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Journal of Liquid Chromatography & Related Technologies
ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc20
Liquid Phase Microextraction of Biomarkers: A Review on Current Methods Samin Hamidi & Nastaran Alipour-Ghorbani To cite this article: Samin Hamidi & Nastaran Alipour-Ghorbani (2017): Liquid Phase Microextraction of Biomarkers: A Review on Current Methods, Journal of Liquid Chromatography & Related Technologies, DOI: 10.1080/10826076.2017.1374291 To link to this article: http://dx.doi.org/10.1080/10826076.2017.1374291
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Date: 21 September 2017, At: 03:33
Liquid Phase Microextraction of Biomarkers: A Review on Current Methods Samin Hamidi1,*, Nastaran Alipour-Ghorbani2,3 1
Food and Drug Safety Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran 3Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Science, Tabriz, Iran
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Corresponding author Samin Hamidi [email protected]
Abstract The detection and quantification of biomarkers has gain more attention in the medical discipline in order to evaluating disease progression to manage medical treatment. Biomarkers range from gases to biological macromolecules. Because of the nanomolar range levels of typical biomarkers in plasma, blood, urine, exhalation samples and other biological fluids as well as complex matrix of biological media, adequate sample preparation methods should be used for quantification of biomarkers. Biomarkers are discussed here generally classified mainly into two subgroups which arisen from disease or exposure compounds. The analytical method is critical for the validity/reliability of a biomarker. Accuracy, precision, reproducibility, recovery, sensitivity and specificity all have high influence to the consistency with the limit and reference values concerned. In this paper, developments in well-established liquid phase microextraction techniques for the clinical analysis of biological samples will be reviewed and discussed. This article presents an overview of microextraction methods for biological samples, focusing especially on biomarkers.
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Graphical Abstract
KEYWORDS: Sample preparation; Liquid phase microextraction; Biomarker; Bioanalysis
List of abbriviations DDLLME: dispersive derivatization liquid–liquid microextraction DLLME: dispersive liquid-liquid microextraction FM2: flunitrazepam HF: hollow fiber HS-SDME: headspace single-drop microextraction ILs: ionic liquids LLE: liquid–liquid extraction LPE: liquid phase extraction LPME: liquid-phase microextraction NNA: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone NPs: nano-particles PAA: phenylacetic acid 2
PPA: phenylpropionic acid SA-SDME: silver nanoparticle assisted single drop microextraction SDME: single drop microextraction SFODME: solidified floating organic drop microextraction SLM: supported liquid membrane SPE: solid phase extraction
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SS-SDME: salt saturated single drop microextraction TC-IL-DLLME: temperature controlled dispersive derivatization liquid–liquid microextraction 3-OHBaP: 3-Hydroxybenzo[a] pyrene USA-DLLME: ultrasound-assisted dispersive liquid–liquid microextraction
1. INTRODUCTION Many important disciplines aiming human health, including medical diagnostics and treatment, medical studies, pharmaceutical research and biochemical research, dealing with the analysis of chemical or biochemical substances present in living organisms or biological fluids. The monitoring of chemical and biochemical substances usually conduct a researcher to elucidate the compound structure and its identification followed by quantification of the target analyte to measure the real concentration level within the matrix. A biomarker is a biological characteristic that is measurable indicator and considered as a representative of biological or pathological status. The major limitation of free metabolites as biological indicators is their rapid clearance from the systemic circulation. In selecting a biomarker as an unambiguous indicator of exposure or disease, elimination profile, chemical stability, and detection sensitivity of the analytical method should be kept in mind. Therefore, implementation of biomarker related studies needs a
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simple and reliable methodology as well. The introduction of suitable biomarkers and bioanalytical technologies for the earlier diagnosis of numerous diseases is currently a hot topic in the clinical medicine [1]. For example, The aldehydes are lipid peroxidation byproducts and have been introduced as biomarkers of tissue damage caused by oxidative stress [2]. Over the years, studies have been proven a relationship between free radicals
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and some diseases, such as cancer, and aldehydes are considered as potential biomarkers of oxidative activity [3]. The noninvasive evaluating of human health status and disease detection and its progression are of the great demands for early disease detection in clinical practices. Urine and breath is potentially a rich source of volatile organic metabolites that can be used as a diagnostic tool in cancer treatment. In addition to biomarkers which are linked to disease there are another group of biomarkers named biomarkers of exposure and biomonitoring studies refers to assess potential health risk of the biomarkers of exposure that is present in body. Examples include the use of 2,5hexanedione and Z-ethoxyacetic acid in urine as indicators of exposure to n-hexane and 2ethoxyethano1, respectively. These two urinary metabolites are ‘active’ metabolites caused to neurotoxic and reproductive effects [4].
The complexity of the biological samples is difficult to handle and sometimes sample treating such as tryptic digests results in even greater complexity. This complexity hinders the reliable analysis in two ways; firstly most of the biological fluids are not amendable with analytical instruments, since all biological samples consist of a huge variety of different ions, proteins, lipids, carbohydrates etc., and cannot be directly subjected to analytical devices and secondly, highly abundant interfering compounds
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disturbed the identification and quantification of trace analytes at sub ng/mL levels. The high salt content of crude urine samples results in increased analysis times and also considerable peak broadening e.g., in CE [5]. Therefore, it seems essential to remove salts and other low-molecular weight compounds in order to avoid column breakage or blockade due to too high currents or pressure. As instance, among the biological fluids
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available, urine seems to have several advantages. Urine is easily accessible in terms of quantities. Collection of urine samples does not require any trained operator. Moreover, the fact that some analytes become significantly more concentrated in urine than in other fluids makes measurements easier. Although direct analysis of analytes or pretreated samples is optimal, additional sample clean-up is usually necessary.
A bioanalytical strategy consists of two integral stages: 1) Sample preparation (treatment/clean-up/extraction) of the target analyte from its complex biological matrix. 2) Separation, detection and quantification of the analyte.
Extraction and purification strategies range from classic sample extraction such as liquid liquid extraction (LLE) to recent developments which are the main concern of present work. Pre-treatment of biological fluids to remove interfering compounds or to hydrolyze the conjugated forms of the target biomarkers is often needed. For bioanalysis of hydrophilic non-persistent chemicals (e.g., phenols, parabens, or phthalates), urine is the sample of choice. Many of these compounds are excreted as urinary glucuronide and sulfate conjugates [6].
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Sample pre-treatment has to be performed taking into account that the major concerns of bioanalysis are to avoid the loss of targeted analytes as well as to guarantee the reproducibility of the process to remove interfering materials.
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Bioanalytical techniques are under the same challenges to meet necessities for matrix effect, accuracy, repeatability and lower limits of sensitivity. Sample pre-treatment has become an important issue of meeting these requirements. As most of the separation equipments are prone to matrix effect, which couldn’t handle complex matrix because of risk of clogging, preparation the clean extract of target analyte is required. Sample preparation methods are subdivided in two main categories; solid phase extraction (SPE) and liquid phase extraction (LPE).
In the most cases in bioanalysis, liquid–liquid extraction (LLE) has been the major sample preparation method and is still very popular. However, LLE methods are timeconsuming, laborious, and utilize large amounts of hazardous and high purity organic solvents. The automation of LLE process is not often practiced. Another issue that must be taken into account is about the sample solution discarding in LLE techniques which should provide distinct and clean extraction droplets. This is an important issue in the case of biological matrices with high content of surfactant-like compounds due to the formation of emulsion in the boundary between two phases. In the past years, the interest in novel extraction strategies with lower sample volume requirements, simpler devices
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and handling, and lower chemical consumption, has led to the appearance of a series of microextraction methods based on extraction solvent in the microliter order.
While there have been significant progresses in bioanalysis for the past several decades, less attention has been paid to the analysis of biomarkers in terms of sample preparation.
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More focus has been placed on substance separation and detection; yet, the sample treatment step in the overall scheme of bioanalysis is of great importance. The main objective of the present work is introduction the liquid phase microextraction (LPME) methods applying in biological samples, focusing especially on biomarkers. In the following sections, the general fundamentals of each methodology will be discussed in summary, followed by the description of some recent modifications and applications for each biomarker.
2. LIQUID PHASE MICROEXTRACTION (LPME) A complement option in miniaturized solvent-minimized sample preparation methods emerged in the middle-to-late 1990s [7–9]; LPME format utilizes only a small volumes of water-immiscible solvent for concentrating analytes from aqueous samples. It overcomes many of the limitations of LLE as well as some of those of solid phase microextraction (e.g. non-dependence on a commercial device and sample carryover). LPME is simple to set-up and use, generally fast, and is characterized by its compatibility by widely available apparatus or materials. In LPME, extraction normally handled into a small amount of extraction solvent (sometimes referred to as the acceptor phase) from an aqueous sample containing analytes.
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2.1. Single Drop Microextraction (SDME) Microextraction methodologies intend to reduce the solvent to aqueous phase ratio. Simplest and earliest established form of solvent based microextraction is single drop microextraction (SDME) which can be carried out by a simple device, i.e. a conventional
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microsyringe [10].
The device directly dipped in the sample, or suspended above the solution into the void space for headspace extraction [8,9]. Later one is known as headspace single-drop microextraction (HS-SDME), is suitable for concentration of the volatile analytes in the solution. Fast determination of the diabetes biomarker, acetone in human blood was implemented using HS-SDME [11]. High pre-concentration factors are found because of the high sample volume-to-extraction phase volume ratio. The extraction framework in SDME is a two-phase system, where analytes are extracted from donor phase to an extraction phase. It is also possible to implement three-phase extraction system in which analytes are extracted from an aqueous phase into an accepter phase, and then “backextracted” into another aqueous phase [12]. This happens by manipulation of pH in the sample solution and acceptor phase. Table 1 shows the bioanalytical methods have been applied in determination of biomarkers so far.
Since the excretion of phenylacetic acid (PAA) changes in serious illnesses such as schizophernia, phenylketonuria and major depressive disorders and due to the importance of phenylpropionic acid (PPA) in some forms of phenylketonuria, PPA determination
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seems to be clinically significant. Three phase liquid phase microextraction methodology coupled with HPLC-UV has been applied as a sensitive and efficient sample pretreatment method to determine PAA as a biomarker of depressive disorders and PPA in biological fluids [13].
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In the direct immersed mode of SDME, the organic phase should be water immiscible and therefore polar solvents cannot be used for extraction of polar or semi polar compounds. In a study, a salt saturated single drop microextraction (SS-SDME) was proposed in order to treat the polar analyte, malondialdehyde, from urine samples [14]. The sample solution was saturated with NaCl, Mg (NO3)2.6H2O and KNO3 salts and 1.3 µL benzyl alcohol was used as an organic phase at the tip of the microsyringe.
In order to improve separation efficiency and selectivity, to save time in extraction process and for environmental concerns, nano-particles (NPs) were integrated for LLE. Wu and his coworkers [15] reported silver nanoparticle assisted single drop microextraction using chemical modification of AgNPs surface with two different capping agents; 1-octadecanethiol/4-aminothiophenol and 1-octadecanethiol/1thioglycerol. The AgNPs have a dual function, and thus they can be used as potent concentrating probes for separation and also for surface assisted laser desorption/ionization mass spectrometry. The separation and identification of cysteine and homocysteine from a urine sample was also reported by this method. In practice, the tip of the syringe which contains 1 mL of organic phase droplet carefully dipped into the 50 mL treated urine solution. The microdroplet was drawn back into the microsyringe
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after 10 min and introduced to matrix-assisted laser desorption/ionization mass spectrometry analysis.
2.2. Dispersive Liquid-Liquid Microextraction (DLLME) However, SDME methods have some limitations: in the case of organic drop, the stirring
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speed needs to be controlled carefully to avoid bubble forming and organic drop broking up. Therefore, these methods need long time but obtain low sensitivity and repeatability. In dispersive liquid-liquid microextraction (DLLME) a mixture of extraction and dispersive solvent were rapidly injected into sample solution and the organic phase which is denser than water was collected at the bottom of tube. This method is widely used in biomedical discipline [17,18]. However, the elimination of dispersive solvent from the procedure and using the disperser-solvent free methods has became challenging issue [19]. Table 2 provides bioanalytical methods for determination of biomarkers using dispersive liquid phase microextraction.
Fuh et al., reported the first application of DLLME in urine samples for determination of aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) coupled with LC–ES-MS/MS. For the analytes which are not amendable with GC, the resultant organic phase obtained by DLLME cannot be introduced directly for reverse phase-liquid chromatography (RP-LC). Hence, evaporation of the water-immiscible phase to dryness and reconstitution of analytes into a more compatible solvent prior to LC was required. In this work the organic phase was evaporated until dryness and then re-constructed it in the mobile phase for subsequent analysis by LC [20]. The blood
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biomarker phosphatidylethanol formed only in the presence of ethanol so it is a promising biomarker to quantify ethanol abuse in drinking. The phosphatidylethanol levels were analyzed in blood samples collected from heavy and social drinkers using LC/MS-MS [21].
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The unbalanced secretion of biogenic amines is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors and in a study DLLME-MEKCUV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children [22].
Shamsipur and his co-workers [23] implemented a dispersive derivatization liquid–liquid microextraction (DDLLME) method as a simple, rapid and sensitive procedure for extraction of some amino acids in urine for the first time. A mixture of acetonitrile + pyridine + carbon tetrachloride was rapidly dispersed into the aqueous solution and shacked vigorously. It is noteworthy to say that pyridine acts as both catalyst and buffering agent in controlling the pH of subsequent chloroformate derivatization process. Then, isobutyl chloroformate was added and after 1 h, N-isobutoxycarbonyl isobutyl esters of the candidate amino acid derivatives were formed. After collecting the extraction phase, the sediment phase was injected directly into GC–MS for subsequent analysis. For LC–MS analysis, the extraction phase was evaporated to dryness under a gentle stream of nitrogen and the residue was re-dissolved in eluent. The agreement
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between two analytical methods was assessed by Bland–Altman plots and shows good alignment in the case of urine levels of sarcosine. They also tried ultrasound-assisted emulsification liquid–liquid microextraction using 150 µL of acetonitrile and other parameters and conditions were similar to DDLLME procedure but the best results were
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attributed to DDLLME.
Extraction of highly hydrophilic compounds with water-immiscible solvents results in low recovery and inefficient process in DLLME. Therefore to overcome this limitation, in-situ derivatization has been emerged as a novel way to enhance the extraction of poorly extracted compounds. A DDLLME–GC–MS method was proposed to determination of 2-chlorovinylarsonous acid as a hydrolysis product and urinary metabolite of lewisite in urine samples. A mixture of methanol (dispersive solvent), chloroform (extraction solvent), and 10 μL neat ethanedithiol (derivatization reagent) was rapidly injected into the urine sample for operating the DLLME process and finally the extraction phase was separated for direct introduction into GC [24]. A low toxic and in situ derivatization-ultrasound-assisted dispersive liquid–liquid microextraction (in situ DUADLLME) has been developed for the simultaneous determination of six neurotransmitters in rat brain microdialysates by UHPLC–MS/MS [25].
In the DLLME procedure, the condition which extraction solvent is dispersed is a key factor affecting the extraction efficiency. In recent years, ionic liquids (ILs) have been emerged as the new replacement to conventional extraction solvents for their outstanding physicochemical properties such as immiscibility with water, low volatility,
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environmental friendly and good extraction capacity for most compounds. Their viscose nature, however, confines the dispersion of ILs into the aqueous solution. Therefore there are some methods papered to prompt the dispersion ability of ILs. In temperature controlled dispersive derivatization liquid–liquid microextraction (TC-IL-DLLME) was used as dispersion of IL instead of organic solvent and was proposed by the Zhou group
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in 2008 [26]. The urinary di(2-ethylhexyl) phthalate and its metabolite mono(2ethylhexyl)phthalate were determined as the biomarkers to monitor the human exposure to the plasticizer followed by HPLC.
3-Hydroxybenzo[a] pyrene (3-OHBaP) is a khown biomarker for evaluating PAHs exposure risks. Due to the pg/mL level in urine excretion, assessing urinary 3-OHBaP suffers from inadequate sensitivity. Gan and his coworkers, reported an IL-DLLME for extraction of 3-OHBaP from urine using [C8MIM][PF6] as extraction solvent. IL and acetone injected to pre-enzymatically hydrolyzed urine and after shaking the IL-reached phase was collected by centrifuge and dansyl chloride was added to enhance MS response. Chemical derivatization using dansyl chloride was performed to enhance MS response [27].
Surfactants are the amphiphilic compounds that caused to increasing the contact area of analyte and extraction solvent by reducing the surface tension between organic phase and aqueous solution.
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Glyoxal and methylglyoxal are the biomarkers of oxidative stress that have been related with diabetic complications and other disorders. Surfactant-assisted dispersive liquid– liquid microextraction was applied for extraction the analytes from urine samples and separated by LC-fluorescence. After derivatization of analytes with 2,3diaminonaphthalene the pH of the solutions adjusted to 10.5 with 0.5 mL of 0.1 M
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carbonate buffer solution. Twenty-five μL of 0.03 M Triton X-114 were added to 2.5 mL of urine; the mixture was reach to 10 mL with water and DLLME was performed by injection of a mixture containing 500 μL ethanol (dispersant) and 75 μL 1-undecanol (extractant) by means of a microsyringe. The upper phase was separated by centrifugation for next analysis [28].
Dispersive-solvent free manners are beneficial in terms of low cost, environmentalfriendly, easy operating and no toxic effects. 4-(methylnitrosamino)-1-(3-pyridyl)-1butanol (NNAL) is the specific metabolite of 4-(Methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) exposure to the carcinogen and used as a biomarker. An efficient and sensitive analytical method based on molecularly imprinted solid-phase extraction (MISPE) and reverse-phase ultrasound-assisted dispersive liquid–liquid microextraction (USA-DLLME) coupled with LC–MS/MS detection was developed and validated for the analysis of urinary NNAL [29]. Phosphatidylethanol in human blood as a biomarker for alcohol intake treated with USA-DLLME and. The extraction efficiencies were increased by two to five times compared with LLE ultrasound-assisted dispersive liquid-liquid microextraction [30]. Voorspoels and his coworkers reported a USA-DLLME for phthalate metabolites in human nails [31].
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Mass transfer of analytes from sample solution into organic phase can be enhanced by nanoparticled as well. Cerium oxide nanoparticles or ceria (CeO2) were introduced in USA-DLLME [32].
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As one of new microectraction methods, solidified floating organic drop microextraction (SFODME) has been developed in recent years. Very small volume of solvent with a melting point near room temperature is utilized during the extraction.
This method was developed for the determination of volatile aldehyde biomarkers (hexanal and heptanal) in human blood samples [33].
Salivary mercury can be used as biomarker for mercury exposure and cold vapor atomic SFODME coupled to fluorescence spectrometry was used for its quantification in ultratrace levels [34].
2.3. Hollow Fiber-Based Liquid Phase Microextraction (HF-LPME) A small organic phase drop hanging at the tip of a needle is very unstable and may be dislodged by elevating the stirring speed or increasing the temperature. Another alternative to cover this limitation, Pedersen-Bjergaard and Rasmussen [36] introduced the use of small segments of polypropylene hollow fiber (HF) membrane to protect the extracting phase. Briefly, an organic solvent was immobilized in the wall pores of the HF, providing a supported liquid membrane (SLM), and an aqueous acceptor phase was
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filled within its lumen. Analytes were extracted into the organic phase on the membrane and then introduced into the aqueous phase called three-phase HF-LPME and is suitable for extraction ionizable compounds from complex matrix because pH gradient is a driving force for diffusion of analytes through the liquid membrane. In two-phase mode of HF-LPME the organic solvent fills both the outer wall pores and the HF lumen.
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Herein, instead of using a fiber, HF was used. The possible memory effect can be eliminated by discarding the HF after single use. Besides, the use of porous polypropylene provides the pure extract by prohibiting the larger interferences enter/adsorb to pores onto the coated material, thus, promotes clean extraction. Table 3 provides the analytical details of those bioanalytical methods which used HF-LPME in sample treatment step for determination of biomarkers.
Nakazawa and coworkers [37] proposed a simultaneously in situ derivatization and extraction of trace amounts of bisphenol A in urine samples using HF-LPME in order to improve the recovery and sensitivity of analysis. In a two phase system they used toluene to fill the membrane pores and lumens. After enzymatic de-conjugation of urine sample and subsequent treatment with acetic acid anhydride as the derivatization reagent extraction was performed at room temperature for 15 min and then 2 µL of extract was carefully withdrawn into the syringe and introduced into the GC–MS system.
In another study of bisphenol A and metabolites in urine phthalates were analyzed by GC/MS.LPME simplifies the derivatization step compared to other preparation techniques [38].
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While in a three-phase system the extraction and desorption processes take place simultaneously, Bartosz and coworkers separated those two processes in time. Determination of 3-phenoxybenzoic acid and 4-hydroxy-3-phenoxybenzoic acid, in human and rat urine was implemented by HF. In the first step HF (filled with dihexyl
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ether) precisely fitted on the Nylon stick (rod) placed in the sample vial for the predefined time. When the extraction is finished the rod is taken out from the sample, and after washing with water placed in an acceptor solution for desorption. Using this setup, gives high cleanup efficiency is and the handling process seems to be simpler than in a routine three-phase hollow fiber microextraction [39].
Chu and coworkers [40] reported hexanal and heptanal determination from urine sample. A HF segment was dipped into the 1-octanol as the organic solvent and after that 10 μL of acceptor solution (0.3 M NaOH) was added carefully into the hollow fiber. After derivatization with an electroactive compound, 2-thiobarbituric acid, the extracted analytes determined by CE-DAD.
Heterocyclic aromatic amines exposure was determined by Cooper group using threephase HF-LPME system [41]. It was the first report of HCA analysis under alkaline conditions extracted into accepter phase in acidic environment.
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Increasing the concentration of endogenous estrogens and their metabolites are accepted risk factors of endometrial cancer and HF-LPME proposed for an enriched pretreatment estrogens and their metabolites in the urine samples quantified by LC-MS [42].
Generally, a microemulsion consists of oil, water, surfactant, and sometimes co-
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surfactant but in a study a surfactant free microemulsion composed of toluene, isopropanol, and water was used to reinforce HF [43]. The HFs were entirely immersed in the microemulsion system and after treating were transferred in sampel vial for extraction for 5 min. they were plunged into a tube containing 100 μL of acetone for desorption via the ultrasonic-assisted procedure for 20 min. In order to using the online stacking method coupled with CE, the desorbed solution was blown to dryness at 20 °C, with a gentle stream of nitrogen. The residue was then dissolved in 0.1 mL water containing 5 mM SDS for subsequent analysis by field-enhanced sample injection with reverse migrating micelles mode in CE. Stacking enhancement factors found in the range of 87-478 for target analytes.
For the first time, three-phase HF used to extract trans-muconic acid, in urine samples of workers who had been exposed to benzene followed by HPLC/UV [44]
Hipu has determined exposure to xylene by determining methyl hippuric acids using HFLPME coupled with HPLC-UV [45]. The work published by Ghamari et al., indicated that HF-LPME based on facilitated pH gradient transport can be used as a sensitive and
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effective method for the quantification of mandelic acid and hippuric acid in urine specimens [46].
However, organic liquid phase immobilized in the wall pores of hollow fiber are not very stable in long time and may run off after relatively long period of time, which in turn
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limits the extraction efficiency. Many research groups deal with this problem by using HF-SPME instead of conventional HF-LPME.
Sol-gel technology provides the suitable way to obtain adsorbent with good properties for extraction purpose. Some advantages of sol-gel process that can cover the limitations of commercial SPME are; porous material with high surface areas for better analytes extraction, coating material is stable towards high temperature and good adhesion towards fiber as support owing to chemical bonding [47,48].
In a work done by Ibrahim et al., [49] a new hybrid silica material based on methyltrimethoxysilane-(3-mercaptopropyl)trimethoxysilane was introduced as a new material in HF-SPME of hexanal and heptanal in the urine samples. Prepared HF was directly immersed in a sample vial containing sample solution and after extraction, analytes were desorbed in 100 µL of methanol solution by vortex for 3 min.
3. CONCLUSION The introduction of biomarkers determination and bioanalytical technologies for the earlier diagnosis of numerous diseases is currently a hot topic in the clinical medicine.
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Sample preparation step is very important stage in bioanalytical procedures as it takes almost 80% of procedure time. Microextraction techniques, since their proposal as beneficial replacement to conventional procedures as LLE or SPE, have revealed a wellestablished field of sample preparation studies, with a growing number of applications. The developments in the field of microextraction have focused to the miniaturization and
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automatization of microextraction set-up in order to reduce the required sample size and staff time. New LPME methods and procedures have been proposed each year in order to discuss the efficiency of these techniques or to overcome some of their inherent limitation. These developments include (but are not limited to) modified (or new) experimental configurations for LPME, the proposal of new materials for the fabrication of DLLME and etc.
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Shamsipur, M., M.T. Naseri, and M. Babri, Quantification of candidate prostate
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Naseri, M.T., et al., Determination of lewisite metabolite 2-chlorovinylarsonous
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He, Y., et al., In situ derivatization-ultrasound-assisted dispersive liquid–liquid
microextraction for the determination of neurotransmitters in Parkinson’s rat brain microdialysates by ultra high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 2016. 1458: p. 70-81.
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Zhou, Q., et al., Trace determination of organophosphorus pesticides in
environmental samples by temperature-controlled ionic liquid dispersive liquid-phase microextraction. Journal of Chromatography A, 2008. 1188(2): p. 148-153. 27.
Hu, H., et al., Sensitive determination of trace urinary 3-hydroxybenzo [a] pyrene
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Campillo, N., et al., Glyoxal and methylglyoxal determination in urine by
surfactant-assisted dispersive liquid–liquid microextraction and LC. Bioanalysis, 2017. 9(4): p. 369-379. 29.
Liu, B.Z., et al., Development of a Sensitive Method for the Determination of
4‐ (Methylnitrosamino)‐ 1‐ (3‐ pyridyl)‐ 1‐ butanol in Human Urine Using Solid‐ phase Extraction Combined with Ultrasound‐ assisted Dispersive Liquid–liquid Microextraction and LC‐ MS/MS Detection. Journal of the Chinese Chemical Society, 2013. 60(8): p. 1055-1061. 30.
Wang, S., et al., Sensitive and precise monitoring of phosphatidylethanol in
human blood as a biomarker for alcohol intake by ultrasound-assisted dispersive liquidliquid microextraction combined with liquid chromatography tandem mass spectrometry. Talanta, 2017. 166: p. 315-320. 31.
Alves, A., et al., Ultrasound assisted extraction combined with dispersive liquid–
liquid microextraction (US-DLLME)—a fast new approach to measure phthalate metabolites in nails. Analytical and bioanalytical chemistry, 2016. 408(22): p. 61696180.
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Table 1. Bioanalytical methods have been applied in determination of biomarkers Analyte
Acetone
Sample/
Organic
Enrich
Analytical
size
phase/
ment
method
volume
factor
Blood/ 1 Decane/ 2
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mL
-
GC/MS
µL
LDR
Validation
Ref.
0.005–
%RSD: 11.9;
[11]
2.0 mM %recovery: 88
Phenylacetic acid
Urine/
1-hexanol/
110 and HPLC/UV
1–5000
%RSD: 1.3-
phenylpropionic
20 µL;
95 µL
104
µg/L
6.5
acid
Serum/ -
Plasma/
Benzyl
204.4
10-
%RSD: 8.7-
250 µL
alcohol/
1000
9.1;
1.3 µL
µg/L
%recovery:
[13]
; Plasma/ Malondialdehyde
GC/FID
[14]
93.2-104 Insulin,
Urine/-
Modefied
-
matrix-
cytochrome C,
Ag
assisted laser
ubiquitin,
nanoparticl
desorption/io
cysteine,
es
nization ion
homocysteine
trap mass spectrometry
28
-
-
[15]
Hexanal and
Serum/
Methyl
heptanal
500 µL
cyanide/
-
HPLC/UV
0.01–10 %RSD: 5.6μM
10 µL
9.8; %recovery:
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75.2- 101.1
29
[16]
Table 2. Bioanalytical methods for determination of biomarkers using dispersive liquidliquid phase microextraction. Analyte
Extracti Matrix
Extracti
Disper Enri
on
on
sive
chm
solvent/
solven
ent
volume
t/
facto
volum
r
/ size
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method
method
LDR
validation Re f.
e 7-
DLLM
Urine/
DCM/
Isoprp
aminoflunitr
E
5 mL
250 µL
yl
azepam
20
LC–ES-
0.05–
%RSD <
MS/MS
2.5
8.6;
ng/mL
accuracy:
alcoho l/ 500
92.3–
µL
103.7%
Phosphatidyl
DLLM
Whole
DCM/
Aceto
ethanol
E
blood/
230 μL
0.2 mL
-
LC/MS-MS
0.03-
%RSD <
ne/
10
15;
630
µg/L
accuracy
μL
[20]
[21]
< 15%; selectivit y
Biogenic
DLLM
Urine/
DCM/
Ethan
amines
E
1 mL
500 μL
-
MEKC-UV
0.5-
%RSD:
ol/
300
0.18-
1 mL
µg/mL
9.68; accuracy
30
[22]
91.9120.5% Amino acids
DDLL
Urine/
Carbon
ACN/
140–
GC–EI/CI–
0.05–
%RSD<
(sarcosine,
ME
5-150
tetrachl
150
155
MS and
0.1 ng/
10;
µL
oride/
µL
LC/ESI–MS
mL
%recover
alanine,
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leucine and
25 µL
y; 93.8–
proline) 2-
[23]
106 DDLL
chlorovinylar ME
Urine/
Chlorof
Metha
5 mL
orm/ 65
nol/
μL
500
sonous acid
250
GC–MS
1–400
%Recove
μg/L
ry: 95–
[24]
103
μL Neurotransm
DUAD
Rat
Bromob ACN/
itters
LLME
brain
enzene/
150 μ
microd
50 μL
L
120–
UHPLC–
0.05−3
%RSD:
286
MS/MS
000.0
3.2-12.8;
nM
accuracy:
ialysat
94.2-
es/
108.6%;
30 μL
%recover
[25]
y; 94.5105.5 di(2-
TC-IL
Urine/
[C6MI
ethylhexyl)p
DLLM
1 mL
M][PF6
and
hthalate and
E
]/ 50
164
mono(2-
-
mL
115
20–1920 µg/L HPLC/ DAD
%RSD: 0.4-7.2; %recover y: 80.4-
31
[26]
ethylhexyl)p
112.5
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hthalate 3-
IL-
Urine/
[C8MI
Aceto
Hydroxyben
DLLM
10 mL
M][PF6
ne/ 1
zo[a]pyrene
E
-
0.6–50.0 pg/mL
]/ 60 µL mL
%RSD:
[27]
HPLC- 2.2-6.8; HRMS
%recover
/MS
y: 87.896.2
Glyoxal and
Surfact
Urine/
1-
Ethan
102
methylglyox
ant-
2.5 mL undecan ol/ 0.5
al
assisted
ol/ 75
dispersi
μL
mL
0.1-25 ng/mL
LC-
%RSD:
and
fluores
5-10.6;
108
cence
%recover
[28]
y; 88–103
ve liquid– liquid microe xtractio n 4-
USA-
Urine/
ethyl
-
(Methylnitro
DLLM
5 mL
acetate/t
samino)-1-
E
23
LC/Tandem
5-1200
%RSD:
pg/mL
3.6-9.7;
oluene
accuracy;
(3-pyridyl)-
(1:2,
88.5-
1-butanol
v/v) / 2
93.7%;
mL
%recover
32
[29]
y; 88.593.7 Phosphatidyl
USA-
Blood/
DCM/
ACN/
ethanol
DLLM
25 µL
80 μL
E
-
LC-MS/MS
5 to
%RSD:
200 μ
500 ng
1.00-
L
/ mL
6.44;
[30]
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%recover y: 95.04105.28 Phthalate
USA-
Nail/
Trichlor Metha
metabolites
DLLM
30 mg
oethyle
nol/ 2
ne/
mL
E
-
UPLC-
0.3–
%RSD:
MS/MS
490 μg
6–17;
/L
accuracy;
180 μL
79-108 %
P.seudomon
Ceria@ Mouse
Chlorob CeO2
as
surfacta and
enz/ 10
@CT
assisted laser
aeruginosa
nt
sheep
µL and
AB/
desorption/ion
and
assisted blood/
chlorof
10–
ization mass
S.taphylococ
DLLM
orm/ 10
20 µL
spectrometry
cus
E
1 mL
[31]
-
µL
matrix
-
-
[32]
0.01–5
%RSD:1.
[33]
µM
85-4.11;
(MALDI-MS
aureus Hexanal and
SFO-
Human 1-
Metha
heptanal
DME
blood/- dodecan nol/ 50 μL
ol/ 50 μL
-
HPLC/UV
%recover y: 67.84-
33
70.28 Mercury SFOD
Saliva/
1-
6 mL
undecan
ME
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3-
USA-
-
182.
Cold vapor
0.025-
%RSD:
4
atomic
10.0
4.1
ol/ 60
fluorescence
ng/mL
μL
spectrometry
Portion Trichlor Metha
-
GC/MS-MS
10–
Phenoxybenz DLLM
of rat
oethyle
nol/
750
oic acid and
brain/
ne/ 100
500
ng/g
0.5 g
µL
µL
4-hydroxy-3-
E
phenoxybenz oic acid DCM: dichloromethane; DLLME: dispersive liquid-liquid microextraction; DUADLLME: derivatization-ultrasound-assisted dispersive liquid-liquid microextraction; TC-IL DLLME: temperature controlled ionic liquied dispersive liquidliquid microextraction; USA-DLLME: ultrasound assisted dispersive liquid-liquid microextraction; SFODME: solidified floating organic drop microextraction;
34
-
[34]
[35]
Table 3. Bioanalytical methods for determination of biomarkers using hollow fiber-based liquid phase microextraction Analyte
Matrix/s
Mode
Extractio
Enrich
ize
of
n
ment
LDR
Validation
Re f.
extrac technique factor
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tion Bisphenol A
Urine/ 1
Two
mL
phase
GC/MS
-
0.1–50
%RSD: 1.8–6.7;
ng/mL
%recovery: 98.8–
[37]
101. Bisphenol A and
Two
Urine
eight phthalate
phase
metabolites
GC/MS
20-
%RSD: 11.7-
/16
1000
19.7%
mL
µg/L
3-phenoxybenzoic
Urine/ 1
Three
HPLC/D
acid and 4-hydroxy-
mL
phase
AD
3-phenoxybenzoic acid
-
-
50–
%RSD, 1.6–12.6;
10,000
%recovery: 49.8–
ng/mL
55.1; accuracy: 93.3– 110.9%; stability under different storage conditions (20 °C for 2 months; 22°C for 6 h; and after three
35
[38]
[39]
freeze–thaw
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cycles). Hexanal and
Urine/3
Three
CZE-
heptanal
mL
phase
DAD
heterocyclic
Plasma/
Three
LC-
aromatic amines
0.2 mL
phase
MS/MS
-
-
1.75-
%RSD: 2.2-8.5%;
355
%recovery: 61–
µM
95%
5-80
%RSD: 4.5 and
pg/mL
8.8;
[40]
[41]
%recovery: 92-94 2-hydroxyestradiol,
Urine/
Two
2-hydroxyestrone, 4-
140 mL
phase
LC/MS
-
1.01 ×
%RSD: 5.8-11.8;
103-
%recovery: 91-
hydroxyestradiol, 4-
10.1
106;
hydroxyestrone,
pg/mL
stability: room
16α-hydroxyestrone,
temperature and
2-methoxyestradiol,
freeze thaw
[42]
and 2methoxyestrone Phthalic acid esters
trans-muconic acid
Two
Urine
phase
Urine/-
CE-UV
-
0.30–
%recovery: 77.65–
/1
80.0
106.86
mL
ng/mL
Two
HPLC/U
153–
0.005
%RSD: 2.7-8.1;
phase
V
182
to 1.2
%recovery: 83–92
mg/mL
36
[43]
[44]
Methyl hippuric acid
Three
Urine
HPLC/U
210-
10-
%RSD: 3.6-8.5;
phase
/ 10
V
312
50,000
%recovery: 83-98
mL
µg/L
Mandelic acid and
Three
Urine
HPLC-
hippuric acid
phase
/ 10
UV
-
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mL Hexanal and
Urine/10 Two
heptanal*
mL
[45]
GC/FID
-
phase
*determined by hollow fiber-based solid phase microextraction.
37
0.02–
%RSD: 2.5-10.7;
195
%recovery: 84–
mg/L
94%
0.020-
%RSD:3.2-9.3;
10.00
%recovery: 91.21–
µg/mL
97.51
[46]
[49]
ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc20
Liquid Phase Microextraction of Biomarkers: A Review on Current Methods Samin Hamidi & Nastaran Alipour-Ghorbani To cite this article: Samin Hamidi & Nastaran Alipour-Ghorbani (2017): Liquid Phase Microextraction of Biomarkers: A Review on Current Methods, Journal of Liquid Chromatography & Related Technologies, DOI: 10.1080/10826076.2017.1374291 To link to this article: http://dx.doi.org/10.1080/10826076.2017.1374291
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Date: 21 September 2017, At: 03:33
Liquid Phase Microextraction of Biomarkers: A Review on Current Methods Samin Hamidi1,*, Nastaran Alipour-Ghorbani2,3 1
Food and Drug Safety Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran 3Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Science, Tabriz, Iran
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Corresponding author Samin Hamidi [email protected]
Abstract The detection and quantification of biomarkers has gain more attention in the medical discipline in order to evaluating disease progression to manage medical treatment. Biomarkers range from gases to biological macromolecules. Because of the nanomolar range levels of typical biomarkers in plasma, blood, urine, exhalation samples and other biological fluids as well as complex matrix of biological media, adequate sample preparation methods should be used for quantification of biomarkers. Biomarkers are discussed here generally classified mainly into two subgroups which arisen from disease or exposure compounds. The analytical method is critical for the validity/reliability of a biomarker. Accuracy, precision, reproducibility, recovery, sensitivity and specificity all have high influence to the consistency with the limit and reference values concerned. In this paper, developments in well-established liquid phase microextraction techniques for the clinical analysis of biological samples will be reviewed and discussed. This article presents an overview of microextraction methods for biological samples, focusing especially on biomarkers.
1
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Graphical Abstract
KEYWORDS: Sample preparation; Liquid phase microextraction; Biomarker; Bioanalysis
List of abbriviations DDLLME: dispersive derivatization liquid–liquid microextraction DLLME: dispersive liquid-liquid microextraction FM2: flunitrazepam HF: hollow fiber HS-SDME: headspace single-drop microextraction ILs: ionic liquids LLE: liquid–liquid extraction LPE: liquid phase extraction LPME: liquid-phase microextraction NNA: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone NPs: nano-particles PAA: phenylacetic acid 2
PPA: phenylpropionic acid SA-SDME: silver nanoparticle assisted single drop microextraction SDME: single drop microextraction SFODME: solidified floating organic drop microextraction SLM: supported liquid membrane SPE: solid phase extraction
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SS-SDME: salt saturated single drop microextraction TC-IL-DLLME: temperature controlled dispersive derivatization liquid–liquid microextraction 3-OHBaP: 3-Hydroxybenzo[a] pyrene USA-DLLME: ultrasound-assisted dispersive liquid–liquid microextraction
1. INTRODUCTION Many important disciplines aiming human health, including medical diagnostics and treatment, medical studies, pharmaceutical research and biochemical research, dealing with the analysis of chemical or biochemical substances present in living organisms or biological fluids. The monitoring of chemical and biochemical substances usually conduct a researcher to elucidate the compound structure and its identification followed by quantification of the target analyte to measure the real concentration level within the matrix. A biomarker is a biological characteristic that is measurable indicator and considered as a representative of biological or pathological status. The major limitation of free metabolites as biological indicators is their rapid clearance from the systemic circulation. In selecting a biomarker as an unambiguous indicator of exposure or disease, elimination profile, chemical stability, and detection sensitivity of the analytical method should be kept in mind. Therefore, implementation of biomarker related studies needs a
3
simple and reliable methodology as well. The introduction of suitable biomarkers and bioanalytical technologies for the earlier diagnosis of numerous diseases is currently a hot topic in the clinical medicine [1]. For example, The aldehydes are lipid peroxidation byproducts and have been introduced as biomarkers of tissue damage caused by oxidative stress [2]. Over the years, studies have been proven a relationship between free radicals
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and some diseases, such as cancer, and aldehydes are considered as potential biomarkers of oxidative activity [3]. The noninvasive evaluating of human health status and disease detection and its progression are of the great demands for early disease detection in clinical practices. Urine and breath is potentially a rich source of volatile organic metabolites that can be used as a diagnostic tool in cancer treatment. In addition to biomarkers which are linked to disease there are another group of biomarkers named biomarkers of exposure and biomonitoring studies refers to assess potential health risk of the biomarkers of exposure that is present in body. Examples include the use of 2,5hexanedione and Z-ethoxyacetic acid in urine as indicators of exposure to n-hexane and 2ethoxyethano1, respectively. These two urinary metabolites are ‘active’ metabolites caused to neurotoxic and reproductive effects [4].
The complexity of the biological samples is difficult to handle and sometimes sample treating such as tryptic digests results in even greater complexity. This complexity hinders the reliable analysis in two ways; firstly most of the biological fluids are not amendable with analytical instruments, since all biological samples consist of a huge variety of different ions, proteins, lipids, carbohydrates etc., and cannot be directly subjected to analytical devices and secondly, highly abundant interfering compounds
4
disturbed the identification and quantification of trace analytes at sub ng/mL levels. The high salt content of crude urine samples results in increased analysis times and also considerable peak broadening e.g., in CE [5]. Therefore, it seems essential to remove salts and other low-molecular weight compounds in order to avoid column breakage or blockade due to too high currents or pressure. As instance, among the biological fluids
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available, urine seems to have several advantages. Urine is easily accessible in terms of quantities. Collection of urine samples does not require any trained operator. Moreover, the fact that some analytes become significantly more concentrated in urine than in other fluids makes measurements easier. Although direct analysis of analytes or pretreated samples is optimal, additional sample clean-up is usually necessary.
A bioanalytical strategy consists of two integral stages: 1) Sample preparation (treatment/clean-up/extraction) of the target analyte from its complex biological matrix. 2) Separation, detection and quantification of the analyte.
Extraction and purification strategies range from classic sample extraction such as liquid liquid extraction (LLE) to recent developments which are the main concern of present work. Pre-treatment of biological fluids to remove interfering compounds or to hydrolyze the conjugated forms of the target biomarkers is often needed. For bioanalysis of hydrophilic non-persistent chemicals (e.g., phenols, parabens, or phthalates), urine is the sample of choice. Many of these compounds are excreted as urinary glucuronide and sulfate conjugates [6].
5
Sample pre-treatment has to be performed taking into account that the major concerns of bioanalysis are to avoid the loss of targeted analytes as well as to guarantee the reproducibility of the process to remove interfering materials.
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Bioanalytical techniques are under the same challenges to meet necessities for matrix effect, accuracy, repeatability and lower limits of sensitivity. Sample pre-treatment has become an important issue of meeting these requirements. As most of the separation equipments are prone to matrix effect, which couldn’t handle complex matrix because of risk of clogging, preparation the clean extract of target analyte is required. Sample preparation methods are subdivided in two main categories; solid phase extraction (SPE) and liquid phase extraction (LPE).
In the most cases in bioanalysis, liquid–liquid extraction (LLE) has been the major sample preparation method and is still very popular. However, LLE methods are timeconsuming, laborious, and utilize large amounts of hazardous and high purity organic solvents. The automation of LLE process is not often practiced. Another issue that must be taken into account is about the sample solution discarding in LLE techniques which should provide distinct and clean extraction droplets. This is an important issue in the case of biological matrices with high content of surfactant-like compounds due to the formation of emulsion in the boundary between two phases. In the past years, the interest in novel extraction strategies with lower sample volume requirements, simpler devices
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and handling, and lower chemical consumption, has led to the appearance of a series of microextraction methods based on extraction solvent in the microliter order.
While there have been significant progresses in bioanalysis for the past several decades, less attention has been paid to the analysis of biomarkers in terms of sample preparation.
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More focus has been placed on substance separation and detection; yet, the sample treatment step in the overall scheme of bioanalysis is of great importance. The main objective of the present work is introduction the liquid phase microextraction (LPME) methods applying in biological samples, focusing especially on biomarkers. In the following sections, the general fundamentals of each methodology will be discussed in summary, followed by the description of some recent modifications and applications for each biomarker.
2. LIQUID PHASE MICROEXTRACTION (LPME) A complement option in miniaturized solvent-minimized sample preparation methods emerged in the middle-to-late 1990s [7–9]; LPME format utilizes only a small volumes of water-immiscible solvent for concentrating analytes from aqueous samples. It overcomes many of the limitations of LLE as well as some of those of solid phase microextraction (e.g. non-dependence on a commercial device and sample carryover). LPME is simple to set-up and use, generally fast, and is characterized by its compatibility by widely available apparatus or materials. In LPME, extraction normally handled into a small amount of extraction solvent (sometimes referred to as the acceptor phase) from an aqueous sample containing analytes.
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2.1. Single Drop Microextraction (SDME) Microextraction methodologies intend to reduce the solvent to aqueous phase ratio. Simplest and earliest established form of solvent based microextraction is single drop microextraction (SDME) which can be carried out by a simple device, i.e. a conventional
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microsyringe [10].
The device directly dipped in the sample, or suspended above the solution into the void space for headspace extraction [8,9]. Later one is known as headspace single-drop microextraction (HS-SDME), is suitable for concentration of the volatile analytes in the solution. Fast determination of the diabetes biomarker, acetone in human blood was implemented using HS-SDME [11]. High pre-concentration factors are found because of the high sample volume-to-extraction phase volume ratio. The extraction framework in SDME is a two-phase system, where analytes are extracted from donor phase to an extraction phase. It is also possible to implement three-phase extraction system in which analytes are extracted from an aqueous phase into an accepter phase, and then “backextracted” into another aqueous phase [12]. This happens by manipulation of pH in the sample solution and acceptor phase. Table 1 shows the bioanalytical methods have been applied in determination of biomarkers so far.
Since the excretion of phenylacetic acid (PAA) changes in serious illnesses such as schizophernia, phenylketonuria and major depressive disorders and due to the importance of phenylpropionic acid (PPA) in some forms of phenylketonuria, PPA determination
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seems to be clinically significant. Three phase liquid phase microextraction methodology coupled with HPLC-UV has been applied as a sensitive and efficient sample pretreatment method to determine PAA as a biomarker of depressive disorders and PPA in biological fluids [13].
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In the direct immersed mode of SDME, the organic phase should be water immiscible and therefore polar solvents cannot be used for extraction of polar or semi polar compounds. In a study, a salt saturated single drop microextraction (SS-SDME) was proposed in order to treat the polar analyte, malondialdehyde, from urine samples [14]. The sample solution was saturated with NaCl, Mg (NO3)2.6H2O and KNO3 salts and 1.3 µL benzyl alcohol was used as an organic phase at the tip of the microsyringe.
In order to improve separation efficiency and selectivity, to save time in extraction process and for environmental concerns, nano-particles (NPs) were integrated for LLE. Wu and his coworkers [15] reported silver nanoparticle assisted single drop microextraction using chemical modification of AgNPs surface with two different capping agents; 1-octadecanethiol/4-aminothiophenol and 1-octadecanethiol/1thioglycerol. The AgNPs have a dual function, and thus they can be used as potent concentrating probes for separation and also for surface assisted laser desorption/ionization mass spectrometry. The separation and identification of cysteine and homocysteine from a urine sample was also reported by this method. In practice, the tip of the syringe which contains 1 mL of organic phase droplet carefully dipped into the 50 mL treated urine solution. The microdroplet was drawn back into the microsyringe
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after 10 min and introduced to matrix-assisted laser desorption/ionization mass spectrometry analysis.
2.2. Dispersive Liquid-Liquid Microextraction (DLLME) However, SDME methods have some limitations: in the case of organic drop, the stirring
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speed needs to be controlled carefully to avoid bubble forming and organic drop broking up. Therefore, these methods need long time but obtain low sensitivity and repeatability. In dispersive liquid-liquid microextraction (DLLME) a mixture of extraction and dispersive solvent were rapidly injected into sample solution and the organic phase which is denser than water was collected at the bottom of tube. This method is widely used in biomedical discipline [17,18]. However, the elimination of dispersive solvent from the procedure and using the disperser-solvent free methods has became challenging issue [19]. Table 2 provides bioanalytical methods for determination of biomarkers using dispersive liquid phase microextraction.
Fuh et al., reported the first application of DLLME in urine samples for determination of aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) coupled with LC–ES-MS/MS. For the analytes which are not amendable with GC, the resultant organic phase obtained by DLLME cannot be introduced directly for reverse phase-liquid chromatography (RP-LC). Hence, evaporation of the water-immiscible phase to dryness and reconstitution of analytes into a more compatible solvent prior to LC was required. In this work the organic phase was evaporated until dryness and then re-constructed it in the mobile phase for subsequent analysis by LC [20]. The blood
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biomarker phosphatidylethanol formed only in the presence of ethanol so it is a promising biomarker to quantify ethanol abuse in drinking. The phosphatidylethanol levels were analyzed in blood samples collected from heavy and social drinkers using LC/MS-MS [21].
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The unbalanced secretion of biogenic amines is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors and in a study DLLME-MEKCUV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children [22].
Shamsipur and his co-workers [23] implemented a dispersive derivatization liquid–liquid microextraction (DDLLME) method as a simple, rapid and sensitive procedure for extraction of some amino acids in urine for the first time. A mixture of acetonitrile + pyridine + carbon tetrachloride was rapidly dispersed into the aqueous solution and shacked vigorously. It is noteworthy to say that pyridine acts as both catalyst and buffering agent in controlling the pH of subsequent chloroformate derivatization process. Then, isobutyl chloroformate was added and after 1 h, N-isobutoxycarbonyl isobutyl esters of the candidate amino acid derivatives were formed. After collecting the extraction phase, the sediment phase was injected directly into GC–MS for subsequent analysis. For LC–MS analysis, the extraction phase was evaporated to dryness under a gentle stream of nitrogen and the residue was re-dissolved in eluent. The agreement
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between two analytical methods was assessed by Bland–Altman plots and shows good alignment in the case of urine levels of sarcosine. They also tried ultrasound-assisted emulsification liquid–liquid microextraction using 150 µL of acetonitrile and other parameters and conditions were similar to DDLLME procedure but the best results were
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attributed to DDLLME.
Extraction of highly hydrophilic compounds with water-immiscible solvents results in low recovery and inefficient process in DLLME. Therefore to overcome this limitation, in-situ derivatization has been emerged as a novel way to enhance the extraction of poorly extracted compounds. A DDLLME–GC–MS method was proposed to determination of 2-chlorovinylarsonous acid as a hydrolysis product and urinary metabolite of lewisite in urine samples. A mixture of methanol (dispersive solvent), chloroform (extraction solvent), and 10 μL neat ethanedithiol (derivatization reagent) was rapidly injected into the urine sample for operating the DLLME process and finally the extraction phase was separated for direct introduction into GC [24]. A low toxic and in situ derivatization-ultrasound-assisted dispersive liquid–liquid microextraction (in situ DUADLLME) has been developed for the simultaneous determination of six neurotransmitters in rat brain microdialysates by UHPLC–MS/MS [25].
In the DLLME procedure, the condition which extraction solvent is dispersed is a key factor affecting the extraction efficiency. In recent years, ionic liquids (ILs) have been emerged as the new replacement to conventional extraction solvents for their outstanding physicochemical properties such as immiscibility with water, low volatility,
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environmental friendly and good extraction capacity for most compounds. Their viscose nature, however, confines the dispersion of ILs into the aqueous solution. Therefore there are some methods papered to prompt the dispersion ability of ILs. In temperature controlled dispersive derivatization liquid–liquid microextraction (TC-IL-DLLME) was used as dispersion of IL instead of organic solvent and was proposed by the Zhou group
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in 2008 [26]. The urinary di(2-ethylhexyl) phthalate and its metabolite mono(2ethylhexyl)phthalate were determined as the biomarkers to monitor the human exposure to the plasticizer followed by HPLC.
3-Hydroxybenzo[a] pyrene (3-OHBaP) is a khown biomarker for evaluating PAHs exposure risks. Due to the pg/mL level in urine excretion, assessing urinary 3-OHBaP suffers from inadequate sensitivity. Gan and his coworkers, reported an IL-DLLME for extraction of 3-OHBaP from urine using [C8MIM][PF6] as extraction solvent. IL and acetone injected to pre-enzymatically hydrolyzed urine and after shaking the IL-reached phase was collected by centrifuge and dansyl chloride was added to enhance MS response. Chemical derivatization using dansyl chloride was performed to enhance MS response [27].
Surfactants are the amphiphilic compounds that caused to increasing the contact area of analyte and extraction solvent by reducing the surface tension between organic phase and aqueous solution.
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Glyoxal and methylglyoxal are the biomarkers of oxidative stress that have been related with diabetic complications and other disorders. Surfactant-assisted dispersive liquid– liquid microextraction was applied for extraction the analytes from urine samples and separated by LC-fluorescence. After derivatization of analytes with 2,3diaminonaphthalene the pH of the solutions adjusted to 10.5 with 0.5 mL of 0.1 M
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carbonate buffer solution. Twenty-five μL of 0.03 M Triton X-114 were added to 2.5 mL of urine; the mixture was reach to 10 mL with water and DLLME was performed by injection of a mixture containing 500 μL ethanol (dispersant) and 75 μL 1-undecanol (extractant) by means of a microsyringe. The upper phase was separated by centrifugation for next analysis [28].
Dispersive-solvent free manners are beneficial in terms of low cost, environmentalfriendly, easy operating and no toxic effects. 4-(methylnitrosamino)-1-(3-pyridyl)-1butanol (NNAL) is the specific metabolite of 4-(Methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) exposure to the carcinogen and used as a biomarker. An efficient and sensitive analytical method based on molecularly imprinted solid-phase extraction (MISPE) and reverse-phase ultrasound-assisted dispersive liquid–liquid microextraction (USA-DLLME) coupled with LC–MS/MS detection was developed and validated for the analysis of urinary NNAL [29]. Phosphatidylethanol in human blood as a biomarker for alcohol intake treated with USA-DLLME and. The extraction efficiencies were increased by two to five times compared with LLE ultrasound-assisted dispersive liquid-liquid microextraction [30]. Voorspoels and his coworkers reported a USA-DLLME for phthalate metabolites in human nails [31].
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Mass transfer of analytes from sample solution into organic phase can be enhanced by nanoparticled as well. Cerium oxide nanoparticles or ceria (CeO2) were introduced in USA-DLLME [32].
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As one of new microectraction methods, solidified floating organic drop microextraction (SFODME) has been developed in recent years. Very small volume of solvent with a melting point near room temperature is utilized during the extraction.
This method was developed for the determination of volatile aldehyde biomarkers (hexanal and heptanal) in human blood samples [33].
Salivary mercury can be used as biomarker for mercury exposure and cold vapor atomic SFODME coupled to fluorescence spectrometry was used for its quantification in ultratrace levels [34].
2.3. Hollow Fiber-Based Liquid Phase Microextraction (HF-LPME) A small organic phase drop hanging at the tip of a needle is very unstable and may be dislodged by elevating the stirring speed or increasing the temperature. Another alternative to cover this limitation, Pedersen-Bjergaard and Rasmussen [36] introduced the use of small segments of polypropylene hollow fiber (HF) membrane to protect the extracting phase. Briefly, an organic solvent was immobilized in the wall pores of the HF, providing a supported liquid membrane (SLM), and an aqueous acceptor phase was
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filled within its lumen. Analytes were extracted into the organic phase on the membrane and then introduced into the aqueous phase called three-phase HF-LPME and is suitable for extraction ionizable compounds from complex matrix because pH gradient is a driving force for diffusion of analytes through the liquid membrane. In two-phase mode of HF-LPME the organic solvent fills both the outer wall pores and the HF lumen.
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Herein, instead of using a fiber, HF was used. The possible memory effect can be eliminated by discarding the HF after single use. Besides, the use of porous polypropylene provides the pure extract by prohibiting the larger interferences enter/adsorb to pores onto the coated material, thus, promotes clean extraction. Table 3 provides the analytical details of those bioanalytical methods which used HF-LPME in sample treatment step for determination of biomarkers.
Nakazawa and coworkers [37] proposed a simultaneously in situ derivatization and extraction of trace amounts of bisphenol A in urine samples using HF-LPME in order to improve the recovery and sensitivity of analysis. In a two phase system they used toluene to fill the membrane pores and lumens. After enzymatic de-conjugation of urine sample and subsequent treatment with acetic acid anhydride as the derivatization reagent extraction was performed at room temperature for 15 min and then 2 µL of extract was carefully withdrawn into the syringe and introduced into the GC–MS system.
In another study of bisphenol A and metabolites in urine phthalates were analyzed by GC/MS.LPME simplifies the derivatization step compared to other preparation techniques [38].
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While in a three-phase system the extraction and desorption processes take place simultaneously, Bartosz and coworkers separated those two processes in time. Determination of 3-phenoxybenzoic acid and 4-hydroxy-3-phenoxybenzoic acid, in human and rat urine was implemented by HF. In the first step HF (filled with dihexyl
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ether) precisely fitted on the Nylon stick (rod) placed in the sample vial for the predefined time. When the extraction is finished the rod is taken out from the sample, and after washing with water placed in an acceptor solution for desorption. Using this setup, gives high cleanup efficiency is and the handling process seems to be simpler than in a routine three-phase hollow fiber microextraction [39].
Chu and coworkers [40] reported hexanal and heptanal determination from urine sample. A HF segment was dipped into the 1-octanol as the organic solvent and after that 10 μL of acceptor solution (0.3 M NaOH) was added carefully into the hollow fiber. After derivatization with an electroactive compound, 2-thiobarbituric acid, the extracted analytes determined by CE-DAD.
Heterocyclic aromatic amines exposure was determined by Cooper group using threephase HF-LPME system [41]. It was the first report of HCA analysis under alkaline conditions extracted into accepter phase in acidic environment.
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Increasing the concentration of endogenous estrogens and their metabolites are accepted risk factors of endometrial cancer and HF-LPME proposed for an enriched pretreatment estrogens and their metabolites in the urine samples quantified by LC-MS [42].
Generally, a microemulsion consists of oil, water, surfactant, and sometimes co-
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surfactant but in a study a surfactant free microemulsion composed of toluene, isopropanol, and water was used to reinforce HF [43]. The HFs were entirely immersed in the microemulsion system and after treating were transferred in sampel vial for extraction for 5 min. they were plunged into a tube containing 100 μL of acetone for desorption via the ultrasonic-assisted procedure for 20 min. In order to using the online stacking method coupled with CE, the desorbed solution was blown to dryness at 20 °C, with a gentle stream of nitrogen. The residue was then dissolved in 0.1 mL water containing 5 mM SDS for subsequent analysis by field-enhanced sample injection with reverse migrating micelles mode in CE. Stacking enhancement factors found in the range of 87-478 for target analytes.
For the first time, three-phase HF used to extract trans-muconic acid, in urine samples of workers who had been exposed to benzene followed by HPLC/UV [44]
Hipu has determined exposure to xylene by determining methyl hippuric acids using HFLPME coupled with HPLC-UV [45]. The work published by Ghamari et al., indicated that HF-LPME based on facilitated pH gradient transport can be used as a sensitive and
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effective method for the quantification of mandelic acid and hippuric acid in urine specimens [46].
However, organic liquid phase immobilized in the wall pores of hollow fiber are not very stable in long time and may run off after relatively long period of time, which in turn
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limits the extraction efficiency. Many research groups deal with this problem by using HF-SPME instead of conventional HF-LPME.
Sol-gel technology provides the suitable way to obtain adsorbent with good properties for extraction purpose. Some advantages of sol-gel process that can cover the limitations of commercial SPME are; porous material with high surface areas for better analytes extraction, coating material is stable towards high temperature and good adhesion towards fiber as support owing to chemical bonding [47,48].
In a work done by Ibrahim et al., [49] a new hybrid silica material based on methyltrimethoxysilane-(3-mercaptopropyl)trimethoxysilane was introduced as a new material in HF-SPME of hexanal and heptanal in the urine samples. Prepared HF was directly immersed in a sample vial containing sample solution and after extraction, analytes were desorbed in 100 µL of methanol solution by vortex for 3 min.
3. CONCLUSION The introduction of biomarkers determination and bioanalytical technologies for the earlier diagnosis of numerous diseases is currently a hot topic in the clinical medicine.
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Sample preparation step is very important stage in bioanalytical procedures as it takes almost 80% of procedure time. Microextraction techniques, since their proposal as beneficial replacement to conventional procedures as LLE or SPE, have revealed a wellestablished field of sample preparation studies, with a growing number of applications. The developments in the field of microextraction have focused to the miniaturization and
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automatization of microextraction set-up in order to reduce the required sample size and staff time. New LPME methods and procedures have been proposed each year in order to discuss the efficiency of these techniques or to overcome some of their inherent limitation. These developments include (but are not limited to) modified (or new) experimental configurations for LPME, the proposal of new materials for the fabrication of DLLME and etc.
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Ghamari, F., et al., Hollow-fiber liquid-phase microextraction based on carrier-
mediated transport for determination of urinary methyl hippuric acids. Toxicological &
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Environmental Chemistry, 2017(just-accepted): p. 01-17. 46.
Bahrami, A., et al., Hollow Fiber Supported Liquid Membrane Extraction
Combined with HPLC-UV for Simultaneous Preconcentration and Determination of Urinary Hippuric Acid and Mandelic Acid. Membranes, 2017. 7(1): p. 8. 47.
Spietelun, A., et al., Recent developments and future trends in solid phase
microextraction techniques towards green analytical chemistry. Journal of Chromatography A, 2013. 1321: p. 1-13. 48.
Mu, L., et al., Robust aptamer sol–gel solid phase microextraction of very polar
adenosine from human plasma. Journal of Chromatography A, 2013. 1279: p. 7-12. 49.
Wahib, S.M.A., W.A.W. Ibrahim, and M.M. Sanagi, NEW
Methyltrimethoxysilane-(3-Mercaptopropyl)-Trimethoxysilane Coated Hollow FiberSolid Phase Microextraction For Hexanal And Heptanal Analysis. Malaysian Journal of Analytical Sciences, 2016. 20(1): p. 51-63.
27
Table 1. Bioanalytical methods have been applied in determination of biomarkers Analyte
Acetone
Sample/
Organic
Enrich
Analytical
size
phase/
ment
method
volume
factor
Blood/ 1 Decane/ 2
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mL
-
GC/MS
µL
LDR
Validation
Ref.
0.005–
%RSD: 11.9;
[11]
2.0 mM %recovery: 88
Phenylacetic acid
Urine/
1-hexanol/
110 and HPLC/UV
1–5000
%RSD: 1.3-
phenylpropionic
20 µL;
95 µL
104
µg/L
6.5
acid
Serum/ -
Plasma/
Benzyl
204.4
10-
%RSD: 8.7-
250 µL
alcohol/
1000
9.1;
1.3 µL
µg/L
%recovery:
[13]
; Plasma/ Malondialdehyde
GC/FID
[14]
93.2-104 Insulin,
Urine/-
Modefied
-
matrix-
cytochrome C,
Ag
assisted laser
ubiquitin,
nanoparticl
desorption/io
cysteine,
es
nization ion
homocysteine
trap mass spectrometry
28
-
-
[15]
Hexanal and
Serum/
Methyl
heptanal
500 µL
cyanide/
-
HPLC/UV
0.01–10 %RSD: 5.6μM
10 µL
9.8; %recovery:
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75.2- 101.1
29
[16]
Table 2. Bioanalytical methods for determination of biomarkers using dispersive liquidliquid phase microextraction. Analyte
Extracti Matrix
Extracti
Disper Enri
on
on
sive
chm
solvent/
solven
ent
volume
t/
facto
volum
r
/ size
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method
method
LDR
validation Re f.
e 7-
DLLM
Urine/
DCM/
Isoprp
aminoflunitr
E
5 mL
250 µL
yl
azepam
20
LC–ES-
0.05–
%RSD <
MS/MS
2.5
8.6;
ng/mL
accuracy:
alcoho l/ 500
92.3–
µL
103.7%
Phosphatidyl
DLLM
Whole
DCM/
Aceto
ethanol
E
blood/
230 μL
0.2 mL
-
LC/MS-MS
0.03-
%RSD <
ne/
10
15;
630
µg/L
accuracy
μL
[20]
[21]
< 15%; selectivit y
Biogenic
DLLM
Urine/
DCM/
Ethan
amines
E
1 mL
500 μL
-
MEKC-UV
0.5-
%RSD:
ol/
300
0.18-
1 mL
µg/mL
9.68; accuracy
30
[22]
91.9120.5% Amino acids
DDLL
Urine/
Carbon
ACN/
140–
GC–EI/CI–
0.05–
%RSD<
(sarcosine,
ME
5-150
tetrachl
150
155
MS and
0.1 ng/
10;
µL
oride/
µL
LC/ESI–MS
mL
%recover
alanine,
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leucine and
25 µL
y; 93.8–
proline) 2-
[23]
106 DDLL
chlorovinylar ME
Urine/
Chlorof
Metha
5 mL
orm/ 65
nol/
μL
500
sonous acid
250
GC–MS
1–400
%Recove
μg/L
ry: 95–
[24]
103
μL Neurotransm
DUAD
Rat
Bromob ACN/
itters
LLME
brain
enzene/
150 μ
microd
50 μL
L
120–
UHPLC–
0.05−3
%RSD:
286
MS/MS
000.0
3.2-12.8;
nM
accuracy:
ialysat
94.2-
es/
108.6%;
30 μL
%recover
[25]
y; 94.5105.5 di(2-
TC-IL
Urine/
[C6MI
ethylhexyl)p
DLLM
1 mL
M][PF6
and
hthalate and
E
]/ 50
164
mono(2-
-
mL
115
20–1920 µg/L HPLC/ DAD
%RSD: 0.4-7.2; %recover y: 80.4-
31
[26]
ethylhexyl)p
112.5
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hthalate 3-
IL-
Urine/
[C8MI
Aceto
Hydroxyben
DLLM
10 mL
M][PF6
ne/ 1
zo[a]pyrene
E
-
0.6–50.0 pg/mL
]/ 60 µL mL
%RSD:
[27]
HPLC- 2.2-6.8; HRMS
%recover
/MS
y: 87.896.2
Glyoxal and
Surfact
Urine/
1-
Ethan
102
methylglyox
ant-
2.5 mL undecan ol/ 0.5
al
assisted
ol/ 75
dispersi
μL
mL
0.1-25 ng/mL
LC-
%RSD:
and
fluores
5-10.6;
108
cence
%recover
[28]
y; 88–103
ve liquid– liquid microe xtractio n 4-
USA-
Urine/
ethyl
-
(Methylnitro
DLLM
5 mL
acetate/t
samino)-1-
E
23
LC/Tandem
5-1200
%RSD:
pg/mL
3.6-9.7;
oluene
accuracy;
(3-pyridyl)-
(1:2,
88.5-
1-butanol
v/v) / 2
93.7%;
mL
%recover
32
[29]
y; 88.593.7 Phosphatidyl
USA-
Blood/
DCM/
ACN/
ethanol
DLLM
25 µL
80 μL
E
-
LC-MS/MS
5 to
%RSD:
200 μ
500 ng
1.00-
L
/ mL
6.44;
[30]
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%recover y: 95.04105.28 Phthalate
USA-
Nail/
Trichlor Metha
metabolites
DLLM
30 mg
oethyle
nol/ 2
ne/
mL
E
-
UPLC-
0.3–
%RSD:
MS/MS
490 μg
6–17;
/L
accuracy;
180 μL
79-108 %
P.seudomon
Ceria@ Mouse
Chlorob CeO2
as
surfacta and
enz/ 10
@CT
assisted laser
aeruginosa
nt
sheep
µL and
AB/
desorption/ion
and
assisted blood/
chlorof
10–
ization mass
S.taphylococ
DLLM
orm/ 10
20 µL
spectrometry
cus
E
1 mL
[31]
-
µL
matrix
-
-
[32]
0.01–5
%RSD:1.
[33]
µM
85-4.11;
(MALDI-MS
aureus Hexanal and
SFO-
Human 1-
Metha
heptanal
DME
blood/- dodecan nol/ 50 μL
ol/ 50 μL
-
HPLC/UV
%recover y: 67.84-
33
70.28 Mercury SFOD
Saliva/
1-
6 mL
undecan
ME
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3-
USA-
-
182.
Cold vapor
0.025-
%RSD:
4
atomic
10.0
4.1
ol/ 60
fluorescence
ng/mL
μL
spectrometry
Portion Trichlor Metha
-
GC/MS-MS
10–
Phenoxybenz DLLM
of rat
oethyle
nol/
750
oic acid and
brain/
ne/ 100
500
ng/g
0.5 g
µL
µL
4-hydroxy-3-
E
phenoxybenz oic acid DCM: dichloromethane; DLLME: dispersive liquid-liquid microextraction; DUADLLME: derivatization-ultrasound-assisted dispersive liquid-liquid microextraction; TC-IL DLLME: temperature controlled ionic liquied dispersive liquidliquid microextraction; USA-DLLME: ultrasound assisted dispersive liquid-liquid microextraction; SFODME: solidified floating organic drop microextraction;
34
-
[34]
[35]
Table 3. Bioanalytical methods for determination of biomarkers using hollow fiber-based liquid phase microextraction Analyte
Matrix/s
Mode
Extractio
Enrich
ize
of
n
ment
LDR
Validation
Re f.
extrac technique factor
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tion Bisphenol A
Urine/ 1
Two
mL
phase
GC/MS
-
0.1–50
%RSD: 1.8–6.7;
ng/mL
%recovery: 98.8–
[37]
101. Bisphenol A and
Two
Urine
eight phthalate
phase
metabolites
GC/MS
20-
%RSD: 11.7-
/16
1000
19.7%
mL
µg/L
3-phenoxybenzoic
Urine/ 1
Three
HPLC/D
acid and 4-hydroxy-
mL
phase
AD
3-phenoxybenzoic acid
-
-
50–
%RSD, 1.6–12.6;
10,000
%recovery: 49.8–
ng/mL
55.1; accuracy: 93.3– 110.9%; stability under different storage conditions (20 °C for 2 months; 22°C for 6 h; and after three
35
[38]
[39]
freeze–thaw
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cycles). Hexanal and
Urine/3
Three
CZE-
heptanal
mL
phase
DAD
heterocyclic
Plasma/
Three
LC-
aromatic amines
0.2 mL
phase
MS/MS
-
-
1.75-
%RSD: 2.2-8.5%;
355
%recovery: 61–
µM
95%
5-80
%RSD: 4.5 and
pg/mL
8.8;
[40]
[41]
%recovery: 92-94 2-hydroxyestradiol,
Urine/
Two
2-hydroxyestrone, 4-
140 mL
phase
LC/MS
-
1.01 ×
%RSD: 5.8-11.8;
103-
%recovery: 91-
hydroxyestradiol, 4-
10.1
106;
hydroxyestrone,
pg/mL
stability: room
16α-hydroxyestrone,
temperature and
2-methoxyestradiol,
freeze thaw
[42]
and 2methoxyestrone Phthalic acid esters
trans-muconic acid
Two
Urine
phase
Urine/-
CE-UV
-
0.30–
%recovery: 77.65–
/1
80.0
106.86
mL
ng/mL
Two
HPLC/U
153–
0.005
%RSD: 2.7-8.1;
phase
V
182
to 1.2
%recovery: 83–92
mg/mL
36
[43]
[44]
Methyl hippuric acid
Three
Urine
HPLC/U
210-
10-
%RSD: 3.6-8.5;
phase
/ 10
V
312
50,000
%recovery: 83-98
mL
µg/L
Mandelic acid and
Three
Urine
HPLC-
hippuric acid
phase
/ 10
UV
-
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mL Hexanal and
Urine/10 Two
heptanal*
mL
[45]
GC/FID
-
phase
*determined by hollow fiber-based solid phase microextraction.
37
0.02–
%RSD: 2.5-10.7;
195
%recovery: 84–
mg/L
94%
0.020-
%RSD:3.2-9.3;
10.00
%recovery: 91.21–
µg/mL
97.51
[46]
[49]