Research article

Received: 5 March 2013, Revised: 3 May 2013, Accepted: 5 May 2013 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2541

Development and validation of a sensitivespectrofluorimetric method for thedetermination of amoxapine in human plasmaand urineAyça Karasakala and Sevgi Tatar Ulub*

ABSTRACT: A selective and sensitive spectrofluorimetricmethodwas developed and validated for the determination of amoxapinein human plasma and urine. The developed method is based on labeling with 5-dimethylaminonaphthalene-1-sulfonyl chloride(dansyl chloride) and monitoring at 397nm (excitation)/514nm (emission). The method was validated for linearity, limit ofdetection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear overa concentration range of 250–2500 and 50–1250ng/mL for plasma and urine, respectively. The LOD values were calculated to be13.31 and 13.17ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in humanplasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: derivatization; spectrofluorimetry; plasma; urine; validation; dansyl chloride

* Correspondence to: S. T. Ulu, Department of Analytical Chemistry, Facultyof Pharmacy, Istanbul University, 34416, Istanbul, Turkey. E-mail:sevgitatar@yahoo.com

a Department of Chemistry, Faculty of Science and Letters, Namik KemalUniversity, Tekirdag, Turkey

b Department of Analytical Chemistry, Faculty of Pharmacy, IstanbulUniversity, 34416, Istanbul, Turkey

IntroductionAmoxapine (AMX) is known chemically as 2-chloro-11-(1-piperazinyl)dibenzo[b,f][1,4]oxazepine (1). It is an N-demethylated dibenzoxazepine, closely related to theneuroleptic loxapine. Its tricyclic structure appears to give itantidepressant properties resembling imipramine andmitriptyline. In uncontrolled trials it has been shown to haveantidepressant activity (2).

Several methods have been described for the determination ofAMX in biological fluids. High-performance liquid chromatography(HPLC) with UV detection has been reported for the determinationof AMX and metobolites in human serum (3). HPLC has been usedto study 14 tricyclicantidepressants and metabolites in humanplasma (4). HPLC with UV detection has been used in thedetermination of AMX and nortriptyline in blood plasma and serum(5). AMX and its metabolites have been determined using HPLC (6).AMX and its metabolites have been analysed using HPLC for theirdetermination in plasma and serum (7,8). Determination of AMX inwhole blood by capillary gas chromatography has been described(9). HPLC-tandemmass spectrometry has been used in the determi-nation of AMX in whole blood (10). Dansyl chloride was chosen as aderivatizing reagent because it forms highly fluorescent derivativeswith primary, secondary amines, imidazoles and phenols usingrelatively mild reaction conditions (11–13).

In this study, a new spectrofluorimetric method was developedfor the determination of AMX in plasma and urine. The method isbased on the derivatization of AMX with dansyl chloride. Dansylchloride was chosen as a derivatizing reagent because it formshighly fluorescent derivatives with primary, secondary amines, imid-azoles and phenols using relativelymild reaction conditions (11–13).

A literature search suggests that this is the first time thatAMX has been derivatized and determined using aspectrofluorimetric method.

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Experimental

Reagents

AMX was from Sigma (St. Louis, MO, USA). Dansyl chloride was pur-chased from Fluka (Buchs, Switzerland). Other chemicals were fromMerck (Darmstadt, Germany). All solvents were of analytical grade.Venous blood samples from healthy volunteers were collected

into a polyethylene tube containing EDTA and centrifuged at4500 rpm for 35min. Human plasma was separated from wholeblood using a pipet.Urine samples were transferred into 50mL polyethylene

tubes. Plasma and urine samples were kept in a refrigerator at-20�C until analysis.

Apparatus

A model RF-1501 spectrofluorimeter from Shimadzu (Kyoto,Japan) was used to record the fluorescence spectrum ofderivatized AMX–dansyl compound.

Solutions

Stock solution of AMX was prepared in methanol at 1mg/mL.This stock solution was further diluted with water to obtainworking solutions of 100mg/mL.

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A. Karasakal and S. T. Ulu

A 0.2mg/mL dansyl chloride solution was prepared daily inacetone. Bicarbonate/carbonate buffer (pH 9.5) was preparedby adjusting the pH of sodium bicarbonate (0.1M) aqueoussolution with 0.1M sodium hydroxide.

Figure 1. The reaction scheme of AMX with dansyl chloride.

Preparation of plasma samples

Blood samples were obtained from healthy male volunteers. Allof the frozen plasma and urine samples were kept frozen at -20 �C and thawed just before the experiment. Aliquots(0.25mL) of plasma were transferred into a series of centrifugetubes. Aliquots of AMX working solution were added so thatthe final concentration was in the range 250–2500 ng/mL. Afterthe addition of 3.0mL of methanol, the mixtures were vortexmixed for ~ 1min, then centrifuged at 4500 rpm for ~ 30min.The organic layer was transferred to another clean glass tubeand evaporated to dryness under a steady stream of nitrogenat 40 �C. Carbonate buffer (100 mL) and dansyl chloride solutions(250 mL) were then added to the residue. The solution was mixedvigorously and kept in a water bath at 40 �C for 30min. The tubewas then cooled. The content of the tube was extracted threetimes with 3.0mL of chloroform. The combined organic phaseswere adjusted to 10mL with chloroform. The mixture was filteredthrough a 0.2mm membrane filter. The fluorescence intensity ofthe resulting solution was measured at 514nm after excitation at397nm against reagent blanks that had been treated similarly.

Figure 2. Emission spectra of plasma spiked with 2500 ng/mL AMX-dansyl deriv-ative (a) and urine spiked with 500 ng/mL AMX–dansyl derivative (b) and blank (c).

Preparation of urine samples

Aliquots of 1mLurine (1:100)were transferred to a screw-cap reactiontube. Aliquots of AMX working solution were added, so that the finalconcentration was in the range 50–1250ng/mL. Carbonate buffer(100mL) and dansyl chloride solutions (250mL) were then added.The solution was mixed vigorously and kept in a water bath at40 �C for 30min. The tube was then cooled. The content of the tubewas extracted three times with 3mL of chloroform. The combinedorganic phases were adjusted to 10mL with chloroform. The fluores-cence intensity of the resulting solutions was measured at 514 nmafter excitation at 397 nm against reagent blanks treated similarly.

Results and discussion

Derivatization

AMX contains an amino group and can therefore react withdansyl chloride to yield a strongly fluorescent product. Thereaction scheme is shown in Fig. 1.

Under the described experimental conditions, the fluorophore(dansyl–AMX derivative) affords favorable characteristics to the excita-tion and emissionwavelengths at 514 and 397nm, respectively (Fig. 2).

To improve the sensitivity and accuracy of AMX analysis, thederivatization conditions concentration of dansyl chloride, pH,temperature and time were optimized.

The effect of the volume and concentration of the dansylchloride reagent was studied using different amounts (50–300 mL) of 0.2% (w/v) reagent solution. It was observed that250 mL of 0.2% (w/v) reagent solution is the optimum for thedansyl–AMX derivative (Fig. 3).

The pH was varied over the range 9.0–11.0 using carbonatebuffers; the maximum fluorescence was obtained at pH9.5 (Fig. 4).

The completeness of the derivatization was investigated at dif-ferent temperatures (room, 40, 50 and 60 �C) and different

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reaction times. AMX–dansyl chloride was completely derivatizedin carbonate buffer pH9.5 after heating at 40 �C for 30min (Fig. 5).

Method validation

The method was validated according to the ICH guidelines withrespect to linearity, limit of detection (LOD), limit of quantifica-tion (LOQ), precision, accuracy, recovery and robustness (14).

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Figure 4. Effect of pH on the derivatization.

Figure 5. Effect of time on the derivatization.

Table 1. Optical characteristics and statistical data (n=3)

Parameters Plasma Urine

lex/em (nm) 397/514 397/514Beer’s law range (ng/mL) 250–2500 50–1250LOD (ng/mL) 13.31 13.17LOQ (ng/mL) 39.93 39.53Regression equation F= 0.2256C –

18.072A= 0.5463C –

120.03Slope (a) 0.2256 0.5463Intercept (b) 18.072 120.03Standard deviation of theintercept

0.91 2.18

Correlation coefficient (r) 0.9997 0.9967Figure 3. Effect of the volume (mL) of 0.2% dansyl chloride added.

The proposed method is highly sensitive

Calibration curves (F = aC+ b) were constructed using plots ofthe fluorescence intensity (F) of AMX versus the concentration(C) of the calibration standards (Table 1).

Linearity was observed in the range 250–2500 ng/mL forplasma and 50–1250 ng/mL for urine. Regression equations werecalculated and found to be:

plasma : F ¼ 0:2256C þ 18:072 r ¼ 0:9997ð Þ;urine : F ¼ 0:5463C þ 120:03 r ¼ 0:9967ð Þ:

The LOD and LOQ of the drug using the proposed methodswere determined using calibration standards. LOD and LOQ were

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calculated to be 3.3s/S and 10s/S, respectively, where S is theslope of the calibration curve and s is the standard deviation ofthe intercept of the regression equation.The intra- and interday precision and accuracy of the

proposed method were determined. Three replicate sampleson the same day, and on three consecutive days were assayedfor intra- and interday accuracy at three different concentrationsfor each analyte. The precision and accuracy of the method wereexpressed by the relative standard deviation (RSD %) andrelative mean (RME %), respectively.The intra- and interday precision values for plasma were

1.05%; intra- and interday accuracy ranged between 6.03and 8.00%.The intra- and interday precision values for urine were< 1.22%;

intra- and interday accuracy ranged between 8.60 and 8.65%.The recovery for plasma and urine at three different concen-

trations of AMX was determined. Mean recovery was 97.85%for urine and 81.03% for plasma.Robustness is a measure of the capacity of a method to

remain unaffected by small, but deliberate, changes in the deriv-atization pH buffer (� 0.1), derivatization temperature (� 2 �C)and derivatization time (� 2min). % RSD values were≤ 1.0 andwere within the limit required for spectrofluorimetric analysis.No significant effect on derivatization parameters was observed.Thus, the method was found to be robust.

ConclusionIn conclusion, a new spectrofluorimetric method has beendeveloped with high reproducibility and sensitivity for thedetermination of AMX based on.Derivatization with dansyl chloride. Compared with existing

procedures, the described method exhibits advantages includingshort analysis time, low derivatization concentration LOD (5) andlow consumption of sample and reagent. In addition, the recoverypercentage of AMX is high (4–10) and derivatization processes arenot time-consuming. The instrument is simple and is not of highcost (3–10).All the analytical reagents are inexpensive and available

in any analytical laboratory. The proposed method is proposedfor both routine pharmaceutical analysis, the observationof AMX concentrations in plasma and urine, and bioavail-ability studies.

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A. Karasakal and S. T. Ulu

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