ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2014, том 69, № 4, с. 395-399
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
DEVELOPMENT OF A DISPERSIVE LIQUID-LIQUID MICROEXTRACTION METHOD BASED ON SOLIDIFICATION OF A FLOATING ORGANIC DROP FOR THE DETERMINATION OF BETA-CAROTENE IN HUMAN SERUM
© 2014 A. Rahimi, P. Hashemi
Department of chemistry, Faculty of sciences, Lorestan university 68151-44316 Khoramabad, Iran Received 26.07.2011; in final form 09.01.2013
A dispersive liquid—liquid microextraction method based on solidification of a floating organic drop (DLLME-SFO) was developed for HPLC determination of P-carotene in human serum. A narrow-neck glass tube was used for simple and rapid collection of the solidified organic phase from aqueous surface after its centrifugation and cooling in a water bath. Acetone and 2-decanol were used as the disperser and organic phases, respectively. Effects of salt concentration and phase volumes on the extraction of the analyte were optimized using a central composite design method. During the optimization, a spectrophotometric method was used for determinations. Under the optimized conditions, an extraction recovery of 99.0 ± 2.4% was obtained for five replicated analyses of P-carotene with an enrichment factor of 40. A detection limit of 0.08 p.g/mL was achieved. The proposed method was successfully applied to the determination of P-carotene in human serum samples.
Keywords: dispersive liquid-liquid microextraction, solidification of organic drop, high performance liquid chromatography, beta-carotene, human plasma, central composite design optimization.
DOI: 10.7868/S0044450214040070
Carotenoids are important biological compounds, not only due to their provitamin A activity but also for preventing free radical formation, resulting from reactions involving singlet oxygen and reducing oxidative stress in the organism [1]. Plasma concentrations of carotenoids are associated with a lower risk of certain epithelial cancers [2]. Incorporating foods containing carotenoids into the diet may help reduce the risk of developing colon cancer [3]. P-Carotene, which is a common carotenoid, is known as a powerful singlet oxygen scavenging antioxidant [4].
A number of different methods have been utilized for the analysis of carotenoids in plasma and serum [2, 5—7]. Among them, HPLC, spectrophotometry and colour evaluation techniques have been of more concern [8]. Determination of carotenoids in serum or plasma generally requires an extraction or pretreat-ment step [2, 7] such as liquid—liquid extraction, solid phase extraction or supercritical fluid extraction. Microextraction techniques have attracted much attention in recent years as alternatives for classic extraction procedures [9]. In recent years, Assadi and co-workers introduced a novel microextraction method called dispersive liquid—liquid microextraction (DLLME) [10]. DLLME is based on a ternary component solvent system containing an extraction solvent, an aqueous sol-
vent and a dispersive solvent that can dissolve in both phases to produce a cloudy solution. The extraction time of DLLME is shortened from several hours to a few minutes with low consumption of organic solvent. However, the extraction solvent used in DLLME is generally toxic and not environmentally friendly. More recently, DLLME was modified by its integrating with solidification of a floating organic drop (DLLME-SFO) [11, 12]. In this modified technique, the disadvantage of the ordinary DLLME is overcome by replacing a low-density and less-toxic solvent with the toxic chlorinated solvents [13]. In DLLME-SFO usually the melting point of the solvent is so that it may be easily solidified by cooling the mixture and easily scooped out.
In the present work, we combine the advantages of a narrow-neck glass tube (NNGT) [14] and a DLLME-SFO method to facilitate withdrawal of the floating organic solvent from the aqueous sample surface. The developed method is applied, for the first time, for cleanup and preconcentration of P-caro-tene from human serum samples after multivariate optimization of the extraction procedure by a response surface method [15].
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Scoop
The NNGT
Ice bath
Solidified phase
Aqueous phase
Fig. 1. The NNGT device inserted in an ice bath.
EXPERIMENTAL
Reagents and materials. P-Carotene was obtained from Merck (Darmstadt, Germany) with the highest available purity. HPLC grade acetonitrile and tetrahy-drofuran (THF), reagent grade 2-decanol, organic solvents and other chemicals were purchased from Merck (Darmstadt, Germany) and used as received. Double-distilled water was used throughout. A 50 mg/L solution of P-carotene in acetone was prepared and used as stock solution for the preparation of working standards.
Apparatus. Spectrophotometric analysis of the samples was performed by a Shimadzu (model UV-160) double-beam instrument. A pair of quartz 350 ^L micro-cells (ES-quartz, Model Q124, Spain) was used for the absorbance determinations of the test solutions against a blank solution. HPLC analysis of the samples was conducted on a Shimadzu (model L-10AD) instrument consisting of two reciprocating pumps, a DGU-14A in-line degasser, a model CT10-10AC oven, a high pressure manual injection valve (20 ^L injection loop) and a UV-Vis (model SPD-10A) detector. The software used for the data acquirement and processing was Class-vp v.R 6.1. The analytical column was a 25 cm x 4.6 mm i.d. RP-18 column (Shim-Pack CLC-C18) packed with 5 ^m particles and equipped with a 1 cm guard column (C18-B197) packed with 10 ^m particles of the same type. A 25 ^L HPLC micro-syringe (F-LC, SGE, Australia) was used for the sample withdrawal and injection.
A totally glass Fisons (UK) double distiller was used for preparation of doubly distilled water. For ultrasonic irradiation of the samples, an ultrasonic water bath (22 kHz, model 5RS, Sonica, Italy) was used. An Eppendorf AG centrifuge (model 5810, Hamburg) was used for centrifugation of the extracts.
For DLLME-SFO with lighter than water organic solvents, a homemade NNGT was used (Fig. 1) [14]. The glass tube was about 10 cm in length and 12 and 4 mm in diameter of the body and neck, respectively. The NNGT was placed inside a 15 mL polyethylene centrifuge tube for the centrifugation and phase separation.
Sample preparation. Human serum samples were obtained from four healthy high school students and stored at —20°C. Stock solutions of the analyte were prepared in acetone. Spiked serum samples were prepared by adding a few microliters of the analyte to 1.0 mL of centrifuged serum. For sample protein precipitation, 3 mL of acetone was mixed with each serum sample (1 mL) to precipitate out heavy proteins. The mixtures were vigorously shaken for 30 s and then centrifuged for 10 min at 4000 rpm in order to form a condensed sediment. After centrifuging, the samples were treated using DLLME-SFO. The analyte concentrations were between 1 and 5 mg/L.
Procedures. For performing the DLLME-SFO method, the ionic strength of the sample was adjusted to 0.2 M respecive to sodium nitrate. A mixture of 30 ^L 2-decanol as the extraction solvent and 1.2 mL acetone as the disperser solvent containing the analyte was rapidly injected into 4-mL aqueous sample in the NNGT device.
After formation of a cloudy solution, the mixture was immediately centrifuged at 4000 rpm for 5 min for the phase separation. Since 2-decanol is a lighter than water solvent, the extraction phase was collected at the solvent top. By careful addition of some water drops, the organic phase was conducted to the narrow neck of the NNGT. As shown in Fig. 1, the NNGT was cooled in an ice bath for a few minutes in order to solidify the organic phase. The solid organic drop was easily
DEVELOPMENT OF A DISPERSIVE LIQUID-LIQUID MICROEXTRACTION 397
Table 1. The factors included in the central composite design and the studied levels for each factor
Parameter Abbreviation Factor's levels
Extractant (2-decanol) volume (p.L) EV 20 25 32 40 45
Disperser (acetone) volume (mL) DV 0.8 1 1.2 1.5 1.7
Ionic strength (M) IS 0.04 0.1 0.2 0.3 0.35
scooped out by a narrow scoop and transferred into a micro-vial. The solid drop was quickly melted at room temperature and mixed with 200 ^L acetone by a syringe. For the analysis, either the whole mixture was transferred to a quartz micro-cell for spectrophotometry determination, or 25 ^L of it was injected into the HPLC column.
For the HPLC separation, an isocratic elution with a 1 : 1 (v/v) mixture of acetonitrile and THF solvents with a flow rate of 1.0 mL/min was used. The chro-matograms were acquired at 450 nm.
For optimization of the extraction conditions by the proposed DLLME-SFO method, a central composite (response surface) design method was used. Minitab statistical software [16] was used for the modeling and calculations. Three factors of 2-decanol (organic solvent) volume, acetone (disperser) volume and ionic strength (sodium nitrate concentration) were included in the design. The extraction recovery (E, %) was considered as the dependent variable to be optimized using the model. Table 1 shows the abbreviations and levels of each factor included in the design.
For calculation of the extraction recovery (E, %) of the proposed method, 100 times of the amount extracted of P-carotene (mmol) into the organic phase was divided by its initial amount in the sample. For calculation of the enrichment factor (EF), the P-caro-tene concentration in the receiving phase was divided by its initial concentration in the sample. The detection limit of the method was calculated from 3a for 10 replicated measurements on the blank.
RESULTS AND DISCUSSION
Since P-carotene is not miscible with water, its samples were prepared in the disperser solvent. Different water miscible organic solvents such as methanol, ethanol and acetone were tested as dispersers for the DLLME-SFO system. Using acetone, a more efficient extraction of the analyte
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