Research article Received: 23 April 2014,
Revised: 25 September 2014,
Accepted: 23 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3396
Determination of three antidepressants in urine using simultaneous derivatization and temperature-assisted dispersive liquid–liquid microextraction followed by gas chromatography–flame ionization detection Ali Akbar Alizadeh Nabila, Nina Nourib and Mir Ali Farajzadehb* ABSTRACT: This paper presents a fast and simple method for the extraction, preconcentration and determination of fluvoxamine, nortriptyline and maprotiline in urine using simultaneous derivatization and temperature-assisted dispersive liquid–liquid microextraction (TA-DLLME) followed by gas chromatography–flame ionization detection (GC-FID). An appropriate mixture of dimethylformamide (disperser solvent), 1,1,2,2-tetrachloroethane (extraction solvent) and acetic anhydride (derivatization agent) was rapidly injected into the heated sample. Then the solution was cooled to room temperature and cloudy solution formed was centrifuged. Finally a portion of the sedimented phase was injected into the GC-FID. The effect of several factors affecting the performance of the method, including the selection of suitable extraction and disperser solvents and their volumes, volume of derivatization agent, temperature, salt addition, pH and centrifugation time and speed were investigated and optimized. Figures of merit of the proposed method, such as linearity (r2 > 0.993), enrichment factors (820–1070), limits of detection (2–4 ng mL1) and quantification (8–12 ng mL1), and relative standard deviations (3–6%) for both intraday and interday precisions (concentration = 50 ng mL1) were satisfactory for determination of the selected antidepressants. Finally the method was successfully applied to determine the target pharmaceuticals in urine. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: antidepressants; derivatization; gas chromatography; temperature-assisted dispersive liquid–liquid microextraction; urine
Introduction Nortriptyline and maprotiline are tricyclic antidepressants (TCAs) and fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) (Scheme 1). Theses pharmaceuticals are used in the treatment of various types of depression and other psychiatric disorders like phobias and anxiety. TCAs are a class of psychoactive drugs that inhibit the re-uptake of neurotransmitters by blocking the serotonin and noradrenalin transporters, which results in an enhancement of neurotransmission. The side-effects, toxicity and severe drug–drug interactions of these compounds led to the introduction of SSRIs. These new generation antidepressants enhance the serotoninergic neurotrasmission process, through selective inhibition of neuronal re-uptake of serotonin. SSRIs exhibit clinical efficacy comparable with TCAs but are less toxic to the heart and generally poses fewer harmful side effects. However, recently, it was reported that even these compounds could exhibit toxicity, resulted in arousing, aggressive character in the patients (Eap and Baumann, 1996). Because there are many reports of accidental poisoning by these antidepressants (including suicide using overdose of TCAs), the development of a rapid and specific method for the determination of these compounds in biological samples could be of great interest. Several analytical methods have been developed for the determination of these antidepressants in biological matrices, such as gas chromatography (GC; Berzas Nevado et al., 2005; Ito et al., 2011;
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Papoutsis et al., 2012; Ulrich et al., 1996; Yazdi et al., 2008), highperformance liquid chromatography (HPLC; Bahrami and Mohammadi, 2007; Addolorata Saracino et al., 2006), capillary electrophoresis (Wu et al., 2000; Kou et al., 2004; Dell’Aquila, 2002; Sasajima et al., 2010) and micellar electrokinetic chromatography (Labat et al., 2002). Because of the difficulties that follow with complex biological matrices, the extraction of the compounds of interest from these samples is an important step in the pharmaceutical analysis process. Liquid–liquid extraction (LLE) is time-consuming and tedious, and requires large amounts of potentially toxic organic solvents that are usually expensive and also form an
* Correspondence to: M. A. Farajzadeh, Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran. Email: [email protected] a
Food and Drug Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran
b
Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran Abbreviations used: DLLME, dispersive liquid–liquid microextraction; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EF, enrichment factor; ER, extraction recovery; FID, flame ionization detection; IL, ionic liquid; LLE, liquid– liquid extraction; LR, linear range; SBSE, Stir bar sorptive extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; SSRI, selective serotonin reuptake inhibitor; TA-DLLME, temperature-assisted tandem dispersive liquid–liquid microextraction; TCA, tricyclic antidepressant.
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A. A. Alizadeh Nabil et al.
Scheme 1. Chemical structure of fluvoxamine (a), nortriptyline (b) and maprotiline (c).
emulsion. In this context, solid-phase extraction (SPE) seems to be better, as smaller amounts of organic solvents are usually used. However, SPE cartridges need conditioning and require further toxic organic solvents for washing and elution steps. LLE (Ishida et al., 1995; Lacassie et al., 2000; Tatar Ulu, 2007) and SPE (Olesen et al., 2000) have been used for the extraction and preconcentration of some antidepressants. Solid-phase microextraction (SPME), introduced by Arthur and Pawliszyn (1990), is a solvent-free process that includes simultaneous extraction and preconcentration of analytes from aqueous samples or the headspace of the samples. Despite the advantages provided by this method, most commercial extractive fibers used in SPME are relatively expensive and fragile, and sample carry-over is also a problem (Mehdinia and Aziz-Zanjani, 2013; Prosen and Zupančič-Kralj, 1999). Stir bar sorptive extraction (SBSE) is another form of sorptive extraction based on the same principles as SPME. In SBSE, the sorbent is coated on a magnetic stir bar and the liquid sample is stirred with this bar. After the extraction, the analytes can be introduced quantitatively into the analytical system by thermal or liquid desorption (Farajzadeh et al., 2010). In 2006, a new liquid phase microextraction method termed dispersive liquid–liquid microextraction (DLLME) was introduced by Assadi and co-workers (Rezaee et al., 2006). In DLLME, the mixture of extraction and disperser solvents is rapidly injected into an aqueous sample to form a cloudy solution. The analytes are extracted into the fine droplets of extraction solvent, which are further separated by centrifugation and are determined by either chromatographic or spectrometric methods. The large contact surface area between phases speeds up mass transfer; hence, the equilibrium state is achieved quickly. This process reduces the extraction time and increase the EFs (Rahnama Kozani et al., 2007). SPME (Ulrich and Martens, 1997), SBSE (Chaves et al., 2007) and DLLME (Yazdi et al., 2008) have been used as sample preparation techniques in the determination of some antidepressants. Combination of derivatiation and DLLME can reduce analysis time and/or increase the extractability of the analytes. Some works on this combination have been reported, such as simultaneous derivatization and DLLME of atranol and chloroatranol (López-Nogueroles et al., 2014), amino acids (Li et al., 2013), and chlorophenols (Wang et al., 2014). Recently a new version of DLLME named temperatureassisted dispersive liquid–liquid microextraction (TA-DLLME) was developed (Farajzadeh et al., 2014). TA-DLLME method is based on injection of the mixture of extraction and disperser solvents into an aqueous sample solution at an elevated temperature. Similar approaches were performed previously using ionic liquids (ILs) as the extractant in DLLME (Zhou et al., 2008; Baghdadi and Shemirani, 2008). However using ILs before GC is difficult. Therefore in this study an organic solvent was used as the extraction solvent in DLLME performed at an elevated temperature. During cooling of
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this solution, the cloudy state of the solution is increased. The movement of extraction solvent droplets through the solution during the cooling process leads to an efficient transition of analytes from the aqueous phase into the extraction solvent. In this method the volume of sample solution used is higher than that of conventional DLLME, so high EFs are achievable. On the other hand, the formation of new droplets of extraction solvent during the cooling step leads to higher extraction recoveries similar to the continuous extraction process. As nortriptyline and maprotiline have a secondary amino group and fluvoxamine has a primary amino group, derivatization can be used to improve sensitivity and to obtain better peak shape. Some derivatization reagents such as acetic anhydride (Ito et al., 2011), heptafluorobutyric anhydride (Papoutsis et al., 2012), 1-(heptafluorobutyryl) imidazole (Wille et al., 2005) and tert-butyldimethylsilane (Gunnar et al., 2004) have been used for this purpose. In this work, a simultaneous TA-DLLME and derivatization method followed by GC-FID is proposed for the determination of fluvoxamine, nortriptyline and maprotiline in urine. Here, a derivatization reagent, an extraction solvent and a disperser were simultaneously injected into the heated sample solution to perform derivatization and extraction in one step. The solubility of the extraction solvent in the aqueous sample depends on the temperature. At an elevated temperature, the solubility of the extraction solvent into aqueous sample increased. By injection of the above mixture, a partially turbid solution was obtained. During cooling, the turbidity of the solution was increased by forming new droplets of extraction solvent which led to an efficient extraction. The analytes were derivatized and extracted into the fine droplets of extraction solvent, which were further separated by centrifugation. The effects of different parameters on the derivatization/microextraction procedure were thoroughly investigated. The performance of the optimized method was then evaluated and applied to determine the selected antidepressant in urine samples.
Materials and methods Chemicals and reagents Fluvoxamine, nortriptyline and maprotiline were a gift from Daana Pharmaceutical Company (Tabriz, Iran). Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, iso-propanol and acetonitrile (as disperser solvents), acetic anhydride (as derivatization agent), 1,2-dibromoethane (as an extraction solvent), hydrochloric acid and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Also, 1,1,2,2-tetrachloroethane and 1,1,2,2-tetrabromoethane (as extraction solvents) were obtained from Janssen Chimica (Beerse, Belgium). Stock solutions of nortriptyline, and maprotiline (1000 mg L1) in methanol and fluvoxamine (as maleate; 1000 mg L1) in water were prepared and stored in a refrigerator at 4 °C. Working solutions were prepared daily by appropriate dilutions of the stock solutions with de-ionized water (Ghazi Company, Tabriz, Iran). A standard solution of the target analytes (each 250 mg L1) in a mixture of acetic anhydride– 1,1,2,2-tetrabromoethane (2:1, v/v) was injected (three times) into the separation system daily for quality control and the obtained peaks areas were used in the calculation of EFs and extraction recoveries (ERs). Sodium carbonate solution (concentration, C = 0.2 mol L1) was prepared by dissolving 1.06 g of sodium carbonate (Merck, Darmstadt, Germany) in 50 mL deionized water.
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Simultaneous derivatization/TA-DLLME of some antidepressants in urine Instrumentation Chromatographic analysis was performed on a Shimadzu 2014 gas chromatograph (Kyoto, Japan) equipped with a split/splitless injector operated at 250 °C in a splitless mode (sampling time 1 min) and a flame ionization detector (FID). Helium (99.999%, Gulf Cryo, United Arab Emirates) was used as the carrier gas at a linear velocity of 30 cm s1. Separation was carried out on a CP-Sil 8 CB capillary column (30 m × 0.25 mm i.d., and film thickness 0.25 μm; poly (5%-diphenyl-95%-dimethylsiloxane; Chrompack, Milan, Italy). The column oven temperature was initially held at 50 °C for 1 min, then raised to 300 °C at a rate of 10 °C min1, and held at 300 °C for 2 min. A 1 μL microsyringe (zero dead volume, Hamilton, Switzerland) was used for the injection of samples into GC. Injection volume was 0.5 μL. The FID temperature was maintained at 250 °C. Hydrogen gas was generated with a hydrogen generator (OPGU-1500S, Shimadzu, Japan) for FID at a flow rate of 30 mL min1. The flow rate of air for FID was 300 mL min1. Gas chromatography–mass spectrometry (GC-MS) analysis was carried out on an Agilent 7890A gas chromatograph with a 5975C quadrupole mass-selective detector (Agilent Technologies, CA, USA) equipped with a split/splitless injector operated at 250 °C in a splitless/split mode (sampling time 1 min). The separation was performed on an HP-5 MS capillary column (30 m × 0.25 mm i.d., and film thickness of 0.25 μm; Hewlett-Packard, Santa Clara, USA). The carrier gas was helium with a flow rate of 1.0 mL min1. The column oven temperature programming was the same as mentioned above for GC-FID analysis. MS operational conditions were: electron ionization, 70 eV; ionic source temperature, 250 °C; transfer line temperature, 260 °C; mass range, m/z 55–350; acquisition rate, 20 Hz; detector voltage, 1700 V. Library searching was performed using the commercial NIST library. pH measurements were performed with a Metrohm pH meter (model 654, Herisau, Switzerland). A ROTOFIX 32A centrifuge from Hettich (Kirchlengern, Germany) was used in extraction/derivatization procedure.
sedimented organic phase (20 ± 2 μL) was removed using a 1 μL GC microsyringe and injected into the GC system for analysis. The derivatization reaction (acylation) between acetic anhydride and the selected pharmaceuticals that have primary (fluvoxamine) or secondary (nortriptyline and maprotiline) amino groups was performed according to the following reaction:
Analytical parameters Two main parameters, namely EF and ER, were employed for evaluation of the proposed method. EF is defined as the ratio of the analyte concentration in the sedimented phase (Csed) to its initial concentration (C0) within the sample: EF ¼ C sed =C 0
(1)
Csed was obtained from the comparison of peaks areas of analytes in two cases: direct injection of standard solution 250 mg L1 in the mixture of acetic anhydride–1,1,2,2-tetrachloroethane (2:1, v/v) and injection of the sedimented organic phase after performing the proposed method on the deionized water or diluted urine sample spiked with 1 mg L1 of each analyte. ER is defined as the percentage of the total analyte amount (n0) that is extracted into the sedimented phase (nsed): ER ¼ ðnsed =n0 Þ 100 ¼ ðC sed V sed Þ= C 0 V aq 100 (2) ER ¼ V sed =V aq EF 100 where Vsed and Vaq are the volumes of sedimented phase and aqueous solution, respectively.
Urine samples Blank urine samples were collected from a healthy female adult who had not taken the selected antidepressants. Also urine samples were obtained from two 26- and 27-year-old females after oral administration of fluvoxamine (50 mg, once in a day) and maprotiline (25 mg, once in a day), respectively, and a female depressed patient (60-year-old) who had ingested a tablet containing 25 mg of nortriptyline (twice in a day, 12 h interval). Urine samples were collected within 24 h from the first oral administration. After reading the volume of collected urine, 5 mL urine was centrifuged at 8000 rpm for 5 min. To reduce the matrix effect of the urine sample the supernatant was diluted 10-fold with sodium carbonate solution (0.2 mol L1) and then subjected to the derivatization/microextraction procedure.
Derivatization/microextraction procedure A 50 mL aliquot of diluted urine sample or standard solution was placed into a 60 mL glass test tube with conical bottom. Then the tube was immersed into a water bath for 10 min at 70 °C. A 1.5 mL aliquot of DMF containing 150 μL acetic anhydride and 80 μL 1,1,2,2-tetrachloroethane was rapidly injected into the solution using a 2 mL glass syringe. Then the solution was cooled to room temperature and the cloudy solution formed was centrifuged for 3 min at 4000 rpm. An aliquot (0.5 μL) of the
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Results and discussion The initial objective of this work was to optimize the derivatization/microextraction procedure. There are several parameters affecting the process, that is, selection of suitable extraction and disperser solvents and their volumes, volume of derivatization agent, temperature, salt addition, pH and centrifugation time and speed. Therefore, all of these parameters were optimized in order to achieve the good performance. Selection of extraction solvent The selection of an appropriate extraction solvent is of primary importance in a DLLME process. Therefore, some factors should be considered, that is, low solubility in water, extraction capability of compounds of interest, good chromatographic behavior and compatibility with the derivatizing reagent (in this study). Based on these facts, 1,1,2,2-tetrachloroethane, 1,2-dibromoethane and 1,1,2,2-tetrabromoethane were tested. Initially experiments were performed with different volumes of the selected solvents to obtain 50 μL of the sedimented organic phase. The volumes needed for each solvent were 100, 110 and 140 μL, respectively. According to obtained resutls, 1,1,2,2-tetrachloroethane provided high extraction efficiency for all analytes. Hence it was selected as the optimum extraction solvent for the subsequent experiments.
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Figure 1. Effect of extraction solvent volume on the derivatization/ microextraction efficiency. Extraction conditions: aqueous phase, sodium car1 1 bonate solution 0.2 mol L (50 mL); analytes concentrations, 500 ng mL of each antidepressant; extraction solvent, 1,1,2,2-tetrachloroethane (100 μL); disperser solvent, dimethylformamide (DMF; 2 mL); extraction temperature, 60 °C; centrifuging time, 3 min; and centrifuging speed, 4000 rpm. The error bars indicate the maximum and minimum of three determinations.
Figure 2. Effect of disperser solvent volume on the microextraction efficiency. Extraction conditions: the same as Fig. 1, except 80 μL extraction solvent was used. The error bars indicate the maximum and minimum of three determinations.
Figure 4. Effect of temperature on the derivatization/microextraction efficiency. Extraction conditions: all conditions are the same as Fig. 3 except 150 μL acetic anhydride was used as derivatization agent. The error bars indicate the maximum and minimum of three determinations.
Figure 5. Effect of sample solution pH on the derivatization/microextraction efficiency. Extraction conditions: all conditions are the same as Fig. 4 except extraction temperature was controlled at 70 °C. The error bars indicate the maximum and minimum of three determinations.
Selection of disperser solvent Miscibility in both extraction solvent and aqueous phase is an essential factor in the selection of a disperser solvent. On the other hand, in this study, relatively high temperature was applied during the extraction step. Therefore the use of disperser solvents having low boiling points such as acetone and methanol is limited. In order to determine a suitable disperser solvent, five disperser solvents with relatively higher boiling points (DMF, DMSO, 1,4dioxane, iso-propanol and acetonitrile) were examined. The derivatization/microextraction of analytes was carried out using 2 mL of each disperser containing 100 μL 1,1,2,2-tetrachloroethane (extraction solvent) and 200 μL acetic anhydride (derivatization reagent). According to the results, DMF was selected as the disperser because of the formation a cloudy state with very fine droplets and high peak areas of the derivatized analytes.
Figure 3. Optimization of derivatizing agent volume. Extraction conditions: the same as Fig. 2 except 1.5 mL DMF was used as disperser solvent. The error bars indicate the maximum and minimum of three determinations.
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Optimization of extraction solvent volume The volume of extraction solvent used can affect the volume of the sedimented phase. Also, increasing extraction solvent volume
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3 4 6 5 5 6 3 3 3 3 4 5 Square of correlation coefficient. Limit of detection, signal-to-noise ration (S/N) = 3. c Limit of quantification, S/N = 10. d Mean enrichment factor ± standard deviation, EF = CSed/C0 (n = 3). e Mean extraction recovery ± standard deviation, (n = 3). f Relative standard deviation intraday (n = 6, C = 50 ng mL1) and interday (n = 4, C = 50 ng mL1). b
Human urine
Fluvoxamine Nortriptyline Maprotiline Fluvoxamine Nortriptyline Maprotiline
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Figure 6. GC-FID chromatograms of (a) drug-free urine sample, (b) urine sample of 26 year-old female volunteer, (c) urine sample of 60-year-old female depressed patient, (d) urine sample of 27-year-old female volunteer, 1 and (e) spiked de-ionized water with 100 ng mL of each antidepressant. In all cases, the derivatization/microextraction method was performed and 0.5 μL of the sedimented phase was injected into GC. Peaks identification: 1, fluvoxamine; 2, nortriptyline; and 3, maprotiline.
a
910 ± 53 1100 ± 69 870 ± 53 91 ± 4 107 ± 5 82 ± 4 0.4 0.3 0.5 3 2 4 1–4000 1–4000 2–4000 8–4000 10–4000 12–4000 De-ionized water
721.31 ± 62 838.58 ± 36 923.32 ± 17 546.44 ± 25 798.38 ± 10 844.4 ± 38
18918 ± 1202 43977 ± 1657 47099 ± 1253 40378 ± 1318 55450 ± 1810 52492 ± 1560
0.999 0.997 0.996 0.996 0.995 0.993
1 1 2 8 10 12
36 ± 4 44 ± 5 35 ± 3 36 ± 3 43 ± 4 33 ± 2
Interday Intraday
RSD (%)f ER (%) ± SDe
EF ± SDd LOQc (ng mL1) LODb (ng mL1) R2a Intercept ± SD Slope ± SD Linear range (ng mL1) Matrix Analyte
Table 1. Analytical features of simultaneous derivatization/temperature-assisted dispersive liquid–liquid microextraction (TA-DLLME) followed by gas chromatography-flame ionization detection (GC-FID) determination of the selected antidepressants
Simultaneous derivatization/TA-DLLME of some antidepressants in urine
would increase the amonuts of analytes extracted, whereas their concentrations in the sedimented phase are decreased. Also, by changing the volume of extraction solvent (at a constant sample size), the volume ratio of sample to extraction phase is varied and hence extraction recoveries of the analytes will also change. To evaluate the effect of extraction solvent volume, different volumes of 1,1,2,2-tetrachloroethane (60, 80, 100 and 120 μL) were subjected to the same derivatization/microextraction procedure. Maximum peaks areas or EFs were obtained when 80 μL of the extraction solvent was added (Fig. 1). By increasing the extraction solvent volume from 80 to 120 μL, the peaks areas decreased owing to the increase in the volume of sedimented phase and its dilution effect on the concentrations of analytes in the extractant. On the other hand, when 60 μL of the extraction solvent was used, only 4 μL sedimented phase was obtained, which made its removal was difficult. Therefore, in this study, 80 μL was selected as the optimal volume of 1,1,2,2-tetrachloroethane, which leads to obtain 20 ± 2 μL sedimented phase volume.
Optimization of disperser volume To study the effect of dispersive solvent volume on the extraction efficiency, different volumes of DMF (from 0.5 to 2.5 mL at 0.5 mL intervals) containing 200 μL of acetic anhydride and 80 μL of extraction solvent were tested. At low volumes of DMF, the cloudy state was not formed properly and the extraction solvent did not disperse well into the aqueous solution, which resulted in low EFs. Meanwhile, at high volumes of DMF, the solubility of derivatized analytes and extraction solvent into aqueous phase was increased, which led to decreased extraction
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Figure 7. (I) Total ion current (TIC) chromatograms of (a) urine sample of 26-year-old female volunteer, (b) urine sample of 60-year-old female 1 depressed patient, (c) urine sample of 27-year-old female volunteer, and (d) de-ionized water (spiked with 100 ng mL of each analyte). In all cases, derivatization/microextraction method was performed and 0.5 μL of the sedimented phase was injected into GC-MS. (ІІ) Mass spectra of (e) derivatized fluvoxamine, (f) scan 1558 (retention time 16.663 min) in urine sample of 26-year-old female volunteer, (g) derivatized nortriptyline, (h) scan 2076 (retention time 20.180 min) in urine sample of 60-year-old female depressed patient, (i) derivatized maprotiline, and (j) scan 2229 (retention time 21.219 min) in urine sample of 27-year-old female volunteer. Peaks identification: 1, derivatized fluvoxamine (tR = 16.711 min); 2, derivatized nortriptyline (tR = 20.166 min); and 3, derivatized maprotiline (tR = 21.246 min).
efficiency. Therefore, 1.5 mL of DMF was selected as the optimum volume for the dispersive solvent (Fig. 2). Optimization of derivatization agent volume In order to study the effect of derivatization agent volume on the performance of the presented method, different volumes of acetic anhydride (25–250 μL) were tested and the obtained results are shown in Fig. 3. The highest derivatization reaction
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yield was observed when 150 μL of acetic anhydride was used, thus it was chosen as the volume of derivatization reagent for the further studies. By increasing the volume of acetic anhydride from 150 to 250 μL, the analytical signals decreased rapidly. This decrease could possibly be attributed to corresponding increase in the sedimented phase volume. It is noted that, at high volumes of acetic anhydride, the unreacted derivatization agent dissolved into extraction solvent and led to increases in the sedimented phase volume. Also, in the
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Simultaneous derivatization/TA-DLLME of some antidepressants in urine increased followed by a gradual decrease (Fig. 4). Different factors such as loss of analytes, extraction and disperser solvents and derivatization agent or hydrolysis of acetic anhydride can be responsible for this decrease. Finally 70 °C was selected as the optimum derivatization/microextraction temperature for further experiments.
Table 2. Relative recoveries obtained by simultaneous derivatization/TA-DLLME in urine samples spiked at different concentrations Spiked concentration (ng mL1) 50 100 500
Relative recovery (%) ± standard deviation (n = 3) Fluvoxamine
Nortriptyline
Maprotiline
79 ± 4 82 ± 3 89 ± 3
76 ± 3 83 ± 2 86 ± 5
78 ± 2 81 ± 4 85 ± 6
Effect of solution pH The selected antidepressants are basic compounds (pKb value 5.3 for fluvoxamine, 4.3 for nortriptyline and 3.5 for maprotiline). Therefore, the pH of the sample solution should be high to prevent their protonation. In these conditions, the analytes will be in neutral form and can react with the derivatization agent. The effect of pH on the extraction efficiency was studied within the pH range of 8–13 with addition of HCl or NaOH 1 M to the analyte solution containing sodium carbonate (0.2 mol L1). The results in Fig. 5 indicate that the peak areas increased with the pH increasing from 8 to 11 and remained constant up to pH 13. To facilitate the pH adjustment, sodium carbonate solution (0.2 mol L1, pH 12.5) without HCl or NaOH addition was used for the following studies.
cases of volumes 0.993. The repeatability and reproducibility of the proposed method, expressed as RSD, were evaluated by performing the method on six repeated samples (for intraday) and four repeated samples (for interday) at a concentration of 50 ng mL1 and RSDs were found to vary between 3 and 6%. High EFs ranging from 820 to 1100 were obtained. LODs and LOQs were in the ranges 2–4 and 8–12 ng mL1, respectively. The obtained ERs (33–43%) are completely reasonable for a microextraction procedure in which 50 mL aqueous phase along with 20 μL organic phase is used. Low LODs and LOQs, high EFs and ERs, and good repeatability and reproducibility are the main advantages of the method.
Comparison of the proposed method with others To assess performance of the new method, its analytical parameters were compared with those of other methods used in the analysis of target analytes. Table 3 summarizes the LR, LOD, LOQ and RSD of some analytical methods along with the proposed method. The repeatability and reproducibility of the proposed method are good and RSDs for this method are comparable or better than those of the mentioned methods. The LODs achieved by the presented method are better than those of the fourth method (Yazdi et al., 2008) whereas both of them used similar microextraction and detection systems, for example, DLLME-GCFID. It should be noted that, in some of the techniques mentioned, MS or nitrogen–phosphorus (NPD) detectors were used, which are inherently more sensitive and selective than FID. Nevertheless the proposed method has lower LODs and LOQs compared with GCNPD (Lacassie et al., 2000) and GC-MS (Gunnar et al., 2004). In the cases which LLE was used as a preconcentration step, the presented TA-DLLME procedure is more efficient and consumes less organic solvents. Hence, the obtained LODs by the presented method are better than those of the fifth method (Gunnar et al., 2004) in spite of its more sensitive detection system (MS). Another important advantage of the current method is the simultaneous derivatization and microextraction of the selected antidepressants in urine samples. Also, the use of heated sample solution led to an efficient extraction of analytes from the aqueous phase into the extraction solvent, which continued during the cooling process. By considering the results, the proposed method proved to be a sensitive, efficient, reliable and easy to use technique in the preparation and preconcentration of the selected antidepressants from urine.
Urine analysis
Conclusions
The proposed method was applied to analysis of urine samples of two 26- and 27-year-old females after oral administration of fluvoxamine (50 mg, once in a day) and maprotiline (25 mg, once in a day), respectively. Also a urine sample from a patient (60-year-old female) who received nortriptyline (25 mg, twice in a day) was analyzed by the optimum proposed method. Figure 6 show typical GC-FID chromatograms of these samples. There was no peak in the retention times of analytes in the blank urine sample. The obtained concentrations of fluvoxamine, nortriptyline and maprotiline in urine samples were 106 ± 11, 68 ± 3 and 82 ± 8 ng mL1, respectively. In all cases three determinations (n = 3) were performed to identify the compounds eluted in the retention times of the derivatized antidepressants in urine samples chromatograms. These samples
A simultaneous derivatization and TA-DLLME method has been reported for the extraction and preconcentration of three antidepressants from urine followed by GC-FID determination. The developed method has numerous advantages such as simplicity, low cost and excellent repeatability and reproducibility. The use of heated sample solution leads to increase dissolution of extraction solvent into aqueous solution. During the cooling process, the formation of tiny droplets of the extraction solvent leads to an effective extraction of the derivatized analytes from aqueous samples. Also, the ratio of sample volume to extraction solvent volume is improved using high sample size, which results in high EFs being obtained. The results revealed that the developed method was suitable for determination of the selected antidepressant at ng mL1 levels in urine.
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Biomed. Chromatogr. 2014
Simultaneous derivatization/TA-DLLME of some antidepressants in urine
Acknowledgments The authors thank the research Council of University of Tabriz for financial support.
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Revised: 25 September 2014,
Accepted: 23 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3396
Determination of three antidepressants in urine using simultaneous derivatization and temperature-assisted dispersive liquid–liquid microextraction followed by gas chromatography–flame ionization detection Ali Akbar Alizadeh Nabila, Nina Nourib and Mir Ali Farajzadehb* ABSTRACT: This paper presents a fast and simple method for the extraction, preconcentration and determination of fluvoxamine, nortriptyline and maprotiline in urine using simultaneous derivatization and temperature-assisted dispersive liquid–liquid microextraction (TA-DLLME) followed by gas chromatography–flame ionization detection (GC-FID). An appropriate mixture of dimethylformamide (disperser solvent), 1,1,2,2-tetrachloroethane (extraction solvent) and acetic anhydride (derivatization agent) was rapidly injected into the heated sample. Then the solution was cooled to room temperature and cloudy solution formed was centrifuged. Finally a portion of the sedimented phase was injected into the GC-FID. The effect of several factors affecting the performance of the method, including the selection of suitable extraction and disperser solvents and their volumes, volume of derivatization agent, temperature, salt addition, pH and centrifugation time and speed were investigated and optimized. Figures of merit of the proposed method, such as linearity (r2 > 0.993), enrichment factors (820–1070), limits of detection (2–4 ng mL1) and quantification (8–12 ng mL1), and relative standard deviations (3–6%) for both intraday and interday precisions (concentration = 50 ng mL1) were satisfactory for determination of the selected antidepressants. Finally the method was successfully applied to determine the target pharmaceuticals in urine. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: antidepressants; derivatization; gas chromatography; temperature-assisted dispersive liquid–liquid microextraction; urine
Introduction Nortriptyline and maprotiline are tricyclic antidepressants (TCAs) and fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) (Scheme 1). Theses pharmaceuticals are used in the treatment of various types of depression and other psychiatric disorders like phobias and anxiety. TCAs are a class of psychoactive drugs that inhibit the re-uptake of neurotransmitters by blocking the serotonin and noradrenalin transporters, which results in an enhancement of neurotransmission. The side-effects, toxicity and severe drug–drug interactions of these compounds led to the introduction of SSRIs. These new generation antidepressants enhance the serotoninergic neurotrasmission process, through selective inhibition of neuronal re-uptake of serotonin. SSRIs exhibit clinical efficacy comparable with TCAs but are less toxic to the heart and generally poses fewer harmful side effects. However, recently, it was reported that even these compounds could exhibit toxicity, resulted in arousing, aggressive character in the patients (Eap and Baumann, 1996). Because there are many reports of accidental poisoning by these antidepressants (including suicide using overdose of TCAs), the development of a rapid and specific method for the determination of these compounds in biological samples could be of great interest. Several analytical methods have been developed for the determination of these antidepressants in biological matrices, such as gas chromatography (GC; Berzas Nevado et al., 2005; Ito et al., 2011;
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Papoutsis et al., 2012; Ulrich et al., 1996; Yazdi et al., 2008), highperformance liquid chromatography (HPLC; Bahrami and Mohammadi, 2007; Addolorata Saracino et al., 2006), capillary electrophoresis (Wu et al., 2000; Kou et al., 2004; Dell’Aquila, 2002; Sasajima et al., 2010) and micellar electrokinetic chromatography (Labat et al., 2002). Because of the difficulties that follow with complex biological matrices, the extraction of the compounds of interest from these samples is an important step in the pharmaceutical analysis process. Liquid–liquid extraction (LLE) is time-consuming and tedious, and requires large amounts of potentially toxic organic solvents that are usually expensive and also form an
* Correspondence to: M. A. Farajzadeh, Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran. Email: [email protected] a
Food and Drug Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran
b
Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran Abbreviations used: DLLME, dispersive liquid–liquid microextraction; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EF, enrichment factor; ER, extraction recovery; FID, flame ionization detection; IL, ionic liquid; LLE, liquid– liquid extraction; LR, linear range; SBSE, Stir bar sorptive extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; SSRI, selective serotonin reuptake inhibitor; TA-DLLME, temperature-assisted tandem dispersive liquid–liquid microextraction; TCA, tricyclic antidepressant.
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Scheme 1. Chemical structure of fluvoxamine (a), nortriptyline (b) and maprotiline (c).
emulsion. In this context, solid-phase extraction (SPE) seems to be better, as smaller amounts of organic solvents are usually used. However, SPE cartridges need conditioning and require further toxic organic solvents for washing and elution steps. LLE (Ishida et al., 1995; Lacassie et al., 2000; Tatar Ulu, 2007) and SPE (Olesen et al., 2000) have been used for the extraction and preconcentration of some antidepressants. Solid-phase microextraction (SPME), introduced by Arthur and Pawliszyn (1990), is a solvent-free process that includes simultaneous extraction and preconcentration of analytes from aqueous samples or the headspace of the samples. Despite the advantages provided by this method, most commercial extractive fibers used in SPME are relatively expensive and fragile, and sample carry-over is also a problem (Mehdinia and Aziz-Zanjani, 2013; Prosen and Zupančič-Kralj, 1999). Stir bar sorptive extraction (SBSE) is another form of sorptive extraction based on the same principles as SPME. In SBSE, the sorbent is coated on a magnetic stir bar and the liquid sample is stirred with this bar. After the extraction, the analytes can be introduced quantitatively into the analytical system by thermal or liquid desorption (Farajzadeh et al., 2010). In 2006, a new liquid phase microextraction method termed dispersive liquid–liquid microextraction (DLLME) was introduced by Assadi and co-workers (Rezaee et al., 2006). In DLLME, the mixture of extraction and disperser solvents is rapidly injected into an aqueous sample to form a cloudy solution. The analytes are extracted into the fine droplets of extraction solvent, which are further separated by centrifugation and are determined by either chromatographic or spectrometric methods. The large contact surface area between phases speeds up mass transfer; hence, the equilibrium state is achieved quickly. This process reduces the extraction time and increase the EFs (Rahnama Kozani et al., 2007). SPME (Ulrich and Martens, 1997), SBSE (Chaves et al., 2007) and DLLME (Yazdi et al., 2008) have been used as sample preparation techniques in the determination of some antidepressants. Combination of derivatiation and DLLME can reduce analysis time and/or increase the extractability of the analytes. Some works on this combination have been reported, such as simultaneous derivatization and DLLME of atranol and chloroatranol (López-Nogueroles et al., 2014), amino acids (Li et al., 2013), and chlorophenols (Wang et al., 2014). Recently a new version of DLLME named temperatureassisted dispersive liquid–liquid microextraction (TA-DLLME) was developed (Farajzadeh et al., 2014). TA-DLLME method is based on injection of the mixture of extraction and disperser solvents into an aqueous sample solution at an elevated temperature. Similar approaches were performed previously using ionic liquids (ILs) as the extractant in DLLME (Zhou et al., 2008; Baghdadi and Shemirani, 2008). However using ILs before GC is difficult. Therefore in this study an organic solvent was used as the extraction solvent in DLLME performed at an elevated temperature. During cooling of
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this solution, the cloudy state of the solution is increased. The movement of extraction solvent droplets through the solution during the cooling process leads to an efficient transition of analytes from the aqueous phase into the extraction solvent. In this method the volume of sample solution used is higher than that of conventional DLLME, so high EFs are achievable. On the other hand, the formation of new droplets of extraction solvent during the cooling step leads to higher extraction recoveries similar to the continuous extraction process. As nortriptyline and maprotiline have a secondary amino group and fluvoxamine has a primary amino group, derivatization can be used to improve sensitivity and to obtain better peak shape. Some derivatization reagents such as acetic anhydride (Ito et al., 2011), heptafluorobutyric anhydride (Papoutsis et al., 2012), 1-(heptafluorobutyryl) imidazole (Wille et al., 2005) and tert-butyldimethylsilane (Gunnar et al., 2004) have been used for this purpose. In this work, a simultaneous TA-DLLME and derivatization method followed by GC-FID is proposed for the determination of fluvoxamine, nortriptyline and maprotiline in urine. Here, a derivatization reagent, an extraction solvent and a disperser were simultaneously injected into the heated sample solution to perform derivatization and extraction in one step. The solubility of the extraction solvent in the aqueous sample depends on the temperature. At an elevated temperature, the solubility of the extraction solvent into aqueous sample increased. By injection of the above mixture, a partially turbid solution was obtained. During cooling, the turbidity of the solution was increased by forming new droplets of extraction solvent which led to an efficient extraction. The analytes were derivatized and extracted into the fine droplets of extraction solvent, which were further separated by centrifugation. The effects of different parameters on the derivatization/microextraction procedure were thoroughly investigated. The performance of the optimized method was then evaluated and applied to determine the selected antidepressant in urine samples.
Materials and methods Chemicals and reagents Fluvoxamine, nortriptyline and maprotiline were a gift from Daana Pharmaceutical Company (Tabriz, Iran). Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, iso-propanol and acetonitrile (as disperser solvents), acetic anhydride (as derivatization agent), 1,2-dibromoethane (as an extraction solvent), hydrochloric acid and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Also, 1,1,2,2-tetrachloroethane and 1,1,2,2-tetrabromoethane (as extraction solvents) were obtained from Janssen Chimica (Beerse, Belgium). Stock solutions of nortriptyline, and maprotiline (1000 mg L1) in methanol and fluvoxamine (as maleate; 1000 mg L1) in water were prepared and stored in a refrigerator at 4 °C. Working solutions were prepared daily by appropriate dilutions of the stock solutions with de-ionized water (Ghazi Company, Tabriz, Iran). A standard solution of the target analytes (each 250 mg L1) in a mixture of acetic anhydride– 1,1,2,2-tetrabromoethane (2:1, v/v) was injected (three times) into the separation system daily for quality control and the obtained peaks areas were used in the calculation of EFs and extraction recoveries (ERs). Sodium carbonate solution (concentration, C = 0.2 mol L1) was prepared by dissolving 1.06 g of sodium carbonate (Merck, Darmstadt, Germany) in 50 mL deionized water.
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Simultaneous derivatization/TA-DLLME of some antidepressants in urine Instrumentation Chromatographic analysis was performed on a Shimadzu 2014 gas chromatograph (Kyoto, Japan) equipped with a split/splitless injector operated at 250 °C in a splitless mode (sampling time 1 min) and a flame ionization detector (FID). Helium (99.999%, Gulf Cryo, United Arab Emirates) was used as the carrier gas at a linear velocity of 30 cm s1. Separation was carried out on a CP-Sil 8 CB capillary column (30 m × 0.25 mm i.d., and film thickness 0.25 μm; poly (5%-diphenyl-95%-dimethylsiloxane; Chrompack, Milan, Italy). The column oven temperature was initially held at 50 °C for 1 min, then raised to 300 °C at a rate of 10 °C min1, and held at 300 °C for 2 min. A 1 μL microsyringe (zero dead volume, Hamilton, Switzerland) was used for the injection of samples into GC. Injection volume was 0.5 μL. The FID temperature was maintained at 250 °C. Hydrogen gas was generated with a hydrogen generator (OPGU-1500S, Shimadzu, Japan) for FID at a flow rate of 30 mL min1. The flow rate of air for FID was 300 mL min1. Gas chromatography–mass spectrometry (GC-MS) analysis was carried out on an Agilent 7890A gas chromatograph with a 5975C quadrupole mass-selective detector (Agilent Technologies, CA, USA) equipped with a split/splitless injector operated at 250 °C in a splitless/split mode (sampling time 1 min). The separation was performed on an HP-5 MS capillary column (30 m × 0.25 mm i.d., and film thickness of 0.25 μm; Hewlett-Packard, Santa Clara, USA). The carrier gas was helium with a flow rate of 1.0 mL min1. The column oven temperature programming was the same as mentioned above for GC-FID analysis. MS operational conditions were: electron ionization, 70 eV; ionic source temperature, 250 °C; transfer line temperature, 260 °C; mass range, m/z 55–350; acquisition rate, 20 Hz; detector voltage, 1700 V. Library searching was performed using the commercial NIST library. pH measurements were performed with a Metrohm pH meter (model 654, Herisau, Switzerland). A ROTOFIX 32A centrifuge from Hettich (Kirchlengern, Germany) was used in extraction/derivatization procedure.
sedimented organic phase (20 ± 2 μL) was removed using a 1 μL GC microsyringe and injected into the GC system for analysis. The derivatization reaction (acylation) between acetic anhydride and the selected pharmaceuticals that have primary (fluvoxamine) or secondary (nortriptyline and maprotiline) amino groups was performed according to the following reaction:
Analytical parameters Two main parameters, namely EF and ER, were employed for evaluation of the proposed method. EF is defined as the ratio of the analyte concentration in the sedimented phase (Csed) to its initial concentration (C0) within the sample: EF ¼ C sed =C 0
(1)
Csed was obtained from the comparison of peaks areas of analytes in two cases: direct injection of standard solution 250 mg L1 in the mixture of acetic anhydride–1,1,2,2-tetrachloroethane (2:1, v/v) and injection of the sedimented organic phase after performing the proposed method on the deionized water or diluted urine sample spiked with 1 mg L1 of each analyte. ER is defined as the percentage of the total analyte amount (n0) that is extracted into the sedimented phase (nsed): ER ¼ ðnsed =n0 Þ 100 ¼ ðC sed V sed Þ= C 0 V aq 100 (2) ER ¼ V sed =V aq EF 100 where Vsed and Vaq are the volumes of sedimented phase and aqueous solution, respectively.
Urine samples Blank urine samples were collected from a healthy female adult who had not taken the selected antidepressants. Also urine samples were obtained from two 26- and 27-year-old females after oral administration of fluvoxamine (50 mg, once in a day) and maprotiline (25 mg, once in a day), respectively, and a female depressed patient (60-year-old) who had ingested a tablet containing 25 mg of nortriptyline (twice in a day, 12 h interval). Urine samples were collected within 24 h from the first oral administration. After reading the volume of collected urine, 5 mL urine was centrifuged at 8000 rpm for 5 min. To reduce the matrix effect of the urine sample the supernatant was diluted 10-fold with sodium carbonate solution (0.2 mol L1) and then subjected to the derivatization/microextraction procedure.
Derivatization/microextraction procedure A 50 mL aliquot of diluted urine sample or standard solution was placed into a 60 mL glass test tube with conical bottom. Then the tube was immersed into a water bath for 10 min at 70 °C. A 1.5 mL aliquot of DMF containing 150 μL acetic anhydride and 80 μL 1,1,2,2-tetrachloroethane was rapidly injected into the solution using a 2 mL glass syringe. Then the solution was cooled to room temperature and the cloudy solution formed was centrifuged for 3 min at 4000 rpm. An aliquot (0.5 μL) of the
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Results and discussion The initial objective of this work was to optimize the derivatization/microextraction procedure. There are several parameters affecting the process, that is, selection of suitable extraction and disperser solvents and their volumes, volume of derivatization agent, temperature, salt addition, pH and centrifugation time and speed. Therefore, all of these parameters were optimized in order to achieve the good performance. Selection of extraction solvent The selection of an appropriate extraction solvent is of primary importance in a DLLME process. Therefore, some factors should be considered, that is, low solubility in water, extraction capability of compounds of interest, good chromatographic behavior and compatibility with the derivatizing reagent (in this study). Based on these facts, 1,1,2,2-tetrachloroethane, 1,2-dibromoethane and 1,1,2,2-tetrabromoethane were tested. Initially experiments were performed with different volumes of the selected solvents to obtain 50 μL of the sedimented organic phase. The volumes needed for each solvent were 100, 110 and 140 μL, respectively. According to obtained resutls, 1,1,2,2-tetrachloroethane provided high extraction efficiency for all analytes. Hence it was selected as the optimum extraction solvent for the subsequent experiments.
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Figure 1. Effect of extraction solvent volume on the derivatization/ microextraction efficiency. Extraction conditions: aqueous phase, sodium car1 1 bonate solution 0.2 mol L (50 mL); analytes concentrations, 500 ng mL of each antidepressant; extraction solvent, 1,1,2,2-tetrachloroethane (100 μL); disperser solvent, dimethylformamide (DMF; 2 mL); extraction temperature, 60 °C; centrifuging time, 3 min; and centrifuging speed, 4000 rpm. The error bars indicate the maximum and minimum of three determinations.
Figure 2. Effect of disperser solvent volume on the microextraction efficiency. Extraction conditions: the same as Fig. 1, except 80 μL extraction solvent was used. The error bars indicate the maximum and minimum of three determinations.
Figure 4. Effect of temperature on the derivatization/microextraction efficiency. Extraction conditions: all conditions are the same as Fig. 3 except 150 μL acetic anhydride was used as derivatization agent. The error bars indicate the maximum and minimum of three determinations.
Figure 5. Effect of sample solution pH on the derivatization/microextraction efficiency. Extraction conditions: all conditions are the same as Fig. 4 except extraction temperature was controlled at 70 °C. The error bars indicate the maximum and minimum of three determinations.
Selection of disperser solvent Miscibility in both extraction solvent and aqueous phase is an essential factor in the selection of a disperser solvent. On the other hand, in this study, relatively high temperature was applied during the extraction step. Therefore the use of disperser solvents having low boiling points such as acetone and methanol is limited. In order to determine a suitable disperser solvent, five disperser solvents with relatively higher boiling points (DMF, DMSO, 1,4dioxane, iso-propanol and acetonitrile) were examined. The derivatization/microextraction of analytes was carried out using 2 mL of each disperser containing 100 μL 1,1,2,2-tetrachloroethane (extraction solvent) and 200 μL acetic anhydride (derivatization reagent). According to the results, DMF was selected as the disperser because of the formation a cloudy state with very fine droplets and high peak areas of the derivatized analytes.
Figure 3. Optimization of derivatizing agent volume. Extraction conditions: the same as Fig. 2 except 1.5 mL DMF was used as disperser solvent. The error bars indicate the maximum and minimum of three determinations.
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Optimization of extraction solvent volume The volume of extraction solvent used can affect the volume of the sedimented phase. Also, increasing extraction solvent volume
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3 4 6 5 5 6 3 3 3 3 4 5 Square of correlation coefficient. Limit of detection, signal-to-noise ration (S/N) = 3. c Limit of quantification, S/N = 10. d Mean enrichment factor ± standard deviation, EF = CSed/C0 (n = 3). e Mean extraction recovery ± standard deviation, (n = 3). f Relative standard deviation intraday (n = 6, C = 50 ng mL1) and interday (n = 4, C = 50 ng mL1). b
Human urine
Fluvoxamine Nortriptyline Maprotiline Fluvoxamine Nortriptyline Maprotiline
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Figure 6. GC-FID chromatograms of (a) drug-free urine sample, (b) urine sample of 26 year-old female volunteer, (c) urine sample of 60-year-old female depressed patient, (d) urine sample of 27-year-old female volunteer, 1 and (e) spiked de-ionized water with 100 ng mL of each antidepressant. In all cases, the derivatization/microextraction method was performed and 0.5 μL of the sedimented phase was injected into GC. Peaks identification: 1, fluvoxamine; 2, nortriptyline; and 3, maprotiline.
a
910 ± 53 1100 ± 69 870 ± 53 91 ± 4 107 ± 5 82 ± 4 0.4 0.3 0.5 3 2 4 1–4000 1–4000 2–4000 8–4000 10–4000 12–4000 De-ionized water
721.31 ± 62 838.58 ± 36 923.32 ± 17 546.44 ± 25 798.38 ± 10 844.4 ± 38
18918 ± 1202 43977 ± 1657 47099 ± 1253 40378 ± 1318 55450 ± 1810 52492 ± 1560
0.999 0.997 0.996 0.996 0.995 0.993
1 1 2 8 10 12
36 ± 4 44 ± 5 35 ± 3 36 ± 3 43 ± 4 33 ± 2
Interday Intraday
RSD (%)f ER (%) ± SDe
EF ± SDd LOQc (ng mL1) LODb (ng mL1) R2a Intercept ± SD Slope ± SD Linear range (ng mL1) Matrix Analyte
Table 1. Analytical features of simultaneous derivatization/temperature-assisted dispersive liquid–liquid microextraction (TA-DLLME) followed by gas chromatography-flame ionization detection (GC-FID) determination of the selected antidepressants
Simultaneous derivatization/TA-DLLME of some antidepressants in urine
would increase the amonuts of analytes extracted, whereas their concentrations in the sedimented phase are decreased. Also, by changing the volume of extraction solvent (at a constant sample size), the volume ratio of sample to extraction phase is varied and hence extraction recoveries of the analytes will also change. To evaluate the effect of extraction solvent volume, different volumes of 1,1,2,2-tetrachloroethane (60, 80, 100 and 120 μL) were subjected to the same derivatization/microextraction procedure. Maximum peaks areas or EFs were obtained when 80 μL of the extraction solvent was added (Fig. 1). By increasing the extraction solvent volume from 80 to 120 μL, the peaks areas decreased owing to the increase in the volume of sedimented phase and its dilution effect on the concentrations of analytes in the extractant. On the other hand, when 60 μL of the extraction solvent was used, only 4 μL sedimented phase was obtained, which made its removal was difficult. Therefore, in this study, 80 μL was selected as the optimal volume of 1,1,2,2-tetrachloroethane, which leads to obtain 20 ± 2 μL sedimented phase volume.
Optimization of disperser volume To study the effect of dispersive solvent volume on the extraction efficiency, different volumes of DMF (from 0.5 to 2.5 mL at 0.5 mL intervals) containing 200 μL of acetic anhydride and 80 μL of extraction solvent were tested. At low volumes of DMF, the cloudy state was not formed properly and the extraction solvent did not disperse well into the aqueous solution, which resulted in low EFs. Meanwhile, at high volumes of DMF, the solubility of derivatized analytes and extraction solvent into aqueous phase was increased, which led to decreased extraction
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A. A. Alizadeh Nabil et al.
Figure 7. (I) Total ion current (TIC) chromatograms of (a) urine sample of 26-year-old female volunteer, (b) urine sample of 60-year-old female 1 depressed patient, (c) urine sample of 27-year-old female volunteer, and (d) de-ionized water (spiked with 100 ng mL of each analyte). In all cases, derivatization/microextraction method was performed and 0.5 μL of the sedimented phase was injected into GC-MS. (ІІ) Mass spectra of (e) derivatized fluvoxamine, (f) scan 1558 (retention time 16.663 min) in urine sample of 26-year-old female volunteer, (g) derivatized nortriptyline, (h) scan 2076 (retention time 20.180 min) in urine sample of 60-year-old female depressed patient, (i) derivatized maprotiline, and (j) scan 2229 (retention time 21.219 min) in urine sample of 27-year-old female volunteer. Peaks identification: 1, derivatized fluvoxamine (tR = 16.711 min); 2, derivatized nortriptyline (tR = 20.166 min); and 3, derivatized maprotiline (tR = 21.246 min).
efficiency. Therefore, 1.5 mL of DMF was selected as the optimum volume for the dispersive solvent (Fig. 2). Optimization of derivatization agent volume In order to study the effect of derivatization agent volume on the performance of the presented method, different volumes of acetic anhydride (25–250 μL) were tested and the obtained results are shown in Fig. 3. The highest derivatization reaction
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yield was observed when 150 μL of acetic anhydride was used, thus it was chosen as the volume of derivatization reagent for the further studies. By increasing the volume of acetic anhydride from 150 to 250 μL, the analytical signals decreased rapidly. This decrease could possibly be attributed to corresponding increase in the sedimented phase volume. It is noted that, at high volumes of acetic anhydride, the unreacted derivatization agent dissolved into extraction solvent and led to increases in the sedimented phase volume. Also, in the
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Biomed. Chromatogr. 2014
Simultaneous derivatization/TA-DLLME of some antidepressants in urine increased followed by a gradual decrease (Fig. 4). Different factors such as loss of analytes, extraction and disperser solvents and derivatization agent or hydrolysis of acetic anhydride can be responsible for this decrease. Finally 70 °C was selected as the optimum derivatization/microextraction temperature for further experiments.
Table 2. Relative recoveries obtained by simultaneous derivatization/TA-DLLME in urine samples spiked at different concentrations Spiked concentration (ng mL1) 50 100 500
Relative recovery (%) ± standard deviation (n = 3) Fluvoxamine
Nortriptyline
Maprotiline
79 ± 4 82 ± 3 89 ± 3
76 ± 3 83 ± 2 86 ± 5
78 ± 2 81 ± 4 85 ± 6
Effect of solution pH The selected antidepressants are basic compounds (pKb value 5.3 for fluvoxamine, 4.3 for nortriptyline and 3.5 for maprotiline). Therefore, the pH of the sample solution should be high to prevent their protonation. In these conditions, the analytes will be in neutral form and can react with the derivatization agent. The effect of pH on the extraction efficiency was studied within the pH range of 8–13 with addition of HCl or NaOH 1 M to the analyte solution containing sodium carbonate (0.2 mol L1). The results in Fig. 5 indicate that the peak areas increased with the pH increasing from 8 to 11 and remained constant up to pH 13. To facilitate the pH adjustment, sodium carbonate solution (0.2 mol L1, pH 12.5) without HCl or NaOH addition was used for the following studies.
cases of volumes 0.993. The repeatability and reproducibility of the proposed method, expressed as RSD, were evaluated by performing the method on six repeated samples (for intraday) and four repeated samples (for interday) at a concentration of 50 ng mL1 and RSDs were found to vary between 3 and 6%. High EFs ranging from 820 to 1100 were obtained. LODs and LOQs were in the ranges 2–4 and 8–12 ng mL1, respectively. The obtained ERs (33–43%) are completely reasonable for a microextraction procedure in which 50 mL aqueous phase along with 20 μL organic phase is used. Low LODs and LOQs, high EFs and ERs, and good repeatability and reproducibility are the main advantages of the method.
Comparison of the proposed method with others To assess performance of the new method, its analytical parameters were compared with those of other methods used in the analysis of target analytes. Table 3 summarizes the LR, LOD, LOQ and RSD of some analytical methods along with the proposed method. The repeatability and reproducibility of the proposed method are good and RSDs for this method are comparable or better than those of the mentioned methods. The LODs achieved by the presented method are better than those of the fourth method (Yazdi et al., 2008) whereas both of them used similar microextraction and detection systems, for example, DLLME-GCFID. It should be noted that, in some of the techniques mentioned, MS or nitrogen–phosphorus (NPD) detectors were used, which are inherently more sensitive and selective than FID. Nevertheless the proposed method has lower LODs and LOQs compared with GCNPD (Lacassie et al., 2000) and GC-MS (Gunnar et al., 2004). In the cases which LLE was used as a preconcentration step, the presented TA-DLLME procedure is more efficient and consumes less organic solvents. Hence, the obtained LODs by the presented method are better than those of the fifth method (Gunnar et al., 2004) in spite of its more sensitive detection system (MS). Another important advantage of the current method is the simultaneous derivatization and microextraction of the selected antidepressants in urine samples. Also, the use of heated sample solution led to an efficient extraction of analytes from the aqueous phase into the extraction solvent, which continued during the cooling process. By considering the results, the proposed method proved to be a sensitive, efficient, reliable and easy to use technique in the preparation and preconcentration of the selected antidepressants from urine.
Urine analysis
Conclusions
The proposed method was applied to analysis of urine samples of two 26- and 27-year-old females after oral administration of fluvoxamine (50 mg, once in a day) and maprotiline (25 mg, once in a day), respectively. Also a urine sample from a patient (60-year-old female) who received nortriptyline (25 mg, twice in a day) was analyzed by the optimum proposed method. Figure 6 show typical GC-FID chromatograms of these samples. There was no peak in the retention times of analytes in the blank urine sample. The obtained concentrations of fluvoxamine, nortriptyline and maprotiline in urine samples were 106 ± 11, 68 ± 3 and 82 ± 8 ng mL1, respectively. In all cases three determinations (n = 3) were performed to identify the compounds eluted in the retention times of the derivatized antidepressants in urine samples chromatograms. These samples
A simultaneous derivatization and TA-DLLME method has been reported for the extraction and preconcentration of three antidepressants from urine followed by GC-FID determination. The developed method has numerous advantages such as simplicity, low cost and excellent repeatability and reproducibility. The use of heated sample solution leads to increase dissolution of extraction solvent into aqueous solution. During the cooling process, the formation of tiny droplets of the extraction solvent leads to an effective extraction of the derivatized analytes from aqueous samples. Also, the ratio of sample volume to extraction solvent volume is improved using high sample size, which results in high EFs being obtained. The results revealed that the developed method was suitable for determination of the selected antidepressant at ng mL1 levels in urine.
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Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous derivatization/TA-DLLME of some antidepressants in urine
Acknowledgments The authors thank the research Council of University of Tabriz for financial support.
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