ISSN 1061 9348, Journal of Analytical Chemistry, 2011, Vol. 66, No. 3, pp. 296300. Pleiades Publishing, Ltd.

., 2011.

ARTICLES

Determination of Biogenic Amines in Lake Water by Micellar Electrokinetic Chromatography with Fluorescence Detection after Derivatization with Fluorescamine1
Ankita Shukla, Sunil Kumar Sanghi, V. Sorna Gowri, Vishal Kumar Baderia, Sushma Lamba, and Deepesh Kumar Singh
Microfluidics and MEMS Centre, Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, 462026 India
Received September 14, 2009; in final form June 3, 2010

AbstractA simple and rapid method has been developed for the determination of biogenic amines in lake water using micellar electrokinetic chromatography with fluorescence detection. Separation of fluorescam ine derivatized biogenic amines was accomplished by using borate buffer of pH 9.5 containing 40 mM of sodium dodecyl sulphate. The method has been optimized with respect to fluorescamine concentration, reaction pH, reaction time, separation voltage and injection time. Detection was performed by using UG 11 excitation filter and 495 nm emission filter. The proposed method for histamine, tyramine and dopamine allowed their separation within 2 min with detection limits in nM range. The interday and intraday reproduc ibility of peak areas were less than 6.5%. Recovery of spiked samples was 95.76116.31%. Keywords: biogenic amines, fluorescamine, derivatization, micellar electrokinetic chromatography, fluores cence detection DOI: 10.1134/S1061934811030038

Amines are organic bases of low molecular weight, which are frequently present in biological materials and environmental samples, formed through decar boxylation of amino acids [1]. Continued consump tion of these amines shows a wide variety of symptoms such as nausea, respiratory distress, heart palpitation, headache and hyper or hypotension. Threshold doses of these can differ with individuals depending on the efficiency of detoxification through the action of monoamine oxidase (MAO) [2]. Patients who are tak ing MAO inhibitors such as antidepressant or antitu bercular drugs and are on tyramine rich diet can suffer from serious hypertensive reaction [36]. Amines can react with nitrite forming nitrosamines, which are car cinogenic [3]. These findings make important to mon itor biogenic amines in foodstuff, beverages [7, 8], and plants. The level of biogenic amines in food is an indi cator of food quality. Analytical methods for the deter mination of biogenic amines are mostly based on high performance liquid chromatography (HPLC) [2, 9], gas chromatography (GC) [10], thin layer chromatog raphy [11], spectrofluorimetry [12] and capillary elec trochromatography [13]. However, trace level occur rence of biogenic amines makes their quantification in real samples complex. Multiple extraction and sample enrichment are required to achieve good analyte recoveries. Capillary electrophoresis (CE) has proved
1 The article is published in the original.

itself extremely useful for the analysis of biological samples [14, 15]. CE has an inherent advantage over GC or HPLC such as high efficiency separations in relatively shorter time, small sample volume require ment and negligible consumption of organic solvents. Moreover, CE is simpler and occurs at utmost speed with various applications and is cost effective. Several methods of detection, viz., UV [1618], mass spec trometry (MS) [19, 20], conductometric [8, 21], indi rect UV [22], electrochemical detection [3] have been successfully applied to biogenic amines. Most of bio genic amines lack native UV absorption or fluores cence, so derivatizing or labeling them becomes nec essary in method development. Fluorescence detec tion not only increases sensitivity but also provides selectivity in determination. Beside CE, amperometic detection has also been a mode of detection but the methods typically lack repeatability when applied to real matrices due to surface poisoning of electrode. The separation of biogenic amines using CE and micellar electokinetic chromatography (MEKC) is documented in literature [8, 15]. Various labeling reagents like fluorescein isothio cynate (FITC) [23], 3 (2 furoyl)quinoline 2 carbox aldehyde (FQ) [23], fluorenylethyl chloroformate (FMOC) [24, 25], o phthaldehyde (OPA) [25], and naphthalene 2,3 dicarboxaldehyde (NDA) [2628] have been used in the determination of biogenic amines. The individual method drawbacks are immi

296

DETERMINATION OF BIOGENIC AMINES IN LAKE WATER 0.035 0.030 Peak area 0.025 0.020 3 0.015 0.010 0.005 0 7.5 8.0 8.5 9.0 Reaction pH 9.5 10.0 2 1

297

Fig. 1. Effect of pH on fluorescence yield. Capillary, 53.3 cm (30.0 cm effective length) 75 m I.D. BGE, borate buffer of pH 9.5 containing 40 mM sodium dodecyl sulphate (SDS). Applied voltage was 20 kV. Peaks: 1histamine; 2tyramine; and 3 dopamine.

nent, e.g., the kinetic and label chemistry in FITC can limit its application, of the method, derivatives formed with OPA are not stable, FQ can only react with pri mary amines, and FMOC can only react with second ary amines. In this paper fluorescamine [2932] has been used as derivatizing reagent for biogenic amines. Fluores camine has advantages over alternative derivatizing agents as fluorescamine reacts both with primary and secondary amines, reaction period is shorter and the derivatives formed are stable. The derivatives have been separated by MEKC with fluorescence detection involving excitation at 390 nm and emission at 495 nm. EXPERIMENTAL Materials and reagents. Tyramine and histamine were obtained from Sigma Aldrich, Steinheim, Germany. Dopamine was from Himedia, Mumbai, India, and sodium dodecyl sulphate (SDS) from BDH, Mumbai, India. Fluo rescamine {4 phenylspiro[furan (3H),1 phthala] 3,3' dione} was obtained from Fluka, Buchs, Switzerland. HPLC grade methanol, and analytical reagent grade hydro chloric acid and acetone were obtained from Ranbaxy, Ropar, India. Stock solution of biogenic amines, 1000 g/L, were prepared by dissolving their accurately weighed amounts in HPLC grade water (Millipore, Bangalore, India). Working standard were prepared by diluting the stock solutions. Fluorescamine, 30 mM, was prepared daily in acetone and kept refrigerated when not in use. Borate buffer of pH 9.0 was made by treating 20 mM boric acid with 0.1 M sodium hydroxide; this buffer was used for conducting the derivatization reaction. The borate buffer of pH 9.5, used for separation of amines, was prepared adjusting 20 mM boric acid that was 40 mM with respect to SDS, to desired pH by addition of 0.1 M sodium hydroxide.
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Instrumentation and separation condition. A Prince C 255 capillary electrophoresis instrument with programma ble injector and high voltage source (Prince Technolo gies, The Netherlands) was used. Separations were carried out at 20 kV applied voltage. Fused silica cap illary with internal diameter of 75 m, 50 cm total length and 30.3 cm effective length, was purchased from composite Metal Services Ltd. (Worcestershire, UK). Samples were introduced hydro dynamically by applying 40 millibar pressure for 6 sec. For fluores cence detection, an ARGOS 250 B instrument (Flux Instruments, Switzerland) was used where the excita tion light was filtered through a Schott glass UG 11 filter and a 495 nm cut off filter was applied for the limited light. The voltage used for photomultiplier tube (PMT) was 800 V. Data processing was done by DAx 7.1 data acquisition and analysis software (Prince Technologies, The Netherlands). Fresh capillary was charged by rinsing with 0.1 M sodium hydroxide for about 40 min followed by rinsing with water for 15 min. Subsequently, daily charging was done with methanol for 10 min, followed by in sequence with water for 5 min, 1 M hydrochloric acid for 10 min, water for 5 min, 0.1 M sodium hydroxide for 20 min, water for 5 min and the background electrolyte for 20 min. Sample preparation. The performance of the method was tested with unspiked and spiked lake water sample. Water samples were collected from a lake nearby Bhopal, Madhya Pradesh, India, to which 100 L of 0.1 M hydrochloric acid was added to each 1 mL portion of sample to avoid loss of amines by vol atilization. The samples were filtered through a 0.45 m nylon membrane filter and subjected to derivatization and analysis by CE. Derivatization procedure. To the 100 L of sample or standards in borate buffer of pH 9.0, 40 L of fluo rescamine solution was added and the mixture was
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298 0.040 0.035 Peak area 0.030 0.025 0.020 0.015 0.010 0.005 0 10 30 40 20 Fluorescamine, mM

ANKITA SHUKLA et al. Effective mobilities, 105 cm2 v 1 s1 45 40 35 3 30 25 20 15 0 10 20 30 40 50 SDS, mM

Fig. 3. Effect of SDS concentration of the BGE on the effective mobilities of the fluorescamine derivatives. Oper ating conditions and peaks identification as in Fig. 1.

2 1

2 1

50

Fig. 2. Effect of fluorescamine concentration on deriva tives of biogenic amines. Operating conditions and peaks identification as in Fig. 1.

allowed to stand at room temperature for 15 min before injection. RESULTS AND DISCUSSION Optimization of the derivatization conditions. It was found that maximum yield, as judged from the peaks areas, was obtained when the selected biogenic amines were derivatized in aqueous solution of pH between 810 (Fig. 1). A 20 mM borate adjusted to pH 9.0 was found to be the best medium for derivatization, and it was used in subsequent studies. The reaction between biogenic amines and fluores camine has been reported to take several min to several hours for completion [33], and fluorescamine concen tration was one of factors responsible for it [34]. In the present work, biogenic amines were reacted with vary ing amounts of fluorescamine. It was found that 30 mM of fluorescamine concentration was sufficient to achieve the optimum peak area. At higher reagent concentration, the peak areas decreased due to fluo rescence quenching by one of the hydrolysis products of fluorescamine. The reaction mixture became turbid when higher concentration of reagent was added to the reaction mixture and attempt to solubilize the reagent by addition of acetone also decreased the peak areas. Under optimized condition in the present work, the reaction completed in 15 min. Effect of fluorescamine concentration on amines is shown in Fig. 2. Optimization of separation conditions. There was no separation observed over the pH range 46. Above this pH range, dopamine separated from tyramine and histamine, the latter two amines overlapped. Addition of SDS at pH 9.5 in MEKC resolved all three amines from each other. SDS is the most common surfactant used for MEKC. Background electrolyte (BGE) sys tem containing 20 mM borate buffer of pH 9.5 con taining SDS concentration up to 50 mM/L was used

to study the effect of SDS concentration on resolu tion. The results obtained are shown in Fig. 3. Baseline resolution of all the fluorescamine derivatized bio genic amines was obtained at SDS concentration at least 40 mM/L. Separation of all the three biogenic amines is depicted in Fig. 4. The plate number under optimum condition of separation ranged 32.000 116.000. Validation of method. The extraction recoveries were determined by comparing the corrected peak areas of amines extracted from spiked lake water with that of unextracted standard containing the same amount of amines. For the determination of biogenic amines in lake water three replicate analyses of sam ples spiked at the concentration of 40 and 80 M were carried out. The same procedure for the sample prep aration and derivatization as described in previous sec tion was used. Figure 5 shows the chromatogram of spiked and unspiked lake water samples. It shows two
2 1 Fluorescence intensity

0.2 a.u.

3 0.5 1.0 1.5 2.0 Migration time, min 2.5 3.0

Fig. 4. Separation of a standard mixture of three deriva tized biogenic amines by MEKC. Applied voltage, 20 kV. Sample concentration: 10 5 M of each compound. Oper ating conditions and peaks identification as in Fig. 1. Vol. 66 No. 3 2011

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DETERMINATION OF BIOGENIC AMINES IN LAKE WATER Fluorescence intersity

299

1 2 0.5 a.u.

3 * 1 a b

0.04 a.u.

0.5

1.0

1.5

2.0

2.5

3.0

Migration time, min

Fig. 5. The electopherogram of water samples (a) spiked with three amines at 40 M each, and (b) Lake water unspiked sample. Operating conditions and peaks identification as in Fig. 1.

unknown peaks (a) and (b) which are due to contami nants present in lake water. The average recovery ranged 96116% (Table 1). Table 2 represents interday and intraday repeatabil ity in terms of relative standard deviation (RSD) in peak area. Interday repeatability was measured within 15 days. Excellent intraday (1%) and interday (6%) reproducibility for peak area were achieved. Calibration curves were found to be rectilinear over the range of 1100 M of amines. The correlation coefficient (r 2), limit of detection (LOD) and limits of quantification (LOQ) are given in Table 3. The LOD was between 0.41520.03 nM of amines. The LOD
Table 1. Recovery of amines in spiked samples Recovery (%) 40 M 80 M

was taken as three times the standard deviation in the analysis of 1 M of amines, and LOQ was average of background multiplied by 10 standard deviations.

***
The derivatization of biogenic amines with fluores camine yielded derivatives, which were found to be stable, and provided their sensitive and selective fluo rescence detection. The derivatization was fast and total analysis time required was less than 2 min. Limits of detection were in nmol range as required for the analysis of real samples. The simple working condi tions of this method make it suitable for application to routine analysis of biogenic amines. However, there is
Table 2. Precision of analytical method RSD (%) of corrected peak area

Amine

Amine

Interdaya

Intradaya

Averagea SD RSD,% Averagea SD RSD,% Histamine Histamine Tyramine Dopamine 116 3 113 2 103 3 2.83 2.21 2.51 96 3 104 3 99 1 3.77 Tyramine 2.83 Dopamine 1.32
Note: a All results are the averages of three replicate analysis.

6.028

0.905

4.335

0.588

3.054

0.226

Note: a All results are the averages of three replicate analyses.

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Table 3. Features of calibration graph and detection limits y = mx + c a Amine Histamine Tyramine Dopamine c 7.89 103 10.17 103 6.79 103 scb 9.13 103 1.79 103 1.21 103 m 711 993 61 smc 17.94 32.74 2.20 r2 0.9968 0.9957 0.9948 LODd 0.4154 0.4237 6.090 LOQd 1.380 4.12 20.03

Note: a x = concentration, M; y = peak area; c = intercept; m = slope; calibration graph constructed over ten concentration levels; results are the aver ages of three replicate analyses. b Standard error in intercept. c Standard error in slope. d LOD = limit of detection (S/N = 3), nM.

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