570 Abdalla A. Elbashir1,2 Sonja Krieger1 Oliver J. Schmitz1 ∗ 1 Applied
Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany 2 University of Khartoum, Faculty of Science, Chemistry Department, Khartoum, Khartoum, Sudan
Received July 23, 2013 Revised October 19, 2013 Accepted October 23, 2013
Electrophoresis 2014, 35, 570–576
Research Article
Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE-LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4-chloro-7-nitro-2,1,3-benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70⬚C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive-MS with an ESI source was used for identification of the bis-derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra- and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples. Keywords: Acetylpolyamines / CE / LIF / Polyamines
1 Introduction Polyamines (PAs) and acetylpolyamines (AcPAs) increase in proliferating tissues and are important for cellular growth and cell division [1, 2]. They possessed two or more primary and secondary amino groups in their long chain, mainly including putrescine (PUT), cadaverine (CAD), spermidine (SPD), spermine (SPM), acetylspermine (ASPM), and acetylspermidine (ASPD). It is well known that the PAs concentration correlate with many diseases [3–5]. Many reports in the literature have described that the PAs and AcPAs at their elevated level in body tissue and biological fluid correlated closely with the cancer [6–9]. Thus, it is of clinical importance to develop a rapid and sensitive method for simultaneous analysis of PAs and AcPAs in body fluid.
Correspondence: Dr. Abdalla A. Elbashir, Chemistry Department, Faculty of Science, University of Khartoum, Khartoum, 11115, Khartoum, Sudan E-mail: [email protected]; [email protected]
Abbreviations: AcPA, acetylpolyamines; ASPD, acetylspermidine; ASPM, acetylspermine; CAD, cadaverine; IS, internal standard; NBD-Cl, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; PA, polyamine; PUT, putrescine; SPD, spermidine; SPM, spermine
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI 10.1002/elps.201300337
Several studies have been carried out on the CE separation of derivatized PAs but they were often related to classical polyamines [10–16]. However, the previous CE methods were not aimed at the simultaneous separation and quantification of PAs and AcPAs. Recently, AcPAs was identified and determined in cancer patients by TOF MS [6]. This motivated us to develop CZE-LIF method for analysis of both PAs and AcPAs in biological fluids. On the literature only few methods for the simultaneous analysis of both PAs and AcPAs were reported, including GC [17, 18] and HPLC [19–22]. However, these methods have some drawbacks such as often long analysis time, band broadening, high solvent consumption, and extensive sample clean up procedure. CE with LIF has been accepted as a powerful analytical technique for the separation of a wide range of analytes, including biogenic amines and amino acids [23]. This has largely been attributed to its remarkable higher sensitivity, efficiency, low consumption of sample, and reagent and versatility. The direct detection of PAs and AcPAs is difficult since they do not have chromophoric or fluophoric groups and
∗
Additional corresponding author: Professor Oliver J. Schmitz, E-mail: [email protected]
Colour Online: See the article online to view Figs. 1–5 in colour.
www.electrophoresis-journal.com
CE and CEC
Electrophoresis 2014, 35, 570–576
hence require derivatization to yield fluorescent product before analysis by CE-LIF. Many labeling reagents have been applied for biogenic amines and PAs as has been reviewed [24, 25]. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a labeling agent that have been used for the determination of catecholamines and amino acids by MEKC-LIF [26, 27]. Uchiyama et al. [28] and Elbashir et al. [29, 30] have made in depth reviews in the application of this labeling reagent for determination of amines and amino acids using various analytical techniques. To best of our knowledge CZE method with (LIF) detection for simultaneous determination PAs and AcPAs was not reported. In this work a simple, fast and sensitive CZE-LIF method for the simultaneous separation and quantification of four PAs and two AcPAs was developed for the first time. The method is based on the derivatization of PAs and AcPAs with NBD-Cl as a fluorescence-labeling reagent.
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the selectivity of the method. The standards solution were obtained from Sigma, and contained the following 14 amino acids: L-alanine, L-arginine, L-cystine, L-glutamic acid, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-valine.
2.3 Optimization of labeling reaction conditions Generally, 10 L of a 50 M working standard solution of PAs and AcPAs, or diluted urine sample and 10 L of IS (1,6-diaminohexane) were mixed with 100 L aqueous buffer and 100 L of NBD-Cl dissolved in acetonitrile. The effects of reaction variables were investigated: buffer pH (pH 7–7.8), reaction temperature (50–80⬚C), and reaction time (3–15 min).
2.4 LC-LTQ-orbitrap analysis
2 Material and method 2.1 Instrumentation A Beckman P/ACE MDQ CE system with an LIF detector was used for all electrophoretic separations. Excitation was at 488 nm (argon ion laser) and the emission intensity was monitored at 520 nm (band-pass filter, bandwidth 10 nm). A 50 mm id fused-silica capillary from Polymicro Technologies (Phoenix, AZ, USA) of 40 cm length (30 cm from inlet to the detector window) was used and thermostated at 25⬚C. The capillary was first conditioned with 1.0 mol/L HCl, 1.0 mol/L NaOH, and methanol for 2 min each as described in the P/ACE MDQ CE instrumental manual. Then the capillary was rinsed with deionized water for 2 min at a pressure of 50 psi and equilibrated with BGE buffer before sample analysis. Samples were injected by pressure at 0.5 psi for 10 s, and separations were performed under 25 kV for 8 min with a positive high voltage. The data were collected and processed by Beckman P/A CE 32 Karat software Version 4.0. The capillary was rinsed 2 min with 0.1 mol/L NaOH, water, and BGE after each run. An Exactive-MS (ThermoFisher, Bremen, Germany) with an ESI source was used for identification of the bis-derivatized product of the ASPM.
2.2 Chemicals Putrescine dihydrochloride (PUT) 98%, spermidine trihydrochloride (SPD) 99.5%, spermine tetrahydrochloride (SPM) 99%; cadaverine (CAD) 99%; N-acetylspermidine (ASPD), 98% N-acetylspermine (ASPM), 97% 1,6-diaminohexane, 99% (used as an internal standard (IS)), and NBD-Cl 98%, were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemical of analytical reagent grade and were used without further purification. Amino acids standard solutions were used to evaluate C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Standard solution of ASPM was derivatized under the optimum conditions with the NBD-Cl. The ASPM-NBDCl derivatized product was injected into the LTQ Orbitrap XL TM hybrid FTMS (exactive, ThermoFisher). Controlled by the Xcalibur software (2.0.7). The ESI source was operated in positive mode with the spray voltage set at 4 Kv, sheath gas flow rate at 12.5 arb, auxiliary gas flow rate at 0.03 arb, capillary voltage and temperature at 54.5 V and 250⬚C, respectively. The tube lens were set at 140 V, mass range m/z was 120–1000 Da.
2.5 Validation study The method was validated with respect to linearity, LOD, repeatability, intra- and interday assays, recovery, and selectivity. The linearity was evaluated by assaying at least six levels of concentrations of the standards in triplicate. The reproducibility was estimated by injection of standard in midrange of the calibration curve (n = 10). For intra- and interday assays, the samples were prepared individually in triplicate at low (0.02 M), medium (0.5 M), and high (0.9 M) concentration. Similarly, the recovery was estimated at three concentrations levels by comparing in triplicate, the values of spiked samples prepared in the linear range. The selectivity was assessed by analysis of 14 amino acids under the optimum procedure conditions.
2.6 Analysis of urine samples Urine samples were obtained from healthy volunteers. The urine samples collected from single morning urination were stored and frozen at −20⬚C until analysis. To 50 L of a urine sample, 450 L of water were added, and the mixture was passed through a disposable filter (0.45 m, 13 mm www.electrophoresis-journal.com
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temperature and time were investigated in order to obtain fluorescent maximum intensity.
3.1.1 Effect of buffer pH on the labeling reaction
Figure 1. Effect of pH on the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (concentration 3 mM), reaction time 10 s.
Since the labeling reaction of PAs and AcPAs with NBD-Cl is a typical nucleophilic reaction, basic reaction conditions will be suitable. NBD-F was reported to react rapidly with both primary and secondary amines under neutral and moderately alkaline conditions [16,30–35]. Therefore in this study the pH was varied over the pH range of 7–7.8, using borate buffer. The results showed that the fluorescence intensity (as indicated by the CE peak area of the derivative) increased as the pH increased from 7.0 to 7.4 and then decrease for all PAs and AcPAs studied, as shown in Fig. 1. The decrease in the peak areas above pH 7.4 may be due to the formation of di- and multilabeled derivatives [16]. So pH 7.4 was selected as optimum, pH 7.2 was reported to be suitable for labeling of PAs using NBD-F [16].
3.1.2 Effect of temperature on the labeling reaction
Figure 2. Effect of temperature on the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (3 mM), reaction time 8 s.
The labeling reaction was found to be very slow at room temperature so the effect of the reaction temperature was investigated in range from 55 to 80⬚C, at pH 7.4. It was found that the fluorescence intensity of the labeling PAs and AcPAs increase with increasing temperature, up to 70⬚C, and then decrease or remain constant for most of the labeled PAs and AcPAs (Fig. 2). Therefore, the labeling reaction was done at 70⬚C.
3.1.3 Effect of time on the labeling reaction The effect of reaction time on fluorescence intensity was investigated in range of 1–15 min at 70⬚C. The results as shown in Fig. 3 indicate that the fluorescence intensity increased with increasing reaction time up to 10 min and then remain constant or slightly decrease, so 10 min was selected as optimum reaction time. Figure 3. Time courses of the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (3 mM) at 70⬚C.
3.2 Optimization of the CZE conditions id, cellulose acetate; Millipore). The filtrate of diluted urine sample was subjected to derivatization.
In order to investigate the separation conditions by CZE-LIF, a mixture containing the six analytes and the IS in concentration range 0.5 and 1.0 M, respectively, was used.
3 Result and discussion
3.2.1 Effect of the buffer pH on the separation
3.1 Optimization of the labeling reaction conditions
The pH is an important parameter to be optimized in CZE as it affects the charge of the analytes and the ionization of the silanol group of the capillary wall, which in turn affects the magnitude of the EOF. For the separation of PAs, AcPAs, and the IS, the effect of the pH of the running buffer was
NBD-Cl itself is not fluorescent but when it reacts with amines (primary and secondary) a fluorescent product is produced. The different experimental parameters such as pH, reaction
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.electrophoresis-journal.com
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Electrophoresis 2014, 35, 570–576
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Figure 4. Effects of the pH of the running buffer on the separation of the six PAs, AcPAs, and IS after labeling with NBD-Cl. At pH 2.0 peaks: SPM (1), SPD (2), bi-ASPM (3’), ASPM (3), PUT (4), CAD (5), IS (6), ASPD (7), CZE conditions Tris-phosphate buffer, 50 mM, applied voltage 25 kV, injection at 5 psi × 10 s, capillary temperature 25⬚C. For the concentration of PAs and AcPAs see Fig. 3.
Table 1. Linearity and LOD of the proposed method
PAs or AcPAs
Calibration range (M)
Regression equation
R2
SPM SPD ASPM PUT CAD ASPD
0.01–1 0.01–1 0.01–1 0.01–1 0.01–1 0.01–1
y = 1.1718 – 0.0012 y = 1.585 + 0.0923 y = 1.0863 + 0.0491 Y = 1.3123 + 0.1675 Y = 1.0727 + 0.0984 Y = 2.5478 + 0.0639
0.9973 0.9953 0.9875 0.9915 0.9958 0.9845
LOD (nM) 8.9 8.8 7.8 10.0 7.6 3.3
studied in the range of 2 to 4 and the results are shown in Fig. 4. It can be seen that the resolution is strongly dependent on the pH of the running buffer. At pH equal to or higher than 3.5, the resolution of the labeled PAs and AcPAs
was very bad because of the high EOF and Joule heating. It was obviously that based line separation of all analytes and the IS was obtained at pH 2.0. Thus, 50 mM Tris-phosphate buffer, pH 2.0, applied voltage 25 kV, injection 5 psi × 10 s, capillary temperature 25⬚C, were determined to be optimum for the analysis of the PAs and AcPAs. The peak identification was performed with standard addition method. The peak 3 was probably a bis-substituted product or impurity of ASPM, as confirmed by labeling only ASPM with NBD-Cl. To characterized peak 3 a mass spectrometric analysis was performed. The ASPM-NBD-Cl derivatized product was injected into the ESI-MS. [M+H]+ at m/z 408.2 is identified as mono-derivatized of ASPM and [M+H]+ at m/z 571.2 was identified as di-derivatized product of ASPM. The MS results clearly identify that peak 3 is the bis-substituted product of ASPM.
Table 2. Intraday and interday precisions for the determination of PAs and AcPAs
PAs or AcPAs
SPM SPD ASPM PUT CAD ASPD
Intraday precision (RSD%)
Interday precision (RSD%)
0.02 M
0.5 M
0.9 M
0.02 M
0.5 M
0.9 M
1.23 1.11 0.42 1.71 1.67 1.38
1.89 0.70 1.45 1.01 1.34 1.34
1.37 0.90 0.62 0.43 0.71 0.75
2.45 2.23 1.58 2.22 2.32 2.20
2.56 2.26 1.61 1.86 1.67 1.77
2.78 1.01 1.59 0.76 0.46 1.33
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 3. Recovery for determination PAs and AcPAs in human urine
PAs or AcPAs
0.02 M
0.5 M
0.9 M
SPM SPD ASPM PUT CAD ASPD
93.41 103.0 96.75 95.66 91.50 104.10
96.80 91.65 109.9 104.10 107.50 95.10
102.80 94.27 95.88 89.80 109.50 103.10
peak area. The RSDs of the migration time, peak area, corrected peak area, and ratio of corrected peak area were less than 0.66, 5.48, 5.51, and 4.89%, respectively, which indicate that the repeatability of the method is satisfactory. Using the optimum analytical conditions, linearity was studied in the concentration range of 0.01–1 M for each PAs and AcPAs. The calibration graphs were constructed by plotting the ratio of peak area (analyte/IS) (y) as a function of analyte concentration (x) in M. Each point of the calibration curve corresponded to the mean value obtained from four measurements. The LOD of the method was estimated to be 8.9, 8.8, 7.8, 10, 7.6, and 3.3 nM for SPM, SPD, ASPM, PUT, CAD, and ASPD, respectively, which was better than those reported in several previous studies for PAs [16, 38]. This was obtained by multiplying the standard deviation of the noise by 3.0. The statistical linear regression data obtained are summarized in Table 1. Intraday precision ranged from 0.42 to 1.89% and interday precision from 0.46 to 2.78% were obtained Table 2. Furthermore, the recoveries ranged from 91.50 to 109.9 for human urine were measured Table 3. Under the proposed labeling reaction conditions, a mixture of 14 amino acids was analyzed to demonstrate the specificity of the method towards PAs and AcPAs. However basic amino acids gave no peaks within 6 min under the present CZE conditions. This demonstrates the selectivity of the method, and suitability for use with biological samples.
3.3 Validation Various studies have shown that the use of ISs is crucial to obtain good reproducibility in CZE and chromatographic techniques in order to compensate injection errors and minor fluctuations of the migration time [36]. In this study, 1,6-diaminohexane, which belongs to the same group of polyamines was selected as the IS. In all cases, 1.0 M of 1,6-diaminohexane was added as IS. The assay of PAs and AcPAs were validated with respect to linearity, LOD, repeatability, intra- and interday assays, recovery, and selectivity [37]. The repeatability of the method was examined by ten consecutive injections of 0.5 M of analytes mixture containing IS. The results were evaluated by considering the migration time, peak area, corrected peak area, and ratio of corrected
Table 4. Concentration of PAs and AcPAs in human urine in M (n = 3)
Sample
SPM
SPD
ASPM
PUT
CAD
ASPD
1 2 3 4 5 Mean ± SD (This work) Mean ± SD ([42])
0.685 0.437
Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany 2 University of Khartoum, Faculty of Science, Chemistry Department, Khartoum, Khartoum, Sudan
Received July 23, 2013 Revised October 19, 2013 Accepted October 23, 2013
Electrophoresis 2014, 35, 570–576
Research Article
Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE-LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4-chloro-7-nitro-2,1,3-benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70⬚C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive-MS with an ESI source was used for identification of the bis-derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra- and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples. Keywords: Acetylpolyamines / CE / LIF / Polyamines
1 Introduction Polyamines (PAs) and acetylpolyamines (AcPAs) increase in proliferating tissues and are important for cellular growth and cell division [1, 2]. They possessed two or more primary and secondary amino groups in their long chain, mainly including putrescine (PUT), cadaverine (CAD), spermidine (SPD), spermine (SPM), acetylspermine (ASPM), and acetylspermidine (ASPD). It is well known that the PAs concentration correlate with many diseases [3–5]. Many reports in the literature have described that the PAs and AcPAs at their elevated level in body tissue and biological fluid correlated closely with the cancer [6–9]. Thus, it is of clinical importance to develop a rapid and sensitive method for simultaneous analysis of PAs and AcPAs in body fluid.
Correspondence: Dr. Abdalla A. Elbashir, Chemistry Department, Faculty of Science, University of Khartoum, Khartoum, 11115, Khartoum, Sudan E-mail: [email protected]; [email protected]
Abbreviations: AcPA, acetylpolyamines; ASPD, acetylspermidine; ASPM, acetylspermine; CAD, cadaverine; IS, internal standard; NBD-Cl, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; PA, polyamine; PUT, putrescine; SPD, spermidine; SPM, spermine
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI 10.1002/elps.201300337
Several studies have been carried out on the CE separation of derivatized PAs but they were often related to classical polyamines [10–16]. However, the previous CE methods were not aimed at the simultaneous separation and quantification of PAs and AcPAs. Recently, AcPAs was identified and determined in cancer patients by TOF MS [6]. This motivated us to develop CZE-LIF method for analysis of both PAs and AcPAs in biological fluids. On the literature only few methods for the simultaneous analysis of both PAs and AcPAs were reported, including GC [17, 18] and HPLC [19–22]. However, these methods have some drawbacks such as often long analysis time, band broadening, high solvent consumption, and extensive sample clean up procedure. CE with LIF has been accepted as a powerful analytical technique for the separation of a wide range of analytes, including biogenic amines and amino acids [23]. This has largely been attributed to its remarkable higher sensitivity, efficiency, low consumption of sample, and reagent and versatility. The direct detection of PAs and AcPAs is difficult since they do not have chromophoric or fluophoric groups and
∗
Additional corresponding author: Professor Oliver J. Schmitz, E-mail: [email protected]
Colour Online: See the article online to view Figs. 1–5 in colour.
www.electrophoresis-journal.com
CE and CEC
Electrophoresis 2014, 35, 570–576
hence require derivatization to yield fluorescent product before analysis by CE-LIF. Many labeling reagents have been applied for biogenic amines and PAs as has been reviewed [24, 25]. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a labeling agent that have been used for the determination of catecholamines and amino acids by MEKC-LIF [26, 27]. Uchiyama et al. [28] and Elbashir et al. [29, 30] have made in depth reviews in the application of this labeling reagent for determination of amines and amino acids using various analytical techniques. To best of our knowledge CZE method with (LIF) detection for simultaneous determination PAs and AcPAs was not reported. In this work a simple, fast and sensitive CZE-LIF method for the simultaneous separation and quantification of four PAs and two AcPAs was developed for the first time. The method is based on the derivatization of PAs and AcPAs with NBD-Cl as a fluorescence-labeling reagent.
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the selectivity of the method. The standards solution were obtained from Sigma, and contained the following 14 amino acids: L-alanine, L-arginine, L-cystine, L-glutamic acid, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-valine.
2.3 Optimization of labeling reaction conditions Generally, 10 L of a 50 M working standard solution of PAs and AcPAs, or diluted urine sample and 10 L of IS (1,6-diaminohexane) were mixed with 100 L aqueous buffer and 100 L of NBD-Cl dissolved in acetonitrile. The effects of reaction variables were investigated: buffer pH (pH 7–7.8), reaction temperature (50–80⬚C), and reaction time (3–15 min).
2.4 LC-LTQ-orbitrap analysis
2 Material and method 2.1 Instrumentation A Beckman P/ACE MDQ CE system with an LIF detector was used for all electrophoretic separations. Excitation was at 488 nm (argon ion laser) and the emission intensity was monitored at 520 nm (band-pass filter, bandwidth 10 nm). A 50 mm id fused-silica capillary from Polymicro Technologies (Phoenix, AZ, USA) of 40 cm length (30 cm from inlet to the detector window) was used and thermostated at 25⬚C. The capillary was first conditioned with 1.0 mol/L HCl, 1.0 mol/L NaOH, and methanol for 2 min each as described in the P/ACE MDQ CE instrumental manual. Then the capillary was rinsed with deionized water for 2 min at a pressure of 50 psi and equilibrated with BGE buffer before sample analysis. Samples were injected by pressure at 0.5 psi for 10 s, and separations were performed under 25 kV for 8 min with a positive high voltage. The data were collected and processed by Beckman P/A CE 32 Karat software Version 4.0. The capillary was rinsed 2 min with 0.1 mol/L NaOH, water, and BGE after each run. An Exactive-MS (ThermoFisher, Bremen, Germany) with an ESI source was used for identification of the bis-derivatized product of the ASPM.
2.2 Chemicals Putrescine dihydrochloride (PUT) 98%, spermidine trihydrochloride (SPD) 99.5%, spermine tetrahydrochloride (SPM) 99%; cadaverine (CAD) 99%; N-acetylspermidine (ASPD), 98% N-acetylspermine (ASPM), 97% 1,6-diaminohexane, 99% (used as an internal standard (IS)), and NBD-Cl 98%, were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemical of analytical reagent grade and were used without further purification. Amino acids standard solutions were used to evaluate C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Standard solution of ASPM was derivatized under the optimum conditions with the NBD-Cl. The ASPM-NBDCl derivatized product was injected into the LTQ Orbitrap XL TM hybrid FTMS (exactive, ThermoFisher). Controlled by the Xcalibur software (2.0.7). The ESI source was operated in positive mode with the spray voltage set at 4 Kv, sheath gas flow rate at 12.5 arb, auxiliary gas flow rate at 0.03 arb, capillary voltage and temperature at 54.5 V and 250⬚C, respectively. The tube lens were set at 140 V, mass range m/z was 120–1000 Da.
2.5 Validation study The method was validated with respect to linearity, LOD, repeatability, intra- and interday assays, recovery, and selectivity. The linearity was evaluated by assaying at least six levels of concentrations of the standards in triplicate. The reproducibility was estimated by injection of standard in midrange of the calibration curve (n = 10). For intra- and interday assays, the samples were prepared individually in triplicate at low (0.02 M), medium (0.5 M), and high (0.9 M) concentration. Similarly, the recovery was estimated at three concentrations levels by comparing in triplicate, the values of spiked samples prepared in the linear range. The selectivity was assessed by analysis of 14 amino acids under the optimum procedure conditions.
2.6 Analysis of urine samples Urine samples were obtained from healthy volunteers. The urine samples collected from single morning urination were stored and frozen at −20⬚C until analysis. To 50 L of a urine sample, 450 L of water were added, and the mixture was passed through a disposable filter (0.45 m, 13 mm www.electrophoresis-journal.com
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temperature and time were investigated in order to obtain fluorescent maximum intensity.
3.1.1 Effect of buffer pH on the labeling reaction
Figure 1. Effect of pH on the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (concentration 3 mM), reaction time 10 s.
Since the labeling reaction of PAs and AcPAs with NBD-Cl is a typical nucleophilic reaction, basic reaction conditions will be suitable. NBD-F was reported to react rapidly with both primary and secondary amines under neutral and moderately alkaline conditions [16,30–35]. Therefore in this study the pH was varied over the pH range of 7–7.8, using borate buffer. The results showed that the fluorescence intensity (as indicated by the CE peak area of the derivative) increased as the pH increased from 7.0 to 7.4 and then decrease for all PAs and AcPAs studied, as shown in Fig. 1. The decrease in the peak areas above pH 7.4 may be due to the formation of di- and multilabeled derivatives [16]. So pH 7.4 was selected as optimum, pH 7.2 was reported to be suitable for labeling of PAs using NBD-F [16].
3.1.2 Effect of temperature on the labeling reaction
Figure 2. Effect of temperature on the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (3 mM), reaction time 8 s.
The labeling reaction was found to be very slow at room temperature so the effect of the reaction temperature was investigated in range from 55 to 80⬚C, at pH 7.4. It was found that the fluorescence intensity of the labeling PAs and AcPAs increase with increasing temperature, up to 70⬚C, and then decrease or remain constant for most of the labeled PAs and AcPAs (Fig. 2). Therefore, the labeling reaction was done at 70⬚C.
3.1.3 Effect of time on the labeling reaction The effect of reaction time on fluorescence intensity was investigated in range of 1–15 min at 70⬚C. The results as shown in Fig. 3 indicate that the fluorescence intensity increased with increasing reaction time up to 10 min and then remain constant or slightly decrease, so 10 min was selected as optimum reaction time. Figure 3. Time courses of the reaction of PAs and AcPAs (concentration 0.5 M) with NBD-Cl (3 mM) at 70⬚C.
3.2 Optimization of the CZE conditions id, cellulose acetate; Millipore). The filtrate of diluted urine sample was subjected to derivatization.
In order to investigate the separation conditions by CZE-LIF, a mixture containing the six analytes and the IS in concentration range 0.5 and 1.0 M, respectively, was used.
3 Result and discussion
3.2.1 Effect of the buffer pH on the separation
3.1 Optimization of the labeling reaction conditions
The pH is an important parameter to be optimized in CZE as it affects the charge of the analytes and the ionization of the silanol group of the capillary wall, which in turn affects the magnitude of the EOF. For the separation of PAs, AcPAs, and the IS, the effect of the pH of the running buffer was
NBD-Cl itself is not fluorescent but when it reacts with amines (primary and secondary) a fluorescent product is produced. The different experimental parameters such as pH, reaction
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Figure 4. Effects of the pH of the running buffer on the separation of the six PAs, AcPAs, and IS after labeling with NBD-Cl. At pH 2.0 peaks: SPM (1), SPD (2), bi-ASPM (3’), ASPM (3), PUT (4), CAD (5), IS (6), ASPD (7), CZE conditions Tris-phosphate buffer, 50 mM, applied voltage 25 kV, injection at 5 psi × 10 s, capillary temperature 25⬚C. For the concentration of PAs and AcPAs see Fig. 3.
Table 1. Linearity and LOD of the proposed method
PAs or AcPAs
Calibration range (M)
Regression equation
R2
SPM SPD ASPM PUT CAD ASPD
0.01–1 0.01–1 0.01–1 0.01–1 0.01–1 0.01–1
y = 1.1718 – 0.0012 y = 1.585 + 0.0923 y = 1.0863 + 0.0491 Y = 1.3123 + 0.1675 Y = 1.0727 + 0.0984 Y = 2.5478 + 0.0639
0.9973 0.9953 0.9875 0.9915 0.9958 0.9845
LOD (nM) 8.9 8.8 7.8 10.0 7.6 3.3
studied in the range of 2 to 4 and the results are shown in Fig. 4. It can be seen that the resolution is strongly dependent on the pH of the running buffer. At pH equal to or higher than 3.5, the resolution of the labeled PAs and AcPAs
was very bad because of the high EOF and Joule heating. It was obviously that based line separation of all analytes and the IS was obtained at pH 2.0. Thus, 50 mM Tris-phosphate buffer, pH 2.0, applied voltage 25 kV, injection 5 psi × 10 s, capillary temperature 25⬚C, were determined to be optimum for the analysis of the PAs and AcPAs. The peak identification was performed with standard addition method. The peak 3 was probably a bis-substituted product or impurity of ASPM, as confirmed by labeling only ASPM with NBD-Cl. To characterized peak 3 a mass spectrometric analysis was performed. The ASPM-NBD-Cl derivatized product was injected into the ESI-MS. [M+H]+ at m/z 408.2 is identified as mono-derivatized of ASPM and [M+H]+ at m/z 571.2 was identified as di-derivatized product of ASPM. The MS results clearly identify that peak 3 is the bis-substituted product of ASPM.
Table 2. Intraday and interday precisions for the determination of PAs and AcPAs
PAs or AcPAs
SPM SPD ASPM PUT CAD ASPD
Intraday precision (RSD%)
Interday precision (RSD%)
0.02 M
0.5 M
0.9 M
0.02 M
0.5 M
0.9 M
1.23 1.11 0.42 1.71 1.67 1.38
1.89 0.70 1.45 1.01 1.34 1.34
1.37 0.90 0.62 0.43 0.71 0.75
2.45 2.23 1.58 2.22 2.32 2.20
2.56 2.26 1.61 1.86 1.67 1.77
2.78 1.01 1.59 0.76 0.46 1.33
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Table 3. Recovery for determination PAs and AcPAs in human urine
PAs or AcPAs
0.02 M
0.5 M
0.9 M
SPM SPD ASPM PUT CAD ASPD
93.41 103.0 96.75 95.66 91.50 104.10
96.80 91.65 109.9 104.10 107.50 95.10
102.80 94.27 95.88 89.80 109.50 103.10
peak area. The RSDs of the migration time, peak area, corrected peak area, and ratio of corrected peak area were less than 0.66, 5.48, 5.51, and 4.89%, respectively, which indicate that the repeatability of the method is satisfactory. Using the optimum analytical conditions, linearity was studied in the concentration range of 0.01–1 M for each PAs and AcPAs. The calibration graphs were constructed by plotting the ratio of peak area (analyte/IS) (y) as a function of analyte concentration (x) in M. Each point of the calibration curve corresponded to the mean value obtained from four measurements. The LOD of the method was estimated to be 8.9, 8.8, 7.8, 10, 7.6, and 3.3 nM for SPM, SPD, ASPM, PUT, CAD, and ASPD, respectively, which was better than those reported in several previous studies for PAs [16, 38]. This was obtained by multiplying the standard deviation of the noise by 3.0. The statistical linear regression data obtained are summarized in Table 1. Intraday precision ranged from 0.42 to 1.89% and interday precision from 0.46 to 2.78% were obtained Table 2. Furthermore, the recoveries ranged from 91.50 to 109.9 for human urine were measured Table 3. Under the proposed labeling reaction conditions, a mixture of 14 amino acids was analyzed to demonstrate the specificity of the method towards PAs and AcPAs. However basic amino acids gave no peaks within 6 min under the present CZE conditions. This demonstrates the selectivity of the method, and suitability for use with biological samples.
3.3 Validation Various studies have shown that the use of ISs is crucial to obtain good reproducibility in CZE and chromatographic techniques in order to compensate injection errors and minor fluctuations of the migration time [36]. In this study, 1,6-diaminohexane, which belongs to the same group of polyamines was selected as the IS. In all cases, 1.0 M of 1,6-diaminohexane was added as IS. The assay of PAs and AcPAs were validated with respect to linearity, LOD, repeatability, intra- and interday assays, recovery, and selectivity [37]. The repeatability of the method was examined by ten consecutive injections of 0.5 M of analytes mixture containing IS. The results were evaluated by considering the migration time, peak area, corrected peak area, and ratio of corrected
Table 4. Concentration of PAs and AcPAs in human urine in M (n = 3)
Sample
SPM
SPD
ASPM
PUT
CAD
ASPD
1 2 3 4 5 Mean ± SD (This work) Mean ± SD ([42])
0.685 0.437
