Study of Solvent extraction of Atomoxetine from Aqueous solutions and Biological fluids

 

Liudmila Yu. Tomarovska¹, Sergii V. Baiurka², Svetlana A. Karpushina³

¹Assistant, Physical and Colloid Chemistry Department, National University of Pharmacy,

Pushkinska Str., 53, Kharkiv, 61002, Ukraine.

²Doctor of Science (Pharmacy), Professor, Head of Drug and Analytical Toxicology Department,

National University of Pharmacy, Pushkinska Str., 53, Kharkiv, Ukraine.

³PhD (Candidate) of Chemical Science, Associate Professor, Drug and Analytical Toxicology Department, National University of Pharmacy, Pushkinska Str., 53, Kharkiv, Ukraine.

 

ABSTRACT:

This article presents the systematic study of the solvent extraction of an antidepressant Atomoxetine and optimization of the drug isolation methods from blood and urine. The dependence of the extraction recovery of Atomoxetine from aqueous solutions on the type of the organic solvent, pH of the aqueous medium and the presence of a salting-out agent was determined. Сhloroform, methylene chloride, 1,2-dichloroethane, diethyl ether, ethyl acetate, tetrachloromethane, benzene, toluene, hexane were tested as organic extragents. The quantitative determination of Atomoxetine was performed by the UV-spectrophotometric method. The maximum extraction recovery value was of 28% at pH of 13 for chloroform. The extraction recovery with diethyl ether at pH of 1-2 was the lowest and equal to 0.2%, that makes possible to recommend this solvent for the extraction purification from co-extractive components of the biological matrix. To increase the extraction recovery sodium chloride and ammonium sulphate were used as salting-out agents. The maximum value of 89% in the extraction recovery of Atomoxetine was obtained for chloroform at pH of 11-12 in the aqueous phase saturation with ammonium sulphate. Recovery values of the solvent drug extraction were of 38.8% (RSD 8.7%) and 69.3% (RSD 6.7%) from blood and urine, respectively. Precipitation of blood cells by trichloroacetic acid (in sample preparation of blood), back-extraction and TLC clean-up step were incorporated into the sample preparation scheme to eliminate the extraction of the matrix components. The results obtained could be used in toxicological study of biological samples for presence of Atomoxetine.

 

 

INTRODUCTION:

Depression and anxiety are the most frequent psychiatric disorders commonly found including adolescences¹. Atomoxetine was the first non-stimulant drug approved by FDA (USA) in the late of 2002 year for treatment of the attention deficit hyperactivity disorder (ADHD) in both children and adults^2,3.

 

Unlike traditional psychostimulants, Atomoxetine does not have a potential for abuse; it is not classified as a controlled substance⁴. Atomoxetine (the trade name is Strattera®), an antidepressant of the class of selective norepinephrine reuptake inhibitors, is (3R)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-1-amine. It has the empirical formula of C₇H₂₁NO. Its molecular weight is 255.4. It is soluble in water (54.64 mg/L)⁵. The dissociation constant (pKa) is 9.8⁵, 10.1⁶. The partition coefficient (log P(octanol/water)) is 4.23⁵. The volume of distribution (Vd) is 0.85 L/kg^6,7, 8.5 L/kg⁵.

 

Atomoxetine can cause a wide range of side effects^3,4,7-9, among them the appearance of suicide thoughts is the most serious complication¹⁰. Cases of acute and lethal poisonings by Atomoxetine have been registered^11,12. The literature review revealed that post-mortem Atomoxetine concentrations were within the following limits: arterial blood – from 0.1 to 8.3mg/L, femoral blood – from 0.33 to 5.4mg/L, vitreous body – from 0.1 to 0.96mg/L, bile – from 1.0 to 33mg/L, liver – from 0.44 to 29mg/kg, stomach contents – from 0.0097 to 16.8mg in the reported sample; the post-mortem concentrations in the urine was of 0.1mg/L^6,12.

 

Most bioanalytical procedures described in the literature for Atomoxetine determination are based on using HPLC and relates to biological fluids as specimens^6,13. Types of detection used in these HPLC methods were as follows: UV^14-16, mass spectrometry^17-19, fluorescence^20,21, while the most common sample preparation procedures included liquid-liquid extraction (LLE)^14,15,17, solid-phase extraction (SPE)¹⁸ and plasma deproteinization^{19, 22}.

 

LLE is widely approved in most toxicological laboratories in sample preparation step^23-26. This procedure combines effective separation of an analyte from the sample, purification from the biological matrix components (back extraction) and pre-concentration of the target substance with a proper choice of an organic solvent and pH of the aqueous layer. Di-tert-butyl ether, diethyl ether and tert-butyl methyl ether were used for extraction of Atomoxetine from plasma^13,14,16. However, data on the extraction recovery of Atomoxetine depending on the type of an organic solvent and pH of the aqueous medium, which are usually necessary to incorporate a back-extraction step into the extraction scheme for more difficult samples than plasma (such as whole blood, tissues), are not available in the literature.

 

The aim of the present work:

Was the systematic study of the extraction recovery of Atomoxetine from aqueous solutions depending on the type of an organic solvent, the pH value and the presence of salting-out agents and optimization of the drug isolation methods from blood and urine.

 

MATERIALS AND METHODS:

Reagents and chemicals:

The Atomoxetine pure substance isolated from the medicinal product Strattera® (seven 60mg capsules) “Lilly” (Czech Republic) was used for the study. Extraction of the Atomoxetine substance from capsules was previously described²⁷. The purity of the substance was tested by TLC, UV spectrophotometry and HPLC.

 

Chloroform, methylene chloride, 1,2-dichloroethane, tetrachloromethane, diethyl ether, ethyl acetate, hexane, benzene, toluene, methanol were of analytical grade and were purchased from Sigma-Aldrich (USA); platinic chloride (99.995% trace metals basis) was obtained from Sigma-Aldrich (USA); hydrochloric acid 37% w/w was of analytical grade (Merck, Darmstadt, Germany). All other reagents (sodium hydroxide, 25% ammonia, anhydrous sodium sulphate, sodium chloride, ammonium sulphate, potassium iodide, concentrated phosphoric acid, concentrated acetic acid, boric acid, trichloroacetic acid) were of analytical grade and were purchased from Chimmed Company (Moscow, Russia). Acidified iodoplatinate solution was prepared by dissolving 0.25g of platinic chloride and 5g of potassium iodide in bidistilled water to produce 100mL, followed by adding 5mL of hydrochloric acid to 100mL of iodoplatinate solution obtained. Drug-free human blood was obtained from the Kharkiv Regional Blood Service Centre (Ukraine). Drug-free human urine samples were acquired from two volunteers.

 

Equipment:

Spectrophotometric measurements were carried out using a single beam UV/VIS-spectrophotometer SPEKOL®1500 (Analytik Jena AG, Germany) with the wavelength scanned from 1100 to 190nm. Data were processed using the WinASPECT software, version 2.3.1.0. The spectral bandwidth was of 1nm. Quarts square cells with a 10mm path length were used. Weighing was carried out using the analytical balances VLR-200 (Gosmetr’ company, Russia) with d=0.00005g. The buffer solution pH was monitored by a pH-meter 5123 (Elvro, Poland). A LW-4 water bath (Bytom, Poland) was used for vaporization. The following glassware was used: 10.00mL, 25.00mL volumetric flasks; 1.00, 2.00, 5.00, 10.00mL volumetric pipettes, Class A (Simax, Czech Republic); 50mL separation funnels (Simax, Czech Republic). Merck (Silica gel 60 F254, 10×20cm in size, Germany) chromatographic plates were used. Glass capillaries were calibrated with the help of a micropipette (0.200mL).

 

Stock solution and model solutions:

The stock solution (1mg/mL) was prepared by dissolving 0.02850g of Atomoxetine hydrochloride (corresponding to 0.02500g of the Atomoxetine base) in 25.0mL of distilled water using a 25.0mL volumetric flask. The model solutions (500µg/mL, 400µg/mL, 200µg/mL and 100µg/mL) were prepared by diluting stock solution 2 times, 2.5 times, 5 times and 10 times, respectively.

 

Extraction procedure:

9.00 mL of Britton-Robinson buffer²⁸ with a definite pH value in the range of 2.0-11.98 (or 0.1mol/L hydrochloric acid solution with pH1.1, or 0.1mol/L sodium hydroxide solution with pH13), 1.00mL of Atomoxetine aqueous solution with the concentration of 500μg/mL (or 200 μg/mL, or 100μg/mL), 10.00mL of the organic solvent were placed into a separation funnel, and the mixture was shaken by means of a mechanical fluid shaker for 5 min. Then the mixture was left to separate the phases for 10 min. The organic solvent layer was separated, filtered through a paper filter containing 0.5g of anhydrous sodium sulphate and transferred into an evaporating cup. The organic solvent was evaporated using a water bath at the temperature not higher than 40°C (diethyl ether was evaporated at room temperature) to dryness. The dry residue was dissolved in 4-5ml of 0.1mol/L hydrochloric acid solution, quantitatively transferred into a 10.00mL volumetric flask, and diluted to the volume with the same solvent. The absorbance of the resulting solution was measured at a wavelength of 270nm using 0.1mol/L hydrochloric acid as a reference solution. Britton-Robinson buffer, 0.1mol/L hydrochloric acid solution and 0.1mol/L sodium hydroxide solution were saturated with sodium chloride or ammonium sulphate before using to determine the extraction recovery in the presence of a salting-out agent.

 

Isolation of Atomoxetine from the biological fluids:

10mL blood samples were spiked with 1mL of aqueous solutions of Atomoxetine hydrochloride containing 200, 400 and 500μg of Atomoxetine as free base and left standing for 24 hours. 20mL of urine samples were spiked with 1 ml of aqueous solutions of Atomoxetine hydrochloride containing 100, 200 and 400μg of Atomoxetine as free base and left standing for 24 hours. The blank experiments were carried out in parallel.

 

Isolation of Atomoxetine from blood:

10mL of 10% trichloroacetic acid solution were added to the blood sample and mixed, the mixture was centrifuged for 15 min in speed of 3000rpm. 5mL of diethyl ether were added to the supernatant layer and the mixture was shaken on a mechanical shaker for 5 min. The aqueous phase was separated with the help of a separating funnel and the organic solvent layer was discarded. This cleaning procedure was performed twice. Then pH of the aqueous phase was adjusted to 11-12 with 20% sodium hydroxide solution, saturated with ammonium sulphate and the drug was extracted with 5mL of chloroform (twice). The resulting extract was filtered through a paper filter containing 0.5g of anhydrous sodium sulphate on top, evaporated to approximately 0.05mL final volume and applied as a band on the chromatographic plate for TLC purification.

 

Isolation of Atomoxetine from urine:

The urine sample was acidified to pH of 1-2 with 0.1mol/L hydrochloric acid, 10mL of diethyl ether were added and the mixture was shaken on a mechanical shaker for 5 min. The aqueous layer was separated with the help of a separating funnel and the organic solvent layer was discarded. This cleaning procedure was performed twice. Then pH of the aqueous phase was adjusted to 11-12 with 20% sodium hydroxide solution, saturated with ammonium sulphate and the drug was extracted with 10 mL of chloroform (twice). The resulting extract was filtered through a paper filter containing 0.5g of anhydrous sodium sulphate on top, evaporated to approximately 0.05mL final volume and subjected to clean-up step using TLC method.

 

TLC purification:

The final chloroform extracts concentrated to the minimum volume which were obtained from the biological fluids spiked with Atomoxetine and from the blank (drug-free) samples were applied as bands on the chromatographic plate, 10µL aliquot of the standard Atomoxetine solution in methanol (1mg/mL) was spotted next with the help of a calibrated capillary. TLC plates were developed in two mobile phases sequentially: chloroform and then ethyl acetate – methanol – 25% ammonia (85:10:5). Then the zone in the chromatogram corresponding to the standard Atomoxetine solution was treated by acidified iodoplatinate solution. Atomoxetine was eluted from the chromatogram (R_f of the drug was of 0.49±0.04 on Merck plate) with 5mL of methanol. The methanol eluate obtained was evaporated to dryness and reconstituted in 5mL of 0.1mol/L hydrochloric acid. The UV spectrum of the resulting solution was measured using blank extract as a reference solution.

 

RESULTS AND DISCUSSION:

Selection of the extracting solvent and the proper pH of the aqueous medium:

To find out the best extraction conditions for Atomoxetine the extraction procedure described above was carried out using various organic solvents and different aqueous pH in the range of 1-13. Nine typical organic solvents usually used for the sample preparation in bioanalytical methods of drug determination were tested. The model aqueous solution with the concentration of 500μg/mL was used in this study. Thus, the concentration of the drug in the aqueous phase was of 50μg/mL. For each organic solvent three experiments were carried out for each pH value.

 

The quantitative determination of Atomoxetine extracted from the aqueous solutions was performed by UV-spectrophotometric method developed in our previous studies²⁹. Quantitative determination was performed at 270nm by the calibration curve of

y=(0.00455±4×10^-5)x+(0.016±0.005). The calibration curve showed linearity in the range of 15.0–210mg/mL (LOD and LOQ were of 1.8μg/mL and 5.5μg/mL, respectively). The extraction recovery of Atomoxetine in all organic solvents tested was low and showed in most cases a tendency to insignificant increase in the alkaline medium (Fig. 1).

 

Chloroform

 

Methylene chloride

 

1,2-Dichloroethane

 

Tetrachloromethane

 

Diethyl ether

 

Ethyl acetate

 

Benzene

 

Toluene

 

Hexane

 

Fig. 1: The extraction recovery of Atomoxetine from aqueous solutions (^……… without a salting-out agent,^{. _ _ _} in the presence of NaCl, ^____ in the presence of (NH₄)₂SO₄)

 

The maximum extraction recovery value was of 28% at pH of 13 for chloroform. The sample preparation stage should be effective enough to provide the required values of detection and quantification limits of the bioanalytical method and give reproducible recoveries. It should be noted that the drugs of forensic interest are often present in the relatively low concentrations in biological samples. Efficiency of the organic solvent chosen should be at least 50%, and preferably much higher, while the extraction of endogenous substances should be minimised²³. Usually, the last requirement is achieved by incorporating the back-extraction step into the extraction scheme to eliminate or minimise the extraction of the matrix components. Diethyl ether had the lowest recovery of 0.2% at pH 1-2, that makes possible to recommend this solvent for the extraction purification from co-extractive components of the biological matrix.

 

Determination of the extraction recovery of Atomoxetine from aqueous solutions in the presence of salting-out agents:

To increase the extraction recovery of Atomoxetine with organic solvents the salting-out agents were added to the aqueous medium. Sodium chloride and ammonium sulphate were used as salting-out agents. The model aqueous solution of Atomoxetine with the concentration of 500μg/mL was used in this study. Thus, the concentration of the drug in the aqueous phase was of 50μg/mL. For each organic solvent three experiments were carried out for each aqueous pH value and each salting-out agent. As can be seen from Fig. 1, the use of salting-out agents has significantly increased the extraction recovery of Atomoxetine. The maximum value of 89% in the extraction recovery was obtained for chloroform at pH of 11-12 in the aqueous phase saturation with ammonium sulphate.

 

The study of reproducibility of the extraction procedure for Atomoxetine at different concentration levels:

Reproducibility of the extraction procedure for Atomoxetine with chloroform at pH of 12 in the presence of ammonium sulphate was studied using three model aqueous solutions with the concentrations of 100μg/mL, 200μg/mL and 500μg/mL. Thus, the concentrations of the drug in the aqueous phase were of 10μg/mL, 20μg/mL and 50μg/mL, respectively. For each concentration three experiments were carried out. Table 1 shows that the mean extraction recovery was approximately the same in the indicated concentration range.

 

Table 1. Reproducibility of the extraction procedure for Atomoxetine with chloroform at pH 12 in the presence of ammonium sulphate

 

Identification and quantitative determination of Atomoxetine in the extracts from biological fluids:

Identification and quantitative determination of Atomoxetine in the extracts from blood and urine was performed after additional clean-up step by the TLC method. Selection of the mobile phase for TLC purification was based on previous study in TLC screening of Atomoxetine²⁷. The UV-spectra of the solutions containing Atomoxetine isolated from blood and urine showed the principal peaks at wavelengths of 270 and 277nm which correspond to the spectrum of the standard Atomoxetine solution in 0.1mol/L hydrochloric acid²⁷.

 

Table 2. Recovery and precision of the solvent extraction of Atomoxetine from blood

Amount of drug added to 10 mL of blood, mg	Absorbance	Amount of drug found		Metrological characteristics
		mg	Recovery, %	n = 5; Р = 0.95	n = 15; Р = 0.95
200	0.075	64.8	32.3	= 35.0 S = 3.537 RSD = 10.1%	= 38.8 S = 3.120 RSD = 8.7%  = 0.805  = 1.7 e = 4.7%
	0.090	81.3	40.7
	0.081	71.4	35.7
	0.079	69.2	34.6
	0.074	63.7	31.9
400	0.161	159.3	39.8	= 36.9 S = 3.251 RSD = 8.8%
	0.148	145.1	36.3
	0.154	151.6	37.9
	0.158	156.0	39.0
	0.131	126.4	31.6
500	0.179	179.1	35.8	= 35.3 S = 2.90 RSD = 8.2%
	0.188	189.0	37.8
	0.157	154.9	31.0
	0.189	190.1	38.0
	0.171	170.3	34.1

 

Table 3. Recovery and precision of the solvent extraction of Atomoxetine from urine

Amount of drug added to 20 mL of urine, mg	Absorbance	Amount of drug found		Metrological characteristics
		mg	Recovery, %	n = 5; Р = 0.95	n = 15; Р = 0.95
100	0.082	72.5	72.5	= 69.2 S = 4.795 RSD = 6.9%	= 69.3 S = 4.388 RSD = 6.7%  = 0.634  = 1.1 e = 3.1%
	0.078	68.1	68.1
	0.072	61.5	61.5
	0.080	70.3	70.3
	0.083	73.6	73.6
200	0.138	134.1	67.0	= 68.9 S = 4.197 RSD = 6.1%
	0.148	145.1	72.5
	0.136	131.9	65.9
	0.134	129.7	64.8
	0.151	148.3	74.2
400	0.287	297.8	74.5	= 69.8 S = 5.117 RSD = 7.3%
	0.266	274.7	68.7
	0.256	263.7	65.9
	0.250	257.1	64.3
	0.292	303.2	75.8

 

Recovery of the drug extraction from blood and urine are shown in Tables 2 and 3. Sample preparation methods developed have allowed to isolate 38.8% of Atomoxetine from blood with the satisfactory precision (RSD 8.7%) and 69.3% of the drug from urine with satisfactory precision (RSD 6.7%).

 

СONCLUSIONS:

The dependence of the extraction recovery of Atomoxetine from aqueous solutions on the type of the organic solvent, pH of the aqueous medium and the presence of a salting-out agent has been determined. The maximum value of 89% in the extraction recovery was obtained for chloroform at pH of 11-12 in the aqueous phase saturation with ammonium sulphate. The extraction recovery of Atomoxetine with diethyl ether at pH of 1-2 was the lowest and equal to 0.2%, it allowed selecting this solvent for the extraction purification from co-extractive components of the biological matrix. The optimal conditions of the solvent extraction of Atomoxetine were applied for optimization of the isolation methods of the drug from blood and urine.

 

Recovery of the drug extraction from blood was of 38.8% (RSD 8.7%). The isolation method included the precipitation of blood cells by trichloroacetic acid, back-extraction using diethyl ether at pH of 1-2, extraction of the drug from aqueous layer saturated by and ammonium sulphate with chloroform at pH of 12 followed by the TLC clean-up step.

 

Recovery of the drug extraction from urine was of 69.3% (RSD 6.7%). The isolation method included the back-extraction using diethyl ether at pH of 1-2, extraction of the drug from aqueous layer saturated by and ammonium sulphate with chloroform at pH of 12 followed by the TLC clean-up step. The results obtained could be used in toxicological study of biological samples for presence of Atomoxetine.

 

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest to disclose.

 

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Accepted on 23.01.2020              © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4303-4309.