2018 Homayon Ahmad Panahi Monireh Chabouk Maryam Ejlali Department of Chemistry, Central Tehran Branch, Islamic Azad University, Tehran, Iran Received February 17, 2014 Revised April 22, 2014 Accepted May 3, 2014
J. Sep. Sci. 2014, 37, 2018–2024
Research Article
Hollow-fiber-supported liquid membrane microextraction of amlodipine and atorvastatin A simple, environmentally friendly, and efficient method, based on hollow-fiber-supported liquid membrane microextraction, followed by high-performance liquid chromatography has been developed for the extraction and determination of amlodipine (AML) and atorvastatin (ATO) in water and urine samples. The AML in two-phase hollow-fiber liquid microextraction is extracted from 24.0 mL of the aqueous sample into an organic phase with microliter volume located inside the pores and lumen of a polypropylene hollow fiber as acceptor phase, but the ATO in three-phase hollow-fiber liquid microextraction is extracted from aqueous donor phase to organic phase and then back-extracted to the aqueous acceptor phase, which can be directly injected into the high-performance liquid chromatograph for analysis. The preconcentration factors in a range of 34–135 were obtained under the optimum conditions. The calibration curves were linear (R2 ࣙ 0.990) in the concentration range of 2.0–200 g/L for AML and 5.0–200 g/L for ATO. The limits of detection for AML and ATO were 0.5 and 2.0 g/L, respectively. Tap water and human urine samples were successfully analyzed for the existence of AML and ATO using the proposed methods. Keywords: Amlodipine / Atorvastatin / High-performance liquid chromatography / Hollow fibers / Liquid membrane microextraction DOI 10.1002/jssc.201400138
1 Introduction Atorvastatin (ATO) calcium is chemically described as [R(R*,R*)]-2-(4-fluorophenyl)-,␦-dihydroxy-5-(1-methylethyl)3-phenyl-4-[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid [1]. ATO is a selective, competitive inhibitor of HMG-CoA reductase, which has been used for increasing high-density lipoprotein cholesterol in the treatment of hyperlipidaemias [2]. Amostatine (amlodipine (AML) besylate and ATO calcium) tablets are intended for oral administration and are available in several different strength combinations. It is, therefore, necessary to develop precise, specific, and sensitive techniques for the rapid detection and identification of AML and ATO in the water and urine samples. A literature survey revealed that extractive spectrophotometry, LC, GC–MS, LC–MS, LC–ESI-MS/MS, and high-performance thin-layer chromatography (HPTLC) [3–9] methods have been reported for the estimation of ATO. Different LC methods have been reported for the estimation of AML. For the estimation of AML and ATO combination, Correspondence: Dr. Homayon Ahmad Panahi, Department of Chemistry, Islamic Azad University, Central Tehran Branch, Tehran, Iran. E-mail: [email protected] Fax: +98-21-44164539
Abbreviations: AML, amlodipine; ATO, atorvastatin; HPTLC, high-performance thin-layer chromatography; PF, preconcentration factor C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
spectrophotometric, HPTLC, and LC methods have been reported [10–12]. Sample preparation is still a bottleneck for overall throughput because the steps involved often employ large volumes of hazardous organic solvents, are time consuming, and/or expensive. Besides, there might also be the problem of contamination and sample loss [13, 14]. Recently, LPME was developed as a novel and disposable method for sample preparation [15–20]. LPME is a solvent-minimized sample preparation procedure, in which only several microliters of solvent is required to concentrate analytes from various samples and also is compatible with GC, CE, and HPLC. The objective of this work was to present the application of two-phase and three-phase hollow-fiber LPME (HF-LPME) for preconcentration of AML and ATO in urine samples, respectively. Optimum conditions for this method were examined. The feasibility of this methodology is also estimated by determining the preconcentration factor (PF), linearity, detection limit and recovery. Overall, this method proves to be an efficient alternative to other procedures having the advantages of being simple, cheap, sensitive, fast, and requiring little solvent. As the cost of analysis per sample is low, the hollow fiber can be disposed of after a single extraction, thus avoiding the possibility of carryover between analyses.
Colour Online: See the article online to view Figs. 1 and 2 in colour. www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2014, 37, 2018–2024
2019
Table 1. The chemical structure, protein binding, logP, and pKa for AML and ATO
Analyte
Chemical structure
Protein binding (%)
log P
pKa
AML
93
3.0
0.4, 9.0
ATO
>98
6.3
4.5, 11.8
2 Materials and methods 2.1 Chemicals and reagents The Q3/2 Accurel polypropylene microporous HF membrane (200 m wall thickness, 600 m inner diameter, 0.2 m pore size, 75% porosity) was obtained from Membrana (Wuppertal, Germany). AML and ATO were purchased from Aldrich (Milwaukee, WI, USA). The protein binding, logP (the octanol/water distribution constant) and pKa (acidic equilibrium constant) for these drugs are shown in Table 1. 1Octanol, 1-decanol, n-dodecane, toluene, and sodium chloride (NaCl) were of the highest purity available from Merck (Darmstadt, Germany). HPLC-grade methanol and acetonitrile were purchased from Caledon (Ontario, Canada). The ultrapure water was prepared by a model Aqua Max-Ultra Youngling ultrapure water purification system (Dongan-gu, South Korea). Stock standard solution of each analyte with concentration of 1000 mg/L was prepared separately in methanol and stored at 4⬚C. Working standard solutions with different concentrations were prepared daily by dilution of stock solutions with ultrapure water. Analytes at spiking levels were used for the optimization study. Standard solution containing 100 g/L of the analytes was used in the optimization experiments.
2.2 Apparatus Chromatographic separations were carried out on a Shimadzu equipped with LC-20AB pump, a 20 L sample loop, and a UV-Vis detector. A Shimpack column (250 × 4.6 mm, with 5 m particle size) was applied to separate the analytes under isocratic elution condition. A mixture of 20 mmol/L phosphate buffer (pH 4.0) and acetonitrile (45:55) at a flow rate of 1.0 mL/min was used as a mobile phase and the analytes were detected at 240 nm. Although HPLC conditions C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Schematic design of the extraction procedure.
for both of two-phase and three-phase LPME are similar, in the case of AML, 100% acetonitrile was used for 10 min to elute the extraction solvent (1-octanol).
2.3 Two-phase HF-LPME procedure Extractions were performed according to the following procedure: a 24.0 mL aliquot of the sample solution was added to the vial, and a magnetic bar was placed into the solution to ensure efficient stirring during the extraction (Fig. 1). The hollow fiber was sonicated for 5 min in acetone to remove any contaminants in the fiber. It was removed from acetone, and the solvent was allowed to evaporate completely. Before extraction, the syringe was rinsed with acetone followed by 1-octanol to avoid carryover and air-bubble formation. About 25 L of the solvent was withdrawn into the syringe. A piece of the hollow fiber (8.5 cm length) was fixed onto the tip of the syringe needle and the assembly was immersed in extraction phase for 2 min to impregnate the pores of the fiber wall. The fiber was inserted into ultrapure water for 10 s to wash extra 1-octanol from the surface of the hollow fiber. Then, www.jss-journal.com
2020
H. A. Panahi et al.
J. Sep. Sci. 2014, 37, 2018–2024
Figure 2. Ionization plots of (A) AML and (B) ATO.
the syringe plunger was depressed to fill the hollow fiber with the extraction phase. Finally, the end of the hollow fiber was sealed by a piece of aluminum foil. The assembly was then directly immersed into the sample. For each extraction, a new hollow fiber was used to avoid any possible memory effects. The extraction was carried out during the prescribed time. After extraction, the organic phase was retracted into the syringe and injected into the 20 L loop of the HPLC.
2.4 Three-phase HF-LPME procedure The hollow fiber was cut manually in 8.5 cm pieces, cleaned in acetone in an ultrasonic bath to remove any contaminants, and dried in air for use. Then, 24.0 mL of the aqueous sample containing the analytes was poured into a 25 mL glass vial. The sample vial was placed on a magnetic heater-stirrer (Heidolph MR 3001K, Germany) and a 50 mL Hamilton syringe (Bondaduz, Switzerland) was applied to introduce the acceptor phase into the hollow fiber. Before extraction, the microsyringe was rinsed ten times with methanol to avoid carryover and air bubble formation. Twenty five microliters of the receiving phase (pH 11.0) was withdrawn into the microsyringe. Then, the microsyringe needle tip was inserted into the hollow fiber and the assembly was immersed in 1-octanol for about 10 s to impregnate the pores of the hollow fiber. The fiber was inserted into the ultrapure water for 10 s to wash extra organic solvent from the surface of the hollow fiber. Then, the acceptor phase was introduced into the hollow fiber with slow pushing of the microsyringe plunger. Finally, the end of the hollow fiber was sealed by a piece of aluminum foil. The assembly was then directly immersed into the sample. For each extraction, a new configuration was used to avoid any possible memory effects. The extraction was carried out during the prescribed period of time. After extraction, the acceptor phase solution was retracted into the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
syringe and injected into the 20 L loop of the HPLC system followed by analysis.
3 Results and discussion Generally, sample solution pH determines the state of analytes in aqueous solution, which plays an important role in extraction of drugs from water samples. The presence of acidic and basic groups could result in a number of ionic states for each compound at various pH values. In order to facilitate the comparison and investigation of the extraction efficiencies, ionization plots of compounds (AML and ATO) were illustrated in Fig. 2 [http://chemicalize.org]. These ionization plots show the relationship between pH and the relative quantities of each species of the compounds. Thus, theoretically AML (Fig. 2A) can be considered as a dication at pH < 6.0, as a cation at 12.0 > pH > 2.0 and in the neutral form at pH > 8.0. In fact, AML has three different forms and the percentage of each form is affected by pH (two cation forms and one neutral form). In the structure of ATO, three ionizable functions are present, and it can be considered as a dianion at pH > 10.0, an anion at pH range of 2.0–14.0, and a neutral compound at pH < 6.0 (Fig. 2B). In other words, AML is a basic compound but ATO is an acidic compound. Therefore, possibility of AML and ATO extraction into organic solvent is maximal in alkalized and acidified solution, respectively. Preliminary experiments strongly recommended that the two-phase and three-phase HF-LPME were suitable for the extraction of AML and ATO, respectively.
3.1 Optimization of two-phase HF-LPME of AML The type of organic solvent immobilized in the pores of the hollow fiber is an essential consideration for efficient analyte preconcentration. As in LLE, the principle “like dissolves www.jss-journal.com
J. Sep. Sci. 2014, 37, 2018–2024
Figure 3. Effect of organic solvent on the extraction efficiency of AML. Extraction conditions: Sample volume, 24.0 mL; pH of sample solution, 11.0; HF length, 8.5 cm; stirring rate, 1000 rpm; extraction time, 45 min; sample solution, 100 g/L.
like” is applied in LPME. The solvent should be of low volatility to prevent evaporation, low viscosity to ensure rapid mass transfer, low polarity to ensure compatibility with the hollow fiber, and to prevent leakage into the sample. In addition, the solvent should provide high distribution constants for the target analytes. Based on the above four considerations, four organic solvents (1-octanol, 1-decanol, n-dodecane, and toluene) were investigated for use in HF-LPME. HPLC peak areas of the drug were evaluated for 45 min extraction period from 24.0 mL of ultrapure water samples spiked at 100 g/L of AML and stirred at 1000 rpm. The highest extraction recovery was obtained with 1-octanol (Fig. 3). No enrichment effect of the analyte was observed, especially with nonpolar solvent (toluene and n-dodecane). Therefore, 1-octanol was selected as the immobilization solvent for further optimization. For two-phase HF-LPME, the pH value of sample solution (donor phase) is important for extraction efficiency. It can change the partition coefficient of analyte between the sample solution and extraction solvent. Five pH values (from 9.0 to 13.0) were investigated to study their influence on the extraction efficiency. The extraction efficiency is the highest when the pH value is 10.0. Thus, pH 10.0 was selected as optimum for donor phase. The results can be explained by the principle that, the higher the pH value, the more inhibited the ionization of the AML. The compound has two ammonium groups in lower pH (pKa values: 9.0 and 0.4). Thus, they are in a less ionized condition at the higher pH than at a lower pH (Fig. 2A). At high pH value, all drugs will be in the neutral form, which facilitates the extraction from donor phase. Therefore, low pH value benefits to extract four drugs in sample to the organic solvent immersed in the hollow fiber membrane. The effect of salt addition on the extraction efficiency of drug by the HF-LPME method was examined by adding NaCl to 24.0 mL aqueous samples at 0, 0.2, 0.5, 1.0, and 2.0 mol/L. The addition of salt to the sample will lead to a higher ionic strength in the sample and decrease the solubility of drug in the aqueous solution. On the other hand, electrostatic in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample Preparation
2021
teraction will resist organic solvent extraction. Therefore, the effect of salt on extraction is indefinite. The analytical signal of the analyte was increased by increasing the concentration of NaCl up to 0.2 mol/L. Accordingly, the extractions of AML were carried out from the samples containing 0.2 mol/L NaCl. Sample agitation is also extremely important in enhancing extraction recovery and reducing extraction time. Agitation enables continuous exposure of the extraction surface to fresh aqueous sample. Different stirring speeds were tested to determine the optimum stirring speed for the extraction. The experiments were carried out at stirring speeds ranging from 500 to 1200 rpm. The results showed that extraction recovery reaches a maximum at 1000 rpm. Stirring speeds exceeding 1200 rpm were not evaluated because of excessive air bubbles on the surface of the hollow fiber, which could lead to poorer precision and possible experimental failure. So experiments were performed with a stirring speed of 1000 rpm. When organic solvent, salt effect, and agitation rate in HF-LPME had been fixed, the effect of extraction time on HF-LPME extraction efficiency should be determined. However, it is not usually practical to increase extraction time until equilibrium is established, because the longer the extraction time, the greater the chance of solvent loss, due to dissolution in the sample solution and formation of air bubbles. In this work, the effect of extraction time was investigated by conducting experiments for 15, 30, 45, 60, and 90 min at a stirring speed of 1000 rpm. The results showed that during the first 60 min, the extraction recovery of the analyte increased with the increased extraction time. However, when the extraction time was >60 min, the extraction recovery decreased. So an extraction time of 60 min was selected as the working optimum. In this study, to ensure complete extraction a two- and three-phase HF-LPME procedure was used for both AML and ATO. Since poor recovery was obtained for ATO in two-phase HF-LPME and AML in three-phase HF-LPME; thus, the twoand three-phase extractions were performed in order to better recovery for AML and ATO, respectively.
3.2 Optimization of three-phase HF-LPME of ATO The pH of donor and acceptor phases plays an important role in HF-LPME. ATO is an acidic drug (pKa : 4.5 and 11.8). Depending on the solution pH, it can exist at various forms and its ionization is controlled by the solution pH (Fig. 2B). According to the pKa values, the donor phase pH should be adjusted to keep the analyte in its neutral form and reduce its solubility in the source phase. Extraction of the analyte was performed from the donor phase containing HCl to adjust acidity (pH 1.0–5.0). The highest efficiency was obtained at pH 4.0. In a more acidic solution, a decrease in the PF was observed. This can be attributed to protonation of the basic functional group of the ATO. Therefore, pH 4.0 was used for subsequent experiments. www.jss-journal.com
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Table 2. Figures of merit for HF-LPME of the target drugs.
Analyte
DLR (g/L)
LOD (g/L)
R2
PF
RSD% (n = 4)a)
RSD% (n = 4)b)
AML ATO
2.0–200 5.0–200
0.5 2.0
0.990 0.997
135 34
5.7 6.5
8.4 10.0
analytes at 14 different concentrations ranging from 2.0 to 200 and 5.0 to 200 g/L for AML and ATO, respectively. The correlation coefficients (R2 ) of the calibration curves were between 0.990 and 0.997 and the LODs for the analytes based on a S/N of 3 varied in the range of 0.5–2.0 g/L. Intraday precision was obtained from five consecutive replicates and expressed as RSDs%. The RSDs% obtained for AML and ATO were 5.7 and 6.5% and interday RSDs% obtained at five different days were 8.4 and 10.0%. The PF was defined as the ratio of the final analyte concentration in the acceptor phase (Cf,ac ) and the initial concentration of analyte (Ci,s ) within the sample solution:
(DLR, dynamic linear range). a) Intraday RSD was calculated based on extraction of 20 g/L of drugs. b) Interday RSD was calculated based on extraction of 20 g/L of drugs.
Strong alkaline aqueous solution is beneficial for the back-extraction of acidic ATO in HF-LPME. So NaOH was selected as the acceptor phase, and the influence of its concentration on the extraction efficiency of target analyte was studied within the range of 0–1.0 mol/L. The results indicated that the extraction efficiency of target analyte increased rapidly with the increase of the NaOH concentration from 0 to 0.001 mol/L−1 , and then kept almost constant by further increasing the NaOH concentration to 0.10 mol/L. Considering that a relative high concentration of NaOH solution would destroy ODS column, the concentration of NaOH was chosen as 0.001 mol/L (pH 11.0). Notably, during optimization of other parameters for three-phase HF-LPME microextraction of ATO (extraction solvent, ionic strength, extraction time, and stirring rate), similar trends to AML were obtained as optimized conditions. According to the overall results of the optimization study, the following experimental conditions were chosen as optimum conditions for ATO: extraction solvent, 1-octanol; NaOH concentration in acceptor phase, 0.001 mol/L (pH 11.0); donor phase pH, 4.0; NaCl concentration, 0.2 mol/L; extraction time, 60 min; and stirring rate, 1000 rpm.
PF =
C f ,ac C i,s
(1)
where Cf,ac was calculated from a calibration graph obtained from direct injection of analyte standard solutions (0.1– 10 mg/L). The obtained PFs were in the range of 34–135. A comparison between the figures of merit of the proposed method and some published methods for extraction and determination of AML and ATO is summarized in Table 3. Clearly, the proposed method has a good sensitivity and precision with a suitable dynamic linear range. Also, the obtained LODs for the analytes by the present method with sample volume of 24 mL are comparable to those obtained by other methods. All of the results reveal that the new developed method is not only a good sample preconcentration technique, but also an excellent sample cleanup procedure that can be used for ultratrace analysis of AML and ATO in biological samples.
3.4 Analysis of real samples The proposed method was also applied to determination of the AML and ATO in tap water and human urine samples. Human urine samples for obtaining calibration curve and figures of merit were collected from healthy male volunteers. The samples were centrifuged for 5 min at 4000 rpm and filtered through a 0.45 m pore size cellulose acetate filter from Millipore (Madrid, Spain). The filtrates were collected in glass containers, which had been carefully cleaned with hydrochloric acid. They were then washed with deionized water and stored at 4⬚C to prevent bacterial growth and proteolysis. Twelve milliliters of this urine sample was spiked by
3.3 Quantitative analysis In order to evaluate the figures of merit of the two- and threephase HF-LPME techniques, linearity, LOD, and repeatability were investigated under optimized conditions using standard solutions of the analytes. The performance of the developed procedure is summarized in Table 2. The linearity of the method was evaluated using urine samples spiked with the
Table 3. Comparison of the proposed method with other developed methods for the determination of target drugs
Analyte
Sample
Instrumentation
LOD (mg/L)
DLR (mg/L)
RSD (%)
Ref.
AML AML ATO ATO AML, ATO
Urine Plasma Plasma Plasma Water, urine
CE HPLC–UV UHPLC–MS/MS HPLC–UV HPLC–UV
3.0 200 0.03 nM 0.4 0.5−20
100−500 500−16 000 0.1−100 nM 1.0−500 2.0−200
14 10 5
J. Sep. Sci. 2014, 37, 2018–2024
Research Article
Hollow-fiber-supported liquid membrane microextraction of amlodipine and atorvastatin A simple, environmentally friendly, and efficient method, based on hollow-fiber-supported liquid membrane microextraction, followed by high-performance liquid chromatography has been developed for the extraction and determination of amlodipine (AML) and atorvastatin (ATO) in water and urine samples. The AML in two-phase hollow-fiber liquid microextraction is extracted from 24.0 mL of the aqueous sample into an organic phase with microliter volume located inside the pores and lumen of a polypropylene hollow fiber as acceptor phase, but the ATO in three-phase hollow-fiber liquid microextraction is extracted from aqueous donor phase to organic phase and then back-extracted to the aqueous acceptor phase, which can be directly injected into the high-performance liquid chromatograph for analysis. The preconcentration factors in a range of 34–135 were obtained under the optimum conditions. The calibration curves were linear (R2 ࣙ 0.990) in the concentration range of 2.0–200 g/L for AML and 5.0–200 g/L for ATO. The limits of detection for AML and ATO were 0.5 and 2.0 g/L, respectively. Tap water and human urine samples were successfully analyzed for the existence of AML and ATO using the proposed methods. Keywords: Amlodipine / Atorvastatin / High-performance liquid chromatography / Hollow fibers / Liquid membrane microextraction DOI 10.1002/jssc.201400138
1 Introduction Atorvastatin (ATO) calcium is chemically described as [R(R*,R*)]-2-(4-fluorophenyl)-,␦-dihydroxy-5-(1-methylethyl)3-phenyl-4-[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid [1]. ATO is a selective, competitive inhibitor of HMG-CoA reductase, which has been used for increasing high-density lipoprotein cholesterol in the treatment of hyperlipidaemias [2]. Amostatine (amlodipine (AML) besylate and ATO calcium) tablets are intended for oral administration and are available in several different strength combinations. It is, therefore, necessary to develop precise, specific, and sensitive techniques for the rapid detection and identification of AML and ATO in the water and urine samples. A literature survey revealed that extractive spectrophotometry, LC, GC–MS, LC–MS, LC–ESI-MS/MS, and high-performance thin-layer chromatography (HPTLC) [3–9] methods have been reported for the estimation of ATO. Different LC methods have been reported for the estimation of AML. For the estimation of AML and ATO combination, Correspondence: Dr. Homayon Ahmad Panahi, Department of Chemistry, Islamic Azad University, Central Tehran Branch, Tehran, Iran. E-mail: [email protected] Fax: +98-21-44164539
Abbreviations: AML, amlodipine; ATO, atorvastatin; HPTLC, high-performance thin-layer chromatography; PF, preconcentration factor C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
spectrophotometric, HPTLC, and LC methods have been reported [10–12]. Sample preparation is still a bottleneck for overall throughput because the steps involved often employ large volumes of hazardous organic solvents, are time consuming, and/or expensive. Besides, there might also be the problem of contamination and sample loss [13, 14]. Recently, LPME was developed as a novel and disposable method for sample preparation [15–20]. LPME is a solvent-minimized sample preparation procedure, in which only several microliters of solvent is required to concentrate analytes from various samples and also is compatible with GC, CE, and HPLC. The objective of this work was to present the application of two-phase and three-phase hollow-fiber LPME (HF-LPME) for preconcentration of AML and ATO in urine samples, respectively. Optimum conditions for this method were examined. The feasibility of this methodology is also estimated by determining the preconcentration factor (PF), linearity, detection limit and recovery. Overall, this method proves to be an efficient alternative to other procedures having the advantages of being simple, cheap, sensitive, fast, and requiring little solvent. As the cost of analysis per sample is low, the hollow fiber can be disposed of after a single extraction, thus avoiding the possibility of carryover between analyses.
Colour Online: See the article online to view Figs. 1 and 2 in colour. www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2014, 37, 2018–2024
2019
Table 1. The chemical structure, protein binding, logP, and pKa for AML and ATO
Analyte
Chemical structure
Protein binding (%)
log P
pKa
AML
93
3.0
0.4, 9.0
ATO
>98
6.3
4.5, 11.8
2 Materials and methods 2.1 Chemicals and reagents The Q3/2 Accurel polypropylene microporous HF membrane (200 m wall thickness, 600 m inner diameter, 0.2 m pore size, 75% porosity) was obtained from Membrana (Wuppertal, Germany). AML and ATO were purchased from Aldrich (Milwaukee, WI, USA). The protein binding, logP (the octanol/water distribution constant) and pKa (acidic equilibrium constant) for these drugs are shown in Table 1. 1Octanol, 1-decanol, n-dodecane, toluene, and sodium chloride (NaCl) were of the highest purity available from Merck (Darmstadt, Germany). HPLC-grade methanol and acetonitrile were purchased from Caledon (Ontario, Canada). The ultrapure water was prepared by a model Aqua Max-Ultra Youngling ultrapure water purification system (Dongan-gu, South Korea). Stock standard solution of each analyte with concentration of 1000 mg/L was prepared separately in methanol and stored at 4⬚C. Working standard solutions with different concentrations were prepared daily by dilution of stock solutions with ultrapure water. Analytes at spiking levels were used for the optimization study. Standard solution containing 100 g/L of the analytes was used in the optimization experiments.
2.2 Apparatus Chromatographic separations were carried out on a Shimadzu equipped with LC-20AB pump, a 20 L sample loop, and a UV-Vis detector. A Shimpack column (250 × 4.6 mm, with 5 m particle size) was applied to separate the analytes under isocratic elution condition. A mixture of 20 mmol/L phosphate buffer (pH 4.0) and acetonitrile (45:55) at a flow rate of 1.0 mL/min was used as a mobile phase and the analytes were detected at 240 nm. Although HPLC conditions C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Schematic design of the extraction procedure.
for both of two-phase and three-phase LPME are similar, in the case of AML, 100% acetonitrile was used for 10 min to elute the extraction solvent (1-octanol).
2.3 Two-phase HF-LPME procedure Extractions were performed according to the following procedure: a 24.0 mL aliquot of the sample solution was added to the vial, and a magnetic bar was placed into the solution to ensure efficient stirring during the extraction (Fig. 1). The hollow fiber was sonicated for 5 min in acetone to remove any contaminants in the fiber. It was removed from acetone, and the solvent was allowed to evaporate completely. Before extraction, the syringe was rinsed with acetone followed by 1-octanol to avoid carryover and air-bubble formation. About 25 L of the solvent was withdrawn into the syringe. A piece of the hollow fiber (8.5 cm length) was fixed onto the tip of the syringe needle and the assembly was immersed in extraction phase for 2 min to impregnate the pores of the fiber wall. The fiber was inserted into ultrapure water for 10 s to wash extra 1-octanol from the surface of the hollow fiber. Then, www.jss-journal.com
2020
H. A. Panahi et al.
J. Sep. Sci. 2014, 37, 2018–2024
Figure 2. Ionization plots of (A) AML and (B) ATO.
the syringe plunger was depressed to fill the hollow fiber with the extraction phase. Finally, the end of the hollow fiber was sealed by a piece of aluminum foil. The assembly was then directly immersed into the sample. For each extraction, a new hollow fiber was used to avoid any possible memory effects. The extraction was carried out during the prescribed time. After extraction, the organic phase was retracted into the syringe and injected into the 20 L loop of the HPLC.
2.4 Three-phase HF-LPME procedure The hollow fiber was cut manually in 8.5 cm pieces, cleaned in acetone in an ultrasonic bath to remove any contaminants, and dried in air for use. Then, 24.0 mL of the aqueous sample containing the analytes was poured into a 25 mL glass vial. The sample vial was placed on a magnetic heater-stirrer (Heidolph MR 3001K, Germany) and a 50 mL Hamilton syringe (Bondaduz, Switzerland) was applied to introduce the acceptor phase into the hollow fiber. Before extraction, the microsyringe was rinsed ten times with methanol to avoid carryover and air bubble formation. Twenty five microliters of the receiving phase (pH 11.0) was withdrawn into the microsyringe. Then, the microsyringe needle tip was inserted into the hollow fiber and the assembly was immersed in 1-octanol for about 10 s to impregnate the pores of the hollow fiber. The fiber was inserted into the ultrapure water for 10 s to wash extra organic solvent from the surface of the hollow fiber. Then, the acceptor phase was introduced into the hollow fiber with slow pushing of the microsyringe plunger. Finally, the end of the hollow fiber was sealed by a piece of aluminum foil. The assembly was then directly immersed into the sample. For each extraction, a new configuration was used to avoid any possible memory effects. The extraction was carried out during the prescribed period of time. After extraction, the acceptor phase solution was retracted into the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
syringe and injected into the 20 L loop of the HPLC system followed by analysis.
3 Results and discussion Generally, sample solution pH determines the state of analytes in aqueous solution, which plays an important role in extraction of drugs from water samples. The presence of acidic and basic groups could result in a number of ionic states for each compound at various pH values. In order to facilitate the comparison and investigation of the extraction efficiencies, ionization plots of compounds (AML and ATO) were illustrated in Fig. 2 [http://chemicalize.org]. These ionization plots show the relationship between pH and the relative quantities of each species of the compounds. Thus, theoretically AML (Fig. 2A) can be considered as a dication at pH < 6.0, as a cation at 12.0 > pH > 2.0 and in the neutral form at pH > 8.0. In fact, AML has three different forms and the percentage of each form is affected by pH (two cation forms and one neutral form). In the structure of ATO, three ionizable functions are present, and it can be considered as a dianion at pH > 10.0, an anion at pH range of 2.0–14.0, and a neutral compound at pH < 6.0 (Fig. 2B). In other words, AML is a basic compound but ATO is an acidic compound. Therefore, possibility of AML and ATO extraction into organic solvent is maximal in alkalized and acidified solution, respectively. Preliminary experiments strongly recommended that the two-phase and three-phase HF-LPME were suitable for the extraction of AML and ATO, respectively.
3.1 Optimization of two-phase HF-LPME of AML The type of organic solvent immobilized in the pores of the hollow fiber is an essential consideration for efficient analyte preconcentration. As in LLE, the principle “like dissolves www.jss-journal.com
J. Sep. Sci. 2014, 37, 2018–2024
Figure 3. Effect of organic solvent on the extraction efficiency of AML. Extraction conditions: Sample volume, 24.0 mL; pH of sample solution, 11.0; HF length, 8.5 cm; stirring rate, 1000 rpm; extraction time, 45 min; sample solution, 100 g/L.
like” is applied in LPME. The solvent should be of low volatility to prevent evaporation, low viscosity to ensure rapid mass transfer, low polarity to ensure compatibility with the hollow fiber, and to prevent leakage into the sample. In addition, the solvent should provide high distribution constants for the target analytes. Based on the above four considerations, four organic solvents (1-octanol, 1-decanol, n-dodecane, and toluene) were investigated for use in HF-LPME. HPLC peak areas of the drug were evaluated for 45 min extraction period from 24.0 mL of ultrapure water samples spiked at 100 g/L of AML and stirred at 1000 rpm. The highest extraction recovery was obtained with 1-octanol (Fig. 3). No enrichment effect of the analyte was observed, especially with nonpolar solvent (toluene and n-dodecane). Therefore, 1-octanol was selected as the immobilization solvent for further optimization. For two-phase HF-LPME, the pH value of sample solution (donor phase) is important for extraction efficiency. It can change the partition coefficient of analyte between the sample solution and extraction solvent. Five pH values (from 9.0 to 13.0) were investigated to study their influence on the extraction efficiency. The extraction efficiency is the highest when the pH value is 10.0. Thus, pH 10.0 was selected as optimum for donor phase. The results can be explained by the principle that, the higher the pH value, the more inhibited the ionization of the AML. The compound has two ammonium groups in lower pH (pKa values: 9.0 and 0.4). Thus, they are in a less ionized condition at the higher pH than at a lower pH (Fig. 2A). At high pH value, all drugs will be in the neutral form, which facilitates the extraction from donor phase. Therefore, low pH value benefits to extract four drugs in sample to the organic solvent immersed in the hollow fiber membrane. The effect of salt addition on the extraction efficiency of drug by the HF-LPME method was examined by adding NaCl to 24.0 mL aqueous samples at 0, 0.2, 0.5, 1.0, and 2.0 mol/L. The addition of salt to the sample will lead to a higher ionic strength in the sample and decrease the solubility of drug in the aqueous solution. On the other hand, electrostatic in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample Preparation
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teraction will resist organic solvent extraction. Therefore, the effect of salt on extraction is indefinite. The analytical signal of the analyte was increased by increasing the concentration of NaCl up to 0.2 mol/L. Accordingly, the extractions of AML were carried out from the samples containing 0.2 mol/L NaCl. Sample agitation is also extremely important in enhancing extraction recovery and reducing extraction time. Agitation enables continuous exposure of the extraction surface to fresh aqueous sample. Different stirring speeds were tested to determine the optimum stirring speed for the extraction. The experiments were carried out at stirring speeds ranging from 500 to 1200 rpm. The results showed that extraction recovery reaches a maximum at 1000 rpm. Stirring speeds exceeding 1200 rpm were not evaluated because of excessive air bubbles on the surface of the hollow fiber, which could lead to poorer precision and possible experimental failure. So experiments were performed with a stirring speed of 1000 rpm. When organic solvent, salt effect, and agitation rate in HF-LPME had been fixed, the effect of extraction time on HF-LPME extraction efficiency should be determined. However, it is not usually practical to increase extraction time until equilibrium is established, because the longer the extraction time, the greater the chance of solvent loss, due to dissolution in the sample solution and formation of air bubbles. In this work, the effect of extraction time was investigated by conducting experiments for 15, 30, 45, 60, and 90 min at a stirring speed of 1000 rpm. The results showed that during the first 60 min, the extraction recovery of the analyte increased with the increased extraction time. However, when the extraction time was >60 min, the extraction recovery decreased. So an extraction time of 60 min was selected as the working optimum. In this study, to ensure complete extraction a two- and three-phase HF-LPME procedure was used for both AML and ATO. Since poor recovery was obtained for ATO in two-phase HF-LPME and AML in three-phase HF-LPME; thus, the twoand three-phase extractions were performed in order to better recovery for AML and ATO, respectively.
3.2 Optimization of three-phase HF-LPME of ATO The pH of donor and acceptor phases plays an important role in HF-LPME. ATO is an acidic drug (pKa : 4.5 and 11.8). Depending on the solution pH, it can exist at various forms and its ionization is controlled by the solution pH (Fig. 2B). According to the pKa values, the donor phase pH should be adjusted to keep the analyte in its neutral form and reduce its solubility in the source phase. Extraction of the analyte was performed from the donor phase containing HCl to adjust acidity (pH 1.0–5.0). The highest efficiency was obtained at pH 4.0. In a more acidic solution, a decrease in the PF was observed. This can be attributed to protonation of the basic functional group of the ATO. Therefore, pH 4.0 was used for subsequent experiments. www.jss-journal.com
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J. Sep. Sci. 2014, 37, 2018–2024
H. A. Panahi et al.
Table 2. Figures of merit for HF-LPME of the target drugs.
Analyte
DLR (g/L)
LOD (g/L)
R2
PF
RSD% (n = 4)a)
RSD% (n = 4)b)
AML ATO
2.0–200 5.0–200
0.5 2.0
0.990 0.997
135 34
5.7 6.5
8.4 10.0
analytes at 14 different concentrations ranging from 2.0 to 200 and 5.0 to 200 g/L for AML and ATO, respectively. The correlation coefficients (R2 ) of the calibration curves were between 0.990 and 0.997 and the LODs for the analytes based on a S/N of 3 varied in the range of 0.5–2.0 g/L. Intraday precision was obtained from five consecutive replicates and expressed as RSDs%. The RSDs% obtained for AML and ATO were 5.7 and 6.5% and interday RSDs% obtained at five different days were 8.4 and 10.0%. The PF was defined as the ratio of the final analyte concentration in the acceptor phase (Cf,ac ) and the initial concentration of analyte (Ci,s ) within the sample solution:
(DLR, dynamic linear range). a) Intraday RSD was calculated based on extraction of 20 g/L of drugs. b) Interday RSD was calculated based on extraction of 20 g/L of drugs.
Strong alkaline aqueous solution is beneficial for the back-extraction of acidic ATO in HF-LPME. So NaOH was selected as the acceptor phase, and the influence of its concentration on the extraction efficiency of target analyte was studied within the range of 0–1.0 mol/L. The results indicated that the extraction efficiency of target analyte increased rapidly with the increase of the NaOH concentration from 0 to 0.001 mol/L−1 , and then kept almost constant by further increasing the NaOH concentration to 0.10 mol/L. Considering that a relative high concentration of NaOH solution would destroy ODS column, the concentration of NaOH was chosen as 0.001 mol/L (pH 11.0). Notably, during optimization of other parameters for three-phase HF-LPME microextraction of ATO (extraction solvent, ionic strength, extraction time, and stirring rate), similar trends to AML were obtained as optimized conditions. According to the overall results of the optimization study, the following experimental conditions were chosen as optimum conditions for ATO: extraction solvent, 1-octanol; NaOH concentration in acceptor phase, 0.001 mol/L (pH 11.0); donor phase pH, 4.0; NaCl concentration, 0.2 mol/L; extraction time, 60 min; and stirring rate, 1000 rpm.
PF =
C f ,ac C i,s
(1)
where Cf,ac was calculated from a calibration graph obtained from direct injection of analyte standard solutions (0.1– 10 mg/L). The obtained PFs were in the range of 34–135. A comparison between the figures of merit of the proposed method and some published methods for extraction and determination of AML and ATO is summarized in Table 3. Clearly, the proposed method has a good sensitivity and precision with a suitable dynamic linear range. Also, the obtained LODs for the analytes by the present method with sample volume of 24 mL are comparable to those obtained by other methods. All of the results reveal that the new developed method is not only a good sample preconcentration technique, but also an excellent sample cleanup procedure that can be used for ultratrace analysis of AML and ATO in biological samples.
3.4 Analysis of real samples The proposed method was also applied to determination of the AML and ATO in tap water and human urine samples. Human urine samples for obtaining calibration curve and figures of merit were collected from healthy male volunteers. The samples were centrifuged for 5 min at 4000 rpm and filtered through a 0.45 m pore size cellulose acetate filter from Millipore (Madrid, Spain). The filtrates were collected in glass containers, which had been carefully cleaned with hydrochloric acid. They were then washed with deionized water and stored at 4⬚C to prevent bacterial growth and proteolysis. Twelve milliliters of this urine sample was spiked by
3.3 Quantitative analysis In order to evaluate the figures of merit of the two- and threephase HF-LPME techniques, linearity, LOD, and repeatability were investigated under optimized conditions using standard solutions of the analytes. The performance of the developed procedure is summarized in Table 2. The linearity of the method was evaluated using urine samples spiked with the
Table 3. Comparison of the proposed method with other developed methods for the determination of target drugs
Analyte
Sample
Instrumentation
LOD (mg/L)
DLR (mg/L)
RSD (%)
Ref.
AML AML ATO ATO AML, ATO
Urine Plasma Plasma Plasma Water, urine
CE HPLC–UV UHPLC–MS/MS HPLC–UV HPLC–UV
3.0 200 0.03 nM 0.4 0.5−20
100−500 500−16 000 0.1−100 nM 1.0−500 2.0−200
14 10 5
