I.S. were dissolved in methanol to prepare the stock solutions. These solutions were kept in dark at 4 ◦ C. Standard solutions and quality control (QC) samples in three levels (i.e. low: 59 nmol L−1 (LQC), medium: 12200 nmol L−1 (MQC), and high: 19,516 nmol L−1 (HQC)) were freshly prepared from the stock solutions for each experiment in deionized water (resistivity not less than 18 M cm) provided using a water purification system (Milli-Q Plus) from Millipore (Bedford, MA, USA). All other used chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany). Dog plasma samples were obtained from AstraZeneca (Stockholm, Sweden). Plasma samples were stored at −20 ◦ C. Before use, the plasma was thawed at room temperature and centrifuged at 3500 rpm for 10 min, and then 50 ␮L of the plasma samples were diluted 5 times with ammonium formate (10 mmol L−1 ). The samples were spiked at 20–25,000 nmol L−1 concentration levels by adding 5–50 ␮L of the analyte standard solution to 0.5–1.0 mL of plasma and 25 ␮L of the internal standard. 2.2. Apparatus and instruments Commercially available MEPS syringes (250 ␮L gastight syringe) from SGE (SGE Analytical Science Pty Ltd, Melbourne, Australia) with mixed mode sorbent (SCX/C8) connected to an automated analytical microsyringe (eVol) from SGE were used.

For LC-ESI–MS/MS measurements, an ultra-performance liquid chromatography (UPLC) was used and obtained from Waters (Manchester, UK). Two washing solutions were used for the autosampler a weak solution (water, 800 ␮L after each injection) and a strong solution (methanol, 400 ␮L after each injection). An XBridge-C8 column (2.1 mm i.d. × 50 mm length, 2.5 ␮m particle size; Waters Corporation, Milford, MA, USA) was used as the analytical column. The LC was directly coupled to a triple quadrupole mass spectrometer Xevo TQ-S (Waters Corporation, Milford, MA, USA) equipped with an electrospray ionization source (ESI) and operated in positive ion mode. For data handling and quantification, MassLynx software (version 4.1) was used.

2.3. LC-ESI–MS/MS conditions A gradient mobile phase system was used: (A) 0.1% formic acid in 10.0 mmol L−1 ammonium formate, and (B) 0.1% formic acid in methanol-isopropanol (85:15, %v/v). The gradient started with 2% of mobile phase B with a hold of 0.5 min and then increased to 90% in 2.0 min with a hold of 2.5 min. Then, the mobile phase B was set to 2% again. For system stability, the next injection was performed after 4 min. The flow rate was 0.4 mL min−1 and the injected sample volume was 3 ␮L. The column oven and autosampler temperatures were set at 40◦ C and 15◦ C; respectively. The MS source block temperature and desolvation temperature were 150 and 650◦ C, respectively. Nitrogen was used as both drying and nebulizing gas and argon was used as the collision gas (0.15 mL min−1 ). The scan mode was multiple reaction monitoring (MRM) using the precursor ions at m/z (M+1) (m/z: 199.99 and 205.00), and after collisional dissociation, the product ions at m/z 171.03 and 174.00 were used for quantification of the pro-drug and the I.S., respectively. Other setting parameters were: capillary voltage, 1.0 kV; cone voltage, 28 V; extractor, 5 V; RF lens, 0.2 V, collision energy, 20 eV.

2.4. MEPS procedure Preconditioning of the sorbent was performed by passing 100 ␮L 10.0 mmol L−1 ammonium formate through the syringes. Then, the diluted samples (100 ␮L) were drawn slowly (20 ␮L s−1 ) into the packed syringe. After loading the sorbent with the investigated drug, the sorbent was washed by 100 ␮L water to remove biological interference, and then the adsorbed analytes were eluted using 3% ammonium hydroxides in methanol-water (60:40%v/v, 50 ␮L). Then, 3 ␮L of the extract was injected into LC-ESI–MS/MS by autosampler for further separation and analysis. In order to reuse the syringe, the sorbent was washed by methanol (4 × 100 ␮L) and water (4 × 100 ␮L) to decrease memory effects and carry-over. The same packing bed was used for about 100 extractions before it was discarded.

Please cite this article in press as: A. El Beqqali, et al., Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.11.004

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Table 1 Back calculated concentrations from calibration curves as a percentage of the nominal value. Calib. Points

S1

S2

S3

S4

S5

S6

S7

S8

Nominal conc. Batch I Bach II

20 99.4 100.2

50 102.3 100.3

200 95.1 96.1

500 108.0 103.0

1000 92.0 97.1

5000 105.0 104.6

20000 95.1 97.8

25000 103.0 100.9

n %Mean %CV

2 99.8 0.6

2 101.3 1.4

2 95.6 0.7

2 105.5 3.4

2 94.6 3.8

2 104.8 0.3

2 96.5 2.0

2 102.0 1.5

Table 2 Accuracy and precision of AZD6118 QC Samples.

Nominal concentration (nmol L−1 ) na Mean SD %Bias %CV a

QCL

QCM

QCH

59.5 4 59.3 1.88 0.4 3.2

12200 4 11402 325 −7.7 2.9

19516 4 19929 373 0.9 1.9

n = 4 assays, each assay includes 6 × QCs.

3. Results and discussion 3.1. MEPS method development MEPS has four main steps, which include preconditioning, sampling, washing and elution. The sample is drawn once or more through the sorbent to load the analyte(s) of interest on the sorbent. Then, the sorbent is washed by a suitable solvent (usually water) to remove the interferences which are loosely adsorbed by sorbent or syringe surface. Finally, the loaded analyte(s) is eluted with microliter volume of a suitable solvent (eluent) directly into the injector of the instrument. In order to achieve maximum recoveries for the extraction of the investigated analytes using MEPS technique, different parameters that could potentially affect the sorption and desorption efficiencies should be optimized. The most important parameters in MEPS performance are the type of sorbent, type of preconditioning and washing solvent, type and volume of desorption solvent (eluent), and sample and eluent flow rate [13]. In this study, mixed mode sorbent (SCX/C8) was utilized for the extraction of the investigated analyte. SCX/C8 is a mixed-mode sorbent based on a strong cation-exchange bonded silica sorbent (C8) provided by SGE. It is usually used for the extraction of basic drugs from urine or plasma samples. In this study, the preconditioning and washing were performed using 100 ␮L 0.01 mol L−1 ammonium formate. After loading and washing the analytes on the sorbent, in the next step, the analytes should be desorbed in a very low volume of a proper eluent to increase enrichment factor. The eluent should be able to quantitatively desorb the adsorbed analytes with high capacity (low volumes), and also should preferably has a similar composition to the used mobile phase of LC instrument. To this end, different eluents containing methanol, isopropanol, water, formic acid, ammonium formate, and ammonium hydroxide were investigated. The results showed that 3% ammonium hydroxides in methanol-water (60:40%v/v, 50 ␮L) provided the best recoveries, and therefore was used as the eluent. The solvents and sample flow rates can affect all of the steps of MEPS protocols. Lower flow rates allow better interaction between the analyte and the sorbent. In this study, the effect of flow rate on extraction and desorption of the investigated analytes was investigated, and the results (the results are not shown) showed that 20 ␮L s−1 could provide the best recoveries.

Fig. 2. Representative calibration MRM transitions obtained from the analysis of AZD6118 at LOD and the internal standard levels in dog plasma samples.

3.2. Method calibration and analytical parameters The method was validated according to the international FDA (Food and Drug Administration) guidelines [19] using dog plasma. The ratios of peak areas of AZD6118 and the I.S. were measured and a standard curve was constructed. Due to the complexity of a sample matrix such as plasma a quadratic calibration curve was used for quantitation (y = Ax2 + Bx + C, where: y is the peak area ratio, x concentration, A curvature, B slope, and C intercept, respectively). It minimizes the percent relative error in the back-calculated values of the calibration points. The calibration curve was prepared in dog plasma in the concentration range of 20–25,000 nmol L−1 . The calibration curve consists of eight calibration points. The coefficient of determination (r2 ) were over 0.999 in all runs (n = 3). Table 1 shows the back-calculated values of the calibration points. The results showed good agreement with the theoretical concentrations (coefficients of variation percent between 0.6 and 3.8). In addition the LLOQ was 20 nM (%CV: 4, n = 6) and the LOD was 2.9 nM (%CV: 6, n = 6). Fig. 2 shows presentative LC–MS/MS chromatograms of AZD6118 at LOD and the internal standard in dog plasma samples. 3.3. Selectivity, precision and accuracy The method selectivity was investigated by injecting dog blank plasma after MEPS extraction. The injected dog blank plasma showed no presence of endogenous interference peaks with the

Please cite this article in press as: A. El Beqqali, et al., Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.11.004

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Table 3 Individual toxicokinetic parameters for AZD6118 calculated from plasma concentration-time data on day 1 and 7 of once daily dosing of AZD6118 to male and female dogs in the repeat dose phase. Day

Dose (mg/kg)

Sex

Subject

tmax (h)

Cmax (␮mol L−1 )

Cmax /Dose (kg/L)

AUCa (h*␮mol L−1 )

AUC/Dose (h*kg/L)

t½ (h)

1 1 1 1 7 7

6 6 12 12 6 6

F M F M F M

4 3 6b 5b 4 3

0.50 1.0 0.50 0.50 0.50 4.0

2.8 3.1 2.7 1.7 6.9 5.7

0.093 0.10 0.046 0.029 0.23 0.19

12 26 16 11 27 74

0.40 0.88 0.27 0.19 0.89 2.5

3.8 5.7 4.0 5.0 3.9 6.5

Vomiting occurred within 2 hours after dose at several dosing occasions. a AUC on day one, AUC(0–24) on day 7. b Both dogs, 5 M and 6F, in the high dose group (12 mg/kg) were sacrificed on Day 4 due to welfare reasons.

3.5. Analyte stability The analyte was stable in dog plasma at room’s temperature for at least 6 hours. The stock solution in methanol was stable up to three weeks at +4 ◦ C. The samples were stored at −70 ◦ C until analysis. All samples were analysed within the evaluated stability duration. 4. Method application to toxicokinetics study

Fig. 3. MRM transitions obtained from blank plasma samples spiked with the internal standard utilizing PPT and MEPS.

quantification of the studied analyte. A good method selectivity was obtained using MEPS as a sample preparation method. The repeatability of the measurements was evaluated by four assays. Each assay contains six consecutive standard solutions at QC concentration levels (low, medium, and high). The obtained coefficients of variation (CV) (Table 2) were found to vary between 1.9% and 3.2%, displaying that acceptable repeatability was achieved. The accuracy values (as%Bias) were in the range of −7.7 and +0.4% (Table 1), indicating the high accuracy of the proposed method for the extraction of the investigated drug.

3.4. Study of carry-over and matrix effect The carry-over is one of the common problems in trace analysis, therefore, it is important to be investigated. To reduce the memory effect and avoid carry-over, the MEPS syringe was washed with methanol (4 × 100 ␮L) and water (4 × 100 ␮L) after every injection. The carry-over after these washing steps was <0.01%. In addition MEPS method was compared with protein precipitation (PPT) as follow: Blank plasma samples were spiked with the I.S. and were extracted using the proposed MEPS and conventional PPT method and then the LC–MSMS chromatograms were compared. The blank plasma sample after PPT showed some interferences peaks whereas the samples after MEPS did not show any significant peaks of interferences (Fig. 3) confirming the high ability of the proposed method to clean extract and eliminate matrix effect. Furthermore, the matrix effect was evaluated using postextraction addition method at three concentration levels (LQC, MQC, and HQC). No significant matrix effects were observed and the variations were less than 3.2%.

The present method was applied for determining of AZD6118 in dog plasma from a study evaluating toxicokinetics in male and female dogs after oral administration of AZD6118. Individual toxicokinetic parameters are presented in Table 3. AZD6118 was given orally once daily as a single dose (a dose volume was 2 mL/kg). A washout period of at least one day was allowed between dose level increments. Blood samples for preparation of plasma, using K2-EDTA as anticoagulant, were taken from a jugular or other peripheral vein. Plasma was prepared by centrifugation (10 min at ca 1500g, approximately +4 ◦ C) and subsequently transferred to polypropylene tubes and was stored at −70 ◦ C or below until bioanalysis. 5. Conclusion In summary, for the first time, a sensitive and selective method was developed to determine AZD6118 in dog plasma samples. The results showed that utilization of MEPS as the sample preparation technique prior to LC-ESI–MS/MS could increase the selectivity, and decrease matrix effect and interference problems. The proposed method can be used for preclinical and clinical studies of the investigated drug to evaluate its pharmacokinetics, toxicity, adverse effects, interactions and therapeutic efficiency. The present method applied to analysis of real samples for toxicity study of AZD6118. References [1] J. Inglese, A practical guide to assay development and high-throughput screening in drug discovery. Edited by Taosheng Chen, ChemMedChem 5 (2010) 1398–1399. [2] N.Y. Ashri, M. Abdel-Rehim, Sample treatment based on extraction techniques in biological matrices, Bioanalysis 3 (17) (2011) 2003–2018. [3] M. Abdel-Rehim, New trend in sample preparation: on-line microextraction in packed syringe for liquid and gas chromatography applications: I. Determination of local anaesthetics in human plasma samples using gas chromatography–mass spectrometry, J. Chromatogr. B 801 (2004) 317–321. [4] M. Abdel-Rehim, Recent advances in microextraction by packed sorbent for bioanalysis, J. Chromatogr. A 1217 (2010) 2569–2580. [5] R. Said, M. Kamel, A. El-Beqqali, M. Abdel-Rehim, Microextraction by packed sorbent for LC–MS/MS determination of drugs in whole blood samples, Bioanalysis 2 (2) (2010) 197–205. [6] P.L. Kole, G. Venkatesh, J. Kotecha, R. Sheshala, Recent advances in sample preparation techniques for effective bioanalytical methods, Biomed. Chromatogr. BMC 25 (2011) 199–217. [7] T. Madrakian, M. Ahmadi, A. Afkhami, M. Soleimani, Selective solid-phase extraction of naproxen drug from human urine samples using molecularly

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Please cite this article in press as: A. El Beqqali, et al., Determination of AZD6118 in dog plasma samples utilizing microextraction by packed sorbent and liquid chromatography-electrospray ionization tandem mass spectrometry, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.11.004