Separation of very hydrophobic analytes by micellar electrokinetic chromatography. I. Optimization...

Carolin Huhn1

Michael Pütz2

Ivie Holthausen1

Ute Pyell1

1Department of Chemistry,University of Marburg,Marburg, Germany

2Bundeskriminalamt – FederalCriminal Police Office,Forensic Science Institute,KT 34 – Toxicology,Wiesbaden, Germany

Received March 2, 2007Revised June 28, 2007Accepted July 18, 2007

Research Article

Separation of very hydrophobic analytes bymicellar electrokinetic chromatography.I. Optimization of the composition of thesample solution for the determination of thearomatic ingredients of sassafras and otheressential oils of forensic interest

A micellar electrokinetic chromatographic method using UV and (UV)LIF detection in-linewas developed for the determination of aromatic constituents, mainly allylbenzenes inessential oils. The method optimization included the optimization of the composition of theseparation electrolyte using ACN and urea to reduce retention factors and CaCl2 to widenthe migration time window. In addition, it was necessary to optimize the composition of thesample solution which included the addition of a neutral surfactant at high concentration.With the optimized method, the determination of minor constituents in essential oils waspossible despite of the presence of a structurally related compound being in a molar ratioexcess of 1000:1. The use of UV and LIF-detection in-line enabled the direct comparison ofthe two detection traces using an electrophoretic mobility x-axis instead of the normal time-based scale. This simplifies the assignment of signals and enhances repeatability. Themethod developed was successfully applied to the determination of minor and major con-stituents in herbal essential oils, some of them being forensically relevant as sources ofprecursors for synthetic drugs.

Keywords:

Electrophoretic mobility scale / Essential oils / Hydrophobic compounds / Infiniteelution range / MEKC DOI 10.1002/elps.200700156

526 Electrophoresis 2008, 29, 526–537

1 Introduction

The aim of this project was to determine the aromatic etherconstituents of essential oils prepared from various plantmaterials. The main focus was set to those plant materialsbeing of forensic interest containing very high amounts ofsafrole. The forensic background is a fast-forward synthesisof the controlled substance methylenedioxymeth-amphetamine (MDMA), the active main ingredient of mostof the clandestinely manufactured Ecstasy tablets, which isbased on safrole (4-allyl-1,2-methylenedioxybenzene). Thisprecursor can be found in the essential oils of differentplants, mainly from the family of lauraceae [1, 2]. Econom-ically interesting are Sassasfras albidum, Cinnamomum cam-phora, and Ocotea pretiosa. These oils, as well as pure safrole,

are subject to precursor monitoring acts in Germany andmany other countries. Small amounts of safrole were alsoreported for the essential oils of spices, e.g., Myristica fragrans(nutmeg) and Illicium verum (star anise). Minor aromaticconstituents reported in these essential oils are, e.g., eugenol,methyleugenol, asaron, and anethol [3, 4]. For the analysis ofessential oils of lauraceae, mainly TLC and GC procedures[5–12] and in more recent times also HPLC procedures [13–15] were developed.

The analysis of safrole came into focus of interest ofpublic authorities when it was banned as a food and fra-grance additive due to its toxic and carcinogenic properties inthe 1960s in the US. In literature safrole analysis is domi-nated by GC and later HPLC techniques [16–20]. For exam-ple, an HPLC-spectrofluorimetric method (lex = 295 nm) forthe determination of safrole in fragrances was described byWisnewski et al. [21].

Very few studies concerning the forensic background ofsafrole were published. Lukaszewski [22] analyzed safrole asan impurity in MDMA samples synthesized from isosafrole.Noggle et al. [23] reported the analysis of samples from aclandestine laboratory involved in the synthesis of Ecstasy.

Correspondence: Professor Ute Pyell, Department of Chemistry,University of Marburg, D-35032 Marburg, GermanyE-mail: [email protected]: 149-6421-2822124

Abbreviations: MDMA, methylenedioxymethamphetamine;TTAB, tetradecyltrimethylammonium bromide

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2008, 29, 526–537 CE and CEC 527

These samples included a sassafras oil as well as reactionmixtures from the first steps of the synthesis of Ecstasy. Thesame group also reported the synthesis of the m-methoxy-derivative of MDMA, 3-methoxy-(3,4-methylenedioxy-methamphetamine) (MMDMA) from myristicin extractedfrom ground nutmeg [24].

The determination of aromatic ether compounds inessential oils, here mainly oils derived from S. albidum, C.camphora, and O. pretiosa provides specific problems. Firstlya method with high separation efficiency and selectivity isneeded to separate safrole from its isomer isosafrole andsecondly a high tolerance towards matrix constituents and ahigh concentration loadability are necessary to avoid cross-contaminations or excessive rinsing steps of columns (asmight be expected in GC and HPLC analysis). Thirdly themethod used must provide the quantitative determination ofminor compounds in a matrix of a structurally related com-pound, e.g., isosafrole in an excess (molar ratio) of about1000:1 of safrole to permit an assignment to the plant sourceof the analyzed oil based on the minor component composi-tion. In this paper we investigate, whether all these require-ments can be met by employing micellar EKC (MEKC) incombination with UV-absorbance and LIF detection (arran-ged in-line). According to our knowledge, there is only onepublication dealing with the analysis of the ingredients ofessential oils, e.g., rose, anise, and geranium essential oilswith MEKC [25].

In this paper figures of merit for the method developedwill be presented and its applicability to real samples (herbalessential oils) will be shown. Further forensically relevantresults have been published elsewhere [26].

2 Materials and methods

2.1 Chemicals

Methanol, NaH2PO4, eugenol, safrole, thymol, piperonal,and asaron were from Fluka, Buchs, Switzerland; urea andSDS were from Roth, Karlsruhe, Germany; sodium tetra-borate, methyl 4-cyanobenzoate, CaCl2, isosafrole, acet-ophenone, propiophenone, butyrophenone, valerophenone,and Triton X-100 were from Merck, Darmstadt, Germany;ACN from J. T. Baker, Deventer, The Netherlands; methyl-eugenol and trans-anethol were from Aldrich, St. Louis,USA; myristicin was from Sigma, Steinheim, Germany.Hexanophenone, heptanophenone, octanophenone, decano-phenone, and dodecanophenone were from Aldrich. Tetra-decyltrimethylammonium bromide (TTAB) was fromAldrich, Steinheim, Germany and CTAB was from Sigma,Deisenhofen, Germany. Structures of the analytes taken intoconsideration are depicted in Fig. 1.

S. albidum and M. fragrans oils were bought from Aldrich– Flavors & Fragrances, Milwaukee, USA. Seized sampleswere provided by the Federal Criminal Police Office,Germany.

Figure 1. Structures of the analytes and the two internal stan-dards selected, only the trans-isomers are shown.

2.2 Buffer and sample preparation

Analytes were dissolved in methanol at a concentration of10 mmol/L. The optimized separation electrolyte consistedof 7.5 mmol/L sodium tetraborate, 60 mmol/L SDS, pH 9.2,4 mol/L urea, 20% v/v ACN, and 0.5 mmol/L CaCl2. Opti-mized sample solutions contained the nonionic surfactantTriton X-100 as solvating agent. Small volumes of the oilsamples or the mixture of standards were first dissolved in250 mL methanol and then diluted to a total volume of1000 mL with a Triton X-100 solution (25 g/L). Overall dilu-tions of 1:50, 1:100, 1:1000, and/or 1:10 000 of the originalsample were obtained with a defined concentration of TritonX-100.

2.3 Instruments

For the determination of the ingredients of essential oils aPrinCE CE instrument (Prince Technologies BV, Emmen,Netherlands) was used. The sample injection was donehydrodynamically at 50 mbar and 3 s. Fused-silica capillariesfrom Polymicro Technologies LLC (Phoenix, AZ, USA) wereused with an inner diameter of 50 mm and an outer diameterof 363 mm. The length was set to 53.5/60/75 cm (for UV andLIF detection). New capillaries were conditioned by flushingthem first with NaOH solution (0.1 mol/L) for 10 min andsubsequently with run buffer for 10 min. A rinsing with runbuffer for 1 min was used for cleaning the capillary betweenruns. A voltage of 30 kV was used for separation.

UV detection was accomplished with a Spectra 100 fromThermo Separation Products, San José, USA, equipped withan in-capillary detection cell using a wavelength of 240 nmand a data sampling rate of 4 Hz. For data acquisition thesoftware Weinanalytik HPLC Mono from Planum, Kirch-hain, Germany was used.


528 C. Huhn et al. Electrophoresis 2008, 29, 526–537

The LIF detection system was a TIDAS LIF 266 systemfrom J&M Analytische Mess- und Regeltechnik, Aalen,Germany which has been described by Pütz [27] and Huhnet al. [28]. The excitation radiation source was an FQSS266-S laser from Crystal, Berlin, Germany, which is a passivelyQ-switched frequency-converted diode-pumped solid-statemicrochip Nd:YAG laser (neodymium:yttrium–aluminumgarnet) operated at an optical quasi-cw power of about50 mW at 266 nm. A photomultiplier detector was used formeasuring the fluorescence intensity. Optical fibers con-nected the excitation source and the photomultiplierdetector to the in-capillary detection cell, placed in a com-mercially available capillary holder (J&M Analytische Mess-und Regeltechnik). The optical arrangement of this capil-lary holder has been optimized with respect to maximizingthe intensity of the excitation radiation in the inner volumeof the detection cell, minimizing instrumental bandbroadening, and maximizing the fraction of emitted fluo-rescence light collected by one of the optical fibers [29].The S/N was improved by optical filters (long pass filterLWP337 from Delta light & Optics, Lyngby, Denmark incombination with the dichroic band pass filter 505FD64from LOT-Oriel, Darmstadt, Germany), which were placedbetween the detection volume and the photomultiplierdetector. The long pass filter (LWP337) absorbs radiation atthe excitation wavelength of 266 nm and the dichroic bandpass filter (505FD64) reflects the wavelength of the secondharmonic (532 nm) of the exciting solid-state laser and theemission wavelength of the pump laser diode (808 nm).The detection window for collecting the fluorescence lightis given by the optical filters used, here a range of 340–500 nm is covered.

For the separation of the homologous series of alkylphenones a Beckman P/ACE 5510 with PACE station soft-ware was used equipped with a DAD set to a wavelength of240 nm at a sampling rate of 10 Hz. In order to maintain lowanalysis times for the separation of alkyl phenones and tomonitor the signal of Triton X-100, a short capillary of 20/27 cm was employed. The injection was done at low pressurefor 3 s. A voltage of 10 kV was used for separations using thisinstrument.

Origin 6.0 Professional software from Microcal Software,Northampton, USA was used for data analysis.

3 Results and discussion

3.1 Selection of analytes

The analytes selected for this study are structurally relatedto safrole (Fig. 1). All analytes contain a benzene ring withmethoxy, methylenedioxy, or hydroxy substituents. Exceptfor thymol, a propylene group is present with a double bondin conjugation to the aromatic ring or a terminal doublebond. All substances are known constituents in essentialoils of S. albidum, C. camphora, or O. pretiosa. We used two

internal standards, one for each detection system: IS1methyl-4-cyanobenzoate (UV), IS2 acridone (LIF) (seeFig. 1).

3.2 Optimization of the composition of the

separation electrolyte

The optimization of the buffer system to achieve baselineseparation of the analytes of interest included the choice ofthe surfactant (SDS, CTAB, and TTAB were tested) and theaddition of different organic modifiers in order to stronglyreduce retention factors (CDs, urea, and the organic solventsACN, methanol, and isopropanol). The optimum buffercomposition regarding the resolution of critical peak pairswas a disodium tetraborate (7.5 mmol/L, pH 9.2) separationbuffer consisting of 60 mmol/L SDS, 4 mol/L urea, and 20%v/v ACN (empirical optimization of the separation electrolytecomposition). The resolution of critical peak pairs was fur-ther improved by using a smaller bore capillary, 50 mminstead of 75 mm. In order to further increase the resolutionof the most closely migrating peak pairs thymol/safrole andasaron/isosafrole, the divalent cation Ca21 (CaCl2) was addedin a low concentration (0.5 mmol/L). In a former publicationwe have shown that the addition of CaCl2 to a separationelectrolyte containing TTAB can be used to reduce the EOFvelocity, thus widening the migration time window [30]. Inour case a restricted elution mode [31] with an infinite elu-tion range was reached.

With this separation electrolyte baseline separation wasachieved for all analytes except the peak pair thymol/safrolewith R = 1.26 as shown in Fig. 2A. Baseline resolution wasachieved for safrole and isosafrole as well as for eugenol andisoeugenol. In addition, the cis- and trans-isomers of thementioned isoforms can be distinguished, the relative peakheights correspond well with NMR data. The separation ofcis- and trans-isomers by MEKC has already been reported byWan et al. [32] for isomeric epoxy fatty acids at a low SDSconcentration. The migration order of the analytes correlateswith their molecular structure: polar keto or phenolichydroxy groups give rise to low retention factors. Analyteshaving an internal double bond in the propylene unit are lesspolar and are more strongly retarded than analytes having aterminal double bond. Six analytes (cis/trans-isoeugenol,asaron, cis-/trans-isosafrole, and anethol), all with an internaldouble bond in the propenyl group, can be detected by LIFdetection at an excitation wavelength of 266 nm. The otheranalytes have too low fluorescence quantum yields.

3.3 Optimization of the composition of the sample

solution

3.3.1 Solubilization versus band broadening

In addition to the optimization of the composition of theseparation electrolyte, an optimization of the composition ofthe sample solution was necessary to provide a sufficiently



Figure 2. Separation of standards employing UV (solid line) andLIF detection (dashed line) in-line; (1) piperonal, (2) eugenol, (3)cis-/trans-isoeugenol, (4) methyleugenol, (5) thymol, (6) safrole,(7) myristicin, (8) asaron, (9) cis-/trans-isosafrole, and (10) ane-thol; IS1: methyl-4-cyanobenzoate, IS2 acridone; separation buf-fer: 7.5 mmol/L sodium tetraborate, 60 mmol/L SDS, 4 mol/Lurea, and 0.5 mmol/L CaCl2 at a pH of 9.2 and 20% v/v ACN;injection 50 mbar63 s, 30 kV; capillary (535, 600)700 mm650 mm; concentrations of the analytes: 125 mmol/L for piperonal,isoeugenol, asaron, isosafrole, and anethol; 250 mmol/L foreugenol, methyleugenol, safrole, and myristicin; 300 mmol/L forthymol; and 150 mmol/L for the internal standards. (A) Time scale,(B) transformation into effective electrophoretic mobility scale.

homogeneous dispersion of the sample constituents withoutcompromising the separation efficiency. The hydrophobicityof the analytes and the high content of safrole in the essentialoils investigated (at a concentration of up to 1 mol/L in thesample) make it impossible to use diluted run buffer as sol-vent for the sample solution. In this case, no homogeneousphase is formed, oil droplets are clearly visible. The use of ahigh amount of an organic solvent (ACN, methanol, iso-propanol, acetone) or organic modifiers (CDs) had a markedimpact on the subsequent separation performance or did notyield a homogeneous solution. In some cases peak splittingof the analyte signals was observed as also described in[33–37].

Surfactants are commonly used as dissolving agents.Altria et al. [36] showed that diluted SDS solutions(10 mmol/L) can be employed as sample diluents withoutreduction of the separation efficiency (excellent peakshapes). However, higher concentrations of up to 500 mmol/L SDS produced peak splitting. Our idea was to produce astable macroemulsion as sample solution (two-phase sys-tem), not a complete dissolution of the oil samples. Bandbroadening occurred using the ionic surfactants SDS, TTAB,and CTAB in the sample solution. Kelly et al. [38] used thenonionic surfactant Polysorbate 80 as dissolving agent forbasic water insoluble drugs in CE at a concentration of100 g/L. Quirino and Terabe [39] mentioned nonionic sur-factants as a feasible alternative to improve the solubility ofhydrophobic neutral analytes, but they used the neutral sur-factant Brij 35 only to stabilize the sample solution and tolower adsorption effects. We used Triton X-100 as a neutralsurfactant to obtain an oil-in-water emulsion, which showedonly a moderate influence on the separation efficiency com-pared to the sample solutions described before. Only themore hydrophobic solutes show a slight peak broadening,but the baseline resolution for all solutes can be maintained.

In a further study we optimized the content of TritonX-100 in the sample solution within the concentration range0–100 mg/L (ca. 0–160 mmol/L) (Fig. 3). To 150 mL of themethanolic solution (see Fig. 2) of standards 350 mL of anaqueous Triton X-100 solution at varying concentration of thenonionic surfactant were added to yield a total volume of500 mL. The electropherograms in Fig. 3 reveal that theseparation efficiency decreases with a higher Triton X-100content, but the influence on peak broadening is acceptableat a concentration lower than 37.5 g/L in the final samplesolution. For the sample preparation of essential oils a Triton

Figure 3. Influence of different concentrations of the Triton X-100solution (0–75 g/L) in the sample solution. For separation condi-tions refer to Fig. 2. Concentrations of Triton X-100 in the injectionsolution as given in the legend.



X-100 concentration in the sample solution of 18.75 mg/mLwas selected. Increasing the Triton X-100 content in thesample solution a reduction of migration times for thehydrophobic analytes was observed. This can be explained bythe formation of mixed micelles in the sample plug with anelectrophoretic mobility lower than the electrophoretic mo-bility of pure SDS micelles. At c(Triton X-100) ,37.5 g/L theplate numbers for the recorded zones are reduced by a factorof 2 relative to a separation with a methanolic/aqueous sam-ple solution (data not shown).

The optimized sample solution contained 33% v/vmethanol and a concentration of Triton X-100 of 18.75 g/L.The sample solutions prepared from essential oils areopaque, indicating a macroemulsion (presumably oil-in-water emulsion) [40]. These emulsions proved to be stablefor months, especially in case of the sassafras oils. Fornutmeg oils a relatively fast (less than 1 h) phase separa-tion was observed, but freshly prepared samples could beanalyzed without problems. Thus, only using a neutralsurfactant as solubilizing agent in the sample solution, theanalysis of the ingredients of essential oils becomes possi-ble.

3.3.2 Influence of Triton X-100 in the sample solution

on peak parameters of analyte zones

We studied the influence of Triton X-100 in the sample solu-tion on the separation process in order to understand theimpact of this modifier during the separation process. Inorder to keep analysis times low, a shorter capillary (20/27 cm) was used. In order to cover a broader range of reten-tion factors of analytes, alkyl phenones, which have a struc-tural similarity to the analytes, were used as model analytes.Figure 4 shows the electropherogram at a detection wave-length of 240 nm corresponding to the absorption minimumof Triton X-100. The peaks of the alkyl phenones(c = 0.25 mmol/L) as well as a band due to Triton X-100(c = 20 g/L) are present. Triton X-100 shows a broad band inthe electropherogram, the beginning of this band is char-acterized by several small maxima indicating the separationof monomers with different alkyl chain length (in literature2–12 polyoxyethylene units [41] are described). Thus, TritonX-100 can be treated as an analyte contained in the samplesolution. On the other hand, however, Triton X-100 also actsas a modifier of the separation of the other analytes presentin the sample solution due to the formation of mixedmicelles, becoming part of the pseudostationary phase andchanging its electrophoretic mobility as well as the phaseratio (micellar phase/aqueous phase). The modification ofthe pseudostationary phase reduces migration times of theanalytes and has a negative impact on the separation effi-ciency including peak tailing which is more pronounced forthe more hydrophobic analytes with higher retention factors.Two processes are involved in the band broadening processobserved: (i) the effective electrophoretic mobility of themixed micelles (the velocity of the pseudostationary phase) is

Figure 4. Separation of alkylphenones with a separation electro-yte of 7.5 mmol/L borate, 60 mmol/L SDS, pH 9.2, 4 mol/L urea,0.5 mmol/L CaCl2, 20% v/v ACN; composition of the injection so-lution: 50 mL of a methanolic mixture of standards of phenones(1 mmol/L each) were dissolved in Triton X-100 solution (c (TritonX-100) = 20 g/L). Concentrations of Triton X-100 in the samplesolution see legend. labs = 240 nm, injection low pressure, 3 s,10 kV, (1) acetophenon, (2) propiophenon, (3) butyrophenon, (4)valerophenon, (5) hexanophenon, (6) heptanophenon, and (7)Triton X-100.

reduced compared to the effective electrophoretic mobility ofSDS micelles. This process affects the apparent retention ofthe analytes. (ii) In the sample plug, the analytes are solubi-lized by Triton X-100 micelles. The CMC is very low(0.32 mmol/L [42]), a high phase ratio is given. When the fastSDS monomers enter the sample plug from the detector sideof the sample plug, they are incorporated into the TritonX-100 micelles, mixed micelles are formed. These mixedmicelles have synergistic effects on the solubilization ofanalytes due to diminished steric and electrostatic repulsionand a high phase ratio. Retention factors in the mixedmicelles are higher than in pure Triton X-100 and in pureSDS micellar phases. The strong tailing effect observedmainly for the more hydrophobic analytes is presumablybased on the electrophoretic heterogeneity of the micellarphase (mixed micelles) and on the synergistic behavior ofmixed micelles concerning their solubilization properties.Analytes have a higher distribution factor in this mediumthan with exclusively SDS containing micellar phase. Thetransition into the micelles with high molar fraction of SDSat the end of the sample plug is retarded by the mixedmicelles. These effects induce peak broadening and peakasymmetry. The higher impact of these effects on the peaksof the more hydrophobic analytes is due to their longer stayin the mixed micelle zone compared to the less hydrophobicanalytes (see Figs. 3 and 4). This peak broadening process iscounteracted by a focusing effect similar to sweeping due to ahigher molar fraction of SDS at the detection side of thesample plug.



3.4 Transformation into the electrophoretic mobility

scale

A direct comparison of the two detection traces in Fig. 2Ais not possible. The difference of 5 cm between the detec-tion windows gives rise to differences in the migrationtimes of more than 1 min. In order to simplify peak iden-tification, the time based x-axis has to be transformed intoa magnitude which is independent of the length of theseparation chamber. In CE this is done by the calculationof the effective electrophoretic mobility meff. The transfer ofmigration times into electrophoretic mobilities has beendescribed as a method to enhance repeatability via nor-malization of data on varying EOF velocity [43]. The calcu-lation of the electrophoretic mobilities was done accordingto Eq. (1):

meff ¼ mint þLdLtðtm � tintÞ

Vtmtint(1)

where meff is the effective electrophoretic mobility of theanalyte, mint the effective electrophoretic mobility of theinternal standard, Ld the capillary length to the detector, Lt

the total length of the capillary, tm and tint are the migrationtimes of the analyte and the internal standard, respectively,and V the applied voltage. The migration time of an internalstandard is used as reference in this calculation. This pro-cedure is preferable to the calculation via the EOF systempeak, because this peak, which can be very broad due tomatrix constituents in real samples, is normally not detec-ted by LIF. An EOF marker for MEKC with LIF detection isnot yet available. The internal standard acridone was chosenas reference. Its signal is present in both UV and LIFdetection. Thus, mint can be determined from the migrationtime of the standard and the migration time of the EOFsystem peak obtained from the UV trace. mint is employed asa constant in all transformations. Deleting mint in Eq. (1) thezero point of the mobility-scale of the electropherogramcoincides with the mobility of the internal standard, atransformation is still possible but the scale then onlyreflects mobility differences relative to the internal stand-ard. The electropherogram presented in Fig. 2A is alsoshown in Fig. 2B, here converted into the electrophoreticmobility scale. A direct comparison of peaks in both tracesis now possible simplifying peak identification. The influ-ence of this transformation on repeatability and interdayprecision is given in Section 3.6. A slight deviation of theeffective electrophoretic mobilities calculated for UV andLIF detection is present in Fig. 2B. This deviation is due tomigration time variations which are more pronounced forthe late eluting compounds (see Tables 3 and 4), thus thedistance between the two detection windows might have amarked influence on the precision of qualitative peak data.In addition, the larger difference between the migrationtimes of these compounds and the internal standard usedfor the mobility determination according to Eq. (1) also hasto be taken into account.

3.5 UV and LIF detection in-line

Recording the absorbance and fluorescence traces withinone run was accomplished in order to simplify peak identifi-cation and to improve detection selectivity. All analytes with aconjugated system, e.g., those having a double bond in con-jugation with the aromatic system show a native fluores-cence at excitation at 266 nm with fluorescence quantumyields adequate for their detection with the setup used in thisstudy. These are asaron, isosafrole, and trans-anethol as wellas isoeugenol with the latter showing only a weak fluores-cence presumably due to the dissociation of the phenolicOH-substituent. Reduction of the fluorescence quantumyield due to the dissociation of phenolic hydroxy groups wasalso observed before for bufotenine and serotonin [28]. Allother analytes exhibit only a very weak fluorescence. It isinteresting to note, that exposing these analytes in thedetection volume to focused UV-irradiation (LIF detection)induced the decomposition of the photolabile analytesasaron, anethol, and isosafrole. The products of this reactioncould be observed in the UV traces, when the LIF detectionwindow is placed in front of the UV detection window.

The use of the UV-LIF detection combination is alsouseful for the determination of minor and major constitu-ents in the same run. In order to detect minor componentsin sassafras oils, a sample dilution of only 1:100 has to beused. For UV detection, the concentration of safrole isbeyond the linear working range for the UV detector asshown in Fig. 5 for a seized sample. In the LIF trace a broadsafrole peak due to concentration overload is present. Safroleshows only a weak native fluorescence excited at 266 nm as aconsequence of the small conjugated p-system. Thus, withthe use of LIF in-line with UV detection, we can show thatsystem overload is present when injecting samples withsafrole concentrations of up to 1 mol/L, whereas the quanti-fication of minor constituents is not disturbed.

Figure 5. Seized sample of a sassafras oil at a dilution of 1:100using a Triton X-100 (25 g/L) solution as diluent; electro-pherogram with both UV (solid); and LIF (dash dot) trace; for fur-ther parameters and peak assignment refer to Fig. 2. Injectionsolution: c(Triton X-100) = 18.75 g/L.



3.6 Figures of merit

3.6.1 Detection limits

An S/N of 3 was used to determine the detection limits ofall analytes. About 200 data points were used for thedetermination of the SD of the noise in an undisturbedpart of the electropherogram. The results are shown inTable 1 for UV and LIF detection. The molar ratio of thecis- and trans-isomers of isosafrole and isoeugenol in thestandard solution was calculated from NMR data. Thismolar ratio was used for subsequent calculation of LODsfor those analytes being available only as a mixture of thecis- and trans-isomer. Anethol was present only in the trans-configuration. Detection limits for all analytes were in thelower mg/L range and adequate for the determination ofthe minor constituents in sassafras oils. A significantlyhigher detection limit was determined for thymol due toits low absorption coefficient at the wavelength used fordetection. Slightly lower detection limits are obtained forthe injection of aqueous solutions of the analytes (withoutTriton X-100, data not shown). LIF detection gives slightlylower detection limits compared to UV detection only forthose analytes having an expanded conjugated p-system(except trans-isoeugenol). In this respect it has to be takeninto account, that the solid-state UV-laser was operated atan optical power of only 50 mW for this study. Lower LODfor LIF detection can be achieved, if required by the ana-lytical problem, by operating the UV-laser system at itsmaximum optical power in the range of 500–800 mW. Adetection limit of 1.8 mmol/L was determined for the LIFdetection of safrole.

Table 1. LOD evaluated from standard electropherograms (x-axis = time)

Analyte LOD/(mmol/L) LOD/(mg/L)

UV LIF UV LIF

Piperonal 1.47 –a) 0.22 –a)

Eugenol 5.68 –a) 0.93 –a)

cis-Isoeugenol 5.91 –a) 0.97 –a)

trans-Isoeugenol 3.47 4.28 0.57 0.70Methyleugenol 4.92 –a) 0.88 –a)

Thymol 46.04 –a) 6.92 –a)

Safrole 8.24 –a) 1.34 –a)

Myristicin 9.94 –a) 1.91 –a)

cis-Isosafrole 3.80 –a) 0.62 –a)

Asaron 3.43 2.08 0.72 0.43trans-Isosafrole 7.31 3.44 1.19 0.56Anethol 5.50 4.46 0.82 0.66

Detection limits in mmol/L and mg/L (calculated as LOD = 3c(s/H),with c concentration,s standard deviation of noise, H peak height)For further parameters refer to Fig. 2.a) Detection limit not sufficient.

3.6.2 Repeatability

Six consecutive runs were taken to determine the run-to-runrepeatability of the method. The series of experiments wasconducted both with a solution of the standard compoundsin water as sample and a sample solution prepared accordingto the procedure developed for the direct injection of oilsamples containing Triton X-100. Calculations were doneusing time-corrected relative peak areas AR (peak area divid-ed by migration time) and by normalization on the internalstandard (tm and AR divided by the appropriate parameters ofthe internal standard, see also legend of Table 2). Analyteconcentrations in the sample are also given in Table 2. Bynormalization on tm or AR of the internal standard the influ-ence of drifting phenomena observed for migration timesand thus changes in peak area due to varied migration ve-locities of analyte zones were diminished. Normalizingtm(analyte) on tm(internal standard) improves the RSD aboutfive-fold. In contrast to this improvement of the precision ofthe migration times, there is no significant reduction of theRSD of peak areas neither by normalization on the migrationtimes of the analytes nor by normalization on the peak areaof the internal standard (see Table 2). From these observa-tions we conclude: (i) Variation of the EOF velocity is themajor source of variance in tm, (ii) variation of the EOF ve-locity and imprecision of the injected sample volume are ofnegligible influence regarding RSD(A), and (iii) RSD(A) isdetermined by the signal-to-noise-ratio of the detection pro-cess.

The precision of quantitative data presented in this studyneeds further improvement. However, for the analysis ofseized oils, the identification of the analytes and the deter-mination of relative amounts of ingredients are moreimportant. They enable batch to batch comparisons of seizedsamples and give indications concerning the origin of theessential oil. The wide spectrum of analyte concentrations inbiological samples makes the evaluation of quantitative dataless suitable for forensically relevant classifications of sei-zures of essential oils.

Data regarding the influence of the composition of thesample solution on RSD(tm) and RSD(A) are also given inTable 2. In general, the RSDs of peak area A, relative peakarea AR, and peak height (data not shown) are not affected bythe presence of Triton X-100 in the sample solution (F-test,a = 0.05). However, there is a significant improvement inRSD(tm) for the Triton X-100 containing samples comparedto pure aqueous samples. Consequently, sample preparationby forming an oil-in-water macroemulsion of a water-insolu-ble sample with Triton X-100 as emulsifier does not have anegative impact on the precision of the qualitative andquantitative data. For LIF detection similar trends wereobserved (data not shown). In general, repeatabilitiesobtained with UV detection are better than for LIF detection.

With LIF-detection, for the migration time the improve-ment by the normalization on the migration time of aninternal standard is higher than observed for UV detection,



Table 2. Evaluation of standard electropherograms (x-axis = time)

Analyte tm(Analyte) tm(Analyte)/tm(IS)

A(Analyte) AR(Analyte) AR(Analyte)/AR(IS)

S1 S2 S1 S2 S1 S2 S1 S2 S1 S2

Piperonal 1.56 1.25 2.83 2.63 3.66 1.92 2.18 2.40 2.39 3.09IS1 1.80 1.34 2.20 2.98 1.09 4.15Eugenol 2.36 1.68 0.55 0.34 3.55 1.56 1.76 2.67 1.69 2.01cis-Isoeugenol 2.59 1.74 0.78 0.42 9.21 3.89 7.33 3.88 7.45 4.29trans-Isoeugenol 4.09 1.68 1.48 0.38 10.18 3.93 6.84 5.39 6.86 2.42Methyleugenol 3.29 1.70 1.47 0.44 6.25 3.69 3.81 5.03 3.31 2.04Thymol 3.66 2.03 1.96 0.79 4.05 8.56 6.07 9.02 6.28 8.14Safrole 3.75 1.93 1.93 0.60 1.74 1.64 4.67 3.11 5.17 2.50Myristicin 3.68 2.75 2.11 1.35 5.72 2.93 2.55 3.46 4.20 3.33Asaron 4.36 2.12 2.53 1.03 5.94 1.19 1.80 1.40 1.75 4.09trans-Isosafrole 4.46 2.97 2.63 1.84 2.00 3.79 3.71 3.86 3.92 6.82Anethol 4.49 2.06 2.87 1.15 2.48 2.05 5.55 3.36 5.97 5.92

Repeatability data (%RSD) of six consecutive runs; UV detection, determined for migration time tm, peak area A, relative peak area AR;compositions of the sample solution see footnote (S1, S2); for further parameters refer to Fig. 2, determined at analyte concentration ofpiperonal 125 mmol/L, eugenol 250 mmol/L, cis-isoeugenol 55 mmol/L, trans-isoeugenol 75 mmmol/L, methyleugenol 125 mmol/L, thymol300 mmol/L, safrole 250 mmol/L, myristicin 250 mmol/L, asaron 125 mmol/L, trans-isosafrole 109 mmol/L, and anethol 125 mmol/L.tm, migration time; IS, internal standard; A, peak area; AR = A/tm; S1, aqueous sample solution; S2, sample solution with c(Triton X-100) = 18.75 g/L.

possibly due to the better mobility matching of analytes andinternal standard. A comparison of the peak height repeat-ability does not show significant differences. However,RSD(A) was significantly higher for LIF detection than forUV detection reflecting the influence of fluctuations in thelaser intensity (signal noise).

In Tables 3 and 4 the results using the electrophoreticmobility-based axis are compared to those using the normaltime axis for UV and LIF detection. The effects due to trans-formation of the time-based scale are the same for water andTriton X-100 containing sample solutions (data not shown).The transformation has a strong positive impact on therepeatability of the migration parameter zm. The calculationof the effective electrophoretic mobility eliminates differ-ences in the EOF velocity. Consequently, the RSDs of thecalculated effective electrophoretic mobilities are up to ten-fold lower than the RSDs of the recorded migration times.Normalization on meff of the internal standard does not evokefurther improvements in RSD(meff ), showing that short-timefluctuations in the EOF velocity can be neglected. Peak areasA (but not AR) based on the electrophoretic mobility scaleshow significantly higher RSDs (F-Test a = 0.05) than peakareas based on migration time electropherograms. Thistrend is similar for LIF detection (see Table 4). While there isgood repeatability of normalized migration data (tm(analyte)/tm(IS) or meff ) the precision of the quantitative parameter Acan neither be improved by normalization on A(IS) nor bytransformation of the time-based electropherogram into anelectrophoretic mobility-based electropherogram. Due to thehigh RSD(A) of the internal standard IS2, its use for peakarea corrections is not advisable for LIF detection.

In general, repeatability can be improved by suitablerinsing steps. Ahuja et al. [44] reported the adsorption of thepolyether surfactant Brij onto the capillary wall. We presumethat within a course of repeated separations, the adsorptionof Triton X-100 might also take place, resulting in the driftingphenomena observed. Fluctuations in temperature and cur-rent fluctuation induced by the different compositions ofsample and BGE can have an additional negative impact onthe repeatability.

The day-to-day reproducibility was determined frommeasurements at 6 days including different buffer batchesand different capillaries. Table 5 shows the RSD calculatedfrom six runs for UV detection for the time-based and theelectrophoretic mobility-based scale (data for LIF detectionnot shown). For both detection schemes the RSD of migra-tion times is higher by a factor of six in the interday studythan for the data obtained under run-to-run repeatabilityconditions. However, using the electrophoretic mobilityscale, there is only a rise in RSD values by a factor of three,with still excellent repeatability. RSD(meff ) does not signifi-cantly depend on the retention factor. This holds true also forRSD(tm). Normalization on tm(IS) clearly improves RSD(tm)due to its correction for EOF variations. This is not the casefor the effective mobility. Best precision of qualitative datacan be obtained by transformation of time-based electro-pherograms into meff-based electropherograms. These dataindicate that a screening of samples is possible using theelectrophoretic mobility scale. An RSD of 0.3% for the effec-tive mobility using a CE method was found by Hudson et al.[45] for an interday repeatability study of 20 drugs of forensicinterest within 4 months. The values presented in our study



Table 3. Comparison of repeatability data (%RSD) of six consecutive runs after transformation of the x-axis data into a mobility scale forTriton X-100 containing sample solutions

Analyte zm(Analyte) zm(Analyte)/zm(IS)

A(Analyte) AR(Analyte) AR(Analyte)/AR(IS)

M1 M2 M1 M2 M1 M2 M1 M2 M1 M2


UV trace; determined for migration parameter zm, peak area A, relative peak area AR; for further parameters refer to Fig. 2; determined atanalyte concentrations as given in Table 2.M1, standard electropherograms, x-axis = time; M2, transformed electropherograms, x-axis = effective electrophoretic mobility; zm,migration parameter tm or meff; tm, migration time; meff, effective electrophoretic mobility; IS, internal standard; A, peak area; AR, A/tm.

Table 4. Repeatability data as %RSD of six consecutive runs


A(Analyte) AR(Analyte) AR(Analyte)/AR (IS)

M1 M2 M1 M2 M1 M2 M1 M2 M1 M2

IS2 2.65 0.3419.57

12.44 20.51 12.53trans-Isoeugenol 3.26 0.26 0.30 0.58 24.54 29.05 26.76 28.91 27.56 27.67Asaron 3.45 0.87 0.49 1.20 7.99 7.58 6.91 6.99 10.19 9.60trans-Isosafrole 3.73 0.85 0.77 1.17 9.60 11.33 9.27 10.66 10.91 10.52Anethol 3.65 0.95 0.72 1.27 9.63 12.67 10.20 11.87 10.12 9.49

LIF trace; RSD values for migration time and peak area, their relative values (with reference to the migration time), and the repeatabilitycalculated using the internal standard using the time-based and the mobility-based scale for Triton X-100 containing sample solutions; forfurther parameters refer to Fig. 2 and Table 3; determined at analyte concentrations as given in Table 2.

are slightly higher using an MEKC method with a complexseparation electrolyte without temperature control of theseparation capillary and additional influences on the separa-tion system by the sample solution. Concerning the inter-mediate precision, quantitative data can be improved bynormalization on the peak area of an internal standard.

In order to determine whether a high concentration ofsafrole in the sample effects the repeatability of qualitativeand quantitative data for analytes present at lower con-centrations, a seized sassafras oil (dilution 1:100) was ana-lyzed (see Fig. 5). The RSDs for six consecutive runs werecalculated for LIF detection (data not shown): the repeat-ability of qualitative (tm(analyte)/tm(IS)) and quantitative datafor the real sample is similar to those of a standard solution.

We conclude that the precision of the developed method isadequate for the determination of a trace constituent in thematrix of a major constituent.

3.6.3 Resolution and plate numbers

Resolution and plate numbers decreased following the addi-tion of Triton X-100 to the sample as shown before (Section3.3). However, baseline resolution was obtained for all ana-lytes except for the peak pair of thymol and safrole with aresolution of only 1.26 also for Triton X-100 containing sam-ples. For the plate numbers of all analyte peaks in UV andLIF traces a negative impact due to the addition of TritonX-100 to the sample solution is present (see Figs. 3 and 4).



Table 5. Intermediate precision (% RSD) of six runs determined from measurements at 6 days with exchanged capillaries


A(Analyte) AR(Analyte) AR(Analyte)/AR (IS)

M1 M2 M1 M2 M1 M2 M1 M2 M1 M2


UV trace; RSD values for migration time and peak area, their relative values (with reference to the migration time), and the intermediateprecision calculated using the internal standard using the time-based and the mobility-based scale for Triton X-100 containing samplesolutions; for further parameters refer to Fig. 2 and Table 3; determined at analyte concentration 30–100 times LOD.

Degrading of the peak efficiency is less pronounced for themore hydrophilic analytes. However, peak broadening due tothe processes discussed in Section 3.3.2 is noticeable for themore hydrophobic solutes. For example, the plate numberfor piperonal is 242 000 injecting an aqueous solution and175 000 with an injection solution containing Triton X-100.Eugenol shows Naq = 249 000 and NT = 104 000 with a 2.4-fold difference, but for anethol, a factor of more than 3 ispresent comparing Naq (171 000) to NT (51 000).

3.6.4 Calibration

The linear working range in case of UV detection is relativelysmall due to the limited working range of the A/D converterin use leading to an upper limit of the working range ofabout 1 mmol/L for all analytes except thymol. In case of LIFdetection the larger linear working range is limited by peakasymmetry due to overload of the pseudostationary phase,not by the restricitions of the detection system used. For theconjugated analytes a lower limit of determination of8 mmol/L was observed with both detection techniques whilefor the unconjugated analytes a limit of determination ofabout 16 mmol/L was reached.

Calibration curves for all analytes were measuredusing eight calibration points (25–750 mmol/L). All ofthem proved to be linear with correlation coefficientshigher than 0.995 except for piperonal (0.986 UV) andsafrole (0.987 LIF). The calibration curve for safrole in LIFdetection is linear in the concentration range from 2.5 to50 mmol/L. Linear regression revealed an intercept of0.109, a slope of 0.081 mmol/L, and a correlation coeffi-cient of 0.9868.

3.7 Real samples

3.7.1 Sample of a commercially available oil of S.

albidum

The UV and LIF traces of the electropherogram of a sampleof a commercially available essential oil prepared from S.albidum are presented in Fig. 6 A. The sample solution wasprepared as described before (Section 2.2). The effectiveelectrophoretic mobility was taken as x-axis scale. The maincomponent of the sassafras oil is safrole with minor contentsof eugenol, methyleugenol, isosafrole, and myristicin (seeTable 6). This sample clearly shows a different pattern ofconstituents compared to the seized sample shown in Fig. 5.It can be assumed that different plants were used for theproduction of the two oils. Further results concerning theforensic results of this study are presented elsewhere [26].The contents of aromatic ether analytes found in our sam-ples correspond well with the concentrations given in litera-ture [3, 8, 11, 15].

As can be seen in Table 6, in this sample the molar ratioof n(safrole)/n(isosafrole) is 1220 corresponding to a differ-ence in concentrations of three orders of magnitude. How-ever, the difference in their migration times makes it possi-ble to obtain a baseline separation of these closely relatedconstituents. As is shown in Fig. 6B, the separation effi-ciency and the concentration loadabilities of the MEKCmethod developed are adequate to permit the quantitativedetermination of myristicin: safrole is baseline separatedfrom the closely migrating myristicin when injecting a solu-tion which contains the essential oil prepared from S. albi-dum at a dilution of 1:1000 in spite of the extreme con-centration difference between these two constituents (see



Figure 6. Electropherogram of a commercial sample of a S. albi-dum oil, recorded by UV (solid line) and LIF (dash-dot-line)detection. For further parameters and peak assignment refer toFig. 2. Injection solution: c(Triton X-100) = 18.75 g/L. (A) At adilution of 1:100; (B) at a dilution of 1:1000.

Table 6. Contents g/L of analytes of interest in a seized sample(Fig. 5), a commercial sample of S. albidum (Fig. 6), anda sample of M. fragrans (Fig. 7)

Seized oil S. albidum M. fragrans

Eugenol –a) 0.4 –b)

trans-Isoeugenol –a) –b) 14.2Methyleugenol –a) 15.8 4.3Safrole 998.1 714.6 8.5Myristicin –a) 14.4 74.2trans-Isosafrole –a) 0.59 –b)

Anethol –a) 0.56 –b)

a) Not detected.b) Partially overlapping constituent.

Table 6). Figure 6B also shows the partial overlap of the peakof methyleugenol with a peak of a matrix constituent, possi-bly elemicin. A baseline separation of these two constituentsmight be achieved with a higher concentration of calcium

ions in the separation electrolyte due to a further widening ofthe migration time window [30], but at the expense of anincrease in the analysis time.

3.7.2 Sample of a M. fragrans oil

Figure 7 shows the UV and LIF trace of an electropherogramof a sample of commercial nutmeg oil (M. fragrans) at adilution of 1:100. In this sample myristicin is the maincomponent, eugenol, isoeugenol, methyleugenol, andsafrole are minor constituents. In Table 6 the quantitativeresults for this sample are listed. These quantitative resultsshow, that in accordance with the supplier’s label, the oilbelongs to the group of nutmeg oils from Eastern India,which are described to contain 1.9–2.9% w/w safrole and ahigh mass fraction of myristicin (14.0–15.8%), in contrast tooils prepared from nutmeg from the Western Indian groupwith a safrole content of 0.28–0.38% and a myristicin contentof 2.6% [8, 13, 46]. With the method presented here, a cleardistinction of the origin of the oils is possible.

Figure 7. Electropherogram of a commercial sample of M. fra-grans oil at a dilution of 1:100, UV (solid line) and LIF (dash-dot-line) detection. For further parameters and peak assignment referto Fig. 2. Injection solution: c(Triton X-100) = 18.75 g/L.

4 Concluding remarks

We were able to show that the identification and quantifica-tion of very hydrophobic analytes in essential oils is possiblewith MEKC with high separation efficiency and LOD ade-quate for the quantification of minor constituents in realsamples. The preparation of homogeneous samples waspossible by the preparation of stable oil-in-water macro-emulsions employing the nonionic surfactant Triton X-100as emulsifier with only moderate impact on the separationefficiency achieved. The use of UV and LIF detection in-lineimproves the identification of constituents and allows thesimultaneous quantification of major and minor constitu-ents without additional dilution steps. The determination of



minor constituents was possible despite the presence of ahigh amount of safrole at a molar ratio of minor to majorconstituent of 1:1000.

We thank Istemi Kuzu and Jan Rittgen for performing some ofthe measurements.Financial support from the Hessian Ministry ofScience and Art is greatly acknowledged. Part of this work was aresearch project funded by the German Bundeskriminalamt.

The authors have declared no conflict of interest.

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