Determination of Underivatized Polyamines: A Review of … · 2014-04-10 · Process Analytical DETERMINATION OF UNDERIVATIZED POLYAMINES: A REVIEW OF ANALYTICAL METHODS AND APPLICATIONS

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Determination of UnderivatizedPolyamines: A Review of AnalyticalMethods and ApplicationsNabil N. AL-Hadithi a & Bahruddin Saad aa School of Chemical Sciences, Universiti Sains Malaysia, Malaysia

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To cite this article: Nabil N. AL-Hadithi & Bahruddin Saad (2011): Determination of UnderivatizedPolyamines: A Review of Analytical Methods and Applications, Analytical Letters, 44:13, 2245-2264

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Process Analytical

DETERMINATION OF UNDERIVATIZEDPOLYAMINES: A REVIEW OF ANALYTICALMETHODS AND APPLICATIONS

Nabil N. AL-Hadithi and Bahruddin SaadSchool of Chemical Sciences, Universiti Sains Malaysia, Malaysia

Putrescine, cadaverine, spermidine, and spermine are ubiquitous polyamines that are

essentially found in diverse organisms (e.g., bacteria, fungi), animals, and higher plants.

Analysis of these analytes is traditionally performed either by chromatographic or electro-

phoretic techniques. The majority of assays employ liquid chromatography with fluorimetric

detection either with pre-column or post-column derivatization. However, the derivatization

procedures have several disadvantages to the whole analytical process. This article describes

the analytical method developments in the determination of underivatized polyamines. This

includes flow injection analysis, high performance liquid chromatography (using conduc-

tivity, condensation nucleation light scattering, chemiluminescence, indirect fluorescence,

and mass spectrometric detection) and electromigration methods. The reported systems

are essentially based on the work developed by the authors since 1995. A comparison of

the methods highlighting their main advantages and disadvantages and sensitivity was also

provided. The most promising system seems to be the liquid chromatography-tandem mass

spectrometric due to its high sensitivity and specificity.

Keywords: Cadaverine; Polyamine; Putrescine; Spermidine; Spermine; Underivatized

INTRODUCTION

The polyamines putrescine, cadaverine, spermidine, and spermine (Fig. 1) aresimple aliphatic primary amines that are fully protonated under physiological con-ditions and are essential constituents of eukaryotic and prokaryotic cells (Morgan1998; Khuhawar and Qureshi 2001). They have been implicated in a variety of cellfunctions involving cell growth and differentiation and receptor function (Baronand Stasolla 2008). They also impact DNA replication, gene expression, protein syn-thesis, stabilization of lipids, brain development, and nerve growth and regeneration.Overproduction or over intake of these polyamines is toxic to the cells and facilitatescell death by oxidative mechanism (Hoet and Nemery 2000) or may cause headaches,

Received 18 March 2010; accepted 22 August 2010.

Nabil N. AL-Hadithi thanks the Universiti Sains Malaysia for providing a postdoctoral research

position.

Address correspondence to Bahruddin Saad, School of Chemical Sciences, Universiti Sains

Malaysia, 11800 Penang, Malaysia. E-mail: [email protected]

Analytical Letters, 44: 2245–2264, 2011

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2010.551686

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nausea, hypo- or hypertension, and cardiac palpitations (Teti, Visalli, and McNair2002). These compounds have also been proposed as possible cancer markers (Maet al. 2004; Gerner and Meyskens 2004; Criss 2003; Wallace and Robert 2001)and are used by the food industry as a measure of the degree of spoilage or agingin fish, meat, and cheese (Yano, Yokoyama, and Karube 1996; Tombelli andMascini 1998; Onal 2007).

Moreover, polyamines are also used in the production of value-added chemi-cals, pharmaceuticals, polymers, and pesticides (Khuhawar and Qureshi 2001). Thus,the identification and quantification of polyamines from different food products,human fluids, and environmental samples is an important and challenging task (Onal2007).

Chromatographic methods, such as thin layer chromatography (TLC), gaschromatography (GC), ion-exchange chromatography and high-performance liquidchromatography (HPLC), have been described for the determination of selectedpolyamines (Teti et al. 2002; Conca et al. 2001; Roger 2001; Muskiet et al. 1995).Several other techniques, including spectrophotometric (Vandenabeele et al. 1998)and electrochemical (Kraly et al. 2006; Ma et al. 2004; Oguri 2000) methods, as wellas fluorescence (Paproski, Roy, and Lucy 2002; Wang et al. 2000) chemilumi-nescence (Liu, Yang, and Wang 2003), and mass spectrometry (Ducros et al. 2008)have also been applied.

For the analysis of polyamines, HPLC remains the preferred method (Gosettiet al. 2007; Gaboriau et al. 2003). However, derivatization is needed to increase thesensitivity of the method when using UV or fluorescence detection, since polyamines

Figure 1. Chemical structures and abbreviations of the polyamines discussed in this study.

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show neither UV absorption nor fluorescence properties (Hakkinen, Keinanen, andVepsalainen 2008; Yang and Tomellini 1999; Draisci et al. 1998; Zhou et al. 1995).The main problems in derivatization are: (1) longer analysis times; (2) the possibilityto have inaccurate results due to incomplete or unstable reactions with required deri-vatization compounds; (3) unselective labeling may lead to interfering by-products;(4) toxicity of some derivatizing agents; and (5) extra derivatization procedures notonly complicate the operations but also tend to increase the probability of humanerrors. Therefore, direct detection without derivatization is preferred whenever poss-ible. Thus, it is the aim of the present review to update readers on the major advancesin the determination of underivatized polyamines in different samples.

ANALYTICAL METHODS FOR THE DETERMINATION OFUNDERIVATIZED POLYAMINES

Flow Injection Analysis (FIA)

The impetus for the adoption of FIA methods is driven by the need to increaselaboratory productivity. The short analysis time based on transient signal measure-ments in a flow-through detector not only helps to increase the sample throughput,but also allows the on-line analysis of difficult operations (e.g., separation, pre-concentration, or physicochemical conversion of analytes into detectable species).

Aliphatic polyamines can be quantitatively electro-oxidized at diamond sur-faces (Jolley et al. 1997). The oxidation is highly dependent on the physicochemicalproperties of the polycrystalline-diamond surface. Specifically, the presence of sur-face boron for adsorbing=coordinating the polyamine near localized non-diamondcarbon impurities, where (OH�) is generated at lower overpotential than the sur-rounding diamond matrix can be exploited for analytical application. The basic elec-trochemical properties of polycrystalline, boron-doped, diamond thin-film electrodeshave been investigated by Koppang et al. (1999). Cyclic voltammetry of the polya-mines was conducted in carbonate buffer (pH 10) and measurements were made witha film deposited from a 0.50% methane-to-hydrogen (C=H) volumetric ratio. Thediamond thin films have been successfully used in amperometric detection schemescoupled with FIA for the determination of the polyamines (PUT, CAD, SPD, andSPM) without derivatization as shown in Fig. 2 (Witek and Swain 2001). The elec-trode performance was evaluated in terms of the linear dynamic range, limit ofquantification, response variability, and response stability. Linear dynamic rangeswere found from 1.0 mM to 1.0mM for PUT, CAD, and SPMD and from0.32 mM to 1.0mM for SPM. The response variability was vastly improved by intro-ducing a 3–6min delay period between injections and typical peak height variabilitieswere in the 2–4% range at extensively used films. Some response attenuation wasoften observed during the first 10 injections at a new diamond film electrode, butafter this, the response generally remained quite stable. The long-term stabilitywas excellent over a 10 h period of continuous use.

Flow injection with chemiluminescence detection (FI-CL) has been used for thedetermination of a wide range of analytes in diverse samples at nano to picomolarconcentrations (Sakamoto-Arnold 1987). Z.-P. Li et al. 2006 found that an unsatu-rated complex of Cu (II) and polyamines (PUT, SPD, and SPM) had a strong

DETERMINATION OF UNDERIVATIZED POLYAMINES 2247

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catalytic effect on luminol-H2O2-chemiluminescence reaction, and that the CL inten-sity is proportional to the concentration of polyamines. The FIAmethod was based onthe formation of an unsaturated complex of polyamines and Cu(II) when a solutioncontaining polyamines are passed through a column packed with solid Cu(OH)2.Under the optimum conditions (1.0mL of 1.0� 10�4molL�1 Cu (II), pH 9.2 ofpolyamine solution adjusted by 5� 10�3molL�1 H3BO3-NaOH buffer, the concen-tration of chemiluminescence reagents were 1.0� 10�2molL�1 of H2O2 and 5.0�10�3molL�1 of NaOH with 1.0m length of mixing coil), the polyamines weredetermined independently with linear range from 1.0� 10�7molL�1 to 1.0�10�5molL�1. The limits of detection (LODs) of the FI-CL method were 0.028,0.0.87, and 0.166 nmolL�1 for SPMSPD, and PUT, respectively, which are lower thanthose achieved with the immobilized enzymatic chemiluminescence detection methodand dansyl chloride or o-phthalaldehyde (OPA) derivatization method. Moreover, themethod offers advantages of being faster and simpler since there was no need for thepreparation of immobilized enzymes or for a derivatization reaction.

However, the bulk of the analytical methods reported are based on some formof separation, and these are discussed in the following sections.

High-Performance Liquid Chromatography (HPLC)

The HPLC method offers unique possibilities for the separation of analytesthat would otherwise be difficult or impossible to separate with other technologies.

Figure 2. Chromatogram for the isocratic, reverse-phase separation of CAD, PUT, SPMD, and SPM on a

C18 column. The solution mixture contained 0.5mM PUT, CAD, SPD, and 5.0mM SPM. The mobile

phase was (2:98 v=v) acetonitrile: 0.01M carbonate buffer, pH 11. Detection potential: þ820mV;

working electrode: 1.0% C=Hfilm; injection volume: 20mL; flow rate: 1.0mLmin�1 (Witek and Swain 2001).


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The separation of underivatized polyamines using reversed-phase HPLC (RP-HPLC) is challenging as these compounds have low column retention and oftenresulted in severe peak tailing when separated using the conventional C18 columns(Feistner 1994). As a result, the separation of underivatized polyamine can beachieved by ion-exchange or ion-pair RP-HPLC. Indeed, the dynamic ion-exchangecapacity of ion-pair system can be modulated through the careful choice of ionpair-reagents as well as mobile phase compositions.

Liquid chromatography-conductivity detection (LC-CD). Since poly-amines are relatively strong base (Perrin 1965) and are consequently ionized overa wide pH range, an ion chromatographic technique coupled with conductivitydetection was developed (Draisci et al. 1993). However, due to their strong hydro-phobic interactions with the chromatographic ion-exchanger stationary phase,separations required the use of a high concentration of acid or salt gradient in theeluent phase (modified with organic solvent) to elute the stronger retained poly-amines (Draisci et al. 1998; Hoekstra and Johnson 1998, 1999; Pineda et al. 2001).To simplify the separation, a weak carboxylic acid functionalized cation-exchangecolumn was introduced (Cinquina et al. 2004). The column consists of an ethylvinyl-benzene–divinylbenzene polymer support that was covered with a hydrophilic poly-mer layer that allows the use of low concentrations of an acidic eluent (no organicsolvent required) to separate the hydrophobic polyamines. This allows the use ofsimple conductimetric detection after the chemical suppression of the eluent usinga continuous automatic regeneration suppressor. The ion chromatographic tech-nique coupled with suppressed conductivity detection (LC-CD) was successfullyapplied to the determination of PUT, CAD, and SPD in tuna fish samples preservedin olive oil (Cinquina et al. 2004). The LODs were 0.15mg kg�1 for CAD,0.15mg kg�1 for PUT, and 0.50mg kg�1 for SPD. The precision was determinedby calculating the relative standard deviation (RSD%) for the repeated measure-ments. The RSD% was in the range between 2.44 and 3.69%.

The LC-CD method was also used to evaluate the influence of storage time andconditions on the evolution of PUT, CAD, SPM, and SPD in alcoholic beverages(De Borba and Rohrer 2007). The linearity of polyamines responses was within0.1–20mgL�1 and peak area precisions were 0.24–4.97%. The recoveries were within85–122%. Amino acids such as hydroxytryptophan, tryptophan, ornithine, and his-tidine interfered with determination of polyamines in alcoholic beverages. This wasresolved by further optimization of the gradient conditions. Figure 3 shows the sep-aration of polyamines using the LC-CD method in wheat beer after two weeks ofstorage at 4�C. The most significant changes in the polyamines concentrations storedfor up to 3 weeks were the detection of CAD in wheat beer and SPM and SPD in allbeer samples that did not have these polyamines prior to storage.

Casella, Palladino, and Contursi (2008) applied the LC-CD method to determi-nation of polyamines (PUT, CAD, SPM, and SPD) in beer and tuna samplesafter solid phase extraction (SPE). A surface modified styrene divinylbenzene poly-meric sorbent, based on a reversed-phase (RP) and strong cation exchange (SCX)mixed mode was used as the sorbent for the SPE of polyamines. The proposedmethod offers good results in terms of preconcentration, recoveries, and cleanupof samples.


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Liquid chromatography-condensation nucleation light scatteringdetection (LC-CNLSD). Condensation nucleation light scattering (CNLSD) isbased on the scatter intensity of desolvated analyte particles produced by continu-ously nebulizing the LC effluent and subsequently evaporating the mobile phasefrom the aerosol droplets introduced to the condensation growth step prior to thelight scattering detection cell (Szostek 1997). This type of detector can potentiallydetect all solutes that are less volatile than the mobile phase. Polyamines arenon-volatile compounds and are therefore suitable for this type of detection.

The use of CNLSD for the detection of polyamines that were separated withion-exchange chromatography was introduced by Sadain and Koropchak (1999).The polyamine cation amino groups that were protonated by an acidic mobilephase (10mM nitric acid in 10% acetonitrile) were separated by ion-exchange andhydrophobic interaction with the stationary phase (polybutadiene-maleic acid coatedsilica). The method was used for the analysis of PUT and CAD in tuna fish samples.Figure 4 shows a typical chromatogram of a diluted extracts of refrigerated tuna and2-day-old spoiled tuna. Detection by PUT in both tuna samples indicated an initialdecomposition process (Sadain and Koropchak 1999).

Liquid chromatography-chemiluminescence (LC-CL) detection. Inrecent years, there is immense interest in the use of chemiluminescence (CL) detectorin HPLC and CE separations not only due to its simplicity but also excellent sensi-tivity and wide linear working range (Z.-P. Li et al. 2006). An enzymatic CL detec-tion system has been described for the determination of polyamines with highspecificity (Kamei 1989). However, the high purity enzyme used (polyamine oxidase)is expensive and can be denatured easily. Wu et al. 2007 found that an unsaturatedcomplex of Cu(II) with polyamines, in which 2 of 4 coordination sites of Cu(II)are occupied by amine groups, had a strong catalytic effect on the luminol-H2O2

Figure 3. Chromatogram of polyamines determined in wheat beer by using LC-CD. Conditions: IonPac

CS18 (290meq=column, 250mm� 2mm) analytical column, 5 mL sample solution, gradient elution with

3mM Methanesulfonic acid. The determined analytes concentration: PUT (6.6mgL�1), CAD

(0.67mgL�1), SPD (1.2mgL�1), and SPM (0.73mgL�1) (De Borba and Rohrer 2007). (Figure

available in color online.)


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chemiluminescence reaction. Based on this catalytic effect, an HPLC-CL methodwas carried out on a RP-HPLC C18 column using methanol:water (25:75, v=v) asthe mobile phase. Figure 5 illustrates a chromatographic profile of polyamines(PUT, SPM, and SPD) standards: SPM and SPD were not separated. The workingcurves shows that the peak height was proportional to the concentration of PUT, butthe total concentration of SPM and SPD is proportional to the peak areas. There-fore, the quantitated PUT was based on the peak height and SPM and SPD wasbased on the peak areas. Correlation coefficients equal to or greater than 0.992 in

Figure 4. Chromatogram of PUT from tuna fish using LC-CNLSD. Dotted line: refrigerated tuna extract.

Solid line: spoiled tuna extract. Eluent: 10mM nitric acid and 10% acetonitrile. The concentrations of

PUT were 0.115mg g�1 and 0.233mg g�1 in refrigerated and spoiled tunes fish, respectively (Sadain and

Koropchak 1999).

Figure 5. Chromatogram of standard polyamines using LC-CL. Conditions: 1.0� 10�5molL�1 Cu(II)

solution in H3BO3-NaOH buffer solution, pH 9.2, 5.0� 10�4molL�1 luminol, 5.0� 10�4molL�1

NaOH with concentration, 1.0� 10-6molL�1 of each analyte (Wu et al. 2007).


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the concentration range from 1.0� 10-7 to 1.0� 10-4mol L�1 was found and theLOD was 2 pmol. The method was applied to the analysis of PUT, SPD, andSPM in apple leaves and strawberry. The recoveries of the selected polyamines usingLC-CL method were between 82 and 91%.

Liquid chromatography-indirect fluorescence (LC-indirect FL) detec-tion. The RP-HPLC method with indirect fluorescence detection (indirect-FL)was developed for the determination of the polyamines SPD and SPM (Yang andTomellini 1999). The principle of the method is based on the quenching of Cu(II)-L-tryptophan complexes; the fluorescence recovers with the addition of analytes (e.g.,SPD and SPM) that have a greater Cu(II) affinity for the complex. Thus, thepresence of these compounds in the HPLC eluent can be determined by monitoringthe fluorescence intensity of the L-tryptophan complex. A representative chromato-gram obtained under the optimized conditions is presented in Fig. 6. The detectionlimits are 5 and 10 pmol for SPM and SPD.

Liquid chromatography-mass spectrometry (LC-MS). The emergence ofalternative techniques such as liquid chromatography (LC) coupled to mass spec-trometry (MS) or tandem mass spectrometry (MS=MS) opens up new prospects inthe determination of polyamines. The advantage of LC-MS is the fact that all non-volatile polar polyamines can be analyzed without derivatization. Moreover, LC=MS=MS technique is very specific and sensitive; sample preparation thus can besimplified, and the chromatographic separation can be reduced to have baseline res-olution only for pairs of molecules that have similar MS=MS characteristics. Thesetechniques, however, need an extensive validation and careful assessment of LC=MS=MS specificity, including studies of the matrix effect and ion suppression.

The online coupling of electrospray mass spectrometry (ESI-MS) with ionchromatography and RP-HPLC for the analysis of underivatized polyamines has

Figure 6. Chromatogram of SPM and SPD using LC-indirect FL. Separation was conducted using a

Hamilton PRP-X200 SCX column at ambient temperature. The mobile phase was 0.8M KCl adjusted

to pH 5.25 with 1.5� 10�3M acetate buffer. The postcolumn reagent was 0.5� 10�4M Cu(L-Trp)2,

buffered at 8.50 with 4� 10�3M sodium borate. The eluent and postcolumn reagent flow-rates were 0.8

and 2.0mLmin�1 respectively. Analyte concentration: 640 pmol for SPD and 320pmol for SPM (Yang

and Tomellini 1999).


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been described by Feistner (1994). The coupling of HPLC and MS for metabolicprofiling of the underivatized polyamines was, however, found to be unsuccessfuldue to the compatibility problems between the MS and the separation methods.Without derivatization, the target compounds give characteristic product ion spectrabut are not amenable to reversed phase chromatography. Whether the correspon-ding dansyl chloride derivatives would give useful tandem mass spectra was uncer-tain because the dansyl derivatives of polyamines have been reported to give risepredominantly, and sometimes exclusively, to dansyl group-related fragment ions.The latter are, of course, useless for structural characterization (Feistner 1995).

The HPLC methods based on an MS=MS detection that employs the separ-ation and detection of underivatized polyamines have been published. The firstmethod uses cation-exchange chromatography and suppressed conductivity coupledwith mass spectrometry detection (Saccani et al. 2005). The method has been appliedto the analysis of PUT, CAD, and SPD in processed meat products. Polyamineswere extracted from muscle tissue with methanesulfonic acid and separated by acation-exchange column (IonPac CS17) used with gradient elution. The MS detec-tion was carried out by a single stage quadrupole detector operated in the positiveelectrospray (ESIþ) ionization mode at 3.0 kV with a probe temperature set at350�C. Linearity of response was obtained in the range 0.25–25 mgmL�1. The LODswere 22 mgmL�1, 15 mgmL�1, and 33 mgmL�1 for PUT, CAD, and SPD, respect-ively. Average recoveries from meat samples ranged from 83 to 95% and RSD%ranged from 5.2 to 7.2%. The fat content and non-protein nitrogenous substances(low molecular weight peptides and free amino acids) did not interfere with thedetermination of the polyamines. Use of a suppressor device before the MS ioniza-tion provided a number of advantages to the amount of information obtained forpolyamines in a single run, including simple extraction procedure and clean-up, elu-tion at low acid concentration, improved chromatographic separation, and longterm stability of the MS signal. However, LODs of the method are quite high andrather time-consuming, taking 40min of running time.

The separation and analysis of PUT, CAD, SPM, and SPD using RP-HPLC-ESI tandem mass spectrometry (ESI-MS=MS) (Hakkinen et al. 2007) was alsodescribed. The separation of these compounds was achieved by using ESIþ modeand linear gradient elution with added heptafluorobutyric acid (HFBA) as volatileion-pair modifier. Signal suppression was prevented by the post-column additionof propionic acid to the aqueous organic eluents (Fig. 7). The RP-LC-ESI-MS=MS is easier to use and substantially faster due to minimal sample pre-treatmentand rapid chromatographic separation (10min). Moreover, the method allows posi-tive identification of the products by the highly sensitive MS and the possibility toeliminate interfering peaks arising from the matrix by the selective MS=MS detector.The method was applied to the analysis of underivatized polyamines (PUT, SPD,and SPM) in plant samples (Sanchez-Lopez et al. 2009). Linearity of response wasobtained in the range of 25–250 mgL�1. The LODs were 8.4 mgL�1, 9.9 mgL�1,and 3.1 mgL�1 for PUT, SPD, and SPM, respectively. Average recoveries from92.6–108.9% from standard compounds in HFBA and RSD% were in the range of10.5–21.8%. The method has been validated in Arabidopsis thaliana samples andpolyamines have been determined in several genotypes that over express or are dis-rupted in the Arginine Decarboxylase2 gene. Consequently, this methodology allows


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the analysis of plant polyamine, opening up opportunities for deeper studies of therole of polyamine in plant-pathogen interactions.

Electromigration Methods

Capillary electrophoresis (CE) is a powerful separation technique that is basedon differences in the mobilities of analytes (normally charged) in an electrophoreticmedium within a narrow bore capillary. CE is an attractive alternative for polya-mines determination because of its many advantageous features, especially in termsof higher efficiency, lower sample volume requirements, minimal solvent consump-tion, and so forth (Ma et al. 2004).

Due to the positive charge under both neutral and acidic conditions, underiva-tized polyamines are readily separated by the electrophoresis technique. The mostfrequently used mode is capillary zone electrophoresis (CZE) (Zhang, Cooper, andMa 1993; Ma, Zhang, and Cooper 1992) and capillary electrochromatography(CEC) (H. Li et al. 2008).

Indirect UV detection offers universal detection for the simultaneous determi-nation of polyamines. An adsorbing co-ion was added to the background electrolyte(BGE), and negative peaks were detected at the migration zones of the charged andnon-UV active solutes (Fekete et al. 2008). Co-ions such as copper sulfate (Arce,Rıos, and Valcarcel 1998, 1997), imidazole (Matchett and Brumley 1997), andquinine sulfate (Zhou et al. 1995) have been used as BGE for CE determination

Figure 7. Positive-ion RP-LC=MS=MS SRM chromatogram of mixture of polyamines. The separation

condition was carried out by linear gradient elution of 0–50% B in 10min (solvent A: 0.1% (v=v)

heptafluorobutyric acid in water, solvent B: 0.1% (v=v) heptafluorobutyric acid in ACN) at flow rate of

0.2mLmin�1. Injection volume of the 100mM sample was 10mL. Sample was diluted with water.

Isocratic post-column addition of a solution of 75% propionic acid and 25% isopropanol in ratio 1:2

was performed through a peek mixing tee at 0.1mLmin�1 into the column flow (Hakkinen et al. 2007).


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of underivatized polyamines. The co-ions concentration was in the range of5–20mM and the pH of the buffer was in the range of 3.0–5.5. Crown ethers areusually added into the BGE to improve the selectivity of the method. The electro-osmotic flow-rate and the velocity of migration of the analytes are proportional tothe applied voltage used for the separation. The analysis time can be shortened byincreasing the applied voltage. Short analysis times (4.5min) were achieved by run-ning the separation at 30 kV, but this voltage was too high for the separation of allthe polyamines. The resolution can be improved by reducing the applied voltage. At10 kV good resolution was obtained for the selected polyamines (Fig. 8; Arce et al.1997). Although the electrophoretic separation of the underivatized polyamines wasdemonstrated, the sensitivity of the technique needs to be further improved.

A CE method with indirect chemiluminescence (CL) detection for the determi-nation of underivatized polyamine was demonstrated by Huang and Ren (2003). Inthis method, a strong background CL signal was generated by the luminol-H2O2-chemiluminescence reaction that was catalyzed by a Co(II) probe ion in the back-ground electrolyte (BGE). Displacement of the Co(II) probe ion in the BGE bymigration of polyamine cations results in a decrease in the background signal. Underthe optimum conditions, PUT, SPD, and SPM were well separated in less than4.5min and LODs were in the range between 5–12 nmol L�1.

Closely related to the CL method is the electrochemiluminescence (ECL) tech-nique. The luminescence generated by the relaxation of exited state molecules thatwere produced during an electrochemically initiated reaction can be exploited foranalytical measurements (Fahnrich, Pravda, and Guilbault 2001). This was demon-strated by the use of tris(2,2-bipyridyl) ruthenium(II)(RuðbpyÞ2þ3 ) (one of the mostwidely used reagent due to its good stability and ECL efficiency in aqueous media)

Figure 8. Electropherogram of a standard mixture of polyamines (0.05–1mgmL�1). The electrolyte

comprised 4mM copper sulfate, 4mM formic acid, and 4mM 18-crown-6 at pH 3.0. The separation

was performed at applied potential 10 kV, wavelength 210 nm, and temperature 20�C (Arce et al. 1997).


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to oxidize polyamines directly by (RuðbpyÞ3þ3 ), as outlined in Scheme 1 (Noffsinger1987; Zu and Bard 2000).

The relative contribution of the two oxidation routes (Eq. 1 and Eq. 2) to theformation of the excited states (Eq. 3) and the observed emission (Eq. 4) depends ona series of parameters such as the electrode potential, the concentration ratio of[RuðbpyÞ2þ3 ] to polyamine and the homogenous reaction rate of [RuðbpyÞ3þ3 ] withpolyamine (Zu and Bard 2000).

Liu et al. (2003) introduced a method based on the coupling of CE with[RuðbpyÞ2þ3 ] ECL for the determination of underivatized polyamines (PUT, CAD,SPD, and SPM) in human urine samples. The separation of polyamines was achievedby using CZE in an uncoated fused-silica capillary (50 cm� 25 mm ID) filled withacidic phosphate buffer (separation voltage of 5 kV), with end-column [RuðbpyÞ2þ3 ]-ECL detection. The analytical conditions were set as: 300 mm Pt disk electrode; sep-aration buffer, 200mmol L�1 phosphate buffer (pH 2.0) and 1mol L�1 phosphoricacid (9:1 v=v); 5 kV separation voltage; 10 kV and 10 s for sample injection; and5mmol L�1 RuðbpyÞ2þ3 þ 200mmol L�1 buffer in the reservoir, pH 11. A typicalelectropherogram is shown in Fig. 9 (Liu et al. 2003). The analytical reproducibility

Scheme 1.

Figure 9. CZE-ECL electropherogram of the four standard polyamines. Working electrode, 300mm Pt

disk electrode; sample solution, 50mmolL�1 PUT and CAD, 0.5 mmolL�1 SPD and SPM; injection,

10 kV for 10 s; separation buffer, 200mmol L�1 phosphate buffer (pH 2.0)–1molL�1 phosphoric acid

(9:1 v=v); capillary, 50 cm� 25 mm ID; separation voltage, 5 kV; RuðbpyÞ2þ3 solution, 5mmolL�1 plus

200mmolL�1 phosphate buffer in the reservoir, pH 11.0; detection potential, þ1.2V (vs. Ag=AgCl)

(Liu et al. 2003).


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of the method was tested by five identical injections in one day and in five days of10 mmolL�1 PUT and CAD, and 0.5 mmolL�1 SPD and SPM standard solution.The relative standard deviations of ECL peak intensities were less than 8%. Thelinearity of peak height response was evaluated using the polyamines standardsolutions and showed correlation factors from 0.9974 to 0.9988. The LODs obtainedwere 1.9� 10�7mol L�1 for PUT and CAD and 7.6� 10�9mol L�1 for SPD andSPM, respectively.

Using CEC coupled with [RuðbpyÞ2þ3 ]-ECL detection has also been applied forthe determination of underivatized polyamines (PUT, SPD, and SPM) in humanurine samples (Fig. 10; H. Li et al. 2008). An efficient CEC separation of PUT,SPD, and SPM was achieved when the pH of the BGE is in the range of 3.5–7.0.The optimum BGE for the CEC separation is much less acidic than that for theCZE separation used by Liu et al. (2003). The separation of polyamines is less than12.5min, which is about half the time needed for the CZE method. Urine samplesusually contain some coreactants of [RuðbpyÞ2þ3 ]-ECL, such as amino acids, amines,ascorbic acid, and oxalate. The unidentified peaks probably resulted from some ofthese coreactants. The concentration of each polyamine calculated by direct interp-olation of the peak height from the corresponding regression equation was 9.82,0.61, and 0.88mM for PUT, SPD, and SPM, respectively. The recoveries formeasurements of 2mM of PUT, SPD, and SPM were 109.6, 85, and 95.4%, respect-ively. The RSD for three consecutive measurements of PUT, SPD, and SPM were2.2, 5.3, and 6.0%, respectively.

Underivatized polyamines (PUT, SPD, and SPM) have also been separatedand quantified by CE with pulsed amperometric detection (Sun, Yang, and Wang2003). Detection potential of the pulsed amperometric method was 0.6V. Optimalseparation of the polyamines was achieved using a BGE 30mM citrate at pH 3.5.It should be pointed out that the optimized pH of the BGE was not suitable for

Figure 10. Electropherogram obtained from hydrolyzed human urine sample by using CEC-ECL.

Conditions: capillary column, 50 cm� 25 mm id; n-octyltriethoxysilane(C8-TEOS)=tetraethoxysilane

(TEOS) organic-inorganic hybrid sol-gel OTCEC column. Mobile phase: 100mmolL�1 pH 6.3

phosphate buffer solution; separation voltage, 15 kV; electrokinetic injection, 10 s at 10 kV. Detection

condition: 100mmolL�1 pH 9.0 phosphate buffer solution containing 5.0mM (RuðbpyÞ2þ3 ) in the

detection cell, 500mm platinum disk electrode, 1.2V (H. Li et al. 2008).


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the optimum functioning of the detector (20mM NaOH; Fig. 11). ExtrapolatedLODs for PUT, CAD, SPD, and SPM were 400, 200 100, and 400 nM for thestandard mixture dissolved in running buffer, respectively. When applying CE-CDto milk analysis, only SPD was found, varying between 0.1 and 0.5mg kg�1.

The coupling of CE with mass spectrometry (MS) has been increasingly usedsince its first report by Olivares et al. (1987). There are three main interfaces tocouple CE to MS, namely: the sheathless, liquid junction, and sheath liquid inter-faces. Sheath liquid has been the most commonly used owing to its high stability,versatility, and commercial availability. Based on the sheath liquid interface couplingof CE to MS, Santos et al. (2004) introduced a capillary electrophoresis-electrospraymass spectrometry (CE-ESI-MS) method for the separation and determination ofselected polyamines. Samples were separated by using 25mM citric acid at pH2.0 as BGE. The proposed method allows CAD to be determined with LOD15 mgL�1 and 4.9% of RSD value. The CE-ESI-MS method was used to analyzeCAD in red and white wines. The detection of CAD was only in red wine with aconcentration of 0.13 mgmL�1.

Comparison of Analytical Methods

Table 1 summarizes the analytical techniques reported for the determination ofunderivatized polyamines. The most commonly used, RP-LC, analyzes nonvolatilecomponents in different matrices. However, low molecular weight polar compoundssuch as hydrophilic polyamines are not sufficiently retained and consequently alter-native strategies are required. Ion-pairing reagents such as perfluorinated carboxylicacids have been proven to improve the separation of polyamines on C18 columns

Figure 11. Electropherogram of the standard solution of PUT, CAD, SPM, and SPD using CE-CD.

Conditions: separation buffer, 50mM citrate with pH of 3.5, detection solution, 20mM NaOH (Sun

et al. 2003).


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Table

1.Comparisonofselected

analyticalmethodsfortheanalysisofunderivatizedpolyamines

Method

Detectedanalytes

Advantages

Disadvantages

LOD

orLOQ

Ref.

FI-CD

PUT,CAD,SPD,

andSPM

Highthrough

putandno

separationneeded

Depositionofdetectionelectrodeby

certain

mobilephase

components

1mm

ol=LforPUT,CAD,

andSPD

(LOQ)

0.32mm

olL

�1forSPM

(LOQ)

Witek

andSwain

2001

Fl-CL

PUT,SPD,and

SPM

Highthrough

putandno

separationneeded

Highconsumptionofsample

and

reagents

5.6–14.0nmolL

�1(LOD)

Z.-P.Liet

al.

2006

LC-C

DPUT,CAD,SPM,

andSPD

Detectionofall

polyamines

Compatibilitybetweenmobilephase

compositionanddetectorresponse

24.0–25.9nmolL

�1(LOD)

DeBorbaand

Rohrer2007

LC-C

NLSD

PUTandCAD

Fast

elution

Highsignalto

noiseratio

74nmolL

�1forPUT

(LOD)

117nmolL

�1forCAD

(LOD)

Sadain

and

Koropchak

1999

LC-C

LPUT,SPD,and

SPM

Goodsensitivity

Specialrequirem

entformobilephase

0.1mm

olL

�1(LOD)

Wuet

al.2007

LC-indirect

fluorescence

SPM

andSPD

Sensitive

Applicable

forlimited

number

of

polyamines

0.5mm

olL

�1forSPM

(LOD)

1mm

ol=LforSPD

(LOD)

Yangand

Tomellini1999

LC-M

SPUT,SPD,and

SPM

Robust

method,

multiple

determinationin

one-step

Ionsuppressionandhighcost

15.3–95.0nmolL

�1(LOD)

Sanchez-Lopez

etal.2009

CE-indirect

UV

PUTandCAD

Donotrequire

additionalcomponent

Matrix

effect

1.04–1.76mm

olL

�1(LOD)

Feketeet

al.2008

CE-C

LPUT,CAD,SPM,

andSPD

Sensitive

Lim

itationin

choosingbuffer

0.19mm

olL

�1forPUTand

CAD

(LOD)

7.6nmolL

�1forSPD

and

SPM

(LOD)

Liu

etal.2003

CE-C

DPUT,CAD,SPM,

andSPD

Fast

separation

Depositionofdetectionelectrodeby

mobilephase

components

0.1–0.4mm

olL

�1(LOD)

Sunet

al.2003

CE-M

SCAD

Sensitive

andlow

sample

consumption

Dilutionoftheanalyte

bythesheath

liquid

andhighcost

0.14mm

olL

�1(LOD)

0.49mm

olL

�1(LOQ)

Santo

etal.2004

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and in LC coupled with ESI-MS. The use of ion-paring reagents in combination withESI-MS might lead to problems such as ion suppression, memory effects, and con-tamination of the MS source. Fortunately, this problem can be solved by addingadditives in the mobile phase (Hakkinen et al. 2007; Sanchez-Lopez et al. 2009).

The LC-CD method offered good sensitivity for all the underivatized poly-amines (PUT, CAD, SPD, and SPM) with LOD ranging from 24–25.9 nmol L�1.However, the reported method requires alkalinization of the eluent phase prior tothe detection.

Detection of underivatized PUT separated by ion-exchange chromatographywith LOD at mmolL�1 was obtained using CNLSD (Table 1). The disadvantageof the ELSD is the use of direct signal measurement from the sensor. This type ofmeasurement has inherent sensor noise and drift that limits the sensitivity andstability of the overall detector.

The LC-indirect fluorescence provides a sensitive and convenient way of detect-ing SPM and SPD following chromatographic separation. However, the limitingfactor for this method is the stability constant for forming the Cu(II) complex.For example PUT, which has two amino groups that are separated by four methyl-ene groups, was found to be much less detectable than either SPM and SPD underthe same experimental conditions due to the fact that substantial structure distortionis required for PUT to coordinate with Cu(II).

The CE method is another technique that was applied to the determination ofunderivatized polyamines that had been coupled with different detectors. The LODsachieved are higher when compared to HPLC due to the lower amounts of samplesinjected and to the short optical path lengths. These drawbacks can be avoided byusing a highly sensitive detection system. Undoubtedly, MS offers the best sensitivityin comparison with the CD and indirect UV detection (Table 1).

The CL detector is interesting due to its simplicity, low cost, providing lowbackground noise, good sensitivity, and wide dynamic range. The coupling of thissensitive detection mode with a high efficient separation technique such as HPLCor CE enhances the selectivity of the CL detection. A problem limiting the wide-spread application of CL to be coupled with HPLC or CE in the determination ofpolyamines is in the interface design. The mixing of the CL reagent and the BGEin CE or the mobile phase of HPLC is critical in order to achieve good separationefficiency while maintaining the sensitivity. As shown in Table 1, the best LODsfor overall analytical methods were achieved by MS detection.

CONCLUDING REMARKS

This review reports the determination of underivatized polyamines (PUT,CAD, SPM, and SPD) in different samples. This has been made possible by themany advances in separation and detection methodologies. The HPLC separationof underivatized polyamines by adding ion pair reagent to RP-C18 columns allowsthe hyphenation of LC to ESI-MS=MS. The latter, however, is subject to matrix andion-suppression effects that impair quantitative accuracy and necessitate the use of avolatile ion pair modifier together with a post-column. Nevertheless, it is anticipatedthat MS based methods will prevail in the future. Interestingly, until now, the advan-tages of the various electrically driven chromatographic systems such as CZE and


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CEC have not been fully exploited. The most promising systems seems to be the tan-dem MS and the CL detector due to their high sensitivity and specificity. It can beexpected that innovations in sample preparations such as microextractions will beused to treat and enrich trace levels of polyamines in complex matrices (Saaidet al. 2009). It is reasonable to expect that the relatively new detectors such asthe capacitively coupled contactless detectors (Makahleh et al. 2010) will also beused together with the fast ultra performance liquid chromatography in the nearfuture.

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