Methodology
For reprint orders, please contact [email protected]
Preclinical pharmacokinetics and biodistribution studies of asenapine maleate using novel and sensitive RP–HPLC method Aim: Asenapine maleate (ASPM) is a newer antipsychotic drug available as a sublingual tablet in the market. Experimental: To investigate the pharmacokinetic and tissue distribution study of ASPM following oral administration in rats, reversed-phase HPLC method was developed and validated. Results: ASPM was extracted from plasma and tissue matrix by liquid–liquid extraction technique and analyzed using mobile phase consisted of phosphate buffer pH 3.0 and acetonitrile (65:35% v/v). The method showed good linearity (10–500 ng/ml) with recovery 83–102%. In pharmacokinetics study, half-life was 32.74 ± 7.51 h due to slow elimination of drug. The biodistribution study indicated preferential distribution of ASPM to highly perfused organs. Conclusion: The current method can be successfully applied for estimating the drug in various biological matrices. First draft submitted: 4 April 2017; Accepted for publication: 23 May 2017; Published online: 24 July 2017 Keywords: asenapine maleate • liquid–liquid extraction • pharmacokinetic • RP–HPLC • tissue distribution
Asenapine maleate (ASPM) is an atypical antipsychotic of dibenzo-oxepino pyrrole class with the chemical name (3aRS,12bRS)5-Chloro-2-methyl-2,3,3a,12b-tetrahydro1H - d i b e n z o [ 2 , 3 : 6 ,7] ox e pi n o [ 4 ,5 - c ] pyrrole(2Z)-2-butenedioate (1:1) [1] . The chemical structure of ASPM is shown in the Figure 1. It is white to off-white nonhygroscopic powder having an MW of 401.84 g/ mol, log K of 8.40 ± 0.08, log P of 4.61 ± 0.05 and intrinsic solubility of 237 ± 18 μg/ ml [2] . ASPM is prescribed for the treatment of schizophrenia and bipolar disorder [1,3] . ASPM is available as sublingual tablet in the market as its oral bioavailability is very low (99%) was obtained from Himedia Laboratories Pvt. Ltd, Mumbai, India. Potassium dihydrogen orthophosphate (purity ≥99%), sodium acetate anhydrous (purity min 99%) and glacial acetic acid (purity ≥99.5%) were obtained from Sisco Research Laboratories Pvt. Ltd (Mumbai, India). Ortho-phosphoric acid (88%) and acetonitrile HPLC grade (purity min 99.8%) were procured from Merck Ltd (Mumbai, India). Methanol HPLC grade (purity min 99.8%) and ethyl acetate (purity min 99%) were procured from Finar Ltd (Ahmedabad, India). Methyl tertbutyl ether (MTBE; purity min 99.8%) was obtained from Spectrochem Pvt. Ltd (Mumbai, India). Ultrapure water, obtained from a Millipore Direct-Q® 3 water purification system (Millipore Corporation, MA, USA) was utilized for the sample and mobile phase preparation. Instrumentation
LC system (LC-2010CHT, Shimadzu Corporation, Kyoto, Japan) was used. It was equipped with two quaternary low-pressure gradient pumps, dual wavelength UV detector, degasser unit, column oven and high-throughput autosampler. LC Solution 5.57 software was used to monitor the chromatography system and process the obtained chromatograms. Sensitive Sartorius CPA 225D analytical balance (Sartorius AG, Germany) was used for weighing standard substances. Mobile phase buffer solution was filtered through 0.22-μm membrane (Pall Pvt. Ltd, Bangalore, India) using a glass vacuum filtration assembly (Millipore) and degassed by sonication in ultrasonic bath (GT SONIC, Guangdong GT Ultrasonic Co. Ltd, China). The pH of the mobile phase buffer was measured with pH meter (CyberScan pH 510, Eutech Instruments, Thermo Fisher Scientific, Mumbai, India) using a glass electrode (Van London Co., TX, USA). Tissues were homogenized in a glass homogenizing tube using a mechanical stirrer (Remi Laboratory Instruments, Mumbai, India). Preparation of plasma matrix & tissue homogenate
Prior to the study, approval from Institutional Animal Ethical Committee, Kasturba Medical College, Manipal, India (Approval no.: IAEC/KMC/42/2014) was
future science group
Preclinical pharmacokinetics & biodistribution studies of asenapine maleate using novel & sensitive RP–HPLC method
obtained and animal handling was carried out as per the institutional and national guidelines for the care and use of animals. Preparation of plasma matrix: blood was collected from rats in Eppendorf tubes containing 20 μl of 10% w/v EDTA disodium anticoagulant. Tubes were gently turned upside down to mix the blood with anticoagulant. Blood sample was centrifuged at 5000 rpm for 10 min in microcentrifuge machine (Spinwin, Tarsons Products Pvt. Ltd, Kolkata, India) to separate the plasma. Clear plasma in the supernatant layer was collected in a fresh tube and stored at -80°C until further usage. Preparation of tissue homogenate: rat was humanely sacrificed and dissected to collect different organs viz. brain, kidneys, liver, spleen and small intestine. The collected organs were weighed on an aluminium foil and then homogenized with saline solution (0.9% w/v of sodium chloride) containing 100 μl of TFA under ice-cold bath. The final volume of homogenate was: brain: 2 ml; spleen: 2 ml; kidneys: 2 ml; liver: 4 ml; and small intestine: 2 ml. The homogenates were centrifuged (18,000 rpm for 10 min at 10°C) in cold centrifuge (Sigma Laborzentrifugen-3K30, Osterode, Germany) and the supernatant obtained was used for further study.
Methodology
Extraction procedure & chromatographic condition
ASPM and its IS LTZ were extracted from the biological matrix by liquid–liquid extraction technique. In 250 μl plasma/750 μl tissue homogenate, 30 μl LTZ (10 μg/ml solution) was added and mixed using the vortex mixer (CM101, Remi Equipment, Mumbai, India). Extracting solvent (MTBE) was added up to 1.5 ml and vortexed for 5 min. The sample was centrifuged at 15,000 rpm and 10°C for 10 min in cold centrifuge and 750 μl of clear supernatant was transferred to evaporating glass tubes. The organic solvent was evaporated until complete dryness using nitrogen evaporator (TurboVap, Zymark, MA, USA) at 50°C. The residue obtained was reconstituted with 500 μl of diluent (70:30% v/v ratio of water–methanol mixture), by the aid of sonication and then 100 μl of resultant solution was injected into the HPLC system. Mobile phase consisted of mixture of potassium phosphate buffer (25 mM, pH adjusted to 3.0 ± 0.05 with 0.01 M ortho-phosphoric acid) and acetonitrile in the ratio of 65:35% v/v. The flow rate of the mobile phase was set to 0.8 ml/min through Phenomenex Kinetex C18 column (250 mm × 4.6 mm id, 5-μm particle size, 100-Å pore size). When the solution of ASPM was scanned in UV/Vis spectrophotometer, it showed maximum
uV
LTZ
80,000 70,000
ASPM
60,000 50,000
a
40,000
b
30,000
c
20,000
d
10,000
e
0 -10,000 0.0
2.5
5.0
7.5
10.0
12.5
Min
Figure 2. Chromatograms of plasma samples and diluent. (A) plasma sample from pharmacokinetic study, (B) plasma spiked with asenapine maleate and LTZ (C) plasma spiked with letrozole, (D) blank plasma and (E) diluent.
future science group
1039
Methodology Managuli, Gourishetti, Shenoy, Koteshwara, Reddy & Mutalik
uV
LTZ
90,000 80,000 70,000 ASPM
60,000 50,000
a
40,000
b
30,000 c 20,000 d 10,000 e
0 2.5
5.0
7.5
10.0
12.5
Min
Figure 3. Chromatograms of asenapine maleate and letrozole extracted from different tissues. (A) brain, (B) kidney, (C) liver, (D) spleen and (E) small intestine.
wavelength values of 230 and 270 nm. However, based on the observed maximum absorbance value, 230 nm wavelength was used to increase the sensitivity of detection. Column temperature and autosampler temperature was maintained at 25 and 10°C, respectively. The run time was fixed to 15 min. Preparation of quality control samples & spiked samples
ASPM standard solution (200 μg/ml) was prepared by dissolving accurately weighed 2 mg of drug into 3 ml of methanol with the aid of sonication. Volume was made up to 10 ml with ultrapure water. Further, series of dilutions were made by diluting sufficient amount of aliquot with diluent. To prepare standard solution (10 μg/ml) of IS, 5 mg of LTZ was dissolved in 5 ml of methanol in a volumetric flask with the help of sonication and volume was made up to 10 ml with methanol. Then 0.2 ml of this solution was further diluted to 10 ml with the diluent. Low quality control (LQC;
75 ng/ml), middle quality control (MQC; 200 ng/ml) and high quality control (HQC) (400 ng/ml) samples were prepared by spiking known concentration of ASPM standard solution in biological matrices followed by extraction and reconstitution of residue obtained upon evaporation of organic solvent. For linearity in plasma, total of eight standard samples were prepared of concentrations: 25, 50, 75, 100, 200, 300, 400 and 500 ng/ml, whereas for linearity in organs, total nine standard samples of concentrations: 10, 25, 50, 75, 100, 200, 300, 400 and 500 ng/ml were prepared. The range of the method was 25–500 ng/ml for plasma and 10–500 ng/ml for organs. LLOQ in plasma was 25 ng/ml and in organs it was 10 ng/ml. Both LLOQs 25 and 10 ng/ml formed the lowest concentrations of calibration curves in plasma and organs, respectively. Chromatographic method optimization
For optimization of analytical method, buffers such as sodium acetate buffer pH 4.5 and potassium phos-
Table 1. System suitability data. System suitability parameters
Acceptance criteria
Observed
RSD of peak area (n = 6)
RSD
For reprint orders, please contact [email protected]
Preclinical pharmacokinetics and biodistribution studies of asenapine maleate using novel and sensitive RP–HPLC method Aim: Asenapine maleate (ASPM) is a newer antipsychotic drug available as a sublingual tablet in the market. Experimental: To investigate the pharmacokinetic and tissue distribution study of ASPM following oral administration in rats, reversed-phase HPLC method was developed and validated. Results: ASPM was extracted from plasma and tissue matrix by liquid–liquid extraction technique and analyzed using mobile phase consisted of phosphate buffer pH 3.0 and acetonitrile (65:35% v/v). The method showed good linearity (10–500 ng/ml) with recovery 83–102%. In pharmacokinetics study, half-life was 32.74 ± 7.51 h due to slow elimination of drug. The biodistribution study indicated preferential distribution of ASPM to highly perfused organs. Conclusion: The current method can be successfully applied for estimating the drug in various biological matrices. First draft submitted: 4 April 2017; Accepted for publication: 23 May 2017; Published online: 24 July 2017 Keywords: asenapine maleate • liquid–liquid extraction • pharmacokinetic • RP–HPLC • tissue distribution
Asenapine maleate (ASPM) is an atypical antipsychotic of dibenzo-oxepino pyrrole class with the chemical name (3aRS,12bRS)5-Chloro-2-methyl-2,3,3a,12b-tetrahydro1H - d i b e n z o [ 2 , 3 : 6 ,7] ox e pi n o [ 4 ,5 - c ] pyrrole(2Z)-2-butenedioate (1:1) [1] . The chemical structure of ASPM is shown in the Figure 1. It is white to off-white nonhygroscopic powder having an MW of 401.84 g/ mol, log K of 8.40 ± 0.08, log P of 4.61 ± 0.05 and intrinsic solubility of 237 ± 18 μg/ ml [2] . ASPM is prescribed for the treatment of schizophrenia and bipolar disorder [1,3] . ASPM is available as sublingual tablet in the market as its oral bioavailability is very low (99%) was obtained from Himedia Laboratories Pvt. Ltd, Mumbai, India. Potassium dihydrogen orthophosphate (purity ≥99%), sodium acetate anhydrous (purity min 99%) and glacial acetic acid (purity ≥99.5%) were obtained from Sisco Research Laboratories Pvt. Ltd (Mumbai, India). Ortho-phosphoric acid (88%) and acetonitrile HPLC grade (purity min 99.8%) were procured from Merck Ltd (Mumbai, India). Methanol HPLC grade (purity min 99.8%) and ethyl acetate (purity min 99%) were procured from Finar Ltd (Ahmedabad, India). Methyl tertbutyl ether (MTBE; purity min 99.8%) was obtained from Spectrochem Pvt. Ltd (Mumbai, India). Ultrapure water, obtained from a Millipore Direct-Q® 3 water purification system (Millipore Corporation, MA, USA) was utilized for the sample and mobile phase preparation. Instrumentation
LC system (LC-2010CHT, Shimadzu Corporation, Kyoto, Japan) was used. It was equipped with two quaternary low-pressure gradient pumps, dual wavelength UV detector, degasser unit, column oven and high-throughput autosampler. LC Solution 5.57 software was used to monitor the chromatography system and process the obtained chromatograms. Sensitive Sartorius CPA 225D analytical balance (Sartorius AG, Germany) was used for weighing standard substances. Mobile phase buffer solution was filtered through 0.22-μm membrane (Pall Pvt. Ltd, Bangalore, India) using a glass vacuum filtration assembly (Millipore) and degassed by sonication in ultrasonic bath (GT SONIC, Guangdong GT Ultrasonic Co. Ltd, China). The pH of the mobile phase buffer was measured with pH meter (CyberScan pH 510, Eutech Instruments, Thermo Fisher Scientific, Mumbai, India) using a glass electrode (Van London Co., TX, USA). Tissues were homogenized in a glass homogenizing tube using a mechanical stirrer (Remi Laboratory Instruments, Mumbai, India). Preparation of plasma matrix & tissue homogenate
Prior to the study, approval from Institutional Animal Ethical Committee, Kasturba Medical College, Manipal, India (Approval no.: IAEC/KMC/42/2014) was
future science group
Preclinical pharmacokinetics & biodistribution studies of asenapine maleate using novel & sensitive RP–HPLC method
obtained and animal handling was carried out as per the institutional and national guidelines for the care and use of animals. Preparation of plasma matrix: blood was collected from rats in Eppendorf tubes containing 20 μl of 10% w/v EDTA disodium anticoagulant. Tubes were gently turned upside down to mix the blood with anticoagulant. Blood sample was centrifuged at 5000 rpm for 10 min in microcentrifuge machine (Spinwin, Tarsons Products Pvt. Ltd, Kolkata, India) to separate the plasma. Clear plasma in the supernatant layer was collected in a fresh tube and stored at -80°C until further usage. Preparation of tissue homogenate: rat was humanely sacrificed and dissected to collect different organs viz. brain, kidneys, liver, spleen and small intestine. The collected organs were weighed on an aluminium foil and then homogenized with saline solution (0.9% w/v of sodium chloride) containing 100 μl of TFA under ice-cold bath. The final volume of homogenate was: brain: 2 ml; spleen: 2 ml; kidneys: 2 ml; liver: 4 ml; and small intestine: 2 ml. The homogenates were centrifuged (18,000 rpm for 10 min at 10°C) in cold centrifuge (Sigma Laborzentrifugen-3K30, Osterode, Germany) and the supernatant obtained was used for further study.
Methodology
Extraction procedure & chromatographic condition
ASPM and its IS LTZ were extracted from the biological matrix by liquid–liquid extraction technique. In 250 μl plasma/750 μl tissue homogenate, 30 μl LTZ (10 μg/ml solution) was added and mixed using the vortex mixer (CM101, Remi Equipment, Mumbai, India). Extracting solvent (MTBE) was added up to 1.5 ml and vortexed for 5 min. The sample was centrifuged at 15,000 rpm and 10°C for 10 min in cold centrifuge and 750 μl of clear supernatant was transferred to evaporating glass tubes. The organic solvent was evaporated until complete dryness using nitrogen evaporator (TurboVap, Zymark, MA, USA) at 50°C. The residue obtained was reconstituted with 500 μl of diluent (70:30% v/v ratio of water–methanol mixture), by the aid of sonication and then 100 μl of resultant solution was injected into the HPLC system. Mobile phase consisted of mixture of potassium phosphate buffer (25 mM, pH adjusted to 3.0 ± 0.05 with 0.01 M ortho-phosphoric acid) and acetonitrile in the ratio of 65:35% v/v. The flow rate of the mobile phase was set to 0.8 ml/min through Phenomenex Kinetex C18 column (250 mm × 4.6 mm id, 5-μm particle size, 100-Å pore size). When the solution of ASPM was scanned in UV/Vis spectrophotometer, it showed maximum
uV
LTZ
80,000 70,000
ASPM
60,000 50,000
a
40,000
b
30,000
c
20,000
d
10,000
e
0 -10,000 0.0
2.5
5.0
7.5
10.0
12.5
Min
Figure 2. Chromatograms of plasma samples and diluent. (A) plasma sample from pharmacokinetic study, (B) plasma spiked with asenapine maleate and LTZ (C) plasma spiked with letrozole, (D) blank plasma and (E) diluent.
future science group
1039
Methodology Managuli, Gourishetti, Shenoy, Koteshwara, Reddy & Mutalik
uV
LTZ
90,000 80,000 70,000 ASPM
60,000 50,000
a
40,000
b
30,000 c 20,000 d 10,000 e
0 2.5
5.0
7.5
10.0
12.5
Min
Figure 3. Chromatograms of asenapine maleate and letrozole extracted from different tissues. (A) brain, (B) kidney, (C) liver, (D) spleen and (E) small intestine.
wavelength values of 230 and 270 nm. However, based on the observed maximum absorbance value, 230 nm wavelength was used to increase the sensitivity of detection. Column temperature and autosampler temperature was maintained at 25 and 10°C, respectively. The run time was fixed to 15 min. Preparation of quality control samples & spiked samples
ASPM standard solution (200 μg/ml) was prepared by dissolving accurately weighed 2 mg of drug into 3 ml of methanol with the aid of sonication. Volume was made up to 10 ml with ultrapure water. Further, series of dilutions were made by diluting sufficient amount of aliquot with diluent. To prepare standard solution (10 μg/ml) of IS, 5 mg of LTZ was dissolved in 5 ml of methanol in a volumetric flask with the help of sonication and volume was made up to 10 ml with methanol. Then 0.2 ml of this solution was further diluted to 10 ml with the diluent. Low quality control (LQC;
75 ng/ml), middle quality control (MQC; 200 ng/ml) and high quality control (HQC) (400 ng/ml) samples were prepared by spiking known concentration of ASPM standard solution in biological matrices followed by extraction and reconstitution of residue obtained upon evaporation of organic solvent. For linearity in plasma, total of eight standard samples were prepared of concentrations: 25, 50, 75, 100, 200, 300, 400 and 500 ng/ml, whereas for linearity in organs, total nine standard samples of concentrations: 10, 25, 50, 75, 100, 200, 300, 400 and 500 ng/ml were prepared. The range of the method was 25–500 ng/ml for plasma and 10–500 ng/ml for organs. LLOQ in plasma was 25 ng/ml and in organs it was 10 ng/ml. Both LLOQs 25 and 10 ng/ml formed the lowest concentrations of calibration curves in plasma and organs, respectively. Chromatographic method optimization
For optimization of analytical method, buffers such as sodium acetate buffer pH 4.5 and potassium phos-
Table 1. System suitability data. System suitability parameters
Acceptance criteria
Observed
RSD of peak area (n = 6)
RSD
