molecules Article
Determination of Biogenic Amines in Seawater Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection Elbaleeq A. Gubartallah 1,2, *, Ahmad Makahleh 3 , Joselito P. Quirino 4 Bahruddin Saad 1,5, * 1 2 3 4 5
*
ID
and
School of Chemical Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia Chemistry Department, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan Department of Chemistry, Faculty of Science, University of Jordan, Amman 11942, Jordan;
[email protected] Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart 7001, Australia;
[email protected] Fundamental & Applied Sciences Department and Institute for Sustainable Living, Universiti Teknologi PETRONAS, Seri Iskandar 32610, Perak, Malaysia Correspondence:
[email protected] (E.A.G.);
[email protected] (B.S.); Tel.: +605-368-7683 (B.S.); Fax: +605-365-5905 (B.S.)
Received: 17 April 2018; Accepted: 3 May 2018; Published: 8 May 2018
Abstract: A rapid and green analytical method based on capillary electrophoresis with capacitively coupled contactless conductivity detection (C4 D) for the determination of eight environmental pollutants, the biogenic amines (putrescine, cadaverine, spermidine, spermine, tyramine, 2-phenylamine, histamine and tryptamine), is described. The separation was achieved under normal polarity mode at 24 ◦ C and 25 kV with a hydrodynamic injection (50 mbar for 5 s) and using a bare fused-silica capillary (95 cm length × 50 µm i.d.) (detection length of 10.5 cm from the outlet end of the capillary). The optimized background electrolyte consisted of 400 mM malic acid. C4 D parameters were set at a fixed amplitude (50 V) and frequency (600 kHz). Under the optimum conditions, the method exhibited good linearity over the range of 1.0–100 µg mL−1 (R2 ≥ 0.981). The limits of detection based on signal to noise (S/N) ratios of 3 and 10 were ≤0.029 µg mL−1 . The method was used for the determination of seawater samples that were spiked with biogenic amines. Good recoveries (77–93%) were found. Keywords: capillary electrophoresis; capacitively coupled contactless conductivity detector; biogenic amines; seawater
1. Introduction Biogenic amines (BAs) are basic organic compounds with aliphatic, aromatic, or heterocyclic structures. They are classified into mono or polyamines according to the number of amino groups they contain. In foods, BAs are formed by microbial decarboxylation processes of related amino acid [1–3]. In low concentrations, polyamines are essential for nucleic acid and protein synthesis [1]. Putrescine (PUT), cadaverine (CAD), spermidine (SPD), spermine (SPM), histamine (HIS), phenylethylamine (PEA), tyramine (TYR) and tryptamine (TRY) are considered to be the most important BAs that are found in foods [1,2]. BAs have also been proposed as indicators of food quality and freshness [2]. BAs can be found in different types of foods, such as fruits, vegetables, dairy products, meat, fish, beverages and fermented food [3]. The presence of BAs in significant concentrations can cause toxicity [3]. Table 1 shows the chemical properties of some of the important BAs. From the environmental point of Molecules 2018, 23, 1112; doi:10.3390/molecules23051112
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BAs can react react with nitrite nitrite to produce produce nitrosamines which which are highly highly carcinogenic compounds compounds [3]. BAs BAs can can react with with nitrite to to produce nitrosamines nitrosamines which are are highly carcinogenic carcinogenic compounds [3]. [3]. Thus, the analysis BAs in samples has started to receive considerable interest lately [4,5]. BAs can react withof nitrite towater produce nitrosamines which are highly carcinogenic compounds [3]. Thus, the analysis of BAs in water samples has started to receive considerable interest lately [4,5]. Thus, the of in samples has receive interest lately Thus,Several the analysis analysis of BAs BAs in water water samples has started started to receive considerable considerable interest lately [4,5]. [4,5]. Several analytical methods have been used used for the theto determination of BAs. BAs. These These methods mainly Thus, the analysis of BAs in water samples has started to receive considerable interest lately [4,5]. analytical methods have been for determination of methods mainly Thus, the analysis of BAs in water samples has started receive considerable interest lately [4,5]. Several analytical methods have been used used for the the to determination of BAs. BAs. These These methods mainly Several analytical methods have been for determination of methods mainly Several analytical methods methods have been used for the the determination determination of BAs. BAs. These These(HPLC) methods mainly used gas chromatography (GC) [6,7], high performance liquid chromatography [8,9] and Several analytical have been used for of methods mainly used gas chromatography (GC) [6,7], high performance liquid chromatography (HPLC) [8,9] and analytical methods have for the BAs.with These methods mainly used gas chromatography (GC) [6,7], highused performance liquid chromatography (HPLC) [8,9] and view, it isSeveral interesting to monitor BAs in been water bodies, as determination some BAs canofreact nitrite to[8,9] produce used gas chromatography (GC) [6,7], high performance liquid chromatography (HPLC) and used gas chromatography (GC) [6,7], high performance liquid chromatography (HPLC) [8,9] and capillary electrophoresis (CE) [10,11]. Most of these techniques required chemical derivatization due used gas gas electrophoresis chromatography (GC) [6,7],Most highofperformance performance liquidrequired chromatography (HPLC) [8,9] [8,9] due and capillary (CE) [10,11]. these techniques chemical derivatization used chromatography (GC) [6,7], high liquid chromatography (HPLC) and capillary electrophoresis electrophoresis (CE) [10,11]. Most of these these techniques techniques required chemical derivatization due nitrosamines which are highly carcinogenic compounds [3]. Thus, the analysis of(UV) BAs in water samples capillary (CE) [10,11]. Most of required chemical derivatization due capillary electrophoresis (CE) [10,11]. Most of these techniques required chemical derivatization due to the lack of chromophores and to increase the sensitivity using ultraviolet and fluorescence capillary electrophoresis (CE)and [10,11]. Most of ofthe these techniques required chemical derivatization due to the lack of chromophores to increase sensitivity using ultraviolet (UV) and fluorescence capillary (CE) [10,11]. Most these techniques required chemical due to the the lack lack of chromophores chromophores and to increase increase the sensitivity using ultraviolet (UV)derivatization and fluorescence fluorescence has started toelectrophoresis receive considerable interest lately [4,5]. to of and to the sensitivity using ultraviolet (UV) to the the lack lack of chromophores chromophores and to increase increase the sensitivity using ultraviolet (UV) and andto fluorescence detection. Recently there has been great interest in the use of direct detection to avoid the to of and to the sensitivity using ultraviolet (UV) and fluorescence detection. Recently there has been great interest in the use of direct detection avoid the to the lack Recently of chromophores andbeen to increase the sensitivity ultraviolet (UV) andtofluorescence detection. there has has great interest in the the using use of of direct detection avoid the detection. Recently there been great interest in direct to avoid the Several analytical methods have been used for the determination BAs.detection These methods mainly detection. Recently there has been great interest in the use use of of direct detection to expensive, avoid the derivatization step. This is mainly due to the fact that (i) most derivatization reagents are expensive, detection. Recently there has been great interest in the use of direct detection to avoid the derivatization step. This is mainly due to the fact that (i) most derivatization reagents are detection. Recently there has been great interest the of direct detection to expensive, avoid the derivatization step. This This is mainly mainly due to the the fact that thatin (i) liquid mostuse derivatization reagents are step. is due to fact (i) most reagents are usedderivatization gasa chromatography [6,7], high performance chromatography (HPLC) [8,9] and derivatization step. This This(GC) is time mainly due to the the fact that that (i) most derivatization derivatization reagents are expensive, expensive, (ii) long derivatization is required, (iii) side products associated with derivatization are derivatization step. is mainly due to fact (i) most derivatization reagents are expensive, (ii) long derivatization is required, side products associated with derivatization are derivatization step. This is time mainly to the (iii) fact (i) most derivatization reagents are expensive, (ii) aaaelectrophoresis long derivatization derivatization time is due required, (iii) that side products associated with derivatization are (ii) long time is required, (iii) side products associated with derivatization are capillary (CE) [10,11]. Most of these techniques required chemical derivatization due (ii) a long derivatization time is required, (iii) side products associated with derivatization are frequently encountered, and (iv) the shelf-life of derivatization reagents are short [4,12]. CE has (ii) aa long long encountered, derivatizationand time(iv) is the required, (iii)ofside side products associated associated with derivatization are frequently shelf-life derivatization reagents are short [4,12]. CE has (ii) derivatization time is required, (iii) products with derivatization are frequently encountered, encountered, and (iv) the shelf-life of derivatization derivatization reagents are are short [4,12]. CE CE has has frequently and (iv) the shelf-life of reagents short [4,12]. frequently encountered, and (iv) the shelf-life of derivatization reagents are short [4,12]. CE has to the lack of chromophores and to increase the sensitivity using ultraviolet (UV) and fluorescence proven to be an interesting separation technique, mainly due to its superb resolving power in frequently encountered, and separation (iv) the the shelf-life shelf-life of derivatization derivatization reagents are short short [4,12]. CE has has proven to be an interesting technique, mainly due to its superb resolving power in frequently encountered, and (iv) of reagents are [4,12]. CE proven to to be be an interesting interesting separation technique, mainly due due to to its superb superb resolving power in proven an separation technique, mainly its resolving power in proven to be an interesting separation technique, mainly due to its superb resolving power in detection. Recently there has been great interest inisomers, the use of direct detection to avoid the derivatization separating closely-related compounds (e.g., isomers, chirals) and the superb consumption of markedly markedly proven to be be an interesting separation technique, mainly due to its its resolving power in in separating closely-related compounds (e.g., chirals) and the consumption of proven to an interesting separation technique, mainly due to resolving power separating closely-related compounds (e.g., isomers, chirals) and the superb consumption of markedly markedly separating closely-related compounds (e.g., isomers, chirals) and the consumption of separating closely-related compounds (e.g., isomers, chirals)the and the consumption of (ii) markedly reduced samples and reagents. Several papers have described the usethe of CE CE forexpensive, the determination determination of step.separating This is samples mainly duereagents. to the fact that (i) most derivatization reagents arefor a long separating closely-related compounds (e.g., isomers, chirals) and consumption of markedly reduced and Several papers have described use of the of closely-related compounds (e.g., isomers, chirals) and consumption of markedly reduced samples samples and reagents. reagents. Several papers papers have described described the usethe of CE CE for the the determination determination of reduced and Several have the use of for of reduced samples and reagents. Several papers have described the usederivatization of CE CE for as theamperometric determination of BAs. As UV detection results in low sensitivities in CE, other detectors, such [5], derivatization time is required, (iii) side products associated with are frequently reduced samples and reagents. Several papers have described the use of for the determination of BAs. As UV detection results in low sensitivities in CE, other detectors, such reduced samples and reagents. papers have use of CE for as theamperometric determination[5], of BAs. As As UV UV detection detection results in inSeveral low sensitivities sensitivities indescribed CE, other otherthe detectors, such as amperometric [5], BAs. results low in CE, detectors, such as amperometric [5], 4D) [4,14], have BAs. As UV detection results in low sensitivities in CE, other detectors, such as amperometric [5], conductometric [13] and capacitively coupled contactless conductivity detections (C 4 BAs. As As UV UV detection results inoflow low sensitivities in CE, CE, other other detectors, such as amperometric [5], 44D) [4,14], encountered, anddetection (iv) theand shelf-life derivatization reagents are detectors, short [4,12]. CEas has to be an conductometric [13] capacitively coupled contactless conductivity detections (C have BAs. results in sensitivities in such amperometric [5], conductometric [13] and capacitively coupled contactless contactless conductivity detections (Cproven D) [4,14], [4,14], have conductometric [13] and coupled conductivity detections (C have conductometric [13] and capacitively capacitively coupled contactless conductivity detections (C4444D) D) [4,14], have been reported. Sensitivity improved by a factor of 100 when the C D was used compared to the indirect 44D conductometric [13] and capacitively coupled contactless conductivity detections (C D) [4,14], have 4 been reported. Sensitivity improved by a factor of 100 when the C was used compared to the indirect interesting separation technique, mainly due to its superb resolving power in separating closely-related conductometric [13] and capacitively contactless detections (C D) have been reported. reported. Sensitivity Sensitivity improved by bycoupled factor of of 100 when whenconductivity the C C444D D was was used used compared to[4,14], the indirect indirect been improved aaaafactor the to the beendetection reported. Sensitivity improved by factorabsorbing of100 100 when when the C C[15]. D was was usedcompared compared to thereagents. indirect UV detection for the determination determination ofby non-UV absorbing amines Areduced further scrutiny of these papers been reported. Sensitivity improved by factor of 100 the D used compared to the indirect 44D UV the of non-UV amines A further scrutiny these papers compounds (e.g.,for isomers, chirals) and the consumption of markedly samplesof and been reported. Sensitivity improved a factor of 100 when the C[15]. was used compared to the indirect UV detection for the determination of non-UV absorbing amines [15]. A further scrutiny of these papers UV for the of absorbing amines A scrutiny of papers UVdetection detection forcomplicated thedetermination determination ofnon-UV non-UV absorbing amines [15]. Afurther further scrutiny ofthese these papers also reveals that that complicated background electrolytes (BGE) that[15]. contain cyclodextrin [15], crown ether UV detection for the determination of non-UV absorbing amines [15]. A further scrutiny of these papers also reveals background (BGE) that contain cyclodextrin [15], crown ether Several papers have described the of use ofelectrolytes CEabsorbing for the determination of BAs. As UV detection UV for the determination non-UV amines [15]. A further scrutiny of these papers alsodetection reveals that that complicated background electrolytes (BGE) that contain contain cyclodextrin [15], crown ether also reveals complicated background electrolytes (BGE) that cyclodextrin [15], crown also reveals reveals that complicated background background electrolytes (BGE) that contain cyclodextrin [15], [15], crown crown ether ether [4,5,15] and α-hydroxyisobutyric acid [16] were used as the chiral selector. also that complicated electrolytes (BGE) that contain cyclodextrin ether [4,5,15] and α-hydroxyisobutyric acid [16] were used as the chiral selector. also that complicated electrolytes contain cyclodextrin [15], crown results inreveals low sensitivities in CE,background other detectors, as amperometric [5], conductometric [13]ether and [4,5,15] and α-hydroxyisobutyric acid [16] weresuch used(BGE) as thethat chiral selector. [4,5,15] and α-hydroxyisobutyric acid [16] were used as chiral selector. 4Dthe [4,5,15] and α-hydroxyisobutyric acid [16] were used as the chiral selector. In this work, we describe a simple and green CE–C method for the separation and quantitation of 4 [4,5,15] and α-hydroxyisobutyric acid [16] were used as the chiral selector. 44D the 4 In this work, we describe a simple and green CE–C method for the separation and quantitation of [4,5,15] and α-hydroxyisobutyric acid [16] were used as chiral selector. capacitively coupled contactless detections (Cmethod D) [4,14], have been reported. Sensitivity In this this work, we we describe describe aaconductivity simple and and green green CE–C44D D method for the the separation andquantitation quantitation of In work, simple CE–C for separation and of In this work, we describe a simple and green CE–C D method for the separation and quantitation of 4 eight BAs (PUT, CAD, SPD, SPM, TYR, HIS, TRY and PEA). The chemical structures of the BAs are shown In this work, we describe simple and green CE–C D method method for theindirect separation and quantitation of 4D eight BAs (PUT, CAD, SPD, SPM, TYR, HIS, TRY and PEA). The chemical structures of the BAs are shown 4and this we aa simple green CE–C the separation quantitation of improved by a work, factor of describe 100 the CHIS, D was compared tofor the UV detection for the eightIn BAs (PUT, CAD, SPD,when SPM, TYR, HIS, TRYused and PEA). Thechemical chemical structures ofand the BAs areshown shown eight BAs (PUT, CAD, SPD, SPM, TYR, and structures of the BAs are eight BAs1. (PUT, CAD, SPD, SPM, TYR, HIS,TRY TRY andPEA). PEA).The The chemical structures of the BAscomponent are shown shown in Table 1. During the course of the method development, malic acid was used as the BGE component eight BAs (PUT, CAD, SPD, SPM, TYR, HIS, TRY and PEA). The chemical structures of the BAs are in Table During the course of the method development, malic acid was used as the BGE eight BAs1.(PUT, CAD, SPM, TYR, HIS, [15]. TRY and PEA). The chemical structures the BAs are shown in Table Table During theSPD, course of the the method development, malic acidof was used asof the BGE component determination of non-UV absorbing amines A further scrutiny these papers also reveals that in 1. the course development, malic was used component in Table Table the 1. During During the course oforganic the method method development, malic acid acid was used as as the the BGE component without the need for for any otherof organic modifiers or chiral chiral selectors, selectors, thus simplifying theBGE procedure. The in 1. During the course of the method development, malic acid was used as the BGE component without need any other modifiers or thus simplifying the procedure. The in Table 1. During the course of the method development, malic acid was used as the BGE component without the the need for for any any other organic organic modifiers or chiralcyclodextrin selectors, thus thus[15], simplifying the procedure. The complicated background electrolytes (BGE) that contain crownthe ether [4,5,15] The and without need other modifiers or selectors, simplifying procedure. without was the need for any any other organic modifiers or chiral chiral selectors, thus thus simplifying the procedure. The method validated and qualified and applied for the determination of BAs in seawater. without the need for other organic modifiers or chiral selectors, simplifying the procedure. The method was validated and qualified and applied for the determination of BAs in seawater. without the need for any other organic modifiers or chiral selectors, thus the procedure. The method was was validated and qualified and applied for the determination ofsimplifying BAs in in seawater. seawater. α-hydroxyisobutyric acid [16] were used as the chiral selector. method validated and qualified and applied for the determination of BAs method was was validated validated and and qualified qualified and and applied applied for for the the determination determination of of BAs BAs in in seawater. seawater. method method was validated and qualified and applied for the determination of BAs in seawater. BAs can react produce nitrosamines which are carcinogenic compounds Molecules 1112with 2[3]. of 9 BAs2018, can react withof nitrite towater produce nitrosamines which are highly highly carcinogenic compounds [3]. Thus, the23, analysis ofnitrite BAs in into water samples has started started to receive receive considerable interest lately [4,5]. [4,5]. BAs can react with nitrite to produce nitrosamines which are highly carcinogenic compounds [3]. Thus, the analysis BAs samples has to considerable interest lately
Table 1. 1. Some Some chemical chemical properties properties of of the the studied studied biogenic biogenic amines amines (BAs) (BAs) [13,17]. [13,17]. Table 1. Some chemical properties of the studied biogenic amines (BAs) [13,17]. Table Table 1. Some Some chemical properties of the the studied biogenicamines amines(BAs) (BAs)[13,17]. [13,17]. Table 1. Some chemical properties of of the studied biogenic Table 1. chemical properties studied biogenic amines (BAs) [13,17]. Table 1. Some chemical properties of the studied biogenic amines (BAs) [13,17]. Table 1. 1. Some Some chemical chemical properties properties of the the studied studied biogenic biogenic amines amines (BAs) (BAs) [13,17]. [13,17]. Structure pK Value Table of
BA BA BA BA BA BA Putrescine BA Putrescine BA BA Putrescine Putrescine Putrescine (PUT) Putrescine (PUT) (PUT) Putrescine (PUT) Putrescine Putrescine (PUT) (PUT) Cadaverine (PUT) Cadaverine (PUT) Cadaverine (PUT) Cadaverine Cadaverine (CAD) Cadaverine (CAD) Cadaverine (CAD) Cadaverine (CAD) Cadaverine (CAD) (CAD) (CAD) Spermidin (CAD) (CAD) Spermidin Spermidin Spermidin Spermidin e (SPD) Spermidin (SPD) Spermidine Spermidin ee (SPD) (SPD) Spermidin eee(SPD) (SPD) (SPD) (SPD) ee (SPD) Spermine Spermine Spermine Spermine Spermine (SPM) (SPM) (SPM) Spermine (SPM) Spermine (SPM) Spermine (SPM) Spermine (SPM) (SPM) (SPM) Histamine Histamine Histamine Histamine Histamine (HIS) (HIS) Histamine (HIS) (HIS) (HIS) (HIS) Histamine Histamine Histamine (HIS) (HIS) (HIS) Tryptamin Tryptamin Tryptamin Tryptamine Tryptamin Tryptamin e (TRY) (TRY) ee (TRY) (TRY) (TRY) ee (TRY) Tryptamin Tryptamin Tryptamin e (TRY) (TRY) ee (TRY) Tyramine Tyramine Tyramine Tyramine Tyramine (TYR) Tyramine (TYR) (TYR) (TYR) (TYR) (TYR) Tyramine Tyramine Tyramine (TYR) Phenylethy (TYR) Phenylethy Phenylethy (TYR) Phenylethylamine Phenylethy Phenylethy lamine lamine lamine (PEA) lamine lamine (PEA) Phenylethy (PEA) Phenylethy (PEA) Phenylethy (PEA) (PEA) lamine lamine lamine (PEA) (PEA) (PEA)
Structure Structure Structure Structure Structure
NH 2 NH 2 NH NH2 2 NH NH22 NH 2 NH 2
Structure Structure Structure
N H 2N H 2N H H2 2N H 22N H N H 2N N H 2
H 2N N H H N H222N N H 22N H H 2N N H
H 2N N H H N H222N N H 22N H H 2N N H 2 H2N N H 2N H H2 2N H H22N N H2 N H2 N
NH2 NH NH NH222 NH NH22 NH 2 NH
2
2
N N N H N H N H N H N H N H H H
H H N H N H N H NH NH N H N N
N N H N H N H N H N HN H N H H
H 2N N H 2 H H2 N N H 22N
H N H 2N H 22 N
NH NH NH NH NH
H H N H N H HN N N
NH NH NNH N N N N N N N
H N H22N N H H2 N H22N
H2N N H 2 H H2N N H22N
NH 2 NH 2 NH NH2 2 NH 2 NH NH 22 NH 2
pK1 == 9.8; 9.8; pK pK2 == 6.0 6.0 pK 1 2 pK 1 = 9.8; pK2 = 6.0 pK == 10.2 10.2 pK pK pK===10.2 10.2 pK 10.2 pK = 10.2
H H N H N N
H N H 2N H22N
NH2 NH NH NH222 NH NH22 NH 2 NH 2
pK Value Value pK pK Value Value pK pK Value Value pK pK Value Value pK11 ==pK 10.8; pK2 == 9.4 9.4 pK 10.8; pK pK = 10.8; pK 1 pK1 == 10.8; 10.8; pK pK222 == 9.4 9.4 pK 11 = 10.8; pK22==9.4 9.4 pK 10.8; pK pK2 == 9.4 9.4 pK1 == 10.8; pK 2 2 = 9.4 pK 11 = 10.8; pK pK = 11.0; pK = 9.9 1 2 pK11 == 11.0; 11.0; pK pK22 == 9.9 9.9 pK pK1 == 11.0; 11.0; pK pK2 == 9.9 9.9 pK pK11 == 11.0; 11.0; pK pK22 == 9.9 9.9 pK 1= 2 = 9.9 pK = 11.0; pK 9.9 pK 11.0; pK 11 2 2 = pK = 9.5; pK = 10.8; pK33 == pK111 == 9.5; 9.5; pK pK222 == 10.8; 10.8; pK pK pK 33 = pK = 9.5; pK = 10.8; pK = 1 2 pK pK pK 33== 11==9.5; 22==10.8; 11.6 pKpK 9.5; pK 10.8; pK 11.6 pK = 9.5; pK = 10.8; pK 3 = = 9.5; pK = 10.8; 1 2 11.6 1 2 pK1 = 9.5; pK 11.6 2 = 10.8; pK3 = 11.6 pK11.6 = 11.6 11.6 3 pK pK11 == 11.50; 11.50; pK = 10.95; 10.95; 2= 11.6 pK pK 11.50; pK 11 = 222 = pK = 11.50; pK = 10.95; 10.95; pKpK pK 1 =3=11.50; 24==10.95; = 9.79; pK 8.90 pK 11.50; pK = 10.95; 1 2 pK =11.50; 9.79; pK pK424 === 8.90 8.90 pK 9.79; pK pK 10.95; 333== 1= pK 9.79; pK pK24 ==== 8.90 8.90 pK 11.50; pK 10.95; 1 = pK === 9.79; pK 11.50; pK 9.79;pK pK = 8.90 42 4 10.95; 1 33 9.79; pK pK4 == 8.90 8.90 pK3 == 9.79; pK 3 4 pK 3 = 9.79; pK4 = 8.90 pK1 == 9.8; 9.8; pK pK22 == 6.0 6.0 pK pK 9.8; pK 22 = pK1111 ====9.8; 9.8; pK = 6.0 6.0 pK 9.8;pK pK = 6.0 pK 1 2 =26.0
pK == 10.2 10.2 pK pK = 10.2 OH OH OH OH OH
pK 9.6 pK 9.6 pK=====9.6 9.6 pK pK 9.6 pK = 9.6
OH OH OH
pK == 9.6 9.6 pK pK = 9.6 pK 10.0 pK===10.0 10.0 pK 10.0 pK pK === 10.0 10.0 pK
H2N N H H22N NH2 NH 2 NH NH2 NH22
NH NH 2 NH2
pK == 10.0 10.0 pK pK = 10.0
In this work, we describe a simple and green CE–C4 D method for the separation and quantitation of eight BAs (PUT, CAD, SPD, SPM, TYR, HIS, TRY and PEA). The chemical structures of the BAs are shown in Table 1. During the course of the method development, malic acid was used as the BGE component without the need for any other organic modifiers or chiral selectors, thus simplifying the procedure. The method was validated and qualified and applied for the determination of BAs in seawater. 2
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2. Results 2.1. Chemicals and Reagents All chemicals and solvents used were of analytical and chromatographic grade, respectively. Spermine tetrahydrochloride (SPM), spermidine trihydrochloride (SPD), cadaverine dihydrochloride (CAD), putrescine dihydrochloride (PUT), histamine dihydrochloride (HIS), tryptamine hydrochloride (TRP) and citric acid were purchased from Sigma–Aldrich (Steinheim, Germany). Tyramine hydrochloride (TYR) and propionic acid (99%) were from Fluka (Buchs, Switzerland). Formic acid (85%) was from QRëC, while acetic acid (99.85%) was from HmbG Chemicals. Tartaric (99.5%) and malic acids, methanol and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany). Succinic acid (99%) was from BDH Chemicals (Bridgeport, PA, USA). Hydrochloric acid was obtained from Lab Scan (Bangkok, Thailand) and trichloroacetic acid (TCA) was from R & M Chemicals (Essex, UK). Milli-Q water was produced from a Nanopure Diamond, Barnstead unit and was used throughout. 2.2. Preparation of Standard Solutions Stock solution (1000 mg L−1 ) of a mixture of the eight BAs was prepared in water in a volumetric flask (10 mL). The solution was stored in the dark at 4 ◦ C. Working solutions were prepared by appropriate dilution of the stock in water. 2.3. Seawater Samples Seawater samples were collected on the 26–28 September 2016 from eight different places (Batu Ferringhi, Tanjung Bunga, Padang Kota, Bayan Lepas, Batu Maung, Teluk Kumbar, Tanjong Assam, Balik Pulau) around Penang Island, Malaysia. 2.4. Preparation of Samples All samples were filtered through 0.22 µm nylon filter before introducing to the CE unit. 2.5. Instrumentation and Electrophoretic Conditions Separations were performed on a 7100 capillary zone electrophoresis system (Agilent Technologies, Waldbronn, Germany) connected with C4 D (eDAQ, Denistone East, Australia). The separations were obtained using a bare fused silica capillary with a capillary size of 95 cm × 50 µm i.d. (detection length, 10.5 cm from the outlet end of the capillary) supplied by Agilent Technologies (Waldbronn, Germany). Standards and samples were introduced hydrodynamically (50 mbar) for 5 s; other conditions are as shown in Table 2. Data acquisition was performed using licensed Power-Chrom software version 2.6.11 (eDAQ, Denistone East, Australia). The new capillary was activated by flushing for 15 min with 1.0 M NaOH, 15 min with 0.1 M NaOH and 20 min with water followed by 15 min with the BGE. Between injections, the capillary was preconditioned with 0.1 M NaOH, water and the BGE (each for 5 min). All standards, samples, BGE, and NaOH solutions were filtered through 0.2 µm nylon filter membranes (Agilent Technologies). Table 2. Adopted capillary electrophoresis (CE)-capacitively coupled contactless conductivity detection (C4 D) conditions. Background electrolyte Applied voltage Capillary temperature Capillary Injection time C4 D parameters
400 mmol L−1 malic acid 25 kV (normal polarity) 24 ◦ C Bare fused silica (50 µm i.d. × 87 cm length) 5s Amplitude, 50 V; frequency, 600 kHz
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3. Results and Discussion 3.1. Capillary Electrophoresis Method Development The initial electrophoretic conditions with C4 D used were adopted from the work of Gong & Hauser [18] and Li et al. [4], who achieved the separation of the eight BAs using 150 mmol L−1 , 18-crown-6 in 500 mmol L−1 acetic acid as the BGE. Thirty minutes of preconditioning and equilibrium time between every two injections was required. Under these conditions, the authors were able to separate the BAs in about 24 min. In order to shorten the run time and simplify the BGE, several parameters affecting the separation of BAs were studied. 3.1.1. Selection of Background Electrolyte Background electrolyte (BGE) is one of the most important parameters in CE method development. Several CE studies using different detectors have been developed for the determination of BAs. For C4 D, it is important to keep the background conductivity as low as possible. This ensures that the small signals due to conductivity changes in the capillary between the excitation and pick-up electrode are amplified [19]. The pH of BGE solution used for the separation of BAs should be significantly different from the pKa values of the BAs. Generally, the pH of the BGE should be 2 units larger or lower than the pKa of the BAs, to ensure that the BAs are in the ionized form for optimum conductivity detection [20]. In this study, different types of weak organic acids were studied, including monocarboxylic acids (formic, acetic and propionic), dicarboxylic acids (oxalic and malonic), tricarboxylic acid (citric), unsaturated dicarboxylic acid (maleic) and hydroxyl dicarboxylic acids (malic and tartaric). These acids were selected based on their solubility in water and low conductivity to ensure high baseline stability [18]. Most of these acids succeeded in achieving the goal of ionization, but unluckily, did not result in satisfactory separation of all of the BAs. The use of acetic acid resulted not only in sensitive signals, but also, fast separations (~14 min). However, HIS and CAD was separated but TRY and TYR were unsolved. Promising separation was achieved using malic acid (Supplementary Figure S1). Therefore, malic acid was selected for further investigations. 3.1.2. Effect of the pH and Concentration of Background Electrolyte Besides the choice of organic acid as the BGE component, the selection of pH is of great importance in CE-C4 D analysis as it can influence the mobility of analytes by modifying the electro-osmotic flow (EOF) velocity and the ionic charge of the analyte molecules [21]. The effect of pH on the separation of the BAs was tested over a pH range of 1.8–2.6, keeping other conditions constant (BGE: malic acid (300 mmol L−1 ); voltage (25 kV); injection time (5 s); capillary temperature, (24 ◦ C); C4 D conditions, frequency (600 kHz) and amplitude (100 V). The best result was obtained when operated at pH 2.0. (See Supplementary Figure S2). Either increasing the pH over 2.0 or decreasing it to 1.8 (mixing with another acid) resulted in very poor sensitivity. The effect of malic acid concentration (100–500 mmol L−1 ) on the separation of BAs was also studied. The results showed that the resolution of the analytes was poor when 300 mmol L−1 was used. Good resolution with baseline separation was obtained when 400 mmol L−1 malic acid was used. When the concentration was increased to 500 mmol L−1 , total overlap between SPM and SPD was observed. Using 350 and 450 mmol L−1 , malic acid did not improve the separation. Therefore, 400 mmol L−1 malic acid was selected for further studies. 3.1.3. Effect of Organic Modifiers The effect of adding different organic modifiers (methanol, ethanol or ACN) to the BGE (5%, v/v) was studied. Both methanol and ethanol showed poor resolution for the BAs, while ACN resulted in acceptable resolution but with low sensitivity, in agreement with an earlier study [22]. Different
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percentages of ACN (1–10%) resulted in satisfactory resolution, but the sensitivity remained poor. Effect of Capillary Temperature Hence, no organic modifier was used for further optimization. The capillary temperature affects the resolution in CE by affecting the viscosity of the BGE [23]. 3.1.4. Effect of Parameters (16–26 °C) were investigated. The best resolution with an The effects of Instrumental different temperatures acceptable run time was obtained when operated at 24 °C. Further increases in temperature Effect of Separation Voltage deteriorated the resolution between SPD and CAD. In order to shorten the separation time and further improve the resolution, the effect of the Optimization of(20–30 C4D Parameters applied voltage kV) was studied. The best resolution with the shortest run time was obtained when 25 kV was applied. Joule heating is not applicable at this applied voltage because of the low In order to improve the sensitivity of the C4D detector, the frequency and amplitude were conductivity of the BGE used. investigated. The frequency of C4D was studied from 300 to 1000 kHz. The highest peak area was obtained when 600Temperature kHz was used. Furthermore, the amplitude was also studied from 20 V to 100 V. Effect of Capillary The highest signals were obtained when operated at 50 V. The the resolution CE by2,affecting viscosity of the aBGE [23]. The capillary adoptedtemperature conditions affects are summarized ininTable while the Figure 1 shows typical ◦ C) were investigated. The best resolution with The effects of different temperatures (16–26 electropherogram of the standard BAs when separated under these conditions. The eight BAs were an acceptable run time was obtained operated at previous 24 ◦ C. Further increases inwhere temperature baseline separated within about 20 min,when compared to the reported methods 24 and deteriorated resolution 29 min were the required [4,5].between SPD and CAD. Optimization of C4 D Parameters Table 2. Adopted capillary electrophoresis (CE)-capacitively coupled contactless conductivity conditions. detection In order (C to4D) improve the sensitivity of the C4 D detector, the frequency and amplitude were investigated. The frequency of C4 D was studied from 300 to 1000−1kHz. The highest peak area was Background electrolyte 400 mmol L malic acid obtained when 600 kHz was used. Furthermore, the amplitude was also studied from 20 V to 100 V. Applied voltage 25 kV (normal polarity) The highest signals were obtained when operated at 50 V. Capillary temperature 24 °C The adopted conditions are summarized in Table 2, while 1 shows a typical electropherogram Capillary Bare fused silicaFigure (50 µm i.d. × 87 cm length) of the standard BAsInjection when separated under these conditions. The eight BAs were baseline separated time 5s within about 20 min, compared to the previous reported50methods where600 24kHz and 29 min were C4D parameters Amplitude, V; frequency, required [4,5].
Figure 1. Typical electropherogram for the separation of eight biogenic amines under the optimum Figure 1. Typical electropherogram for the separation of eight biogenic amines under the optimum conditions mentioned in Table 2. Peak identity: SPM (1), SPD (2), HIS (3), CAD (4), PUT (5), PHE (6), conditions mentioned in Table 2. Peak identity: SPM (1), SPD (2), HIS (3), CAD (4), PUT (5), PHE (6), TYR (7), and TRY (8). TYR (7), and TRY (8).
3.2. 3.2. Analytical Analytical Characteristics Characteristics of of the the Method Method The linearityofofthe the method forBAs all was BAs studied was studied overrange wide of concentrations The linearity method for all over wide of range concentrations (1.0–100.0 −1 for PUT, SPD and SPM; 2.0–100.0 mg L−1 for PEA and TRY; 5.0–100.0 mg L−1 (1.0–100.0 mg L mg L−1 for PUT, SPD and SPM; 2.0–100.0 mg L−1 for PEA and TRY; 5.0–100.0 mg L−1 for HIS and TYR; for HIS and 1.0–50.0 mgThe L−1results for CAD). results are shown in Table 3. Good linearity, with 1.0–50.0 mgTYR; L−1 for CAD). are The shown in Table 3. Good linearity, with correlation 2 correlation coefficients (R ) between 0.981 and 0.996, were obtained (n = 3). The limits of detection 2 coefficients (R ) between 0.981 and 0.996, were obtained (n = 3). The limits of detection (LODs) for the −1 (LODs) analytes at signal-to-noise ratios three and ten ranged0.016 between mg L3). analytesfor atthe signal-to-noise ratios of three andof ten ranged between and 0.016 0.029and mg 0.029 L−1 (Table (Table 3). Thestandard relative standard (RSD) for migration The relative deviationdeviation (RSD) values forvalues migration time weretime lesswere than less 6%. than 6%.
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Table 3. Analytical characteristics of the developed CE-C4 D method. BAs
Linear Range (mg L−1 )
Regression Equation
R2
LOD (µg L−1 )
PUT CAD HIS SPD SPM PEA TYR TRY
1.0–100 1.0–50 5.0–100 1.0–100 1.0–100 2.0–100 5.0–100 2.0–100
y = 0.375x + 3.715 y = 0.465x + 2.400 y = 0.367x + 6.082 y = 0.362x + 3.519 y = 0.430x + 3.725 y = 0.490x + 2.849 y = 0.626x + 0.104 y = 0.473x + 1.193
0.988 0.989 0.981 0.982 0.991 0.982 0.985 0.996
27 22 28 29 24 21 16 27
The intra-day and inter-day precisions were tested using three different concentrations of standard mixture solutions. The intra-day precision was tested with six replicates in one day and the inter-day precision was tested by assays over six days. The intra-day and inter-day RSD were 5.8–9.1% and 4.0–9.7%, respectively. The recovery study was examined using seawater that was spiked with three different concentrations of BA mixture (10, 25 and 50 µg mL−1 ). It is indeed very encouraging to find that satisfactory recovery for all BAs was obtained (77–93%) (Table 4). It must also be pointed out that the seawater was only filtered before the CE-C4 D analysis, and yet, there was no significant interference from the complex matrix. This is mainly due to the fact that conductivities of the background ions were suppressed when operated under the adopted conditions. Table 4. Percent recoveries of BAs obtained from seawater that was spiked with different BA standards (n = 6). Spiked Concentration, mg L−1 BAs PUT CAD HIS SPD SPM PEA TYR TRY
10
25
50
78.6 80.5 86.0 76.6 82.5 84.3 87.6 90.0
83.4 82.3 87.4 80.0 80.6 87.8 88.4 91.6
89.3 92.7 85.7 89.9 88.4 91.9 90.8 89.2
3.3. Analysis of Seawater Samples The developed method was applied for the analysis of BAs in eight seawater samples collected around Penang Island. Before the analysis, these samples were filtered through 0.2 µm nylon membrane filter. BAs were not detected in the analyzed samples. The results obtained were in agreement with those reported for other environmental waters [4,5] where most BAs were not detected. At the moment, BAs are not subjected to any environmental regulations. A comparison of LODs and recoveries of CE-C4 D with other direct CE reported methods is summarized in Table 5. The results show that by using this method, the LODs obtained were lower when compared with previously reported methods using electrochemical and C4 D detectors [4,21]. Also the LOD values were higher compared to HPLC combined with derivatization [8]. Meanwhile, the analysis time (~20 min) was shorter than the CE and HPLC methods reported by Li et al. (~24–29 min) [4,5] and Saaid et al. (~27 min) [8].
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Table 5. Some capillary electrophoresis methods for the determination of BAs without derivatization. Matrix
BAs
Detector
LOD 10−7
L−1
Recovery (%)
References
102
[24]
Conductivity Cyclic Voltammetry Electrochemical
1.8 × mol 2.3 × 10−7 mol L−1 0.15–50 mg kg−1 2.5 nM 4.3 mg L−1
92–102 -
[25] [26] [22]
PUT, CAD, HIS, SPD, SPM, PEA, TYR, TRY
MS/MS
1–2 µg L−1
87–113
[27]
PUT, CAD, HIS, SPD, SPM, PEA, TYR, TRY PUT, CAD, SPD, SPM CAD, HIS, SPD, SPM, PEA, TYR, TRY
C4 D Amperometry Amperometry
44.3–149 µg L−1 10−7 –4 × 10−7 M 10.1–42.6 µg L−1
86.9–104 71.6–101
[4] [28] [5]
PUT, CAD, HIS, TRY, TYR
Conductometry
2–5 µmol L−1
86–103
[13]
TRY, TYR
Amperometry
97.596
[29]
CAD, HIS, SPD, TYR, PUT PUT, CAD, HIS, SPD, SPM, PEA, TYR, TRY
C4 D C4 D
89–103 77–93
[16] This study
Rice spirit
TYR, TRY
Electrochemical
Tuna fish Drosophila brains Beer Beer Wine Water Milk Water Beer Wine Salami Cheese Beer Wine Fermented dairy products Sea water
PUT, CAD, SPD, HIS TYR TYR
5.8 × 10−7 M 15.0 × 10−7 M 41–98 µg L−1 16–29 µg L−1
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4. Conclusions The simultaneous determination of eight BAs without derivatization using CE-C4 D was demonstrated. These analytes were separated in about 20 min. Unlike the earlier CE-C4 D work, our proposed method uses a very simple BGE (malic acid) and requires no sample pretreatment before the analysis. Earlier work used 18-crown-6 as the BGE component and SPE for the treatment of environmental water samples. The BAs were separated in about 25 min. The proposed CE-C4 D thus offers an interesting alternative to replace the common method for BA analysis that involves the HPLC separation of derivatized analytes for UV or fluorescence detection, which requires significant amounts of environmentally unfriendly organic solvents. The proposed method offers remarkable selectivity, enabling the BAs to be analyzed in complex seawater samples without any pretreatment. Supplementary Materials: The following are available online, Figure S1: Effect of different organic acids as BGE on the separation of the BA, Figure S2: Effect of pH of BGE on the separation of BAs. Author Contributions: E.A.G. performed the CE analysis; A.M. provided the seawater samples; B.S designed the experiments; E.A.G., J.P.Q., and B.S. wrote the paper. All authors reviewed the manuscript. Acknowledgments: The authors would like to thank Universiti Sains Malaysia and Universiti Teknologi PETRONAS for financial support of this work. J.P.Q. acknowledges the Australian Research Council Discovery Grant (DP180102810). Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
