Manuel MolinaManuel Silva

Department of AnalyticalChemistry, Edificio MarieCurie (Anexo), Campus deRabanales, University ofCórdoba, Córdoba, Spain

In-capillary derivatization and analysis of aminoacids, amino phosphonic acid-herbicides andbiogenic amines by capillary electrophoresiswith laser-induced fluorescence detection

This paper describes a general approach for the in-capillary derivatization of amino com-pounds and the subsequent sensitive determination of the derivatives by micellar electro-kinetic chromatography (MEKC) or capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection. Amino acids, biogenic amines and amino phospho-nic acid-herbicides were chosen as model analytes to evaluate the analytical potential ofthis approach. Fulfilment of the in-capillary reaction of the analytes using LIF detectionhinged on the excellent labeling chemistry of 5-(4,6-dichloro-s-triazin-2-ylamino)fluores-cein (DTAF) and the good resolution achieved in the separation of derivatized analytes.Careful optimization of the electrophoretic conditions in the mixing step of this protocolallowed the determination of amino acids, biogenic amines and phosphorus-containingamino acid-herbicides with concentration limits of detection at the �g/L level and relativestandard deviations from 3.5 to 5.8%. The whole analysis is carried out within 20 min,resulting in a very simple, fast and practical approach for the fully automated analysis ofamino acids and related compounds in low-volume and low-concentration samples.

Keywords: Amino acids / Amino phosphonic acid-herbicides / Biogenic amines / In-capillaryreactions / Laser-induced fluorescence EL 4997

1 Introduction

Within the last decade CE has become a versatile analyt-ical tool for the routine determination of a great variety ofcompounds [1–3]. As with most analytical techniques,one of the limiting factors to high sensitivity and selectivityin CE is the detection method employed. Most CEseparations are carried out using UV absorption detectiondue to its easy to use; however, detection limits andselectivity are poor. Moreover, an inherent shortcomingarises in the separation and determination of amino com-pounds, specially amino acids, because only very few ofthem give responses to current UV absorption detector.Thus, amino compounds are generally determined usingderivatization procedures to increase the sensitivity of thedetection. So far, more than 50 various derivatizationagents have been reported for CE separations of aminoacids, the typical amino compounds used in these stu-dies [4–6]. However, the use of o-phthalaldehyde (OPA),dansyl chloride and 9-fluorenylmethylchloroformate is by

far prevailing as these labels allow highly sensitive deter-minations on account of their fluorescent properties. Inrecent years, LIF has proven to be an excellent alternativefor the sensitive detection of very low concentrations ofthese compounds after their derivatization [7, 8]. Amongthe most frequent excitation sources, the argon ion laseremitting at 488 nm is employed for exciting a wide rangeof fluorescent derivatives, which are formed by reaction ofthe analytes with suitable reagents. FITC [9–13] is verypopular due to the high-quantum yield of its moiety.Recently, 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein(DTAF) has been receiving attention as an alternative toFITC due to its faster labeling reaction, higher purity andlower cost [14–16]. Other advantage of this compoundwith respect to reported LIF alternatives based on non-fluorescein probes [17–21] is the lower temperaturerequired for the derivatization.

As stated above, using LIF detection the sensitivity pro-blems involved in the determination of amino compoundsby CE can be overcome; however, the procedure requiresa pre- or postcapillary derivatization step. On account ofthe good results achieved by the electrophoreticallymediated microanalysis in the determination of enzymaticactivities and enzyme and substrate concentrations [22–24], the natural progression of in-capillary reaction tech-niques has led to include nonenzymatic (derivatization)reactions over the recent years [25–33]. In-capillary elec-trophoresis reactions use differences in the electrophoret-

Correspondence: Dr. Manuel Silva, Department of AnalyticalChemistry, Edificio Marie Curie (Anexo), Campus of Rabanales,University of Córdoba, E-14071 Córdoba, SpainE-mail: qa1sirom@uco.esFax: �34-957-218614

Abbreviations: AMPA, aminomethylphosphonic acid; DTAF,5-(4,6-dichlorotriazinyllamino) fluorescein; MRB, micellar runningbuffer

Electrophoresis 2002, 23, 2333–2340 2333

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/1407–2333 $17.50�.50/0

CE

and

CE

C

2334 M. Molina and M. Silva Electrophoresis 2002, 23, 2333–2340

ic mobilities to merge distinct zones of analyte and label-ing reagent under the effect of an electric field, which canbe accomplished by using three techniques: at-inlet,zone-passing and throughout capillary derivatization.The at-inlet strategy is the most widely used because aspecific time is very often required to complete the label-ing reaction before applying the running potential. In-cap-illary derivatization offers some relevant advantages overconventional batch procedures, such as: full automationof the derivation step in an easy way, improvement of thereproducibility of the peak areas, and low consumption ofsample and reagents.

Although in-capillary derivatization with fluorescencedetection has lately emerged as a powerful alternative toperform sensitive determinations of species by CE in asimple way, only a few papers have so far been devotedto its use for the determination of amino compounds, ingeneral with amino acids as model compounds. OPA[30, 31] or a mixture of OPA with N-acetylcysteine [32,33] are used as derivatization reagents for amino acids,whose subsequent CE separation can require the addi-tion of �-cyclodextrin to the running buffer depending onthe type and number of amino acids present in the sample[32]. In any case, the resulting limits of detection for thesecompounds are at �M level. In this field, it is worthy to notethe recently reported method for N-methylcarbamates, inwhich the capillary was consecutively utilized for MEKCseparation using CTAB as hydrophobic pseudophases,CTAB-catalyzed thermal decomposition of pesticides,derivatization with OPA of the N-methylamine releasedand fluorescence detection [34]. To our knowledge, thereis no reference about the use of in-capillary derivatizationand LIF detection for the determination of amino com-pounds. This may be attributed to the extremely slowkinetics of the derivatization reaction with FITC, the morecommonly used label for the determination of amino com-pounds by LIF detection.

Herein, the possibilities of LIF detection for in-capillaryderivatization of amino compounds are examined. DTAFwas selected as label reagent due to its reactivity, whichmay be suitable for in-capillary derivatization as high yieldof amino compound derivatives can be obtained insidethe capillary in a relatively short time. Amino acids, bio-genic amines and amino phosphonic acid-herbicideswere chosen as model analytes to demonstrate the suit-ability of this procedure as a general approach for thedetermination of amino compounds. Thus, plugs of ana-lyte and DTAF solutions were injected successively intothe inlet of the separation capillary under hydrodynamicconditions. Owing to their different electrophoretic mobil-ities, electrokinetic mixing of the two zones takes placeand then they react for 10 min in the absence of an

applied potential. Separation of the derivatives is per-formed by CE or MEKC depending on the selectivityrequirements of the mixture under study, and LIF detec-tion with commercial air-cooled argon-ion laser (488 nm)detector.

2 Materials and methods

2.1 Reagents

All chemicals used were of analytical-reagent grade andMilli-Q water was used throughout. The amino acid stan-dards (phenylalanine, arginine, lysine, valine, histidine,alanine, proline, glutamic acid and aspartic acid), glycine,and the biogenic amines (putrescine, cadaverine andspermidine), were purchased from Sigma-Aldrich Quí-mica (Madrid, Spain). Glyphosate, glufosinate-ammo-nium and aminomethylphosphonic acid (AMPA, hydroly-sis product of glyphosate), were obtained from Riedel-deHaën (Seelze, Germany), Brij-35 (polyoxyethylene dode-cyl ether) from Merck (Darmstadt, Germany), and DTAFfrom Fluka (Buchs, Switzerland). Other chemicals andsolvents were common brands such as Sigma and Fluka.Standard solutions containing 200 �g/mL of each analytewere prepared by dissolving the required amount in waterand stored at 4�C in a refrigerator. Standard mixtures wereprepared by dilution of the corresponding stock solutionswith Milli-Q water as required. The micellar running buffer(MRB) employed for the separation of the amino acidsand herbicides, 50 mM borate adjusted to pH 9.5 with0.5 M sodium hydroxide (Merck), contained 12 and 50 mM

Brij-35, respectively. BGE was 50 mM borate adjusted topH 9.5.

2.2 Apparatus

Experiments were conducted using a Beckman P/ACE5500 (Beckman, Fullerton, CA, USA) unit interfaced witha PC-Pentium 75 MHz compatible computer utilizing Sys-tem Gold software (Version 8.1 Beckman) for control ofthe instrument and data collection. The capillary was57 cm (effective length 50 cm) by 50 �m ID (375 �m OD)fused-silica capillary tubing purchased from Beckman,which was accommodated in a cartridge configured forLIF detection. An argon ion laser (3 mW) was used asexcitation source (488 nm) and the electropherogramswere recorded by monitoring the emission intensity at520 nm. The sample end (inlet) was anodic. The newfused-silica capillary was conditioned by rinsing with0.1 M sodium hydroxide (5 min), Milli-Q water (10 min)and running buffer (15 min), in order.

Electrophoresis 2002, 23, 2333–2340 Analysis of amino compounds by CE 2335

2.3 In-capillary derivatization and separationby CZE and MEKC

The in-capillary derivatization of amino acids was as fol-lows: to 45 �L of the sample were added 5 �L of a stockbuffer solution (0.5 M NaHCO3/Na2CO3 adjusted to pH9.5, derivatization buffer) in a microvial (vial 1). The mixturewas homogenized and left till the analysis was conducted.In the vial 2, 5 �L of derivatization buffer were mixed with40�L of water, and immediately before the analysis, 5�L ofthe DTAF stock solution (0.5 mM in 9:1 ethanol:1,2-di-chloroethane, stored at –20�C) were added and thoroughlymixed. Plugs of these solutions were sequentially injectedunder hydrodynamic conditions (5 s at 0.5 psi, 3.45 kPa),followed by the hydrodynamic injection of a BGE plug for35 s (0.5 psi, 3.45 kPa) to avoid the neutral micelles fromreaching the reaction zone during the mixing step. Vialscontaining MRB and BGE were respectively placed in theinlet and outlet of the capillary, and the sample and DTAFzones were electrokinetically mixed by applying a poten-tial of 5 kV for 0.75 min (mixing time). The reaction wasallowed to proceed within the region of mixed reagents for10 min (derivatization time) in the absence of appliedpotential, and the separation was performed at 35�C, usinga running voltage of 20 kV. For the determination of phos-phoros-containing amino acid-herbicides and biogenicamines, the procedure had to be slightly modified. Thus,for herbicides the mixing time was 1.5 min and the MRB50 mM boric acid and 30 mM Brij-35 (adjusted to pH 9.5with 0.5 M sodium hydroxide). The procedure for biogenicamines requires a 5 mM DTAF concentration in the stocksolution and a mixing time of 0.5 min. In this case, thehydrodynamic injection sequence was inverted so thatthe amines (noncharged at the working pH) could passthrough the DTAF zone when the voltage was applied.Moreover, the separation of derivatives was successfullyachieved by CZE with the BGE. At the beginning of eachexperimental session, the capillary was rinsed with 0.1 M

NaOH for 5 min, followed by 10 min with Milli-Q water. Thecapillary was equilibrated with the MRB (1 min) and BGE(2 min) before each sample injection and rinsed with0.1 M sodium hydroxide for 1 min and flushed with Milli-Q water for 1 min after each run. Special care was exer-cised to ensure adequate regeneration and equilibrationof the capillary to obtain reproducible results.

3 Results and discussion

Figure 1 shows the scheme of the derivatization reactionbetween DTAF and amino compounds. On account of therelatively slow kinetics of this reaction (following batchpreliminary experiments we discovered that the maxi-mum signal-to-noise ratio can be achieved for the

Figure 1. Scheme of the derivatization reaction betweenDTAF and amino compounds.

assayed compounds in about 1 h at 35–40�C in an alka-line medium at pH 9.0–9.5), the at-inlet strategy is chosento perform the in-capillary derivatization. In this approach,the injected sample and reagent zones are allowed tostand for a specified time to develop the reaction beforeapplying the running voltage. During this period, the twozones are mixed by diffusion so that they start to react.The resulting derivatives are immediately determined byapplying a running voltage.

Several changes have been introduced to enhance thefeatures of the in-capillary derivatization of amino com-pounds with DTAF, namely: (i) a 5 kV voltage was appliedfor a predetermined time (mixing time) after the injection ofsample and reagent plugs to improve the mixing of ana-lytes and DTAF; (ii) MEKC was introduced to avoid theoverlapping of the excess of DTAF with several labeledamino acids and herbicides and that of amino acid deriva-tives, such as Phe, His, Val and Pro. Thus, Brij-35 wasadded to the MRB, because it has been recently reportedthat nonionic surfactants can significantly improve theseparation of closely related anionic analytes withoutaffecting the ionic current during the run and thus avoidingexcessive Joule heating [13]. (iii) The use of neutralmicelles allowed to achieve a good separation for theamino compound derivatives; however, this pseudosta-tionary phase shows an important inhibitory effect on thelabeling reaction. To overcome this shortcoming, a runningbuffer without neutral micelles (BGE) was employed asleading electrolyte and a plug of it was injected after theDTAF zone so that the micelles did not reach the reactionzone before the derivatization reaction had been carriedout. (iv) For biogenic amines, due to the fact that their elec-trophoretic mobility is lower than that of DTAF (these com-pounds are neutral at basic pH), the sequence of the injec-tion was inverted to achieve an efficient mixing of the DTAFand the analyte zones before the labeling reaction wasconducted. The subsequent separation of the derivativeswas carried out in the BGE because a good resolution wasfound, and therefore, MEKC running buffers are notrequired. The resulting at-inlet capillary derivatization pro-cedures are outlined in Fig. 2.

2336 M. Molina and M. Silva Electrophoresis 2002, 23, 2333–2340

Figure 2. Scheme for in-capillary derivatization with DTAF of (A) amino acids and herbicides and (B) biogenic amines.

3.1 Optimization of the in-capillaryderivatization and separation conditions

When determining amino compounds with in-capillaryderivatization with DTAF, the pH of the running buffer is adominant factor because this buffer serves both as aseparation and derivatization buffer. In this work, a pHvalue of 9.5, provided by boric acid, was chosen for bothprocesses, as a compromise between the above statedresults on the batch derivatization of these compoundswith DTAF and those found for the MEKC separation oflabeled amino acids and herbicides and CZE separationof biogenic amine derivatives. The in-capillary derivatiza-tion process was carried out at 35�C, using a mixing timeof 0.5 min and a derivatization time of 7.5 min (fixed fromprevious experiences). The subsequent study of the effectof the boric acid and Brij-35 concentrations in MRB aswell as the boric acid concentration in BGE on the separa-tion of labeled analytes provides the following optimumcomposition for these electrolytes: MRB, 50 mM boricacid adjusted to pH 9.5 containing 12 and 50 mM Brij-35for amino acids and herbicides, respectively; BGE, 50 mM

boric acid adjusted to pH 9.5.

Typical electropherograms for the LIF detection of aminoacids, phosphorus-containing amino acid-herbicides andbiogenic amines are presented in Figs. 3 and 4. It showsthat satisfactory separation of all analytes was achieved;in addition, Fig. 3 shows clearly the strong effect of the

Brij-35 micelles on the separation of the amino acid deriva-tives. Due to the nonionic character of the surfactant, adecrease in the migration times of all the peaks wasobserved though no significant change in the electroos-motic flow was found. Thisgeneral effect is more importantfor the derivatives with longer hydrocarbon chains in theamino acid tail or aromatic structure, such as Ala, Val andPhe, since their interaction with the neutral micelle is stron-ger and their partitioning between the micellar phase andthe aqueous one results in increasing of their effectivemobility (the electroosmotic flow mobility is higher thanthe effective mobility of the negatively charged free spe-cies) and the subsequent decreasing of the migrationtime. Several inversions in the migration pattern can beobserved according to this mechanism (see pairs Ala/Pro,Val/His and Phe/Arg). With respect to herbicides, the use ofnonionic micelles avoided the overlapping of peaks fromthe reagent (unidentified peaks in Figs. 3 and 4 correspondto the excess and hydrolysis products of DTAF [16, 35])with labeled analytes, yielding their suitable separation asshown in Fig. 4A. In summary, the employment of the non-ionic surfactant not only made possible a satisfactoryseparation but also increased the peak heights andimproved the peak shape for some of the analytes.

In the case of biogenic amines, it is worthy to note thegood sensitivity reached for spermidine, which can beattributed to the low fluorescence of the background inits migration window (Fig. 4B), in spite of the fact that it

Electrophoresis 2002, 23, 2333–2340 Analysis of amino compounds by CE 2337

Figure 3. (A) CZE and (B) MEKC electropherogramsrecorded for amino acids. Derivatization conditions: mix-ing time, 0.5 min, applied voltage, 5 kV; stand-by time,7.5 min. Separation at 35�C; running voltage, 20 kV; run-ning buffer in (A) 50 mM boric acid adjusted to pH 9.5 and(B) 50 mM boric acid, 12 mM Brij-35, adjusted to pH 9.5.The concentration for all the analytes in the aqueous stan-dard solution was 10 �g mL–1 but 1 �g mL–1 for Gly andPro. Further details are given in Section 2.3.

yields three derivatives (probably corresponding to themono-, di- and tri-DTAF-labeled amine, according to theirexpected mobilities). Thus, though it is difficult to assignthe mobility of each derivative to a particular structure, itcan be assumed that the higher substitution ratio lead toincreased charge/mass relation, and consequently highermobility towards the anodic tip of the capillary. However,taking into account the electroosmotic flow, this behaviorinvolves a longer migration time for the polysubstitutedderivatives. Furthermore, the peak height ratios arecoherent with the steric impediment caused in polysubsti-tutions by the structure of the DTAF, which may favorthe formation of the mono-substituted derivative. FromFig. 4B, it is clear that all these three peaks could be

Figure 4. Electropherograms recorded for (A) herbicidesand (B) biogenic amines. Running buffer in (A) 50 mM

boric acid, 30 mM Brij-35, adjusted to pH 9.5 and (B)50 mM boric acid adjusted to pH 9.5. The concentrationfor all the analytes in the aqueous standard solution was1 �g�mL–1 but 10 �g�mL–1 for spermidine. Peak assign-ment: 1, glufosinate; 2, AMPA; 3, glyphosate; 4, spermi-dine (the peak used for quantitation is denoted with anasterisk); 5, cadaverine; 6, putrescine. Other conditionsas in Fig. 3.

used for quantification, but only the first of them(assumed as the monosubstituted derivative) was consid-ered for simplicity reasons. The good performance of theseparation is also testified by the fact that putrescine andcadaverine DTAF-derivatives were satisfactory separateddespite they differ just in one methylene group.

Once the separation of the DTAF-derivatives had beenoptimized, the mixing time was investigated up to 3 minusing a voltage of 5 kV. Results are shown in Fig. 5. Thepeak height of the derivatives was strongly dependent onthis variable, showing that the migration zones for eachanalyte behave differentially. For amino acids, a compro-mise between optimum conditions for acidic (Glu andAsp), neutral (Gly, Pro, Ala, Phe, and Val) and basic (His,Arg and Lys) amino acids may be assumed to achieve the

2338 M. Molina and M. Silva Electrophoresis 2002, 23, 2333–2340

Figure 5. Influence of the mixing time on the peak height of the DTAF-derivatives of (A) amino acids: (�) Phe, (�) Arg,(�) Lys, (�) Val, (�) His, (�) Ala, (�) Pro, (�) Gly, () Glu, () Asp; (B) amino phosphonic acid-herbicides: (�) glufosinate,(�) AMPA, (�) glyphosate; and (C) biogenic amines: (�) spermidine, (�) cadaverine, (�) putrescine. Other conditions as inFigs. 3 and 4.

overall maximum sensitivity (Fig. 5A). 0.75 min wasselected as the optimum value although the peak heightfor Arg was significantly decreased. For the determinationof phosphorus containing amino acid-herbicides (glufosi-nate and glyphosate) and AMPA, a mixing time of 1.5 minyielded maximum signals for all these analytes since theyhave similar mobilities at this pH, so it was chosen forfurther studies (Fig. 5B). Finally, this variable was studiedup to 2 min for biogenic amines. It is clear from Figure 5Cthat a value of 0.5 min can be selected as optimum for thederivatization of these amines.

The subsequent optimization of the so-called derivatiza-tion time was performed to obtain the maximum signal-to-noise ratio within the minimum analysis time. Theobtained results demonstrate that the maximum reactionyield is reached within ca. 20 min for most of the analytes.However, practical reasons demand a shorter analysistime, so 10 min was selected as optimum to achieve afully automated and fast analysis. At this time, the aver-age labeling efficiency is close to ca. 75%, which allowsa very sensitive determination of all the analytes. Longerderivatization times could be employed for the determina-tion of lower concentrations of the analytes, since there isno significant diffusion of the derivatives during this step,but it should imply a much lower sampling frequency;moreover, the attained sensitivity fits common require-ments for the determination of these analytes in food andagricultural products.

The DTAF concentration used for the in-capillary derivati-zation of the assayed amino compounds depends stronglyon the overlapping of its excess with labeled analytes.Thus, no improvement in the limits of detection for aminoacids and herbicides was found at higher DTAF concentra-tions (up to a 5 mM) as the fluorescent background in theblank increased as much as the analytical signals, andtherefore, a value of 0.5 mM was selected as optimum.However, for biogenic amines a significant decreasein the limit of detection (LOD) for spermidine was foundusing the maximum concentration of DTAF that can beemployed (5 mM) as there is no interfering peaks from thelabeling reagent. In this case, this concentration wasselected as optimum for following experiences.

3.2 Analytical performance characteristics

Standard aqueous solutions were analyzed under condi-tions described in Section 2.3 and the corresponding an-alytical curves were obtained by plotting peak height ver-sus analyte concentration. Table 1 gives the least-squaresparameters for the working curves of the analytes andtheir concentration LODs, calculated as the concentrationof analyte in the sample providing a chromatographic sig-nal equal to three times the peak-to-peak noise; concen-tration LODs ranging from 2 to 65 �g/L (except Phe andArg, 160 and 230 �g/L, respectively) testify to the excel-lent performance of the developed method. The within-day precision (repeatibility) for peak heights, expressed

Electrophoresis 2002, 23, 2333–2340 Analysis of amino compounds by CE 2339

Table 1. Characteristic parameters of the calibration graphs and analytical figures of merit of thedetermination of amino compounds

Compound Linear range(mg/L)

Regressionequationa)

Correlationcoefficient(n = 11)

LOD(�g/L)

RSD (%)

Amino acidsPhe 0.3–30 H = 0.05 � 1.24�C 0.9960 160 5.1Arg 0.5–50 H = –0.01 � 0.28�C 0.9975 230 5.8Lys 0.12–25 H = 0.04 � 1.26�C 0.9974 56 5.7Val 0.03–7.0 H = –0.02 � 6.22�C 0.9995 19 4.3His 0.07–20 H = 0.03 � 2.07�C 0.9978 47 5.4Ala 0.05–12 H = 0.08 � 3.48�C 0.9998 28 4.8Pro 0.04–1.7 H = 0.12 � 33.1�C 0.9994 25 5.3Gly 0.04–2.6 H = 0.07 � 19.4�C 0.9997 20 3.8Glu 0.09–13 H = 0.03 � 3.13�C 0.9990 55 5.2Asp 0.04–16 H = 0.03 � 2.43�C 0.9997 26 5.1

Phosphorus containing amino acid-herbicidesGlufosinate 0.1–20 H = 0.07 � 2.00�C 0.9987 65 4.0AMPA 0.004–2.0 H = 0.11 � 20.1�C 0.9998 2 3.5Glyphosate 0.08–17 H = –0.02 � 2.41�C 0.9996 45 3.8

Biogenic aminesSpermidine 0.03–30 H = 0.01 � 0.92�C 0.9997 17 4.8Cadaverine 0.07–2.0 H = 0.16 � 34.4�C 0.9973 43 5.6Putrescine 0.06–1.7 H = –0.12 � 35.5�C 0.9984 36 5.2

a) H, peak height; C, analyte concentration (mg/L)

as the RSD, was calculated from 11 standards containing0.5 mg/L (except to Phe and Arg at 5.0 mg/L) of each ana-lyte in water.

At this point, it should be interesting to evaluate the fea-tures of the proposed in-capillary reaction method for thedetermination of amino compounds by comparison withreported LIF alternatives, which are carried out afterbatch derivatization with different labels, because to ourknowledge, this is the first report devoted to in-capillary-CE-LIF. In this context, it should be noted that the expres-sion of concentration LODs in LIF detection is not clear inthe literature. In fact, the authors usually ascribe the con-centration LOD to the minimum analyte concentrationinjected into the CE instrument (instrumental concentra-tion LOD), and not to that derivatized (derivatization con-centration LOD), which is 2–3 orders of magnitude higher.It is clear that derivatization concentration LODs are thekey to evaluate the analytical potential of a developedLIF method, and therefore, it was used in this study. Sev-eral papers have been reported so far for the determina-tion of amino acids, some of them including biogenicamines, by CZE or MEKC using LIF detection with differ-ent fluorescence labels such as FITC [9, 11, 15], 4-fluoro-7-nitro-2,1,3-benzoxadiaxole (NBD-F) [19], 4-(3-iso-thiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole

[17], 5-furoylquinoline-3-carboxaldehyde [20], and 3-(4-carboxybenzoyl) quinoline-2-carboxyaldehyde [21]. Tak-ing into account the dilution of the derivatized amino acidsolution prior to injection reported in these papers, whichranged from 50 to 10 000 times according to the fluores-cent probe used, the derivatization concentration LODsprovided by these methods can be estimated over therange of 50–300 �g/L, which are, in many cases, higherthan that afforded by the proposed method. Among thesereagents, the most competitive alternative to DTAF, thelabel reagent used in this work, is NBD-F, introduced byWatanabe and Imai [36] as a useful and sensitive fluores-cent label for amino acids. Although NBD-F reacts fasterthan DTAF with amino acids (reaction time 1 min), which isan important feature for in-capillary derivatization reac-tions, it offers several drawbacks: (i) the labeling reactionmust be carried out at 60�C, which involves the use ofnon-commercial heating systems in the CE instrument;(ii) the separation of NBD-F labeled amino acids can beaccomplished within 25 min whereas using DTAF thisseparation can be performed in ca. 16 min including thederivatization time (10 min) as shown in Fig. 3B; and (iii)NBD-F is about 10–12 times more expensive than DTAF.On the other hand, both reagents react with commonamino acids, including primary and secondary ones, andoffer few interfering side products.

2340 M. Molina and M. Silva Electrophoresis 2002, 23, 2333–2340

4 Concluding remarks

DTAF has been used for in situ synthesis of molecular tar-gets of amino compounds by in-capillary derivatization,with the subsequent MEKC or CZE separation of labeledanalytes and LIF detection using a commercial CE instru-ment with potential advantages of significant improve-ments in detection sensitivity, selectivity and analysistime. An electrokinetic step was introduced in the in-cap-illary derivatization reaction to enhance the efficiency ofthe mixing of the analyte and reagent plugs. DTAF hasproved to be the most useful alternative for the determi-nation of these compounds, thanks to its high derivatiza-tion efficiency at relatively low temperature (35�C), whichallows to achieve high detection sensitivity for aminocompounds when excited with a commercial argon ionlaser. In addition, this label offers few fluorescent sideproducts and fast separation of derivatized analytes (6–10 min). Furthermore, the strategy used here could beof great interest in many application fields for the fluores-cent determination of numerous compounds with similarstructural features.

The authors gratefully acknowledge financial support fromSpain’s Dirección General de Investigación del Ministeriode Ciencia y Tecnología for the realization of this work aspart of Project BQU2000–0905.

Received February 19, 2002

5 References

[1] Beale, S. C., Anal. Chem. 1998, 70, 279R–300R.[2] Issaq, H. J., Electrophoresis 2000, 21, 1921–1939.[3] Swinney, K., Bornhop, D., Crit. Rev. Anal. Chem. 2000, 30, 1–

30.[4] Waterval, J. C. M., Lingeman, H., Bult, A., Underberg, W. J.

M., Electrophoresis 2000, 21, 4029–4045.[5] Smith, J. T., Electrophoresis 1999, 20, 3078–3083.[6] Prata, C., Bonnafous, P., Fraysse, N., Treilhou, M., Poinsot, V.,

Couderc, F., Electrophoresis 2001, 22, 4129–4138.[7] Swinney, K., Bornhop, D. J., Electrophoresis 2000, 21, 1239–

1250.[8] McWhorter, S., Soper, S. A., Electrophoresis 2000, 21, 1267–

1280.

[9] Nouadje, G., Simeon, N., Dedieu, F., Nertz, M., Puig, P., Cou-derc, F., J. Chromatogr. A 1997, 765, 337–343.

[10] Dabek-Zlotorzynska, E., Maruszak, W., J. Chromatogr. B1998, 714, 77–85.

[11] Paez, X., Rada, P., Hernandez, L., J. Chromatogr. B 2000,739, 247–254.

[12] Vandenabeele-Trambouze, O., Albert, M., Bayle, C., Cou-derc, F., Commeyras, A., Despois, D., Dobrijevic, M., Gre-nier-Loustalot, M. F., J. Chromatogr. A 2000, 894, 259–266.

[13] Molina, M., Silva, M., Electrophoresis 2001, 22, 1175–1181.[14] Lalljie, S. P. D., Sandra, P., Chromatographia 1995, 40, 513–

518.[15] Lalljie, S. P. D., Sandra, P., Chromatographia 1995, 40, 519–

526.[16] Molina, M., Silva, M., Electrophoresis 2002, 23, 1096–1103.[17] Liu, Y. M., Schneider, M., Sticha, C. M., Toyooka, T., Sweed-

ler, J. V., J. Chromatogr. A 1998, 800, 345–354.[18] Kuroda, N., Nomura, R., Al-Dirbashi, O., Akiyama, S., Naka-

shima, K., J. Chromatogr. A 1998, 798, 325–334.[19] Hu, S., Li, P. C. H., J. Chromatogr. A 2000, 876, 183–191.[20] Chen, Z., Wu, J., Baker, G. B., Parent, M., Dovichi, N. J.,

J. Chromatogr. A 2001, 914, 293–298.[21] Boulat, O., McLaren, D. G., Arriaga, E. A., Chen, D. D. Y.,

J. Chromatogr. B 2001, 754, 217–228.[22] Regnier, F. E., Patterson, D. H., Harmon, B. J., Trends Anal.

Chem. 1995, 14, 177–181.[23] Bao, J. J., Fujima, J. M., Danielson, N. D., J. Chromatogr. B

1997, 699, 481–497.[24] Kwak, E., Esquivel, S., Gomez, F. A., Anal. Chim. Acta 1999,

397, 183–190.[25] Watanabe, T., Terabe, S., J. Chromatogr. A 2000, 880, 295–

301.[26] Zhang, Y., Gomez, F. A., Electrophoresis 2000, 21, 3305–

3310.[27] Jankovskiene, G., Daunoravicius, Z., Padarauskas, A., J.

Chromatogr. A 2001, 934, 67–73.[28] Latorre, R. S., Hernández-Cassou, S., Saurina, J., J. Chro-

matogr. A 2001, 934, 105–112.[29] Larsson, M., Sundberg, R., Folestad, S., J. Chromatogr. A

2001, 934, 75–85.[30] Taga, A., Honda, S., J. Chromatogr. A 1996, 742, 243–250.[31] Taga, A., Sugimura, M., Honda, S., J. Chromatogr. A 1998,

802, 243–248.[32] Oguri, S., Yokoi, K., Motohase, Y., J. Chromatogr. A 1997,

787, 253–260.[33] Oguri, S., Ohta, Y., Suzuki, C., J. Chromatogr. B 1999, 736,

263–271.[34] Wu, Y. S., Lee, H. K., Li, S. F. Y., Anal. Chem. 2000, 72, 1441–

1447.[35] Siegler, R., Sternson, L. A., Stobaugh, J. F., J. Pharm.

Biomed. Anal. 1989, 7, 45–55.[36] Watanabe, Y., Imai, K., Anal. Chem. 1983, 55, 1786–1791.