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Ultrasound-assisted dispersive liquid–liquid microextraction combined with high-performance liquid chromatography-fluorescence detection for sensitive determination of biogenic amines in rice wine samples
Journal of Chromatography A, 1216 (2009) 6636–6641
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Ultrasound-assisted dispersive liquid–liquid microextraction combined with high-performance liquid chromatography-fluorescence detection for sensitive determination of biogenic amines in rice wine samples Ke-Jing Huang a,∗ , Cai-Yun Wei a , Wei-Li Liu a , Wan-Zhen Xie a , Jun-Feng Zhang b , Wei Wang c,∗∗ a
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China Research Center for Agricultural Standards and Inspection Technology, Henan Academy of Agricultural Sciences, Chengzhou 450002, China c School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng 224003, China b
a r t i c l e
i n f o
Article history: Received 26 May 2009 Received in revised form 25 July 2009 Accepted 31 July 2009 Available online 5 August 2009 Keywords: Ultrasound-assisted dispersive liquid–liquid microextraction Biogenic amines 2,6-Dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester High-performance liquid chromatography-fluorescence detection Rice wine samples
a b s t r a c t Ultrasound-assisted dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography-fluorescence detection was used for the extraction and determination of three biogenic amines including octopamine, tyramine and phenethylamine in rice wine samples. Fluorescence probe 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester was applied for derivatization of biogenic amines. Acetonitrile and 1-octanol were used as disperser solvent and extraction solvent, respectively. Extraction conditions including the type of extraction solvent, the volume of extraction solvent, ultrasonication time and centrifuging time were optimized. After extraction and centrifuging, analyte was injected rapidly into high-performance liquid chromatography and then detected with fluorescence. The calibration graph of the proposed method was linear in the range of 5–500 g mL−1 (octopamine and tyramine) and 0.025–2.5 g mL−1 (phenethylamine). The relative standard deviations were 2.4–3.2% (n = 6) and the limits of detection were in the range of 0.02–5 ng mL−1 . The method was applied to analyze the rice wine samples and spiked recoveries in the range of 95.42–104.56% were obtained. The results showed that ultrasound-assisted dispersive liquid–liquid microextraction was a very simple, rapid, sensitive and efficient analytical method for the determination of trace amount of biogenic amines. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biogenic amines (BAs) are nitrogenous low molecular weight organic compounds, which have been recognized biological activity [1]. They have been found to occur in many foods and beverages including cheese, sausage, wine, beer, fish, soy sauces, aged meat, etc. [2–8]. The major pathway of BAs formation is the decarboxylation of free amino acids mainly by microbial enzymatic activity. The amounts of biogenic amines usually increase during controlled or spontaneous microbial fermentation of food or in the course of food spoilage, so they are considered indicators of food quality and freshness. It also has been reported that high amounts of biogenic amines not only damage the quality of food but can also give rise to carcinogenic compounds [9–11]. In view of the possible harmful effects of biogenic amines for human health and food safety, the development of sensitive and routine methods for their analysis is of great importance.
∗ Corresponding author. Tel.: +86 376 6390611; fax: +86 376 6390597. ∗∗ Corresponding author. E-mail address: [email protected] (K.-J. Huang). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.07.070
Various methods have been developed for the analysis of BAs such as thin-layer chromatography (TLC) [12], gas chromatography [13,14], capillary electrophoretic method (CE) [15–17] and high-performance liquid chromatography (HPLC) [8,18–22]. HPLC has been the most generally used technique in determination of BAs in different kinds of food due to its high selectivity, sensitivity and simple sample treatment. Since many BAs in food show neither satisfactory absorption in the visible or ultraviolet range, nor have fluorescence properties, chemical pre- or postcolumn derivatization has become widely accepted and usually shows great sensitivity and selectivity. For the fluorescence labeling of BAs, the fluorescence labeling reagent 2,6-dimethyl-4-quinolinecarboxylic acid Nhydroxysuccinimide ester (DMQC-OSu) not only reacts readily with primary and secondary amines with good selectivity in aqueous solution, but also provides the advantages of few by-products and mild reaction conditions [23]. To our best knowledge, DMQC-OSu has not been validated for the analysis of BAs. The analysis of BAs in food has two problems: the complexity of the sample matrix and the low concentration levels at which the compounds are present in the samples. So a pretreatment step for sample enrichment and cleanup is very necessary. Many
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sample preparation techniques such as liquid–liquid extraction (LLE) [24,25], solid-phase extraction (SPE) [19,26,27], solid-phase microextraction (SPME) [14,28–30] and the single drop microextraction (SDME) [29,30] for this propose have been developed. Recently, a novel sample preparation technique dispersive liquid phase microextraction (LPME) or dispersive liquid–liquid microextraction (DLLME) was developed by Assadi and co-workers. It is based on a ternary component solvent system like homogeneous liquid–liquid extraction (HLLE) and cloud point extraction (CPE) [31]. The advantages of the LPME technique are simple, fast, high recovery, simplicity of operation and low cost. Especially, the sample extracted in L-level solvent can be directly injected in HPLC for analysis. In this paper, a novel method for the detection and quantification of three biogenic amines including octopamine, tyramine, phenethylamine (Fig. 1) using DMQC-OSu derivatization followed by ultrasound-assisted dispersive liquid–liquid microextraction (UDLLME) and high-performance liquid chromatographyfluorescence detection (HPLC-FL) detection was presented. The derivatization procedure and extraction conditions were optimized in detail. The developed method was applied to rice wine samples. The method displayed good sensitivity, linearity as well as excellent recoveries.
1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6 ]) 97% was purchased from Jingchun Chemical Reagent Co., Ltd. (Shanghai, China). Unless otherwise specified, all reagents were of analytical reagent grade and used without further purification. All solutions were prepared with double-distilled water and were stored in the refrigerator at 4 ◦ C and filtered through 0.45 m nylon filters (Automaticscience, Instrument Co., Ltd., Tianjin, China) before used.
2. Experimental
Rice wine samples were purchased at local supermarkets. The procedure for rice wine samples preparation was as follow. In brief, 1 mL of the homogenized rice wine sample and 4 mL trichloroacetic acid (TCAA) were added in a 10-mL vial and then were vigorously mixed by vortexing for 2 min. After centrifuged at 5000 rpm for 10 min, the supernatant was collected and added in 4 mL n-hexane to remove fat. The extracts were filtered and adjusted to the pH of 8.0 with NaOH solution. Aliquots of the solution were derivatized and analyzed with the proposed method.
2.1. Instrumentation An Agilent 1200 Series of high-performance liquid chromatography system, which consisted of a quaternary pump, a vacuum degasser and a fluorescence detector was used. A reversed-phase Eclipse XDB-C18 column (150 mm × 4.6 mm i.d., 5 m, Agilent, USA) was used for separation at ambient temperature and a manual sample injector with a 20 L loop was applied for sample injection. Agilent ChemStation for HPLC system was employed to acquire and process chromatographic data. The mobile phase was a mixture of methanol–water (60/40, v/v) delivered at a flow rate of 1.0 mL min−1 , and the detection wavelength was set at ex /em = 326/412 nm. 2.2. Reagents 2,6-Dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (DMQC-OSu) was synthesized according to Lu et al. [22] and a 2 mM solution prepared with pretreated acetonitrile (dried with P2 O5 ). Octopamine, tyramine, phenethylamine were purchased from Sigma (St. Louis, MO, USA). A stock solution of this compound was prepared in double-distilled water. Working solutions were prepared daily by proper dilution of the stock solution with double-distilled water. H3 BO3 –Na2 B4 O7 buffer was prepared by mixing 0.2 M H3 BO3 solution with 0.05 M Na2 B4 O7 solution to the required pH value.
Fig. 1. The chemical structures of octopamine, tyramine and phenethylamine.
2.3. Ultrasound-assisted dispersive liquid–liquid microextraction 100 L of mixed amine in water, 400 L of DMQC-OSu in acetonitrile (2 mM), 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0) and 300 L of double-distilled water were added in a 1.5 mL conical tube and vigorously mixed. Then the resulted solution was incubated at 20 ◦ C for 40 min. A volume of 50 L 1-octanol was added into the conical tube and sonicated for 1 min. The 1-octanol was dispersed into the aqueous solution, and nearly homogenous solution was achieved. Further the solution was centrifuged at 5000 rpm for 5 min and the organic phase was aspirated into a microsyringe and injected into the HPLC system for direct analysis. 2.4. Sample preparation
3. Results and discussion 3.1. Optimization of derivatization The chemical structure of DMQC-OSu and its reaction with BAs are shown in Fig. 2. In order to achieve the maximum derivatization yield, the derivatization conditions including the concentration of DMQC-OSu, pH of the buffer, the volume of buffer, the reaction temperature and the reaction time were carefully optimized. The effect of DMQC-OSu concentration on the derivatization yield was evaluated from 0.1 to 1.5 mmol L−1 . The subsequent HPLC analysis showed that the peak areas of derivatives were maximum and stable when the concentration of DMQC-OSu was 0.8 mmol L−1 . Then this concentration was used in the following experiments. H3 BO3 –Na2 B4 O7 buffers at pH range of 7.0–9.0 were tested for the derivatization. The maximum and constant peak areas of the derivatives were obtained at pH 8.0. Therefore, pH 8.0 was selected. The influence of the buffer amount was also investigated. It can be found that the peak areas of the derivatives reached the maximum at 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0). Thus, 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0) was selected in the derivatization reaction.
Fig. 2. The reactions of DMQC-OSu with amine.
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Fig. 3. Effect of type of extraction solvent. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , ultrasonic agitation time for 3 min and 5000 rpm centrifuging for 5.0 min. (1) Octopamine; (2) tyramine; and (3) phenethylamine.
The effect of the temperature on derivatization yield was also studied in the range of 4–50 ◦ C. The results showed that the yield of derivatives reached the maximum at 20 ◦ C. Then 20 ◦ C was selected as the optimum temperature value for the derivatization. With respect to the reaction time, optimization was performed in the range of 5–60 min. The results showed that derivatization yield reached the largest and invariable when the reaction time was 40 min. Therefore, 40 min was selected in this work. 3.2. Optimization of ultrasound-assisted dispersive liquid–liquid microextraction In order to obtain the maximal extraction efficiency, parameters that may influence the enrichment performance should be investigated in detail. A series of experiments was designed for this goal. 3.2.1. Type of extraction solvent Choosing the most suitable extraction solvent was great important for ultrasound-assisted dispersive liquid–liquid microextraction (UDLLME). Efficiencies of UDLLME of DMQC-OSu derivatives with carbon tetrachloride, chloroform, hexane, chlorobenzene, 1octanol and 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6 ]) have been evaluated. The relative peak areas are shown in Fig. 3. Chloroform and 1-octanol had higher extraction efficiency than others, and they almost achieve the same recoveries. Though 1-octanol had lower density than water, good chromatographic behavior and good extraction properties for the target compound made it to be the best extraction solvent. Thereby, 1octanol was selected in this work. 3.2.2. Effect of the volume of extraction solvent During UDLLME process, extraction solvent volume was an essential factor which can influence the occurrence of the cloudy state and also determined the enrichment performance. To examine the effect of extraction solvent volume, different volumes of 1-octanol (0, 25, 50, 60, 75, 100, and 150 L) were subjected to the same UDLLME procedures. The results are shown in Fig. 4. As can be seen, peak areas of derivatives increased with the increase of 1-octanol volume in the range of 0–50 L, and then decreased when the volume was continuously increased. Therefore, 50 L of 1-octanol was selected in order to achieve higher enrichment factor, better repeatability and lower limit of detection (LOD).
Fig. 4. Effect of 1-octanol volume. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , ultrasonic agitation time for 3 min and 5000 rpm centrifuging for 5.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
3.2.3. Effect of sonication time Enough time will make the extraction solvent well dispersed into the aqueous solution and resulted in the excellent enrichment. The effect of ultrasonic agitation time was evaluated in the range of 0–10 min and the results showed that the peak areas of the derivatives increased in the first 1 min and then decreased slowly. Hence, 1 min was enough for the dispersive procedure (Fig. 5). 3.2.4. Effect of centrifuging time In UDLLME, ultrasonic agitation makes the extractant completely disperse in aqueous phase and form vast organic vesicles to achieve efficient extraction. Centrifugation was substantial in order to obtain two distinguishable phases in the extraction tubes. The effect of centrifuging time on the extraction efficiency has been evaluated. In Fig. 6, it was found that the extraction performance all reached the best when the solution was centrifuged at 5000 rpm for 5 min. When the centrifugation time was longer or shorter than 5 min, the peak areas decreased, So 5 min was chosen in the following study. The chromatograms obtained by UDLLME–HPLC and direct HPLC analysis under the optimized conditions are shown in Fig. 7. Comparing with the chromatograms obtained by UDLLME–HPLC (Fig. 7A) to that of the direct injection (Fig. 7B), a dramatic peak
Fig. 5. Effect of ultrasonic agitation time on the peak area. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , 1octanol volume for 50 L, 5000 rpm centrifuging for 5.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
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enhancement was presented. This exhibited the remarkable preconcentration ability of the UDLLME. 3.3. Analytical performance of the proposed method
Fig. 6. Effect of time of centrifuging at 5000 rpm on the peak area. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , 1-octanol volume for 50 L, ultrasonic agitation time for 1.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
Under the above-mentioned optimal conditions, a series of experiments were designed for obtaining linear calibration ranges, regression equations, detection limits and other characteristics of the method. The detection limits of biogenic amines were calculated as the amounts of these latter that resulted in a peak area three times larger than that of the baseline noise. The results are listed in Table 1. All the analytes showed good linearity with correlation coefficient (r) ranging from 0.9972 to 0.9998. The precisions were also important for the proposed method. The intra-day precisions ranged from 2.4 to 3.2% and the inter-day precisions ranged from 3.2 to 5.5%. The limits of detection (LODs) were in the range of 5–0.02 ng mL−1 . These excellent results indicated that the present approach was a simple and sensitive procedure to determine biogenic amines at trace level. Comparison of the different methods with other derivatization reagents for the determination of biogenic amines is summarized in Table 2. The proposed method in this work showed high sensitivity and was expected as the most used analysis method for detecting the low concentration of biogenic amines. 3.4. Rice wine samples analysis
Fig. 7. Chromatograms for BAs derivatives obtained by UDLLME (A) and direct HPLC analysis (B). Peaks: (a) octopamine, (b) tyramine, and (c) phenethylamine. UDLLME conditions outlined in Section 2 and HPLC conditions: mobile phase, methanol/water (60/40, v/v); flow rate, 1 mL min−1 ; detection wavelength fluorescence (326/412 nm).
Under the optimized experimental conditions, the proposed method was applied for the determination of biogenic amines in three rice wine samples. The results are shown in Table 3. Octopamine, tyramine and phenethylamine were all found in the samples. Tyramine was the most abundant amine in these samples while phenethylamine was the least. The samples were spiked with three biogenic amines at three levels to investigate the effect of sample matrices (Table 3). The recoveries of the three amines ranged from 95.42 to 104.56%. The typical chromatograms of blank sample and spiked with amines were illustrated in Fig. 8. The results showed the method enabled the precise and sensitive determination of standards and can be applied to detect these biogenic amines in real samples.
Table 1 The performance characteristics of the proposed method. DMQC-amine derivatives
Octopamine Tyramine Phenethylamine
Calibration range (g mL−1 )
Regression equation
5–500 5–500 0.025–2.5
Y = 0.9941X + 83.9888 Y = 1.6204X + 11.1648 Y = 0.2951X + 41.3648
0.9972 0.9998 0.9972
RSD
Detection limit (ng mL−1 )
Intra-day (n = 5)
Inter-day (n = 5)
2.6 3.2 2.4
3.2 4.0 5.5
5 5 0.02
Table 2 Experimental presentation of various methods for biogenic amines. Reagents
Mode
Reaction conditions
LODs
Reference
DnsCl HFBA FBQCA DTAF CNBF DnsCl SFP OPA AQC DMQC-OSu
TLC Ion-pair extraction GC–MS CE–LIF CE–LIF HPLC–UV HPLC–DAD HPLC–MS SPE–HPLC-FL SPE–HPLC-FL UDLLME–HPLC-FL
NaHCO3 , 40 ◦ C, 1 h 80 ◦ C, 60 min Borate buffer, pH 9.6, 50 ◦ C, 20 min 50 mM boric acid, 0.5 min H3 BO3 –Na2 B4 O7 buffer (pH 9.5), 60 ◦ C, 30 min NaOH, saturated NaHCO3 , 40 ◦ C, 0.5 h Borate buffer, pH 9, 60 min Na2 B4 O7 buffer, pH 10.5 Na2 B4 O7 buffer, pH 8.8 Borate buffer, pH 8.0, 20 ◦ C, 40 min
500–105 g L−1 5–30 g L−1 0.4–12 nmol L−1 17–43 g L−1 0.056–0.87 mol L−1 0.05–0.25 mg L−1 1.2–19.0 mg kg−1 0.07–0.4 mg L−1 15–50 g L−1 0.02–5 g L−1
[12] [13] [17] [15] [21] [22] [8] [26] [27] This work
DnsCl = dansyl chloride; TFAA = trifluoroacetylacetone; FBQCA = 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde; DTAF = 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein; CNBF = 4-chloro-3,5-dinitrobenzotrifluoride; SFP = succinimidylferrocenyl propionate; OPA = o-phthaldialdehyde; AQC = 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate.
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Table 3 Analytical results for the three biogenic amines in rice wine samples. Samples
Biogenic amines
Octopamine
Rice wine 1
Tyramine
Phenethylamine
Octopamine
Rice wine 2
Tyramine
Phenethylamine
Octopamine
Rice wine 3
Tyramine
Phenethylamine
Added (g mL−1 )
Found (g mL−1 )
Recovered (g mL−1 )
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.0
128.30 133.16 179.51 321.60 15.30 20.22 64.04 313.7 0.160 0.186 0.668 2.145
128.30 4.86 51.21 193.30 15.30 4.92 48.74 298.4 0.160 0.026 0.508 1.985
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.000
126.80 131.57 178.59 322.30 15.80 20.73 64.54 323.40 0.150 0.175 0.637 2.232
126.80 4.77 51.79 195.50 15.80 4.93 48.74 307.56 0.150 0.025 0.487 2.082
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.000
127.50 132.46 179.78 324.80 16.00 20.87 67.11 313.45 0.154 0.178 0.647 2.201
127.50 4.96 52.28 197.30 16.00 4.87 51.11 297.45 0.154 0.024 0.493 2.047
Recovery (%)
97.21 102.41 96.65 98.42 97.48 99.46 103.25 101.56 99.24 95.42 103.58 97.75 98.63 97.48 102.52 98.62 97.36 104.12 99.12 104.56 98.65 97.47 102.21 99.15 97.45 98.65 102.36
RSD (%) (n = 6) 2.01 3.25 2.68 3.48 3.14 4.56 5.23 8.72 5.35 4.32 4.68 8.51 2.32 4.56 3.65 2.85 3.42 5.62 4.21 5.63 4.52 3.25 4.56 6.45 3.62 4.52 2.85 3.15 2.83 3.69 5.02 4.32 5.12 6.23 3.58 4.82
real samples (95.42–104.56%) showed that the method was sufficiently applicable to determine biogenic amines in real samples. Therefore, it had the potential of practical applications and could be either a complementary or a parallel method for the biogenic amines determination. Acknowledgments
Fig. 8. Typical chromatograms of blank sample (A) and sample spiked with biogenic amines (B), respectively. Peaks: (a) octopamine, (b) tyramine, and (c) phenethylamine. Rice wine samples (B): spiked with octopamine 20 g mL−1 , tyramine 50 g mL−1 , phenethylamine 0.1 g mL−1 . Other conditions were the same as depicted in Fig. 6.
4. Conclusions The proposed method outlined the successful development and application of ultrasound-assisted dispersive liquid–liquid microextraction combining with HPLC-FL for the analysis of biogenic amines. The proposed method had many practical advantages, including simplicity of the extraction method, using of a small volume of organic solvent for extraction, high sensitivity, low cost and rapidity. The LODs of biogenic amines were in the range of 5–0.02 ng mL−1 , which revealed the proposed method had high sensitivity. The excellent spiked recoveries of biogenic amines in
This work was supported by the National Natural Science Foundation of China (20805040, 20875080), Natural Science Foundation of He’nan Province of China (082300420140), Foundation of He’nan Educational Committee (2008A150020), Excellent Youth Foundation of He’nan Scientific Committee (084100410024) and sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province (2010HASTIT025). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Ultrasound-assisted dispersive liquid–liquid microextraction combined with high-performance liquid chromatography-fluorescence detection for sensitive determination of biogenic amines in rice wine samples Ke-Jing Huang a,∗ , Cai-Yun Wei a , Wei-Li Liu a , Wan-Zhen Xie a , Jun-Feng Zhang b , Wei Wang c,∗∗ a
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China Research Center for Agricultural Standards and Inspection Technology, Henan Academy of Agricultural Sciences, Chengzhou 450002, China c School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng 224003, China b
a r t i c l e
i n f o
Article history: Received 26 May 2009 Received in revised form 25 July 2009 Accepted 31 July 2009 Available online 5 August 2009 Keywords: Ultrasound-assisted dispersive liquid–liquid microextraction Biogenic amines 2,6-Dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester High-performance liquid chromatography-fluorescence detection Rice wine samples
a b s t r a c t Ultrasound-assisted dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography-fluorescence detection was used for the extraction and determination of three biogenic amines including octopamine, tyramine and phenethylamine in rice wine samples. Fluorescence probe 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester was applied for derivatization of biogenic amines. Acetonitrile and 1-octanol were used as disperser solvent and extraction solvent, respectively. Extraction conditions including the type of extraction solvent, the volume of extraction solvent, ultrasonication time and centrifuging time were optimized. After extraction and centrifuging, analyte was injected rapidly into high-performance liquid chromatography and then detected with fluorescence. The calibration graph of the proposed method was linear in the range of 5–500 g mL−1 (octopamine and tyramine) and 0.025–2.5 g mL−1 (phenethylamine). The relative standard deviations were 2.4–3.2% (n = 6) and the limits of detection were in the range of 0.02–5 ng mL−1 . The method was applied to analyze the rice wine samples and spiked recoveries in the range of 95.42–104.56% were obtained. The results showed that ultrasound-assisted dispersive liquid–liquid microextraction was a very simple, rapid, sensitive and efficient analytical method for the determination of trace amount of biogenic amines. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biogenic amines (BAs) are nitrogenous low molecular weight organic compounds, which have been recognized biological activity [1]. They have been found to occur in many foods and beverages including cheese, sausage, wine, beer, fish, soy sauces, aged meat, etc. [2–8]. The major pathway of BAs formation is the decarboxylation of free amino acids mainly by microbial enzymatic activity. The amounts of biogenic amines usually increase during controlled or spontaneous microbial fermentation of food or in the course of food spoilage, so they are considered indicators of food quality and freshness. It also has been reported that high amounts of biogenic amines not only damage the quality of food but can also give rise to carcinogenic compounds [9–11]. In view of the possible harmful effects of biogenic amines for human health and food safety, the development of sensitive and routine methods for their analysis is of great importance.
∗ Corresponding author. Tel.: +86 376 6390611; fax: +86 376 6390597. ∗∗ Corresponding author. E-mail address: [email protected] (K.-J. Huang). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.07.070
Various methods have been developed for the analysis of BAs such as thin-layer chromatography (TLC) [12], gas chromatography [13,14], capillary electrophoretic method (CE) [15–17] and high-performance liquid chromatography (HPLC) [8,18–22]. HPLC has been the most generally used technique in determination of BAs in different kinds of food due to its high selectivity, sensitivity and simple sample treatment. Since many BAs in food show neither satisfactory absorption in the visible or ultraviolet range, nor have fluorescence properties, chemical pre- or postcolumn derivatization has become widely accepted and usually shows great sensitivity and selectivity. For the fluorescence labeling of BAs, the fluorescence labeling reagent 2,6-dimethyl-4-quinolinecarboxylic acid Nhydroxysuccinimide ester (DMQC-OSu) not only reacts readily with primary and secondary amines with good selectivity in aqueous solution, but also provides the advantages of few by-products and mild reaction conditions [23]. To our best knowledge, DMQC-OSu has not been validated for the analysis of BAs. The analysis of BAs in food has two problems: the complexity of the sample matrix and the low concentration levels at which the compounds are present in the samples. So a pretreatment step for sample enrichment and cleanup is very necessary. Many
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sample preparation techniques such as liquid–liquid extraction (LLE) [24,25], solid-phase extraction (SPE) [19,26,27], solid-phase microextraction (SPME) [14,28–30] and the single drop microextraction (SDME) [29,30] for this propose have been developed. Recently, a novel sample preparation technique dispersive liquid phase microextraction (LPME) or dispersive liquid–liquid microextraction (DLLME) was developed by Assadi and co-workers. It is based on a ternary component solvent system like homogeneous liquid–liquid extraction (HLLE) and cloud point extraction (CPE) [31]. The advantages of the LPME technique are simple, fast, high recovery, simplicity of operation and low cost. Especially, the sample extracted in L-level solvent can be directly injected in HPLC for analysis. In this paper, a novel method for the detection and quantification of three biogenic amines including octopamine, tyramine, phenethylamine (Fig. 1) using DMQC-OSu derivatization followed by ultrasound-assisted dispersive liquid–liquid microextraction (UDLLME) and high-performance liquid chromatographyfluorescence detection (HPLC-FL) detection was presented. The derivatization procedure and extraction conditions were optimized in detail. The developed method was applied to rice wine samples. The method displayed good sensitivity, linearity as well as excellent recoveries.
1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6 ]) 97% was purchased from Jingchun Chemical Reagent Co., Ltd. (Shanghai, China). Unless otherwise specified, all reagents were of analytical reagent grade and used without further purification. All solutions were prepared with double-distilled water and were stored in the refrigerator at 4 ◦ C and filtered through 0.45 m nylon filters (Automaticscience, Instrument Co., Ltd., Tianjin, China) before used.
2. Experimental
Rice wine samples were purchased at local supermarkets. The procedure for rice wine samples preparation was as follow. In brief, 1 mL of the homogenized rice wine sample and 4 mL trichloroacetic acid (TCAA) were added in a 10-mL vial and then were vigorously mixed by vortexing for 2 min. After centrifuged at 5000 rpm for 10 min, the supernatant was collected and added in 4 mL n-hexane to remove fat. The extracts were filtered and adjusted to the pH of 8.0 with NaOH solution. Aliquots of the solution were derivatized and analyzed with the proposed method.
2.1. Instrumentation An Agilent 1200 Series of high-performance liquid chromatography system, which consisted of a quaternary pump, a vacuum degasser and a fluorescence detector was used. A reversed-phase Eclipse XDB-C18 column (150 mm × 4.6 mm i.d., 5 m, Agilent, USA) was used for separation at ambient temperature and a manual sample injector with a 20 L loop was applied for sample injection. Agilent ChemStation for HPLC system was employed to acquire and process chromatographic data. The mobile phase was a mixture of methanol–water (60/40, v/v) delivered at a flow rate of 1.0 mL min−1 , and the detection wavelength was set at ex /em = 326/412 nm. 2.2. Reagents 2,6-Dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (DMQC-OSu) was synthesized according to Lu et al. [22] and a 2 mM solution prepared with pretreated acetonitrile (dried with P2 O5 ). Octopamine, tyramine, phenethylamine were purchased from Sigma (St. Louis, MO, USA). A stock solution of this compound was prepared in double-distilled water. Working solutions were prepared daily by proper dilution of the stock solution with double-distilled water. H3 BO3 –Na2 B4 O7 buffer was prepared by mixing 0.2 M H3 BO3 solution with 0.05 M Na2 B4 O7 solution to the required pH value.
Fig. 1. The chemical structures of octopamine, tyramine and phenethylamine.
2.3. Ultrasound-assisted dispersive liquid–liquid microextraction 100 L of mixed amine in water, 400 L of DMQC-OSu in acetonitrile (2 mM), 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0) and 300 L of double-distilled water were added in a 1.5 mL conical tube and vigorously mixed. Then the resulted solution was incubated at 20 ◦ C for 40 min. A volume of 50 L 1-octanol was added into the conical tube and sonicated for 1 min. The 1-octanol was dispersed into the aqueous solution, and nearly homogenous solution was achieved. Further the solution was centrifuged at 5000 rpm for 5 min and the organic phase was aspirated into a microsyringe and injected into the HPLC system for direct analysis. 2.4. Sample preparation
3. Results and discussion 3.1. Optimization of derivatization The chemical structure of DMQC-OSu and its reaction with BAs are shown in Fig. 2. In order to achieve the maximum derivatization yield, the derivatization conditions including the concentration of DMQC-OSu, pH of the buffer, the volume of buffer, the reaction temperature and the reaction time were carefully optimized. The effect of DMQC-OSu concentration on the derivatization yield was evaluated from 0.1 to 1.5 mmol L−1 . The subsequent HPLC analysis showed that the peak areas of derivatives were maximum and stable when the concentration of DMQC-OSu was 0.8 mmol L−1 . Then this concentration was used in the following experiments. H3 BO3 –Na2 B4 O7 buffers at pH range of 7.0–9.0 were tested for the derivatization. The maximum and constant peak areas of the derivatives were obtained at pH 8.0. Therefore, pH 8.0 was selected. The influence of the buffer amount was also investigated. It can be found that the peak areas of the derivatives reached the maximum at 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0). Thus, 200 L of H3 BO3 –Na2 B4 O7 buffer (pH 8.0) was selected in the derivatization reaction.
Fig. 2. The reactions of DMQC-OSu with amine.
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Fig. 3. Effect of type of extraction solvent. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , ultrasonic agitation time for 3 min and 5000 rpm centrifuging for 5.0 min. (1) Octopamine; (2) tyramine; and (3) phenethylamine.
The effect of the temperature on derivatization yield was also studied in the range of 4–50 ◦ C. The results showed that the yield of derivatives reached the maximum at 20 ◦ C. Then 20 ◦ C was selected as the optimum temperature value for the derivatization. With respect to the reaction time, optimization was performed in the range of 5–60 min. The results showed that derivatization yield reached the largest and invariable when the reaction time was 40 min. Therefore, 40 min was selected in this work. 3.2. Optimization of ultrasound-assisted dispersive liquid–liquid microextraction In order to obtain the maximal extraction efficiency, parameters that may influence the enrichment performance should be investigated in detail. A series of experiments was designed for this goal. 3.2.1. Type of extraction solvent Choosing the most suitable extraction solvent was great important for ultrasound-assisted dispersive liquid–liquid microextraction (UDLLME). Efficiencies of UDLLME of DMQC-OSu derivatives with carbon tetrachloride, chloroform, hexane, chlorobenzene, 1octanol and 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6 ]) have been evaluated. The relative peak areas are shown in Fig. 3. Chloroform and 1-octanol had higher extraction efficiency than others, and they almost achieve the same recoveries. Though 1-octanol had lower density than water, good chromatographic behavior and good extraction properties for the target compound made it to be the best extraction solvent. Thereby, 1octanol was selected in this work. 3.2.2. Effect of the volume of extraction solvent During UDLLME process, extraction solvent volume was an essential factor which can influence the occurrence of the cloudy state and also determined the enrichment performance. To examine the effect of extraction solvent volume, different volumes of 1-octanol (0, 25, 50, 60, 75, 100, and 150 L) were subjected to the same UDLLME procedures. The results are shown in Fig. 4. As can be seen, peak areas of derivatives increased with the increase of 1-octanol volume in the range of 0–50 L, and then decreased when the volume was continuously increased. Therefore, 50 L of 1-octanol was selected in order to achieve higher enrichment factor, better repeatability and lower limit of detection (LOD).
Fig. 4. Effect of 1-octanol volume. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , ultrasonic agitation time for 3 min and 5000 rpm centrifuging for 5.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
3.2.3. Effect of sonication time Enough time will make the extraction solvent well dispersed into the aqueous solution and resulted in the excellent enrichment. The effect of ultrasonic agitation time was evaluated in the range of 0–10 min and the results showed that the peak areas of the derivatives increased in the first 1 min and then decreased slowly. Hence, 1 min was enough for the dispersive procedure (Fig. 5). 3.2.4. Effect of centrifuging time In UDLLME, ultrasonic agitation makes the extractant completely disperse in aqueous phase and form vast organic vesicles to achieve efficient extraction. Centrifugation was substantial in order to obtain two distinguishable phases in the extraction tubes. The effect of centrifuging time on the extraction efficiency has been evaluated. In Fig. 6, it was found that the extraction performance all reached the best when the solution was centrifuged at 5000 rpm for 5 min. When the centrifugation time was longer or shorter than 5 min, the peak areas decreased, So 5 min was chosen in the following study. The chromatograms obtained by UDLLME–HPLC and direct HPLC analysis under the optimized conditions are shown in Fig. 7. Comparing with the chromatograms obtained by UDLLME–HPLC (Fig. 7A) to that of the direct injection (Fig. 7B), a dramatic peak
Fig. 5. Effect of ultrasonic agitation time on the peak area. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , 1octanol volume for 50 L, 5000 rpm centrifuging for 5.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
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enhancement was presented. This exhibited the remarkable preconcentration ability of the UDLLME. 3.3. Analytical performance of the proposed method
Fig. 6. Effect of time of centrifuging at 5000 rpm on the peak area. Conditions: Coctopamine = 100 g mL−1 , Ctyramine = 100 g mL−1 , Cphenethylamine = 1 g mL−1 , 1-octanol volume for 50 L, ultrasonic agitation time for 1.0 min. () octopamine; (䊉) tyramine; and () phenethylamine.
Under the above-mentioned optimal conditions, a series of experiments were designed for obtaining linear calibration ranges, regression equations, detection limits and other characteristics of the method. The detection limits of biogenic amines were calculated as the amounts of these latter that resulted in a peak area three times larger than that of the baseline noise. The results are listed in Table 1. All the analytes showed good linearity with correlation coefficient (r) ranging from 0.9972 to 0.9998. The precisions were also important for the proposed method. The intra-day precisions ranged from 2.4 to 3.2% and the inter-day precisions ranged from 3.2 to 5.5%. The limits of detection (LODs) were in the range of 5–0.02 ng mL−1 . These excellent results indicated that the present approach was a simple and sensitive procedure to determine biogenic amines at trace level. Comparison of the different methods with other derivatization reagents for the determination of biogenic amines is summarized in Table 2. The proposed method in this work showed high sensitivity and was expected as the most used analysis method for detecting the low concentration of biogenic amines. 3.4. Rice wine samples analysis
Fig. 7. Chromatograms for BAs derivatives obtained by UDLLME (A) and direct HPLC analysis (B). Peaks: (a) octopamine, (b) tyramine, and (c) phenethylamine. UDLLME conditions outlined in Section 2 and HPLC conditions: mobile phase, methanol/water (60/40, v/v); flow rate, 1 mL min−1 ; detection wavelength fluorescence (326/412 nm).
Under the optimized experimental conditions, the proposed method was applied for the determination of biogenic amines in three rice wine samples. The results are shown in Table 3. Octopamine, tyramine and phenethylamine were all found in the samples. Tyramine was the most abundant amine in these samples while phenethylamine was the least. The samples were spiked with three biogenic amines at three levels to investigate the effect of sample matrices (Table 3). The recoveries of the three amines ranged from 95.42 to 104.56%. The typical chromatograms of blank sample and spiked with amines were illustrated in Fig. 8. The results showed the method enabled the precise and sensitive determination of standards and can be applied to detect these biogenic amines in real samples.
Table 1 The performance characteristics of the proposed method. DMQC-amine derivatives
Octopamine Tyramine Phenethylamine
Calibration range (g mL−1 )
Regression equation
5–500 5–500 0.025–2.5
Y = 0.9941X + 83.9888 Y = 1.6204X + 11.1648 Y = 0.2951X + 41.3648
0.9972 0.9998 0.9972
RSD
Detection limit (ng mL−1 )
Intra-day (n = 5)
Inter-day (n = 5)
2.6 3.2 2.4
3.2 4.0 5.5
5 5 0.02
Table 2 Experimental presentation of various methods for biogenic amines. Reagents
Mode
Reaction conditions
LODs
Reference
DnsCl HFBA FBQCA DTAF CNBF DnsCl SFP OPA AQC DMQC-OSu
TLC Ion-pair extraction GC–MS CE–LIF CE–LIF HPLC–UV HPLC–DAD HPLC–MS SPE–HPLC-FL SPE–HPLC-FL UDLLME–HPLC-FL
NaHCO3 , 40 ◦ C, 1 h 80 ◦ C, 60 min Borate buffer, pH 9.6, 50 ◦ C, 20 min 50 mM boric acid, 0.5 min H3 BO3 –Na2 B4 O7 buffer (pH 9.5), 60 ◦ C, 30 min NaOH, saturated NaHCO3 , 40 ◦ C, 0.5 h Borate buffer, pH 9, 60 min Na2 B4 O7 buffer, pH 10.5 Na2 B4 O7 buffer, pH 8.8 Borate buffer, pH 8.0, 20 ◦ C, 40 min
500–105 g L−1 5–30 g L−1 0.4–12 nmol L−1 17–43 g L−1 0.056–0.87 mol L−1 0.05–0.25 mg L−1 1.2–19.0 mg kg−1 0.07–0.4 mg L−1 15–50 g L−1 0.02–5 g L−1
[12] [13] [17] [15] [21] [22] [8] [26] [27] This work
DnsCl = dansyl chloride; TFAA = trifluoroacetylacetone; FBQCA = 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde; DTAF = 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein; CNBF = 4-chloro-3,5-dinitrobenzotrifluoride; SFP = succinimidylferrocenyl propionate; OPA = o-phthaldialdehyde; AQC = 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate.
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Table 3 Analytical results for the three biogenic amines in rice wine samples. Samples
Biogenic amines
Octopamine
Rice wine 1
Tyramine
Phenethylamine
Octopamine
Rice wine 2
Tyramine
Phenethylamine
Octopamine
Rice wine 3
Tyramine
Phenethylamine
Added (g mL−1 )
Found (g mL−1 )
Recovered (g mL−1 )
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.0
128.30 133.16 179.51 321.60 15.30 20.22 64.04 313.7 0.160 0.186 0.668 2.145
128.30 4.86 51.21 193.30 15.30 4.92 48.74 298.4 0.160 0.026 0.508 1.985
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.000
126.80 131.57 178.59 322.30 15.80 20.73 64.54 323.40 0.150 0.175 0.637 2.232
126.80 4.77 51.79 195.50 15.80 4.93 48.74 307.56 0.150 0.025 0.487 2.082
0 5.0 50.0 200.0 0 5.0 50.0 300.0 0 0.025 0.5 2.000
127.50 132.46 179.78 324.80 16.00 20.87 67.11 313.45 0.154 0.178 0.647 2.201
127.50 4.96 52.28 197.30 16.00 4.87 51.11 297.45 0.154 0.024 0.493 2.047
Recovery (%)
97.21 102.41 96.65 98.42 97.48 99.46 103.25 101.56 99.24 95.42 103.58 97.75 98.63 97.48 102.52 98.62 97.36 104.12 99.12 104.56 98.65 97.47 102.21 99.15 97.45 98.65 102.36
RSD (%) (n = 6) 2.01 3.25 2.68 3.48 3.14 4.56 5.23 8.72 5.35 4.32 4.68 8.51 2.32 4.56 3.65 2.85 3.42 5.62 4.21 5.63 4.52 3.25 4.56 6.45 3.62 4.52 2.85 3.15 2.83 3.69 5.02 4.32 5.12 6.23 3.58 4.82
real samples (95.42–104.56%) showed that the method was sufficiently applicable to determine biogenic amines in real samples. Therefore, it had the potential of practical applications and could be either a complementary or a parallel method for the biogenic amines determination. Acknowledgments
Fig. 8. Typical chromatograms of blank sample (A) and sample spiked with biogenic amines (B), respectively. Peaks: (a) octopamine, (b) tyramine, and (c) phenethylamine. Rice wine samples (B): spiked with octopamine 20 g mL−1 , tyramine 50 g mL−1 , phenethylamine 0.1 g mL−1 . Other conditions were the same as depicted in Fig. 6.
4. Conclusions The proposed method outlined the successful development and application of ultrasound-assisted dispersive liquid–liquid microextraction combining with HPLC-FL for the analysis of biogenic amines. The proposed method had many practical advantages, including simplicity of the extraction method, using of a small volume of organic solvent for extraction, high sensitivity, low cost and rapidity. The LODs of biogenic amines were in the range of 5–0.02 ng mL−1 , which revealed the proposed method had high sensitivity. The excellent spiked recoveries of biogenic amines in
This work was supported by the National Natural Science Foundation of China (20805040, 20875080), Natural Science Foundation of He’nan Province of China (082300420140), Foundation of He’nan Educational Committee (2008A150020), Excellent Youth Foundation of He’nan Scientific Committee (084100410024) and sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province (2010HASTIT025). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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