Surfactant enhanced liquid-phase microextraction of basic drugs of abuse in hair combined with high performance liquid chromatography

Surfactant enhanced liquid-phase microextraction of basic drugs of abuse in hair combined with high performance liquid chromatography
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  See discussions, stats, and author profiles for this publication at: Surfactant enhanced liquid-phasemicroextraction of basic drugs of abuse in haircombined with high...  Article   in  Journal of Chromatography A · December 2005 DOI: 10.1016/j.chroma.2005.07.110 · Source: PubMed CITATIONS 74 READS 75 2 authors:Some of the authors of this publication are also working on these related projects: fabrication of new electrochemical sensors based on the novel nanocomposites for drug assay   ViewprojectExtraction of phthalate ester with different solid sorbent and determination by chromatography ViewprojectAli Sarafraz YazdiFerdowsi University Of Mashhad 74   PUBLICATIONS   1,735   CITATIONS   SEE PROFILE Zarrin Es'haghiPayame Noor University 87   PUBLICATIONS   1,241   CITATIONS   SEE PROFILE All content following this page was uploaded by Zarrin Es'haghi on 14 April 2014. The user has requested enhancement of the downloaded file.  Journal of Chromatography A, 1094 (2005) 1–8 Surfactant enhanced liquid-phase microextraction of basic drugsof abuse in hair combined with high performanceliquid chromatography Ali Sarafraz Yazdi a , ∗ , Zarrin Es’haghi a , b a  Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Azadi Sq., Mashhad, Khorasan 91775, Iran b  Department of Chemistry, Faculty of Sciences, Payame Noor University, Iran Received 16 June 2005; received in revised form 17 July 2005; accepted 25 July 2005Available online 19 August 2005 Abstract The aim of this study was to evaluate the performance of a technique for simultaneous testing of hydrophilic abuse drugs in hair. Theanalysis of, codeine and methadone in morphine hair included incubation in methanol (5h, 50 ◦ C), Surfactant enhanced liquid-phase microex-traction (SE-LPME) and HPLC analysis. This study has demonstrated that SE-LPME constitute a real alternative to the other liquid-phasemicroextraction methods, for pre-concentration and extraction of hydrophilic drugs in biological samples and has shown the advantages of these optimized methodologies over the traditional microextraction techniques. For these drugs recoveries in the range of 57.5–93.7 wereobtained from hair. The drugs were enriched by a factor of 61–128 during SE-LPME. Linearity ( r  2 , 0.9982–0.9997) was obtained in the rangeof 50–500  g/l for morphine and 10–500  g/l for codeine and methadone.© 2005 Elsevier B.V. All rights reserved. Keywords:  Basic drug of abuse; Surfactant enhanced liquid-phase microextraction; High performance liquid chromatography; Hair analysis 1. Introduction Hair analysis to detect drug abuse is a new perspective inforensic toxicology [1,2]. In 1979, for the first time Baum- gartner et al. [3] reported the detection of opiates in hair and this was followed by several reports on the detectionof other drugs in hair by different methods like, solid phaseextraction, solid phase microextraction and GC–MS, or tan-dem MS [4–7]. As biological matrix such as plasma and urine, hair gives particular advantages such as; the stabilityof a specimen, non-invasive sampling, broad time detectionwindow and it can be stored and transported without spe-cific tanks [8]. But, drug determination in the human hairand/or biological fluids is often complicated by low ana-lyte concentration and the complex sample matrix. Becauseof this, sample preparation is crucial in drug analysis and ∗ Corresponding author. Tel.: +98 511 8403811; fax: +98 511 8438032.  E-mail address: (A. Sarafraz Yazdi). includesbothanalytepre-concentrationandsampleclean-up.Recently, Pedersen–Bjergaard and Rasmussen introduced analternativeconceptforthree-phasemicroextraction,asapow-erful sample preparation technique for drug analysis, basedon the use of disposable low-cost porous hollow fibers madeofpolyproylene[9–11].Inthissamplepreparationtechnique, analytes are extracted through an aqueous solution (donorphase) into an organic liquid immobilized within the poresof the hollow fiber before they are trapped with the aque-ous acceptor phase, that is contained within the lumen of theporous hollow fiber and thus microextracts are not in directcontact with the sample solution.TheextractioninvolvespHadjustmentofthesamplesolu-tion to a pH where the analytes are uncharged. The analytesare extracted through the organic phase immobilized in theporesofthehollowfiberandintotheaqueousacceptorphase,that has a pH where the analytes are charged preventingthem from back diffusion into the organic solvent [12,13].Hydrophobic analytes are easily extracted into organic sol- 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2005.07.110  2  A. Sarafraz Yazdi, Z. Es’haghi / J. Chromatogr. A 1094 (2005) 1–8 Fig. 1. Structure of the tested drugs and their p K  a  and log P o/w  values [8,38]. vents from the donor aqueous phase, but hydrophilic andpolar analytes have low solubility in the water immiscibleorganic solvents. Therefore, these analytes are difficult toextract by three-phase LPME. For enhancing the analyte sol-ubilityintheorganicsolvents,weusednon-ionicsurfactants.It is well known that surfactant, or surface-active agents,are amphiphilic molecules, the head of which is polar, orhydrophilic, and the tail hydrophobic. The tail is generallya hydrocarbon chain with different member of carbon atomsand may be linear or branched, and also contain aromaticrings. The surfactant molecules can be associated in aque-ous solution to form molecular aggregates called micelle,the minimum concentration of surfactant required for thisphenomenon to occur is called critical micellar concentra-tion (CMC). One of the most important properties of thesecompounds is their good capacity to solubilize solutes of different character and nature [14–16]. These solutes may interact electrostatically, hydrophobically or by a combi-nation of both effects. This capacity of the surfactants tosolubilize different compounds has been used to developthe extraction and the pre-concentration of organic com-pounds and for bio-analysis of different basic drugs as modelcompounds.Themodeldrugs,morphine,codeineandmethadonewereselected to present a broad range of hydrophilicity, seelog P o/w  values, Fig. 1. 2. Experimental 2.1. Chemicals and reagents The drugs, methadone hydrochloride, codeine phosphate,were obtained from Sigma (St., Louis, Mo, USA). Morphinesulfate was obtained from H. Lundbeck (Copenhagen, Den-mark)andalldrugsweregiftsfromtheMinistryofhealthandcure (Center of Khorasan, Iran) and administration of Justifi-cation(Khorasan,Iran).MethanolwaspurchasedfromFluka(Buchs SG, Switzerland). The other compounds were fromMerck (Darmstadt, Germany). Triton X-100 was obtainedfrom Merck, Tween 20 and Nonoxynol-9 were from Sigma.These compounds were all of analytical grade. The Deion-ized water and solutions were filtered by a Milli-Q filteringsystem (Millipore). 2.2. Hair samples A bulk of blank hair, necessary for method developmentand validation, was obtained from a men hairdresser’s shop.The absence of opiate was verified.Hair samples were collected from 20 men ranging from16 to 45 years old. They were captured by the police andfor most of them, screening tests were positive for drug of abuse. Some of the addicted persons were under therapeutictreatment.A standard of hair of about 5mm in diameter was cutfrom close to the scalp at the vertex posterior area, foldedin aluminium foil, and the proximal and distal ends marked.Samples 2–4cm long was selected for analysis. 2.3. Hair analysis The hair, was washed with different solvents as follow:20mldichloromethane,15mlacetone,15mlmethanol,10mlmethanol,atroomtemperaturefor5minandthenitwasdried.The last washing solvent was tested with GC for checkingresidual content of opiates. 2.4. Digestion of hair matrix  The washed and dried hairs was finally cut into approxi-mately1mmpiecesanddigestedbythefollowingprocedure;2mlmethanolasanextractingsolventwasaddedto50mgof hair, in a 10ml screw-cap tube. The pH was adjusted to 7.4by phosphate buffer solution. The samples were incubated at50 ◦ Cfor5h[17].Incaseofaremainingsolidmatrix,extracts were filtered. The remaining was rinsed with 0.5ml ethanoland both fractions were evaporated to dryness at 40 ◦ C undera steam of nitrogen.   A. Sarafraz Yazdi, Z. Es’haghi / J. Chromatogr. A 1094 (2005) 1–8  3 2.5. Stock and working solutions Stock solutions containing 1mg/ml of morphine sulfate,codeine phosphate and methadone hydrochloride were pre-pared, in methanol and stored at 4 ◦ C. Standard calibrationcurves were obtained by adding calculated amounts of thestandards into methanolic solution of 50mg finely cut blank hair. These spiked samples were digested and the calibra-tion curves were obtained. Limit of detection (LOD) andlimit of quantification (LOQ) of the analytes were deter-mined by decreasing concentrations of spiked samples untilsignal to noise ratio (S/N) of 3 and 10 were obtained,respectively.The concentration of analytes in the hair blank samplesfor validation were 20, 50, 100, 300, 500, and 1000ng/ml.All solutions stored at 4 ◦ C and protected from light. 2.6. HPLC system The HPLC system used in this work was a Waters (Mil-lipore. Co, Milford, MA, USA) and consisted of a Waters(488) Tune able absorbance detector and a Waters 746integrator.Themonolithicsilicacolumnswereevaluatedinreversed-phase HPLC. These showed lower plate heights and muchlower pressure drops [18,19] than the conventional columns packed with the 5  m C 18  silica particles. Therefore, weused of a Chromolith performance RP-18e column (4.6mmdiameter 100mm length, 2  m macro-pore size and 13nmmeso-pore size) from Merck (Darmstadt, Germany). A RP-18 guard column was fitted upstream of the analyticalcolumn.The mobile phase consisting of 10mM KH 2 PO 4  at pH3-acetonitrile (93:7) which was filtered by Milli-Q filteringsystem, was delivered by a Waters LC-600 HPLC pump.The flow rate of the mobile phase was 3ml/min and theUV detection wavelength was set at 211nm. 2.7. LPME equipment  The experimental setup is illustrated in Fig. 2. 2.0cmlength of polypropylene hollow fiber (1200  m I.D., a wallthickness of 150  m, a pore size of 0.2  m and a porosityof 70%) flame-sealed at the one end and was plunged intothe organic solvent for 5min to immobilize the pores, andthen the excess of the solvent was removed. 3.0ml samplesolution (first phase) was held in a 5.0ml sample vial, andthepolypropylenehollowfiber,impregnatedwiththeorganicsolvent (second phase) was adjusted and immersed in thesample solution, perfectly. Then, 10  l the acceptor phasewas added into the internal hole of the hollow fiber by amicrosyringe.Thehollowfiberwasshapedwithastarlikedprofileusingheat press. This shape was selected for increasing the contactarea of the hollow fiber with donor and acceptor solutionsand keeping volume of internal hole of the fiber at minimum. Fig. 2. SE-LPME extraction device: (a) HPLC syringe; (b) vial cover; (c)conical guide; (d) acceptor phase (pH 2.0); (e) 2 Cm, hollow fiber with starliked profile; (f) donor phase (pH 10.0); (g) glass vial and (h) stirring bar. The length of the hollow fiber was reduced to 2.0cmand the reduced length was compatible with small sam-ple volumes, which are highly relevant in some analytes inthe biomedical and environmental applications. In addition,enrichment of the analyte increases with increasing the vol-ume ratio of sample solution to acceptor solution [20].A conical guide was placed on the top of the fiber toensure that the microsyringe needle was effectively guidedintothefiber.A25  lmicro-syringe,withaconetip(0.49mmO.D.) (Hamilton, Reno, NV, USA) was used for delivery andremoval of the acceptor phase. Before each extraction, thesyringe was rinsed with acetone and then with de-ionizedwater for 10 times to avoid the analyte carry-over and airbubble formation. Prior to use the fiber was dept into ace-tone for 3h to remove the contaminations. An aluminiumfoil was used to cover the vial during extraction to preventthe evaporation of the organic phase. The solution was agi-tated with a stirring rate of 1000rpm during the extractionprocess. Because of the fiber is very inexpensive, we usedfrom any fiber in one period of extraction, thus was avoidedthe sample carry-over. 2.8. LPME procedure Three millilitres of donor phase, with pH 10.0 (asdescribed in Sections 2.4 and 2.5) was added into a 5.0ml vial. The hollow fiber was dipped into  n -octanol for 5.0minand then the excess of the solvent was carefully removed.Subsequently, 10.0  l of HCl solution (acceptor phase, pH2) were injected into the lumen of the hollow fiber with amicrosyringe. This fiber was placed into the sample solutionpresent in the vial.Thesampleswerestirredat1000rpmfor40min.Aftertheextraction, the total volume of acceptor phase was injectedinto HPLC with monolithic column for further analysis.  4  A. Sarafraz Yazdi, Z. Es’haghi / J. Chromatogr. A 1094 (2005) 1–8 2.9. Calculation of extraction recoveries and analysesenrichments Theextractionrecovery(  R ),wascalculatedbythefollow-ing equation: R  =   n a , final n s , initial  100%  =  V  a C a , final V  s C s , initial  100% (1)where  n s,initial  and  n a,final  are the number of moles of ana-lytes srcinally present in the sample and finally collectedin the acceptor solution, respectively.  V  a , is the volume of acceptor phase and  V  s , the volume of sample,  C  a,final , thefinal concentration of analyte in the acceptor phase, and C  s,initial , is the initial concentration of analyte within thesample.The analyte enrichment factor (EF) was calculated by thefollowing equation:EF  = C a , final C s , initial (2)These calculations are previously reported [4,21]. 3. Results and discussion 3.1. Theoretical notations The main aim of three-phase LPME is to increase enrich-ment and clean-up of analytes from environmental samplesand biological fluids, prior to HPLC or CE. In this technique,theaqueousacceptorphaseisinjecteddirectlyintotheHPLCor CE without further changes. These considerations affectthe selection of phases as the most important factor in thistechnique.In the LPME device, sample solution and acceptor phaseare separated by membrane. The contact area between donorand organic phases is limited because of the large volumeof sample respect to the organic phase immobilized in thepores of the hollow fiber. This situation limited the extrac-tion and furthermore, some ionic analytes are highly water-solubleandhaveaninsignificanttendencytomigratetowardsa lipophilic membrane. Therefore, the approach of addingthe surfactants directly to the sample solution was investi-gated.Surfactant enhanced transport through a liquid membranedepends on a number of parameters such as the nature andconcentration of the surfactant, partition coefficient of theanalyte under these conditions, properties of the organic sol-vent, sample agitation rate, etc.The extraction in this mode is separated into three stages.The first involves extraction of the analyte from the samplesolution to the organic phase immobilized in the pores of hollow fiber. The sample solution is added to buffer solutioncontainingnon-ionicsurfactant.ThebufferadjustedthepHof thesamplesolutiontoapHwheretheanalytesareunchargedand neutral, to advance formation of a hydrophobic species.In this experiment the sample solution has a pH of 10.Thesecondstageisextractionoftheanalyteintothemem-brane phase and diffusion of the analyte-surfactant throughthe membrane.The third stage involves back extraction of analyte to theacceptor phase. In the meantime, at the interface between thedonor and the organic phase, non-ionic surfactant molecules(near,butunderthecriticalmicellarconcentrationlimit)gath-eredandenhancedtheanalytetransferintotheorganicphase.The fraction of surfactant molecules, according to their sizeandtendencyfororganicsolvent,alsoaretransferredintotheorganic phase and in this phase, form reverse micelle. Thisphenomenon occurs because the volume of the organic sol-vent is smaller than the sample solution (donor phase) andsurfactant enriched in this phase and raise up to the CMC.Thus the analyte dissolve strongly by the micelles and is pre-vented from the back extraction into the donor phase.At the interface between the organic phase and the accep-tor phase, the micelle releases the analyte into the acceptorphase.TheacceptorsolutionhasanacidicpHandtheanalytemolecules are ionized within the acceptor solution, they areprevented from re-entering the organic solvent in the poresof the hollow fiber. Since the volume of donor phase is verysmall, the analyte is pre-concentrated within the acceptorsolution,thenon-ionicsurfactanthasnotinclinationforgoingto the strong acidic acceptor phase.Inordertotestthismodelseveralexperimentswerecarriedout. The initial experiments were based on mixing 3.0ml of working solution containing the 1  g/ml of analytes with pH10.0.Thevolumeofacceptorsolutionwas10.0  l.Through-out the experiments the sample solution were stirred for40.0min. 3.2. Optimization of surfactant enhanced liquid-phasemicroextraction (SE-LPME) In SE-LPME, extraction needs to be carried out underconditions in which the pre-concentration factor will be themaximum or the extraction yield will be 100%. This goaldepends on the various factors and the extraction process canbe also altered by different factors such as: organic solvent,extraction time, stirring rate, pH, concentration and nature of thesurfactants,etc.Theeffectofthesefactorsonthepercent-age of extraction of the analyses studied therefore needs tobe established. 3.2.1. Organic solvent  Organic solvent to be immobilized as liquid membrane isan important agent in SE-LPME, and several factors have tobe taken into consideration.The analyte in the sample solution (donor phase) shouldhave high partition coefficients into the organic solvent inthe pores of the membrane. Solvents of low viscosity arepreferred as low viscosity provides large diffusion throughthe membrane. In addition, the water solubility should be as
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