Journal of Chromatography B, 967 (2014) 69–74
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of primary and secondary phenethylamines in biological samples by high-performance liquid chromatographic method with fluorescence detection Xiao-Feng Guo a,b , Jie-Yu Wang a , Hong Wang a,∗ , Hua-Shan Zhang a a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China b Suzhou Institute of Wuhan University, Suzhou, China
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Article history: Received 29 September 2013 Accepted 13 July 2014 Available online 21 July 2014 Keywords: Fluorescence detection High-performance liquid chromatography Phenethylamines 1,3,5,7-Tetramethyl-8-(Nhydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene
a b s t r a c t Phenylalanine is an essential amino acid and its metabolites relate to various physiological and immune functions of living organisms. To monitor the alteration of concentration of primary and secondary phenethylamines including N-methyltyramine, octopamine, tyramine, tyrosine and phenylalanine in the metabolic pathway of phenylalanine, a sensitive and selective reversed-phase high-performance liquid chromatographic method has been developed in this study. The identification and quantification of phenethylamines were performed by fluorescent detection after pre-column derivatization with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene, an excellent fluorescent probe which could react with both primary and secondary amino groups simultaneously. The derivatization was carried out at 25 ◦ C for 25 min, and the separation was performed on a C18 column within 20 min. The linear ranges were from 2.0 to 100 nM for phenylalanine and tyramine to 5.0 to 250 for tyrosine and octopamine, with the detection limits of 0.1 nM for octopamine, tyramine, tyrosine and phenylalanine and 0.2 nM for N-methyltyramine (signal-to-noise ratio = 3), which allowed for the sure determination of phenethylamines at trace levels in the real samples without complex pretreatment or enrichment during multitudinous samples analysis. The proposed method has been validated by the analysis of the five target compounds in biological samples with spiked recoveries of 96.4–104.4% and the relative standard deviation of 1.0 and 4.4%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phenethylamines, such as phenylalanine (Phe), tyrosine (Tyr), tyramine (Tyra), N-methyltyramine (NMT) and octopamine (Oct) (Fig. 1), are important biological compounds. In these compounds, phenylalanine is an essential amino acid and could only be acquired from food, while the others are nonessential and could be both acquired from food and converted from phenylalanine. These phenethylamines relate to various physiological and immune functions of living organisms. Phenylalanine and tyrosine have several metabolites including octopamine and tyramine, which have been considered as neurotransmitters in invertebrates to regulate many actions such as growth, foraging, moving, learning and so on [1–3]. Tyrosine is also the precursor of catecholamines which are major brain and peripheral neurotransmitters in mammals. While for
∗ Corresponding author. Tel.: +86 27 87218924; fax: +86 27 68754067. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.07.017 1570-0232/© 2014 Elsevier B.V. All rights reserved.
human, octopamine and tyramine used to be considered functionally unimportant in the central nervous system in a long period because of their low physiological concentrations and the absence of specific receptors. However, two G protein-coupled receptors of octopamine were discovered recently in brains and tonsils of mammals, which suggest that octopamine may play important roles in several physiological and pathological procedures of human like cirrhosis, schizophrenia and Parkinson’s disease [4–6]. Moreover, octopamine could also be applied for the prophylaxis and treatment of adiposis and Type II diabetic nephropathy [7–9]. Besides, N-methyltyramine is a metabolite of tyramine and has many physiological functions including stimulates gastric and pancreatic secretions [10], and it is also a molecule which has a similar function to ephedrine alkaloids as dietary supplements but more safe [11]. Therefore, a quantification method of phenethylamines with high sensitivity and selectivity is always desired. By far, several analytical methods have been developed for the quantitative analysis of phenethylamines in biological samples, including high-performance liquid chromatography (HPLC)
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deriving with primary and secondary amines [26]. In the present work, to verify the applicability of TMBB-Su for the determination primary and secondary phenethylamines, a new HPLC method with fluorescence detection was developed using TMBB-Su as pre-column derivatization reagent and the results indicated that the proposed method has obvious advantages in sensitivity and selectivity with mild derivatization conditions. 2. Experimental 2.1. Apparatus An LC-20A HPLC system (Shimadzu, Tokyo, Japan) with RF10AXL fluorescence detector (Shimadzu, Tokyo, Japan) and Lab Solutions/LC Solution Lite chromatography chemstation (Shimadzu, Tokyo, Japan) were used in the experiments. Sample injection volume was 20 L. The separation was performed on an ODS column (5 m, 150 mm × 4.6 mm, i.d., Inertsil, GL Sciences, Tokyo, Japan). The pH values of solutions were measured using Mettler Toledo Delta 320 pH meter (Mettler-Toledo, Shanghai, China). Fig. 1. Structures of phenethylamines and their derivatization using TMBB-Su.
[12–15], capillary electrophoresis [16,17], molecularly imprinted solid-phase extraction (MISPE) [18] and electrochemical detection (ECD) [19]. Among these methods, HPLC followed by fluorescence detection is one of frequently used analytical techniques owing to its high sensitivity and selectivity. Since phenethylamines have intrinsic fluorescence, HPLC with fluorescence detection method can be used to analyze them directly without chemical derivatization. However, short absorbance and emission wavelength, small molar absorption coefficient, low fluorescence quantum yield and background interference from complex matrix [14] which could be resolved using fluorescent labeling reagents. Hence, precolumn derivatization combined with HPLC-fluorescence detection is regarded as an ideal analytical method for trace amounts of phenethylamines in complex matrix of bio-samples. Since there are both primary and secondary amines in phenethylamines, the fluorescent reagent used for phenethylamines analysis should label all of them. However, the reports for determination of primary and secondary phenethylamines simultaneously using HPLC-fluorescence detection were not many. Till now, typical fluorescent derivatization reagents which can label primary and secondary aliphatic amines simultaneously are 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) [20], fluorescein isothiocyanate (FITC) [21], 4-fluoro-7-nitrobenzofurazan (NBDF) [22], 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) [23] and N-hydroxysuccinimidyl 4,3,2 -naphthapyrone-4-acetate (NPA-OSu) [24]. However, all of these reagents have a variety of drawbacks. For example, long derivatization time is need for FITC and high temperature (60 ◦ C) is required for NBD-F, and operation procedure will be more complex than operating at room temperature. The excess FMOC-Cl in the derivatization procedure has to be removed by extraction or derivatization with a specific amine to form derivatives having no interference to the analysis of target compounds in order to prevent the generation of derivative with hydroxyl group of methanol. AQC and NPA-OSu have short absorbance and emission wavelengths which cannot avoid background interference from complex matrix. 1,3,5,7-Tetramethyl-8-(N-hydroxy-succinimidyl butyric ester)difluoroboradiaza -s-indacene (TMBB-Su) has been synthesized and applied for amine determination in our group [25] and has high fluorescence quantum yield, long emission wavelength, shorter reaction time, milder derivatization conditions (like lower temperature and weaker alkaline environment) when
2.2. Chemicals and reagents TMBB-Su was synthesized in our laboratory [23]. Phenylalanine, tyrosine, tyramine and N-methyltyramine were purchased from Biochemical Reagents Company (Shanghai, China). Octopamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise specified, all other reagents were of analytical grade. Water used for preparing solutions was purified by a Milli-Q system (Millipore, Bedford, MA, USA). The TMBB-Su stock solution was prepared by dissolving TMBBSu in acetonitrile to give a concentration of 1.0 × 10−4 M. The stock solutions of the analytes (1.0 × 10−4 M) were prepared by dissolving phenethylamines in water. Dilution of these stock solutions to appropriate concentrations was performed before use. H3 BO3 –Na2 B4 O7 buffers were prepared by mixing 0.025 M Na2 B4 O7 solution with 0.10 M H3 BO3 solution to the required pH value. Citric acid (H3 Cit)–Na2 HPO4 buffers were prepared by mixing 0.10 M H3 Cit solution with 0.10 M Na2 HPO4 solution to the required pH value. When not in use, all standards were stored at 4 ◦ C in a refrigerator. 2.3. Derivatization procedure To a 0.5 mL of Eppendorf tube containing 175 L of mixed amines and 125 L of H3 BO3 –Na2 B4 O7 buffer (pH 7.8), 135 L 1.0 × 10−4 M TMBB-Su and 65 L acetonitrile were added. The whole solution was diluted to the mark with water and kept at 25 ◦ C for 25 min. Then, an aliquot (20 L) of the reaction mixture was diluted with mobile phase and injected into the chromatographic system. 2.4. Chromatographic separation The chromatographic separations were performed at ambient temperature on an Inertsil ODS-SP column with gradient elution. Eluent A was methanol and eluent B was 10 mM, pH 4.5, citric acid–Na2 HPO4 buffer solution. Before the analysis, the column was pre-equilibrated for 30 min with mobile phase (A:B = 62:38, v/v). Then 20 L of the prepared sample solution was injected and the derivatives were eluted at a flow rate of 0.7 mL/min. The gradient elution program was used as following: 0–9 min, 62% A; 10–25 min, 75% A. The chromatographic system was re-equilibrated with mobile phase (A:B = 62:38, v/v) for 20 min between runs. The
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detection wavelengths were set at ex /em = 490/510 nm. All the solvents were filtered with 0.45 m membrane filter before use. 2.5. Method performance The linearity of peak areas against concentration of the five phenethylamines was tested under the optimized derivatization and separation conditions. The precision was assessed by repeating six sequential runs within-day and between-day using standard phenethylamines and reflected as relative standard deviations (RSDs). The linear analysis was performed using seven standard levels. Taking tyrosine as presentation, the seven standard levels are 5, 50, 90, 130, 170, 210 and 250 nM. 2.6. Sample preparation Crucian carp was killed, then the flesh on the back of crucian and roe were collected immediately and stored at −40 ◦ C. Just before the analysis, about 0.2 g of each sample was homogenized in 8.0 mL of water. After addition of 2.0 mL of 10% Cl3 CCOOH (w/v), the homogenate was centrifuged at 1430 × g for 10 min at room temperature. One milliliter of the clear solution was neutralized with 27 L of 1.0 M NaOH. The resulted solution was proceeded according to Sections 2.3 and 2.4. The recovery was evaluated by spiking standard mixture of amines in the sample preparation procedure, and the resulting solution was determined as the procedure of Sections 2.3 and 2.4. 3. Results and discussion 3.1. Optimization of separation conditions The separation of TMBB-Su derivatives with isocratic elution mode was tried first because it is simple. If the methanol content is lower than 56%, the separation time will be longer than 60 min and the peak of the hydrolyzed TMBB-Su broadens seriously. When the methanol content is higher than 64%, the peaks of hydrolyzed TMBB-Su, TMBB-Oct and TMBB-Tyr become overlapped. Therefore, 62% was chosen as the optimum methanol content in the mobile phase. The effect of pH value of the buffer in mobile phase on separation was also investigated. In this work, H3 Cit–Na2 HPO4 buffer was chosen to control the pH value in the range of pH 2.6–7.0. After experiments, it is found that the derivatives are baseline separated in the range of pH 4.1–4.9. Therefore, pH 4.5 was chosen to be the optimum value. The effect of the H3 Cit–Na2 HPO4 buffer concentration on the separation was also examined. When the concentration is between 8.0 and 15.0 mM, the satisfactory separation can be obtained. Finally, 10.0 mM pH 4.5 H3 Cit–Na2 HPO4 buffer solution was chosen as eluent B. However, under the optimum separation conditions mentioned above, the separation using isocratic elution mode seems timeconsuming. To obtain better separation efficiency, a gradient elution mode was adopted. By trial and error, gradient elution program as shown in Section 2.4 was used, and baseline separation of five derivatives of phenethylamines including tyrosine, octopamine, phenylalanine, tyramine and N-methyltyramine was achieved in 20 min (Fig. 2a) with the retention time of 8.38, 13.96, 16.84, 17.49 and 19.19 min, respectively.
Fig. 2. Typical chromatogram of the phenethylamines and the interference investigation. Mobile phase was as gradient program in Section 2.4. Detection: fluorescence (490/510 nm). Flow rate: 0.7 mL/min. Injection volume: 20 L. Standard phenethylamines: 0.1 M. Chromatograms: (a) phenethylamines; (b) phenethylamines spiked with amino acids and biogenic amines. Peaks: (1) tyrosine, (2) octopamine, (3) phenylalanine, (4) tyramine, (5) N-methyltyramine, (6) TMBB-Su.
buffer concentration, reaction time and temperature [25]. Therefore, these factors have to be optimized. The effect of the TMBB-Su content was investigated first. A mixture of phenethylamines with a concentration of 2.0 × 10−7 M for each was reacted with various concentrations of TMBB-Su. As shown in Fig. 3A, the peak areas of derivatives are maximum and constant when the concentration of reagent is in the range of 2.7 × 10−5 –3.3 × 10−5 M in the 0.5 mL derivatization solution. As a result, 2.7 × 10−5 M was selected as the optimal concentration. Buffer pH and buffer concentration play critical roles in the derivatization. It is well known that the labeling reaction for amines using succinimidyl ester is usually performed in alkalescence medium. Therefore, the pH in the range of 7.2–8.4 with boric acid and sodium tetraborate was studied in this experiment. As shown in Fig. 3B, the peak areas of the derivatives are the maximum at pH 7.8. With the increase in the pH, the peak areas of some phenethylamines decrease slightly, which is probably due to the increasing hydrolytic speed of TMBB-Su. Therefore, pH 7.8 was used for further investigation. Correspondingly, the buffer concentration also influenced the fluorescence intensity, and the results show that the maximum fluorescence intensity appeared at 25 mM (Fig. 3C). Reaction temperature and time are interdependent and the effects of these two factors on peak areas are shown in Fig. 3D and E. From Fig. 3D, the derivatization efficiencies of the phenethylamines are decreased with the rise in temperature, since high temperature can accelerate the hydrolysis of the reagent as well as derivatization reaction. Also, Fig. 3E indicated that 25 min was enough for the derivatization of all the phenethylamines. Finally, the reaction was performed at 25 ◦ C for 25 min. In summary, the optimized derivatization reaction of TMBBSu with phenethylamines was proceeded at room temperature for 25 min using 2.7 × 10−5 M TMBB-Su and 25 mM pH 7.8 H3 BO3 –Na2 B4 O7 buffer. 3.3. Interference
3.2. Optimization of derivatization conditions The derivatization efficiency is of great importance in precolumn derivatization strategy. Our previous work has proved that the derivatization efficiency for labeling amines using TMBB-Su was determined by the amount of labeling reagent, buffer pH,
Since TMBB-Su is an amine-reactive probe, other amino acid and biogenic amine that widely distributed in biological samples can also be derivatized. In order to assess the selectivity of the proposed method for phenethylamines, the interferences from common amino acids (glycine, alanine, leucine, isoleucine,
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Fig. 3. Optimization of derivatization conditions. (A) Concentration of TMBB-Su; (B) buffer pH; (C) buffer concentration; (D) derivatization temperature; (E) derivatization time. Separation and detection conditions were the same as in Fig. 2. Standard phenethylamine concentrations: 2.0 × 10−7 M for each. Symbol assignation: () tyrosine, () octopamine, () phenylalanine, () tyramine, () N-methyltyramine.
phenylalanine, valine, histidine, cysteine, lysine, asparagine, methionine, arginine, glutamine, proline, threonine, tyrosine, serine, aspartic acid and glutamic acid) and biogenic amines (spermidine, spermine, tyramine, histamine and cadaverine) were investigated. All these amino compounds were added into the mixture of standard phenethylamines and analyzed with them together under optimized derivatization and separation conditions. As shown in Fig. 2, although the peaks of these amino acids and biogenic amines were not assigned in the figure since it is not our purpose to identify and separate them, it is still well proved that they had no interference to the separation of the five target derivatives because of the same shapes of their peaks and variation of less than 2% for the peak areas between chromatograms in Fig. 2a and b.
phenethylamines in flesh and roe of crucian. The chromatograms of the flesh of crucian unspiked and spiked with the standard phenethylamines are shown in Fig. 4. The analytical results are summarized in Table 3. From the results, all the phenethylamines except N-methyltyramine can be detected in flesh, while octopamine and N-methyltyramine were not found in the roe, and the results of octopamine in flesh are in good agreement with previous reports [27]. The recoveries range from 96.4 to 104.4% and the RSDs for peak area of the derivatives are between 1.0 and 4.4%.
3.4. Analytical performance The results including the linear calibration ranges, regression equations and limits of detection (LODs) were listed in Table 1. The correlation coefficients (R2 ) are from 0.9972 to 0.9997, indicating good linearity. The RSD values vary from 1.2 to 2.7% for intraday determination (n = 6) and from 3.1 to 4.3% for interday determination (n = 6). The LODs for the labeled phenethylamines range from 0.1 to 0.2 nM. In order to comment on the attributes of the proposed method, the comparison between the proposed method and reported methods is shown in Table 2. From which, it could be found that the proposed method has advantages on milder derivatization conditions, higher sensitivity and the simultaneous analysis of both primary and secondary phenethylamines. 3.5. Sample analysis Diet is an important source of phenethylamines. Therefore, the developed method was applied to the determination of
Fig. 4. Chromatogram of flesh of crucian (a) and the same sample spiked with 0.03 M of standard phenethylamines. Mobile phase was as gradient program in Section 2.4. Detection: fluorescence (490/510 nm). Flow rate: 0.7 mL/min. Injection volume: 20 L. Standard phenethylamines: 0.1 M. Peaks: (1) tyrosine, (2) octopamine, (3) phenylalanine, (4) tyramine, (5) N-methyltyramine, (6) TMBB-Su.
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Table 1 Linear calibration range, regression equation and detection limits of TMBB-phenethylamine derivatives. Analyte
Calibration Range (nM)
Regression equation
Correlation coefficient
Tyr Oct Phe Tyra NMT
5.0–250 5.0–250 2.0–100 2.0–100 5.0–200
y = 5.5 × 107 x + 1.2 × 105 y = 7.5 × 107 x + 3.8 × 103 y = 9.9 × 107 x + 7.2 × 104 y = 8.6 × 107 x + 1.3 × 105 y = 5.0 × 107 x + 2.6 × 104
0.9925 0.9988 0.9901 0.9971 0.9921
LODsa (nM)
RSD (%, n = 6) Intraday
Interday
1.4 2.3 1.2 2.7 1.9
3.2 3.4 2.5 4.3 3.1
0.1 0.1 0.1 0.1 0.2
x: concentration of phenethylamines (M); y: peak area of TMBB-phenethylamine derivatives. a S/N = 3, per 20 L injection volume. Table 2 Comparison of the proposed method with reported methods for phenethylamines. Analytical method
Analytes
Reagent
Derivatization conditions
LOD
Ref.
HPLC-FD
Oct, Tyra, Tyr Tyr, Tyra Tyr, Phe Tyr, Phe Tyr, Phe Tyr, Phe Oct, Tyra, Phe l-Tyr, d-Tyr Tyr Tyr, Phe, Oct, Tyra, NMT
OPA PBC DBD-F FMOC-Cl NDA FITC DMQC-OSu DBD-PyNCS FITC TMBB-Su
pH 9.5, 25 ◦ C, 1 min 60 ◦ C, 60 min pH 10.0, 60 ◦ C, 3 h pH 12, 37 ◦ C, 1 h pH 10, 37 ◦ C, 30 min pH 9.2, 25 ◦ C, 20 h pH 8.0, 20 ◦ C, 40 min 55 ◦ C, 20 min pH 9.75, 25 ◦ C, 20 h pH 7.8, 25 ◦ C, 25 min
2.5–3.5 nM 0.13–0.23 nM 422, 690 nM 2.3, 5.0 nM 5.0, 5.0 nM 0.051, 0.081 nM 1.5-326 nM 2.9, 2.2 M 0.06 nM 0.1–0.2 nM
[28] [29] [30] [30] [30] [31] [32] [33] [34] This work
HPLC-ICOF-LIF IL-UALLME-HPLC-FD CZE-LIF CE-LIF HPLC-FD
HPLC-ICOF-LIF, HPLC-in-capillary optical fiber laser-induced fluorescence; IL-UALLME-HPLC-FL, ionic liquid-based ultrasound-assisted liquid–liquid microextraction and high-performance liquid chromatography–fluorescence detection; OPA, o-phthalaldehyde; DMQC-OSu, 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester; PBC, 4-(1-pyrene)butanoyl chloride; NDA, 2,3-naphthalenedialdehyde; DBD-F, 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; DBD-PyNCS, R(−)-4(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylamino-sulfonyl)-2,1,3-benzoxadiazole.
Table 3 Analytical results of samples. Samples
Tyr Oct Phe Tyra NMT
Crucian
Role of crucian
Added (mg/g)
Found (mg/g)
RSD (%, n = 6)
Recovery (%)
Added (mg/g)
Found (mg/g)
RSD (%, n = 6)
Recovery (%)
0 0.091 0 0.076 0 0.083 0 0.076 0 0.077
2.0 2.1 0.043 0.116 0.38 0.46 0.042 0.12 0 0.075
1.3 3.4 2.5 4.3 1.1 1.8 1.4 2.8 – 2.3
– 104.4 – 96.1 – 96.4 – 102.6 – 97.4
0 0.14 0 0.114 0 0.12 0 0.13 0 0.11
1.1 1.2 0 0.113 0.26 0.39 0.033 0.17 0 0.12
1.6 2.5 – 4.4 1.6 2.4 2.5 3.8 – 4.1
– 98.4 – 98.9 – 103.8 – 104.2 – 103.1
4. Concluding remarks
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A sensitive method was developed for the determination of both primary and secondary phenethylamines simultaneously in biological samples based on the pre-column fluorescent derivatization using TMBB-Su. The derivatization conditions were milder than most of the reports, and the detection limits were in the range of 0.1–0.2 nM (signal-to-noise ratio = 3), which were better than the reported HPLC methods. The results indicated that TMBB-Su has good potential to investigate the metabolism of biological compounds containing amino group. However, the proposed method may be not appropriate for all the biological samples considering the diversity of biological samples.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of primary and secondary phenethylamines in biological samples by high-performance liquid chromatographic method with fluorescence detection Xiao-Feng Guo a,b , Jie-Yu Wang a , Hong Wang a,∗ , Hua-Shan Zhang a a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China b Suzhou Institute of Wuhan University, Suzhou, China
a r t i c l e
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Article history: Received 29 September 2013 Accepted 13 July 2014 Available online 21 July 2014 Keywords: Fluorescence detection High-performance liquid chromatography Phenethylamines 1,3,5,7-Tetramethyl-8-(Nhydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene
a b s t r a c t Phenylalanine is an essential amino acid and its metabolites relate to various physiological and immune functions of living organisms. To monitor the alteration of concentration of primary and secondary phenethylamines including N-methyltyramine, octopamine, tyramine, tyrosine and phenylalanine in the metabolic pathway of phenylalanine, a sensitive and selective reversed-phase high-performance liquid chromatographic method has been developed in this study. The identification and quantification of phenethylamines were performed by fluorescent detection after pre-column derivatization with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene, an excellent fluorescent probe which could react with both primary and secondary amino groups simultaneously. The derivatization was carried out at 25 ◦ C for 25 min, and the separation was performed on a C18 column within 20 min. The linear ranges were from 2.0 to 100 nM for phenylalanine and tyramine to 5.0 to 250 for tyrosine and octopamine, with the detection limits of 0.1 nM for octopamine, tyramine, tyrosine and phenylalanine and 0.2 nM for N-methyltyramine (signal-to-noise ratio = 3), which allowed for the sure determination of phenethylamines at trace levels in the real samples without complex pretreatment or enrichment during multitudinous samples analysis. The proposed method has been validated by the analysis of the five target compounds in biological samples with spiked recoveries of 96.4–104.4% and the relative standard deviation of 1.0 and 4.4%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phenethylamines, such as phenylalanine (Phe), tyrosine (Tyr), tyramine (Tyra), N-methyltyramine (NMT) and octopamine (Oct) (Fig. 1), are important biological compounds. In these compounds, phenylalanine is an essential amino acid and could only be acquired from food, while the others are nonessential and could be both acquired from food and converted from phenylalanine. These phenethylamines relate to various physiological and immune functions of living organisms. Phenylalanine and tyrosine have several metabolites including octopamine and tyramine, which have been considered as neurotransmitters in invertebrates to regulate many actions such as growth, foraging, moving, learning and so on [1–3]. Tyrosine is also the precursor of catecholamines which are major brain and peripheral neurotransmitters in mammals. While for
∗ Corresponding author. Tel.: +86 27 87218924; fax: +86 27 68754067. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.07.017 1570-0232/© 2014 Elsevier B.V. All rights reserved.
human, octopamine and tyramine used to be considered functionally unimportant in the central nervous system in a long period because of their low physiological concentrations and the absence of specific receptors. However, two G protein-coupled receptors of octopamine were discovered recently in brains and tonsils of mammals, which suggest that octopamine may play important roles in several physiological and pathological procedures of human like cirrhosis, schizophrenia and Parkinson’s disease [4–6]. Moreover, octopamine could also be applied for the prophylaxis and treatment of adiposis and Type II diabetic nephropathy [7–9]. Besides, N-methyltyramine is a metabolite of tyramine and has many physiological functions including stimulates gastric and pancreatic secretions [10], and it is also a molecule which has a similar function to ephedrine alkaloids as dietary supplements but more safe [11]. Therefore, a quantification method of phenethylamines with high sensitivity and selectivity is always desired. By far, several analytical methods have been developed for the quantitative analysis of phenethylamines in biological samples, including high-performance liquid chromatography (HPLC)
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deriving with primary and secondary amines [26]. In the present work, to verify the applicability of TMBB-Su for the determination primary and secondary phenethylamines, a new HPLC method with fluorescence detection was developed using TMBB-Su as pre-column derivatization reagent and the results indicated that the proposed method has obvious advantages in sensitivity and selectivity with mild derivatization conditions. 2. Experimental 2.1. Apparatus An LC-20A HPLC system (Shimadzu, Tokyo, Japan) with RF10AXL fluorescence detector (Shimadzu, Tokyo, Japan) and Lab Solutions/LC Solution Lite chromatography chemstation (Shimadzu, Tokyo, Japan) were used in the experiments. Sample injection volume was 20 L. The separation was performed on an ODS column (5 m, 150 mm × 4.6 mm, i.d., Inertsil, GL Sciences, Tokyo, Japan). The pH values of solutions were measured using Mettler Toledo Delta 320 pH meter (Mettler-Toledo, Shanghai, China). Fig. 1. Structures of phenethylamines and their derivatization using TMBB-Su.
[12–15], capillary electrophoresis [16,17], molecularly imprinted solid-phase extraction (MISPE) [18] and electrochemical detection (ECD) [19]. Among these methods, HPLC followed by fluorescence detection is one of frequently used analytical techniques owing to its high sensitivity and selectivity. Since phenethylamines have intrinsic fluorescence, HPLC with fluorescence detection method can be used to analyze them directly without chemical derivatization. However, short absorbance and emission wavelength, small molar absorption coefficient, low fluorescence quantum yield and background interference from complex matrix [14] which could be resolved using fluorescent labeling reagents. Hence, precolumn derivatization combined with HPLC-fluorescence detection is regarded as an ideal analytical method for trace amounts of phenethylamines in complex matrix of bio-samples. Since there are both primary and secondary amines in phenethylamines, the fluorescent reagent used for phenethylamines analysis should label all of them. However, the reports for determination of primary and secondary phenethylamines simultaneously using HPLC-fluorescence detection were not many. Till now, typical fluorescent derivatization reagents which can label primary and secondary aliphatic amines simultaneously are 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) [20], fluorescein isothiocyanate (FITC) [21], 4-fluoro-7-nitrobenzofurazan (NBDF) [22], 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) [23] and N-hydroxysuccinimidyl 4,3,2 -naphthapyrone-4-acetate (NPA-OSu) [24]. However, all of these reagents have a variety of drawbacks. For example, long derivatization time is need for FITC and high temperature (60 ◦ C) is required for NBD-F, and operation procedure will be more complex than operating at room temperature. The excess FMOC-Cl in the derivatization procedure has to be removed by extraction or derivatization with a specific amine to form derivatives having no interference to the analysis of target compounds in order to prevent the generation of derivative with hydroxyl group of methanol. AQC and NPA-OSu have short absorbance and emission wavelengths which cannot avoid background interference from complex matrix. 1,3,5,7-Tetramethyl-8-(N-hydroxy-succinimidyl butyric ester)difluoroboradiaza -s-indacene (TMBB-Su) has been synthesized and applied for amine determination in our group [25] and has high fluorescence quantum yield, long emission wavelength, shorter reaction time, milder derivatization conditions (like lower temperature and weaker alkaline environment) when
2.2. Chemicals and reagents TMBB-Su was synthesized in our laboratory [23]. Phenylalanine, tyrosine, tyramine and N-methyltyramine were purchased from Biochemical Reagents Company (Shanghai, China). Octopamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise specified, all other reagents were of analytical grade. Water used for preparing solutions was purified by a Milli-Q system (Millipore, Bedford, MA, USA). The TMBB-Su stock solution was prepared by dissolving TMBBSu in acetonitrile to give a concentration of 1.0 × 10−4 M. The stock solutions of the analytes (1.0 × 10−4 M) were prepared by dissolving phenethylamines in water. Dilution of these stock solutions to appropriate concentrations was performed before use. H3 BO3 –Na2 B4 O7 buffers were prepared by mixing 0.025 M Na2 B4 O7 solution with 0.10 M H3 BO3 solution to the required pH value. Citric acid (H3 Cit)–Na2 HPO4 buffers were prepared by mixing 0.10 M H3 Cit solution with 0.10 M Na2 HPO4 solution to the required pH value. When not in use, all standards were stored at 4 ◦ C in a refrigerator. 2.3. Derivatization procedure To a 0.5 mL of Eppendorf tube containing 175 L of mixed amines and 125 L of H3 BO3 –Na2 B4 O7 buffer (pH 7.8), 135 L 1.0 × 10−4 M TMBB-Su and 65 L acetonitrile were added. The whole solution was diluted to the mark with water and kept at 25 ◦ C for 25 min. Then, an aliquot (20 L) of the reaction mixture was diluted with mobile phase and injected into the chromatographic system. 2.4. Chromatographic separation The chromatographic separations were performed at ambient temperature on an Inertsil ODS-SP column with gradient elution. Eluent A was methanol and eluent B was 10 mM, pH 4.5, citric acid–Na2 HPO4 buffer solution. Before the analysis, the column was pre-equilibrated for 30 min with mobile phase (A:B = 62:38, v/v). Then 20 L of the prepared sample solution was injected and the derivatives were eluted at a flow rate of 0.7 mL/min. The gradient elution program was used as following: 0–9 min, 62% A; 10–25 min, 75% A. The chromatographic system was re-equilibrated with mobile phase (A:B = 62:38, v/v) for 20 min between runs. The
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detection wavelengths were set at ex /em = 490/510 nm. All the solvents were filtered with 0.45 m membrane filter before use. 2.5. Method performance The linearity of peak areas against concentration of the five phenethylamines was tested under the optimized derivatization and separation conditions. The precision was assessed by repeating six sequential runs within-day and between-day using standard phenethylamines and reflected as relative standard deviations (RSDs). The linear analysis was performed using seven standard levels. Taking tyrosine as presentation, the seven standard levels are 5, 50, 90, 130, 170, 210 and 250 nM. 2.6. Sample preparation Crucian carp was killed, then the flesh on the back of crucian and roe were collected immediately and stored at −40 ◦ C. Just before the analysis, about 0.2 g of each sample was homogenized in 8.0 mL of water. After addition of 2.0 mL of 10% Cl3 CCOOH (w/v), the homogenate was centrifuged at 1430 × g for 10 min at room temperature. One milliliter of the clear solution was neutralized with 27 L of 1.0 M NaOH. The resulted solution was proceeded according to Sections 2.3 and 2.4. The recovery was evaluated by spiking standard mixture of amines in the sample preparation procedure, and the resulting solution was determined as the procedure of Sections 2.3 and 2.4. 3. Results and discussion 3.1. Optimization of separation conditions The separation of TMBB-Su derivatives with isocratic elution mode was tried first because it is simple. If the methanol content is lower than 56%, the separation time will be longer than 60 min and the peak of the hydrolyzed TMBB-Su broadens seriously. When the methanol content is higher than 64%, the peaks of hydrolyzed TMBB-Su, TMBB-Oct and TMBB-Tyr become overlapped. Therefore, 62% was chosen as the optimum methanol content in the mobile phase. The effect of pH value of the buffer in mobile phase on separation was also investigated. In this work, H3 Cit–Na2 HPO4 buffer was chosen to control the pH value in the range of pH 2.6–7.0. After experiments, it is found that the derivatives are baseline separated in the range of pH 4.1–4.9. Therefore, pH 4.5 was chosen to be the optimum value. The effect of the H3 Cit–Na2 HPO4 buffer concentration on the separation was also examined. When the concentration is between 8.0 and 15.0 mM, the satisfactory separation can be obtained. Finally, 10.0 mM pH 4.5 H3 Cit–Na2 HPO4 buffer solution was chosen as eluent B. However, under the optimum separation conditions mentioned above, the separation using isocratic elution mode seems timeconsuming. To obtain better separation efficiency, a gradient elution mode was adopted. By trial and error, gradient elution program as shown in Section 2.4 was used, and baseline separation of five derivatives of phenethylamines including tyrosine, octopamine, phenylalanine, tyramine and N-methyltyramine was achieved in 20 min (Fig. 2a) with the retention time of 8.38, 13.96, 16.84, 17.49 and 19.19 min, respectively.
Fig. 2. Typical chromatogram of the phenethylamines and the interference investigation. Mobile phase was as gradient program in Section 2.4. Detection: fluorescence (490/510 nm). Flow rate: 0.7 mL/min. Injection volume: 20 L. Standard phenethylamines: 0.1 M. Chromatograms: (a) phenethylamines; (b) phenethylamines spiked with amino acids and biogenic amines. Peaks: (1) tyrosine, (2) octopamine, (3) phenylalanine, (4) tyramine, (5) N-methyltyramine, (6) TMBB-Su.
buffer concentration, reaction time and temperature [25]. Therefore, these factors have to be optimized. The effect of the TMBB-Su content was investigated first. A mixture of phenethylamines with a concentration of 2.0 × 10−7 M for each was reacted with various concentrations of TMBB-Su. As shown in Fig. 3A, the peak areas of derivatives are maximum and constant when the concentration of reagent is in the range of 2.7 × 10−5 –3.3 × 10−5 M in the 0.5 mL derivatization solution. As a result, 2.7 × 10−5 M was selected as the optimal concentration. Buffer pH and buffer concentration play critical roles in the derivatization. It is well known that the labeling reaction for amines using succinimidyl ester is usually performed in alkalescence medium. Therefore, the pH in the range of 7.2–8.4 with boric acid and sodium tetraborate was studied in this experiment. As shown in Fig. 3B, the peak areas of the derivatives are the maximum at pH 7.8. With the increase in the pH, the peak areas of some phenethylamines decrease slightly, which is probably due to the increasing hydrolytic speed of TMBB-Su. Therefore, pH 7.8 was used for further investigation. Correspondingly, the buffer concentration also influenced the fluorescence intensity, and the results show that the maximum fluorescence intensity appeared at 25 mM (Fig. 3C). Reaction temperature and time are interdependent and the effects of these two factors on peak areas are shown in Fig. 3D and E. From Fig. 3D, the derivatization efficiencies of the phenethylamines are decreased with the rise in temperature, since high temperature can accelerate the hydrolysis of the reagent as well as derivatization reaction. Also, Fig. 3E indicated that 25 min was enough for the derivatization of all the phenethylamines. Finally, the reaction was performed at 25 ◦ C for 25 min. In summary, the optimized derivatization reaction of TMBBSu with phenethylamines was proceeded at room temperature for 25 min using 2.7 × 10−5 M TMBB-Su and 25 mM pH 7.8 H3 BO3 –Na2 B4 O7 buffer. 3.3. Interference
3.2. Optimization of derivatization conditions The derivatization efficiency is of great importance in precolumn derivatization strategy. Our previous work has proved that the derivatization efficiency for labeling amines using TMBB-Su was determined by the amount of labeling reagent, buffer pH,
Since TMBB-Su is an amine-reactive probe, other amino acid and biogenic amine that widely distributed in biological samples can also be derivatized. In order to assess the selectivity of the proposed method for phenethylamines, the interferences from common amino acids (glycine, alanine, leucine, isoleucine,
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Fig. 3. Optimization of derivatization conditions. (A) Concentration of TMBB-Su; (B) buffer pH; (C) buffer concentration; (D) derivatization temperature; (E) derivatization time. Separation and detection conditions were the same as in Fig. 2. Standard phenethylamine concentrations: 2.0 × 10−7 M for each. Symbol assignation: () tyrosine, () octopamine, () phenylalanine, () tyramine, () N-methyltyramine.
phenylalanine, valine, histidine, cysteine, lysine, asparagine, methionine, arginine, glutamine, proline, threonine, tyrosine, serine, aspartic acid and glutamic acid) and biogenic amines (spermidine, spermine, tyramine, histamine and cadaverine) were investigated. All these amino compounds were added into the mixture of standard phenethylamines and analyzed with them together under optimized derivatization and separation conditions. As shown in Fig. 2, although the peaks of these amino acids and biogenic amines were not assigned in the figure since it is not our purpose to identify and separate them, it is still well proved that they had no interference to the separation of the five target derivatives because of the same shapes of their peaks and variation of less than 2% for the peak areas between chromatograms in Fig. 2a and b.
phenethylamines in flesh and roe of crucian. The chromatograms of the flesh of crucian unspiked and spiked with the standard phenethylamines are shown in Fig. 4. The analytical results are summarized in Table 3. From the results, all the phenethylamines except N-methyltyramine can be detected in flesh, while octopamine and N-methyltyramine were not found in the roe, and the results of octopamine in flesh are in good agreement with previous reports [27]. The recoveries range from 96.4 to 104.4% and the RSDs for peak area of the derivatives are between 1.0 and 4.4%.
3.4. Analytical performance The results including the linear calibration ranges, regression equations and limits of detection (LODs) were listed in Table 1. The correlation coefficients (R2 ) are from 0.9972 to 0.9997, indicating good linearity. The RSD values vary from 1.2 to 2.7% for intraday determination (n = 6) and from 3.1 to 4.3% for interday determination (n = 6). The LODs for the labeled phenethylamines range from 0.1 to 0.2 nM. In order to comment on the attributes of the proposed method, the comparison between the proposed method and reported methods is shown in Table 2. From which, it could be found that the proposed method has advantages on milder derivatization conditions, higher sensitivity and the simultaneous analysis of both primary and secondary phenethylamines. 3.5. Sample analysis Diet is an important source of phenethylamines. Therefore, the developed method was applied to the determination of
Fig. 4. Chromatogram of flesh of crucian (a) and the same sample spiked with 0.03 M of standard phenethylamines. Mobile phase was as gradient program in Section 2.4. Detection: fluorescence (490/510 nm). Flow rate: 0.7 mL/min. Injection volume: 20 L. Standard phenethylamines: 0.1 M. Peaks: (1) tyrosine, (2) octopamine, (3) phenylalanine, (4) tyramine, (5) N-methyltyramine, (6) TMBB-Su.
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Table 1 Linear calibration range, regression equation and detection limits of TMBB-phenethylamine derivatives. Analyte
Calibration Range (nM)
Regression equation
Correlation coefficient
Tyr Oct Phe Tyra NMT
5.0–250 5.0–250 2.0–100 2.0–100 5.0–200
y = 5.5 × 107 x + 1.2 × 105 y = 7.5 × 107 x + 3.8 × 103 y = 9.9 × 107 x + 7.2 × 104 y = 8.6 × 107 x + 1.3 × 105 y = 5.0 × 107 x + 2.6 × 104
0.9925 0.9988 0.9901 0.9971 0.9921
LODsa (nM)
RSD (%, n = 6) Intraday
Interday
1.4 2.3 1.2 2.7 1.9
3.2 3.4 2.5 4.3 3.1
0.1 0.1 0.1 0.1 0.2
x: concentration of phenethylamines (M); y: peak area of TMBB-phenethylamine derivatives. a S/N = 3, per 20 L injection volume. Table 2 Comparison of the proposed method with reported methods for phenethylamines. Analytical method
Analytes
Reagent
Derivatization conditions
LOD
Ref.
HPLC-FD
Oct, Tyra, Tyr Tyr, Tyra Tyr, Phe Tyr, Phe Tyr, Phe Tyr, Phe Oct, Tyra, Phe l-Tyr, d-Tyr Tyr Tyr, Phe, Oct, Tyra, NMT
OPA PBC DBD-F FMOC-Cl NDA FITC DMQC-OSu DBD-PyNCS FITC TMBB-Su
pH 9.5, 25 ◦ C, 1 min 60 ◦ C, 60 min pH 10.0, 60 ◦ C, 3 h pH 12, 37 ◦ C, 1 h pH 10, 37 ◦ C, 30 min pH 9.2, 25 ◦ C, 20 h pH 8.0, 20 ◦ C, 40 min 55 ◦ C, 20 min pH 9.75, 25 ◦ C, 20 h pH 7.8, 25 ◦ C, 25 min
2.5–3.5 nM 0.13–0.23 nM 422, 690 nM 2.3, 5.0 nM 5.0, 5.0 nM 0.051, 0.081 nM 1.5-326 nM 2.9, 2.2 M 0.06 nM 0.1–0.2 nM
[28] [29] [30] [30] [30] [31] [32] [33] [34] This work
HPLC-ICOF-LIF IL-UALLME-HPLC-FD CZE-LIF CE-LIF HPLC-FD
HPLC-ICOF-LIF, HPLC-in-capillary optical fiber laser-induced fluorescence; IL-UALLME-HPLC-FL, ionic liquid-based ultrasound-assisted liquid–liquid microextraction and high-performance liquid chromatography–fluorescence detection; OPA, o-phthalaldehyde; DMQC-OSu, 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester; PBC, 4-(1-pyrene)butanoyl chloride; NDA, 2,3-naphthalenedialdehyde; DBD-F, 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; DBD-PyNCS, R(−)-4(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylamino-sulfonyl)-2,1,3-benzoxadiazole.
Table 3 Analytical results of samples. Samples
Tyr Oct Phe Tyra NMT
Crucian
Role of crucian
Added (mg/g)
Found (mg/g)
RSD (%, n = 6)
Recovery (%)
Added (mg/g)
Found (mg/g)
RSD (%, n = 6)
Recovery (%)
0 0.091 0 0.076 0 0.083 0 0.076 0 0.077
2.0 2.1 0.043 0.116 0.38 0.46 0.042 0.12 0 0.075
1.3 3.4 2.5 4.3 1.1 1.8 1.4 2.8 – 2.3
– 104.4 – 96.1 – 96.4 – 102.6 – 97.4
0 0.14 0 0.114 0 0.12 0 0.13 0 0.11
1.1 1.2 0 0.113 0.26 0.39 0.033 0.17 0 0.12
1.6 2.5 – 4.4 1.6 2.4 2.5 3.8 – 4.1
– 98.4 – 98.9 – 103.8 – 104.2 – 103.1
4. Concluding remarks
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20835004, 31170344 and 21105074, Beijing, China) and Basic Research Program of Jiangsu Province (Natural Science Foundation, No. BK20130369, Jiangsu, China).
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