Comparison of conventional hollow fiber based liquid phase microextraction and electromembrane extraction efficiencies for the extraction of ephedrine from biological fluids

Journal of Chromatography A, 1218 (2011) 8581–8586

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Comparison of conventional hollow fiber based liquid phase microextraction and electromembrane extraction efficiencies for the extraction of ephedrine from biological fluids Lida Fotouhi a,∗ , Yadollah Yamini b , Saeideh Molaei a , Shahram Seidi b a b

Department of Chemistry, School of Sciences, Alzahra University, P.O. Box 1993891176, Vanak, Tehran, Iran Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 9 July 2011 Received in revised form 26 September 2011 Accepted 28 September 2011 Available online 6 October 2011 Keywords: Electromembrane Ephedrine Hollow fiber Microextraction Plasma Urine

a b s t r a c t In the present study, hollow fiber liquid phase microextraction (HF-LPME) based on pH gradient and electromembrane extraction (EME) coupled with high-performance liquid chromatography (HPLC) was compared for the extraction of ephedrine from biological samples. The influences of fundamental parameters affecting the extraction efficiency of ephedrine were studied and optimized for both methods. Under the optimized conditions, preconcentration factors of 120 and 35 for urine and 51 and 8 for human plasma were obtained using EME and HF-LPME, respectively. The calibration curves showed good linearity for urine and plasma samples by both methods with the coefficient of estimations higher than 0.98. The limits of detection were obtained 5 and 10 ng mL−1 using EME and 60 and 200 ng mL−1 by HF-LPME for urine and plasma samples respectively. The relative standard deviations of the analysis were found in the range of 5.2–8.6% (n = 3). The results showed that in comparison with HF-LPME based on pH gradient, EME is a much more effective transport process, providing high extraction efficiencies in very short time.

1. Introduction Ephedrine hydrochloride (Eph) is a sympathomimetic amine which determines an increase of cardiac rate and contractility, peripheral vasoconstriction and bronchodilatation [1]. It is administered to relieve pain for long-term treatment of moderate to severe cancer pains. It can also make patients excited and feel exhilarant. Ephedrine has been marketed in pharmaceutical formulations to be used in the cold, hypersensitivity [2] and symptomatic treatment of asthma and spasms, as a stimulant and diaphoretic [3]. Ephedrine as an illicit drug may be used for doping in sports and is therefore included in the list of pharmacological substances prohibited by the Medical Commission of the International Olympic Committee (MCIOC) [4]. Recently, MCIOC has established a threshold value of Eph in urine above which an athlete is considered as “positive”. Adverse effects attributed to consumption of products containing ephedrine alkaloids have led to the development of several methods for the quantitative determination of Eph in dosage forms containing these drugs either in pharmaceutical products or in human fluids. A variety of analytical methods including gas

∗ Corresponding author. Tel.: +98 21 88044040; fax: +98 21 88035187. E-mail addresses: lida [email protected], [email protected] (L. Fotouhi). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.09.078

© 2011 Elsevier B.V. All rights reserved.

chromatography (GC) [5] and HPLC, with UV [6] or MS [7] detection and micellar electrokinetic capillary chromatography (MEKC) [8] have been successfully applied to determine illicit drugs. Nevertheless, the analysis of illicit drugs in complex matrixes such as plasma or urine is very difficult and due to the low concentration of drugs and the presence of high number of interferences existing in human fluids, sample preconcentration and preparation must be carried out before the analysis of drugs using HPLC. Analyte extraction and pretreatment are the most challenging and time-consuming steps in an analytical procedure. Many extraction procedures such as liquid–liquid extraction (LLE) [9] and solid-phase extraction (SPE) [10] techniques are available. However, LLE is time consuming and requires large quantities of expensive, toxic and environmentally unfriendly organic solvents. To reduce the solvent usage in sample preparation, a miniaturized format of LLE, called liquid phase microextraction (LPME), has been introduced. Hollow fiber liquid phase microextraction is a sample preparation technique introduced by Pedersen-Bjergaard and Rasmussen [11]. This technique divides two and three-phase groups. The three-phase HF-LPME is based on gradient of pH between donor phase (DP) and acceptor phase (AP). Despite of these advantages, HF-LPME method is time consuming [12]. In order to increase the extraction speed, a new concept has recently been introduced by Pedersen-Bjergaard et al. [13]. According to which, mass transfer across the supported liquid membrane (SLM) is accomplished by application of an electrical

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potential difference as the driving force called electromembrane extraction (EME). This technique has been used for extraction of many compounds so far [13–23]. To best of our knowledge, there are few reports concerning the comparison between HF-LPME and EME methods [14,15]. Recently we studied the microextraction of mebendazole by both EME and HF-LPME methods and it was shown that EME has a strong potential as a future sample preparation technique [16]. In continuation of our interest in the microextraction, both HF-LPME and EME were applied for the extraction and preconcentration of Eph as a polar drug (log P 1.1 and pKa 9.6) in the human fluids [24]. In the present study the influences of different parameters on the extraction efficiency of ephedrine by applying EME and HF-LPME were investigated and optimized. Finally, both of the developed methods were compared for extraction of ephedrine from human fluids. 2. Experimental 2.1. Chemicals Ephedrine (Eph) was kindly donated by the Department of Pharmacy, Tehran University (Tehran, Iran). HPLC-grade methanol and acetonitrile were purchased from Caledon (Georgetown, Ont., Canada). n-Dodecane, 2-nitrophenyl octyl ether (NPOE), 1undecanol, benzyl alcohol, toluene, 1-decanol, 1-octanol, n-dihexyl ether, di-(2-ethylhexyl) phosphoric acid (DEHP), tris(2-ethylhexy) phosphate (TEHP), methyltrioctylammonium chloride (Aliquat 336) and trioctylphosphine oxide (TOPO) were purchased from Fluka (Buchs, Switzerland). HCl, NaOH, NaCl, and ortho-phosphoric acid were purchased from Merck (Darmstadt, Germany). All the used reagents were of analytical grade. The water used in the experiment was purified on a MilliQ ultra-pure water purification system purchased from Millipore (Bedford, MA, USA). 2.2. HPLC analysis Chromatographic separations and analysis of extracted drug from the aqueous samples were carried out on a Cecil HPLC including a CE4100 HPLC pump (Cambridge, England), a six-port two-position Rheodyne HPLC valve (Oak Harbor, WA, USA) with a 20 ␮L sample loop and equipped with a CE 4300 HPLC UV–vis detector (version R0050). Chromatographic data were recorded and analyzed using Power Stream software (version 3.2). A C18 column (25 cm × 4.6 mm, with particle size of 5 ␮m) from Hichrom (Berkshire, England) was applied to separate the analytes under isocratic elution conditions. A mixture of 10 mmol L−1 phosphate buffer with pH 4.5 and acetonitrile (80:20, v/v) with a flow rate of 1.0 mL min−1 was used as the mobile phase. The injection volume was 20 ␮L for all of the standards and the samples, and detection was performed at wavelength of 210 nm. All of the pH measurements were made using an 827 Metrohm pH meter (Herisau, Switzerland). 2.3. Equipment for LPME and EME The setup used for EME was identical to the HF-LPME unit except for the potential application system. The sample compartments with same volumes, internal diameter, and heights were used in HF-LPME and EME methods. The porous hollow fiber used for the SLM and for housing the acceptor solution was a PPQ 3/2 polypropylene hollow fiber from Membrana (Wuppertal, Germany) with an inner diameter of 600 ␮m, wall thickness of 200 ␮m, and pore size of 0.2 ␮m. The sample compartment was stirred during the experiments with a magnetic stirrer model MR 3001 from Heidolph (Kelheim, Germany). The platinum wires with diameter of 500 and

200 ␮m were prepared from Pars Pelatine (Tehran, Iran) and used as electrodes in the sample and acceptor solutions, respectively. They were both connected to the power supply model PTS 1002 with programmable voltage in the range of 0–300 V, and to a current output in the range of 0–2.5 A from Akhtarian (Tehran, Iran). A 25 ␮L syringe model 702 NR from Hamilton (Bonaduz, Switzerland) was employed during extraction procedure and also to inject the extracted analyte into the HPLC. 2.4. Extraction procedure for HF-LPME and EME Both HF-LPME and EME extractions were carried out according to our previous paper [16] with some modifications. An 8 mL glass vial containing a 5 mm × 3 mm magnetic stirring bar was used for both EME and HF-LPME methods. Also, the whole fiber was cut into small segments with length of 7.5 cm and all the experiments were conducted at room temperature. The concentration of 500 ng mL−1 of ephedrine aqueous solution was used throughout the optimization process. In HF-LPME, 7 mL of alkaline sample solution (containing 1 mmol L−1 NaOH) as a DP was filled into the 8 mL glass vial. Toluene containing 10% (w/v) TEHP was used as organic solvent and the lumen of the fiber was filled with acidic solution containing 1 mmol L−1 of HCl. In EME, the donor and acceptor phases were 10 mmol L−1 HCl and 100 mmol L−1 of HCl, respectively. NPOE containing 10% (v/v) of DEHP was used as organic solvent and an electrical potential (100 V, DC) was applied for a predetermined period of time. Schematic representations of HF-LPME (A) and EME (B) are shown in Fig. 1. The preconcentration factors (PFs) and extraction recoveries (ERs) were calculated based on the previous papers [15,16]. 2.5. Standard solution and real samples Ephedrine stock solution at concentration of 1000 ␮g mL−1 was prepared in aqueous solution. Standard solutions were freshly prepared from the stock solution by proper dilution with ultra-pure water. All of the standard solutions were stored at 4 ◦ C. Urine sample was collected from a volunteer. One milliliter of the urine sample was diluted to 7 mL with ultra-pure water and its pH was adjusted by dropwise addition of HCl and/or NaOH solutions, such that the final pH of samples was adjusted at 11 and 2 in HF-LPME and EME experiments, respectively. Plasma sample was obtained from the Iranian Blood Transfusion Organization (Tehran, Iran) and stored at −20 ◦ C prior to use. Frozen plasma sample was thawed and allowed to reach room temperature. To precipitate the protein contents of the plasma, a known concentration of ephedrine (500 ng mL−1 ) was spiked into 1 mL of plasma. After 30 min, 500 ␮L of 60% trichloroacetic acid was added and centrifuged for 5 min at the rate of 8000 rpm. After decantation of the plasma sample, it was transferred into the 8 mL glass vial and diluted to 7 mL with ultra-pure water. By dropwise addition of 4 mol L−1 NaOH and/or HCl solutions, pH of the real samples was adjusted at 11 and 2 in HF-LPME and EME experiments, respectively. 3. Results and discussion 3.1. Effect of the organic solvent in HF-LPME and EME In order to further investigate the two systems, experiments with different organic solvents as the artificial liquid membrane were conducted. There are specific requirements for a solvent to be used as a SLM in both HF-LPME and EME methods [21]. In preliminary experiments, Eph was transported by passive diffusion from the DP adjusted at pH 11 through different organic

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Table 1 Effect of impregnation solvent on PF of Eph in (a) HF-LPME and (b) EME. Solvent (w/v, %)

HF-LPMEa

EMEb

pH of donor solution

Solvent (v/v, %)

11 1-Decanol n-Dodecane 1-Undecanol Benzylalcohol n-Hexylether 1-Octanol Toluene Toluene + 5% Aliquat 336 Toluene + 5% DEHP Toluene + 5% TOPO Toluene + 5% TEHP Toluene + 5% Aliquat 336 Toluene + 5% DEHP Toluene + 5% TOPO Toluene + 5% TEHP Toluene + 10% TEHP Toluene + 15% TEHP

3

2.6 2.8 3.8 4.6 8.3 10.5 15.0

NPOE NPOE + 10% TEHP NPOE + 10% DEHP NPOE + 15% DEHP

8.7 25.6 64.7 52.4

0.0 0.5 0.0 0.0 5.1 8.9 20.9 27.5 38.0 36.4

a VDP , 7 mL, CEph , 500 ng mL−1 ; VAP , 20 ␮L of 1 mmol L−1 HCl (pH 3); stirring rate, 1000 rpm; time, 20 min. b VDP , 7 mL of 10 mmol L−1 HCl (pH 2); CEph , 500 ng mL−1 ; VAP , 20 ␮L of 100 mmol L−1 HCl (pH 1); stirring rate, 1000 rpm; time, 15 min; voltage, 100 V.

Eph and increase its extraction efficiency. Further addition of DEHP decreased the extractability of the Eph. This may be attributed to the decrease in electrical resistance of SLM and the increase in the current level and bubble formation. 3.2. Effect of pH of donor and acceptor phases in HF-LPME and EME

Fig. 1. Schematic representations of HF-LPME (A) and EME (B) systems.

solvents immobilized in the pores of a HF into an AP with pH 3. As can be seen in Table 1, low PF values in the range of 2.6 for 1-decanol to 15.0 for toluene were obtained. Therefore, toluene was selected as the optimum organic solvent for the HF-LPME. In the next step, the effects of three types of carriers including TOPO and TEHP (as neutral carriers), DEHP (as anionic carrier) and Aliquat 336 (as cationic carrier) were investigated (Table 1). As shown in Table 1, high PF values were obtained for TOPO and TEHP as neutral carriers with pH of 3 and 11 for DP and AP, respectively. The enhancement of PF value for TOPO and TEHP as carrier could be attributed to two possible reasons: first, increase in polarity of organic phase due to addition of a polar carrier; second, the unprotonated analyte might associate with the carrier through non-ionic forces, e.g., hydrophobic interaction between the carrier’s alkyl group and the analyte’s aromatic ring [25]. Finally, toluene containing 10% of TEHP was selected as the best SLM for subsequent experiments with HF-LPME. Based on previous studies for basic drugs using EME, NPOE solely or in combination with DEHP and TEHP are good candidates for SLM [15,17]. As provided in Table 1, a high PF value was obtained with NOPE containing 10% (v/v) DEHP as the SLM. The acquired results indicated that the presence of the anionic carrier DEHP is crucial for the extraction of Eph. It is in good agreement with the literature for polar drugs [17]. DEHP can form ion-pair complexes with

A crucial step in the HF-LPME is choosing the pH of donor and acceptor solutions. The effect of pH of donor and acceptor solutions on PF was investigated in the ranges of 8–12 and 2–5, respectively. The results are summarized in Fig. 2. In the acceptor solution, by keeping the pH of DP constant at 11, the highest PF was obtained at pH 3 (Fig. 2A, curve a). At higher pH values, the PFs decreased, because the protonation of drug was not complete and a large portion of the analyte existed in neutral form. In DP (acceptor phase pH 3), the PFs increased as the pH increased to 11, whereas it decreased as the pH exceeded this level (Fig. 2B, curve a). In EME, the effect of different pH values in both acceptor and donor solutions was also investigated. While the pH of DP was kept constant at 2, the highest PF was obtained at pH 1 in the acceptor solution (Fig. 2A, curve b). Also, the effect of different pH values in the donor solution was examined. The highest PF was obtained at pH 2 of donor solution while the pH of AP was kept at 1 (Fig. 2B, curve b). Thus, the pH values of 1 and 2 in the AP and DP were selected respectively for the subsequent experiments. As can be concluded, two different extraction mechanisms exist in HF-LMPE and EME methods. In HF-LPME, for extraction of basic drugs, the donor phase should be alkaline to effectively deionize the analyte and consequently transfer it into the organic phase, while in the acceptor phase; the analyte should be converted into its ionized form for its release into the acceptor phase. EME is based on electrokinetic migration of ionizable compounds under electrical field toward cathodic (for positive charged compounds) or anodic electrode (for negative charged compounds). For converting a basic compound into its ionized form (protonation of basic compounds) the pH of donor phase should be decreased whereas in HF-LPME the pH of donor phase should be increased for converting the basic analyte into its neutral form (deporotonation of analyte) as was mentioned above. On the other

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Fig. 2. Effect of pH on the PF of Eph in (A) acceptor phase and (B) donor phase. (a) HF-LPME and (b) EME. Conditions of (A, a): VDP , 7 mL of 1 mmol L−1 NaOH (pH 11); CEph , 500 ng mL−1 ; VAP , 20 ␮L; stirring rate, 1000 rpm; time, 20 min. Conditions of (A, b): donor, 7 mL of 10 mmol L−1 HCl (pH 2); CEph , 500 ng mL−1 ; VAP , 20 ␮L; stirring rate, 1000 rpm; voltage, 100 V; time, 15 min. Conditions of (B, a): VAP , 20 ␮L of 1 mmol L−1 HCl (pH 3); CEph , 500 ng mL−1 ; VDP , 7 mL; stirring rate, 1000 rpm; time, 20 min. Conditions of (B, b): VAP , 20 ␮L of 100 mmol L−1 HCl (pH 1); CEph , 500 ng mL−1 ; VDP , 7 mL; stirring rate, 1000 rpm; voltage, 100 V; time, 15 min.

hand, like HF-LPME, the acceptor phase in EME should be acidified. This increases releasing of analyte into the acceptor phase. Therefore, in EME both donor and acceptor phases should be acidified for basic compounds while the pHs of donor and acceptor phases in HF-LPME are opposite. In addition, the concentration of H+ in donor phase should not be increased so much in EME because the proton ions can compete with basic drugs under electrical field which results in reduction of extraction efficiency. An acidic donor solution that effectively protonates the basic analytes is sufficient. The concentration of proton ions in the acceptor phase should not also be increased so much because the probability of bubble formation on the electrode surface into the lumen of hollow fiber increases which leads to increase in system instability and RSDs% of determination [21]. 3.3. The effect of salt addition in HF-LPME and EME The salting out effect can often improve the extraction performance by reducing the solubility of polar analytes [26]. The effect of salt was examined by adding sodium chloride to the DP in the range of 1–5 mol L−1 . The obtained results revealed that there was a slight increase in PF by increasing the NaCl concentration. The efficiency was decreased slightly at concentrations higher than 2 mol L−1 of NaCl because of the change in physical properties of Nernst’s diffusion layer on the hollow fiber [27]. Hence, the concentration of 2 mol L−1 of NaCl was chosen as optimum value for subsequent analysis in HF-LPME. In EME system according to previous studies [18,19,21], the presence of high contents of ionic substances causes an increase in the value of the ion balance () which is defined as the ratio of the total ionic concentration in the sample solution to that in the acceptor solution [19]. Indeed, increasing other ions in donor solution causes an increase in the competition among analyte and interference ions which in turn decreases the flux of analytes across the SLM. Our observations were completely in accordance with previous studies [18,19,21]. Thus, electrical migration of the analyte would be more efficient in the absence of salt and all of the subsequent analyses in EME were performed in the absence of salt. 3.4. The effect of stirring rate in HF-LPME and EME Magnetic stirring of donor solution enhances diffusion of analyte by accelerating the mass transfer in DP and reducing the thickness of Nernst’s diffusion film around the interface between the sample solution and the SLM. The results showed that the PFs increased by increase of the stirring rate to 1200 and 1000 rpm for HF-LPME and EME, respectively.

3.5. The effect of extraction time in HF-LPME and EME In three-phase microextraction, mass transfer is a timedependent process; therefore, the amount of extracted analyte is expected to increase with extraction time until reaching the equilibrium between the DP, membrane phase and AP. In HF-LPME, the PF increased with extraction time in the range of 15–40 min and reached its maximum at 25 min, but showed a slight decline afterwards, which is due to slight loss of the organic solvent that occurs at longer extraction times. In EME, extraction time was investigated in the range of 5–20 min. The results showed that 15 min is the optimum extraction time in EME. The observed decrease in PF values after 15 min is probably due to the saturation of the analyte in AP [14] or due to instability of the electrical current in the system and experimental inaccuracies or small loss of artificial liquid membrane [7,15]. To summarize the experiments, EME was found to provide significantly improved extraction kinetics as compared with HF-LPME. 3.6. The effect of applied voltage in EME The main mass transfer mechanism in EME is the electrokinetic migration of the analyte across the SLM into AP. Thus, the applied voltage across a SLM is an important factor for efficient extraction of basic drugs in EME. Experiments with different applied potentials were performed over the range of 50–200 V. The results showed that PFs increased up to 100 V however, beyond this voltage, a decrease in the EME performance was observed. This phenomenon most probably was caused by analyte back-extraction into the SLM and sample solution due to slight increase in the acceptor solution’s pH as a result of electrolysis, bubble formation and the gradual suppression of the net transfer of the analyte due to the heat generated at a higher voltage [20]. Based on the obtained results, electrical potential of 100 V was applied for the rest of the work. 3.7. Evaluation of the three-phase HF-LPME and EME performances To evaluate the practical applicability of the HF-LPME and EME techniques, under optimized extraction conditions, the figures of merit of both methods were investigated in non-spiked human urine and plasma samples. The results are shown in Table 2. Comparison of the proposed methods with different existing methods for extracting and determining Eph is provided in Table 3 in terms of validation and precision. As can be concluded, in comparison with other extraction methods, the proposed HF-LPME and EME methods have some advantages including low consumption

L. Fotouhi et al. / J. Chromatogr. A 1218 (2011) 8581–8586 Table 2 Figures of merit of the proposed HF-LPME and EME methods followed by HPLC-UV for extraction and determination of Eph. HF-LPME

LOD (ng mL−1 ) LOQ (ng mL−1 ) DLR (ng mL−1 ) R2 RSD%a PF ER% a

Table 4 Determination of Eph in different samples. HF-LPME

EME

Plasma

Urine

Plasma

Urine

200 250 250–4000 0.988 8.6 8 2

60 100 100–3000 0.991 7.5 35 10

10 30 30–1000 0.993 7.3 51 15

5 15 15–750 0.994 5.2 120 37

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Cinitial (ng mL−1 ) Cadd (ng mL−1 ) Cfound (ng mL−1 ) RR%c RSD% (n = 3) a b c

EME

Plasma

Urine

Plasma

Urine

– 600 560 93 9.2

150a 100 240 96 7.8

– 600 580 96 6.5

220b 100 330 103 5.3

The sample was taken after 15 h. The sample was taken after 10 h. Relative recovery.

Standard deviation for three-replicate measurements.

of organic solvents and reagents, simplicity and producing a clean extracting phase for the analysis. However, in comparison with HF-LPME, EME demonstrated high sensitivities with an important emphasis on the extraction time which seems to be noticeably short. Nevertheless, the initial set-up for EME is more expensive than HF-LPME and also the second one is safer than EME. 3.8. Analysis of Eph in urine and plasma samples by HF-LPME and EME systems In order to study the influence of the biological fluids, both methods were applied to extraction and analysis of Eph in plasma and urine samples. Especially in the case of polar drugs, HF-LPME does not show noticeable extraction efficiency from plasma samples without sample preparation (i.e., precipitation of protein, etc.) because in HF-LPME, pH gradient does not have enough potential for extraction of analyte from complex matrices. Moreover it was reported that in EME system, electrical potential can act as a powerful force for breaking and reduction of analyte–protein binding [14]. It seems that true criteria for comparison of HF-LPME and EME cannot be obtained if EME and HF-LPME were carried out without and with precipitation of protein, respectively. Therefore, sample pretreatment was done on plasma sample for both methods. The preparation steps of real samples were performed according to Section 2.5. The results of real sample analysis are shown in Table 4, and indicate a satisfactory agreement among the obtained results with spiked values and also between two microextraction methods. Fig. 3 shows the chromatograms corresponding to (a) a nonspiked urine sample; (b) direct injection of Eph at concentration

Fig. 3. The chromatograms corresponding to (a) non-spiked urine sample; (b) direct injection of Eph at concentration of 500 ng mL−1 ; (c) extraction of a urine sample related to a volunteer who treated with Eph, using HF-LPME; and (d) using EME, respectively. The urine sample was taken from the volunteer after 10 h and 15 h for analyzing with EME and HF-LPME, respectively.

of 500 ng mL−1 ; (c) extraction of a urine sample related to a volunteer who treated with Eph, using HF-LPME; and (d) using EME, respectively.

Table 3 Comparison of the proposed methods with other reported methods for extraction and determination of Eph. Method/instrumentation

Matrix

LOD (ng mL−1 )

DLR (ng mL−1 )

R2

RSD%

Ref.

LLE/LC-UVa LPME/HPLC-UVb SPME/CE-DADc HS-SPME/GC-FIDd PMME/CE-UVe

Plasma Urine Urine Urine Urine Plasma Urine/serum Urine Urine Urine Plasma Urine Plasma

– 50.00 3.00 0.33 8.00 53.00 0.15–0.25 5.0 50 60.00 200.00 5.00 10.00

2–300 100–10000 20–5000 20–20000 50–5000 50–5000 5/10–200 10–50000 100–10000 100–3000 250–4000 15–750 30–1000

0.998 0.999 0.996 0.999 0.999 0.999 0.9988–0.9994 0.998 0.992 0.991 0.988 0.994 0.993

3.0 3.0 7.57 3.9 4.0 2.6 5.3–8.9 8.4 5 7.5 8.6 5.2 7.3

[9] [26] [28] [29] [30]

CME/OLBE-FASIf LLLME/HPLC-UVg SPME-IMSh HF-LPME/HPLC-UV EME/HPLC-UV a b c d e f g h

Liquid–liquid extraction–liquid chromatography ultraviolet detection. Liquid-phase microextraction. Solid-phase microextraction–capillary electrophoresis-photo diode array. Headspace solid-phase microextraction–gas chromatography flame ionization. Poly monolith microextraction. Centrifuge microextraction and on-line backextraction field-amplified sample injection. The reported data have been achieved in buffer samples. Liquid–liquid–liquid microextraction. Ion mobility spectrometry.

[31] [32] [33] This work This work

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4. Conclusions The aim of the present work was to compare the extraction of Eph as a polar drug from biological samples, across a supported liquid membrane based on (1) passive diffusion in a pH gradient sustained over the SLM (HF-LPME), and (2) electrokinetic migration in an electrical field sustained over the SLM (EME). The proposed methods were successfully developed for the extraction and analysis of Eph from plasma and urine. These techniques demonstrated several advantages over the other extraction methods especially high sample clean-up. As compared to HF-LPME, EME provides high preconcentration factor within short extraction times from biological samples. Accordingly, EME can be an efficient method to extract and preconcentrate ephedrine from complicated matrices in comparison with HF-LPME. Acknowledgments The authors wish to thank for the partial financial support from the Research Council of Alzahra University. The authors also gratefully acknowledge Women Research Center of Department of Biomedical Science for their cooperation. References [1] W.A. Konig, K. Ernst, J. Chromatogr. 280 (1983) 135. [2] P.J.V. Merwe, S.E. Hendrikz, J. Chromatogr. B 663 (1995) 160. [3] J.E.F. Reynolds, Martindale, The Extra Pharmacopoeia, 29th ed., Pharmaceutical Press, London, 1989, pp. 1462–1526. [4] International Olympic Committee Medical Code and Explanatory Document, International Olympic Committee, Lausanne, 2003. [5] S.M. Wang, R.J. Lewis, D. Canfield, T.L. Li, C.Y. Chen, R.H. Liu, J. Chromatogr. B 825 (2005) 88. [6] N. Okamura, H. Miki, T. Harada, S. Yamashita, Y. Masaoka, Y. Nakamoto, M. Tsuguma, H. Yoshitomi, A. Yagi, J. Pharm. Biomed. Anal. 20 (1999) 363.

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