Microchim Acta 154, 281–286 (2006) DOI 10.1007/s00604-006-0566-1
Original Paper Derivatization of Hydrophobic Amino Acids in Nonaqueous Media and Separation by Nonaqueous Capillary Electrophoresis with Laser-Induced Fluorescence Detection Yuming Dong1;2 , Xingguo Chen1; , and Zhide Hu1 1 2
Department of Chemistry, Lanzhou University, Lanzhou 730 000, P.R. China Department of Pharmacy, Lanzhou University, Lanzhou 730 000, P.R. China
Received September 20, 2005; accepted January 19, 2006; published online April 18, 2006 # Springer-Verlag 2006
Abstract. A method of nonaqueous capillary electrophoresis was established for the separation and determination of alanine, phenylalanine, isoleucine and valine after derivatization with 4-chloro-7-nitrobenzo2-oxa-1, 3-diazol in nonaqueous media. The derivatization and separation conditions were optimized. The optimum derivatization conditions were 70 min for the reaction time, 55 C for the reaction temperature and 20 mM ammonium acetate in methanol for the derivatization buffer. The most suitable running buffer was composed of 60 mM ammonium acetate, 10% acetonitrile in methanol with a fused-silica capillary column (47 cm75 mm i.d.), 20 kV applied voltage and 20 C capillary temperature. The correlation coefficients were better than 0.995 in the investigated concentration ranges. The relative standard deviation (R.S.D.) (n ¼ 5) of the migration times and peak areas were 2.0–4.3% and 1.9–4.5%, respectively. The method was applied to the determination of four compounds in two compound amino acid injections, with the exception of phenylalanine, the recoveries for the other three compounds ranging between 88–116%. Key words: Nonaqueous capillary electrophoresis; laser-induced fluorescence; alanine; isoleucine; phenylalanine; valine. Author for correspondence. E-mail:
[email protected] or
[email protected]
Amino acids, the basic units of proteins, play an important role in the metabolic processes of a living organism [1]. Alanine (Ala), phenylalanine (Phe), isoleucine (Ile) and valine (Val) are hydrophobic and always found in the interior of proteins [2]. The detection of native amino acids is difficult because most of them do not possess strong spectrophotometric or fluorogenic properties. Although conductivity detection [3], electrochemical detection [4], refractometric detection [5] and indirect UV [6] have been used for the determination of amino acids by capillary electrophoresis (CE) without derivatization, chemical derivatization in aqueous media is still a frequently used method for the detection of amino acids [7]. Prior to their derivatization, the analytes and derivatization reagents should be completely dissolved in a suitable solvent. Hydrophobic amino acids are more easily dissolved in nonaqueous than in aqueous solvent. Therefore, the efficiency of derivatization for hydrophobic amino acids was poor when they were derivatizatized in aqueous solvent. On the other hand, the derivatives of analytes in aqueous solution can not be directly analyzed by nonaqueous capillary electrophoresis. The main reason for this is that the derivatives in water could cause a current breakdown when nonaqueous capillary electrophoresis (NACE) was used. NACE was the most suitable method to analyze the derivatives in
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nonaqueous solvent. Laser-induced fluorescence (LIF) detection has been used in many modes of CE. However, the applications of LIF in NACE are still very few. Derivatization in nonaqueous media can extend the application area of LIF detection in NACE. Common methods for the determination of physiological amino acids, such as classic ion-exchange chromatography [8, 9] and reversed-phase high performance liquid chromatography [10–13], are characteristically expensive. Capillary electrophoresis, using aqueous buffers as background electrolytes, is now a widely employed separation technique which affords a high separation speed and a high separation efficiency, utilizes relatively inexpensive and long lasting capillary columns, and consumes small volumes of sample and reagents [14]. Recently, NACE has become an active area of study. Compared with the system of water, the different chemical and physical properties of solvents (viscosity, dielectric constant, polarity, auto-protolysis constant, conductivity, etc.) have shown several advantages in terms of selectivity, efficiency, rapidity, mass spectrum compatibility and analyte solubility and stability [15]. NACE has been successfully applied to the analysis of a large number of pharmaceuticals, including acidic and basic drugs, chiral compounds, peptides, ions and preservatives [16, 17]. LIF is a very sensitive detection method for CE that has become widely applicable due to the availability of a large variety of fluorescent tags and lasers [18]. Free amino acids in human plasma have been separated by CE with LIF [19]. Micellar capillary zone electrophoresis [20] and capillary zone electrophoresis [21] with LIF have been used for the separation of amino acids. Continuous on-line derivatization and determination of amino acids using a microfluidic capillary electrophoresis system with a continuous sample introduction interface [22] and microemulsion electrokinetic chromatography with LIF for amino acids [23] was performed in our laboratory. With these methods, derivatization was carried out in aqueous media. Because the four hydrophobic amino acids have relatively higher derivatization efficiency and the other amino acids can almost not be derivatized in nonaqueous media, a method for the separation and determination of the analytes based on derivatization in nonaqueous media by NACE should have relatively high selectivity. However, there have been no publications on the separation of hydrophobic amino acids by nonaqueous capillary electrophoresis with
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LIF (NACE-LIF) after derivatization in nonaqueous media. In this study, a NACE-LIF method was developed for the separation and determination of Ala, Phe, Ile and Val derivatizatized with 4-chloro-7-nitrobenzo-2oxa-1, 3-diazol (NBD-Cl) in methanol containing ammonium acetate. Experimental Instruments All separations were performed on a P=ACE 5510 system (Beckman Coulter, Fullerton, CA, USA, www.beckmam.com) equipped with a LIF detector. The excitation light from an argon ion laser (3 mW) was focused on the capillary window by means of a fiber-optic connection. The excitation was performed at 488 nm, and a 520 nm band-pass filter was used for emission. The system was controlled by P=ACETM station software. The separation was carried out on a 47 cm (40 cm to the detector) 75 mm ID fused-silica capillary (Yongnian Photoconductive Fiber Factory, Handan, Hebei, China). The capillary was treated prior to its first use by flushing with methanol for 5 min, with distilled water for 2 min, 1.0 M HCl for 5 min, distilled water for 2 min, 0.1 M NaOH for 15 min, distilled water for 5 min and run buffer for 15 min, respectively. Between two runs, a rinse cycle, 0.1 M NaOH, distilled water and run buffer for 2 min, respectively, were used. The capillary was maintained at 20 C. The sample was injected by applying a pressure of 0.5 psi for 2 s. To avoid buffer evaporation, the buffer reservoirs must have lids.
Materials Standard of Ala, Ile, Phe and Val were obtained from the National Institute for the Control of Pharmaceuticals and Bioproducts of China (Beijing, China, www.nicpbp.org.cn). NBD-Cl was from Acros Organics Company (Geel, Belgium, www.acros.com). Ammonium acetate and acetic acid were purchased from Tianjin First Chemical Factory (Tianjin, China, www.tj-cr.com). Acetonitrile was purchased from Tianjin Secondary Chemical Factory (Tianjin, China, TJ-chemreagent.com.cn). Methanol was purchased from Shanghai Zhenxing First Chemical Factory (Shanghai, China). Compound Amino Acid Injection (9 AA) and Compound Amino Acid Injection (18 AA) were obtained from a local pharmaceutical store (Lanzhou, China). All reagents were of analytical grade, unless otherwise specified. 600 mg L1 stock solutions of Ala, Ile, Phe and Val were prepared in distilled water, respectively, and a 27.8 mM stock solution of NBD-Cl was prepared in acetonitrile. All stock solutions were stored at 4 C and kept stable for several weeks.
Preparation of the Electrolytes The derivatization buffers were prepared from 2.5 mL of 0.2 M ammonium acetate (methanol medium) in a 25 mL flask and diluted to 25 mL with methanol. The run buffer was prepared from 7.5 mL of 0.2 M ammonium acetate (methanol medium) and 2.5 mL acetonitrile in a 25 mL flask and diluted to 25 mL with methanol. Prior to use, all solutions and run buffers were degassed by ultrasound for 10 min and filtered through a 0.45 mm membrane filter.
Derivatization of Hydrophobic Amino Acids in Nonaqueous Media and Separation by NACE Sample Preparation Compound Amino Acid Injection (9 AA) and Compound Amino Acid Injection (18 AA) were diluted 5-fold with distilled water, and filtered through a 0.45 mm membrane filter, respectively. The resulting solution was derivatized directly for analysis.
Derivatization Procedure In a 1.5 mL plastic tube, 50 mL standard solutions of Ala, Ile, Phe and Val with 200 mL NBD-Cl solutions were added to the derivatization buffer. The blank solutions were prepared by mixing derivatization buffer and 200 mL NBD-Cl. The mixtures were diluted to 1.0 mL with derivatization buffer and kept in a hot-water bath to react. Prior to analysis, the derivatization solutions were diluted with run buffer to the desired concentration.
Results and Discussion Derivatization of Ala, Ile, Phe and Val Derivatization of Ala, Ile, Phe and Val with NBDCl were investigated in methanol. The effects of derivatization conditions, which have a predominant effect on the fluorescence intensity, are highlighted below. Effect of Reaction Time and Temperature on the Derivatization The effect of reaction time on the fluorescence intensity was investigated in the range of 10–90 min at
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55 C. The results showed that the fluorescence intensity increased with increasing reaction time in the range of 10–70 min, but the fluorescence intensity decreased slightly when the reaction time was longer than 70 min. The effect of reaction temperatures on the fluorescence intensity was also investigated at 25, 35, 55 C. The electropherograms of the analytes at different temperatures are shown in Fig. 1. It was found that the fluorescence intensity of the four analytes at 25 C and 35 C was much smaller than that at 55 C. The results indicated that a higher reaction temperature provided higher derivatization efficiency. Considering the boiling point of methanol (64 C), 55 C was chosen as the optimum. Effect of Ammonium Acetate Concentration and Percentage of Acetonitrile on the Derivatization The effect of ammonium acetate concentrations were investigated in the range of 10–50 mM. The results indicated that the concentration of ammonium acetate has little effect on the derivatization of the four analytes. The fluorescence intensity of the analytes decreased slightly with the increase of ammonium acetate. Therefore, 20 mM was selected in subsequent experiments because it could provide the most appropriate ionic strength for the derivatization. The percent of acetonitrile (ACN) in the derivatization was investigated in the range of 0–20% (v=v). The results indicated that acetonitrile has hardly any effect on the fluorescence intensity of the analytes. So no acetonitrile was added to the derivatization solution. Separation of the Analytes
Fig. 1. Effect of reaction temperature on the fluorescence intensity. (1) 25 C, (2) 35 C, (3) 55 C. Derivatization conditions: 20 mM ammonium acetate (methanol medium), reaction time 70 min; separation conditions: 60 mM ammonium acetate (methanol medium), 10% acetonitrile; applied voltage: 20 kV; the capillary (75 mm ID) is 47 cm (40 cm to the detector), sample injection time 5 s with 0.5 psi pressure
To achieve good sensitivity and satisfactory separation, the optimization of the separation condition was of primary importance. An electrophoretic medium containing a mixture of solvents was found particularly advantageous to achieve high selectivity. The dielectric constant and the viscosity of the media are parameters that affect the electrophoretic and the electroosmotic mobilities. The change of the solvent properties has a significant influence on solute solvation and ion–ion interactions. In media with a low ratio of dielectric constant to viscosity, less current and therefore less Joule heating are
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generated, and consequently the zone broadening is minimized. Although the dielectric constant-to-viscosity ratio of methanol (60.6) is much lower than water (89.9) or ACN (110.3), the migration time, too, is longer using undiluted methanol as solvent. Therefore, good separation performances can be expected from mixing amphiprotic methanol and dipolar aprotic ACN.
Effect of Ammonium Acetate on the Separation The buffer concentration markedly affects the separation because it can influence the EOF, Joule heating, ionic strength and the viscosity of electrolytes, the adsorption of capillary wall for the analytes, and the current produced in the capillary. The effect of ammonium acetate on the separation of the four analytes was investigated in the range of 40–80 mM (Fig. 2). It can be seen that when increasing the concentrations of ammonium acetate, the migration times and resolution of the analytes as well as the current increase. Consid-
Fig. 2. Effect of buffer concentration on the separation. Derivatization conditions: 20 mM ammonium acetate (methanol medium), reaction time was 70 min at 55 C. Separation conditions: 60 mM ammonium acetate, 10% acetonitrile. (&) running current. Other conditions as in Fig. 1
ering the effect of Joule heating and band broadening phenomena caused by higher ionic strength, analysis time, resolution and sensitivity, 60 mM ammonium acetate was chosen as the optimum concentration.
Effect of ACN Concentration on the Separation In previous studies on the application of NACE to the analysis of pharmaceutical drugs it was demonstrated that the organic solvent composition has a critical effect on the resolution, efficiency, and migration time [25, 26]. Thus, an ACN percentage of 0 to 20% in buffer was examined. The effect of ACN concentration on migration time of the analytes is demonstrated in Fig. 3. As shown in Fig. 3, the migration times of the four analytes considerably decreased when the ACN percentage in buffer was increased. This behavior was mainly due to the modification of the dielectric constant-to-viscosity ratio. When the concentration of ACN was 10% (v=v), the fluorescence intensity of the four analytes was highest and the resolution was
Fig. 3. Effect of ACN concentration on the migration time of analytes. Buffer, 60 mM ammonium acetate, 0 to 20% (v=v) ACN. (!) migration time. Other conditions as in Fig. 1
Table 1. Results of regression analysis on calibration curves and detection limits Analyte
Regression equation Y ¼ a þ bca
Correlation coefficient
Linear range (mg L1 )
Detection limit (mg L1 )b
Val Ile Ala Phe
Y ¼ 88407 þ 26737.07c Y ¼ 391910 þ 14856.43c Y ¼ 47740 þ 9857.51c Y ¼ 70185 þ 9014.82c
0.9991 0.9987 0.9995 0.9954
75–800 60–660 70–730 110–870
0.8 2.7 4.9 5.5
a b
Y and c stand for the peak area and the concentration (mg L1 ) of the analytes, respectively. The detection limit was defined as the concentration where the signal-to-noise ratio is 3.
Derivatization of Hydrophobic Amino Acids in Nonaqueous Media and Separation by NACE
good. Considering the total analysis time and peak area, 10% (v=v) ACN was chosen as the optimum concentration.
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and the peak area were 2.0% and 4.5% for Val, 2.2% and 1.6% for Ile, 3.2% and 1.9% for Ala, 4.3% and 2.6% for Phe, respectively.
Effect of Applied Voltage and Capillary Temperature on the Separation The effect of applied voltage on the migration times of the four analytes was investigated in the range of 15–25 kV. With increasing applied voltage, the migration time was shortened and the theoretical plate number (N) increased markedly. However, a decrease of resolution was also apparent. The breakdown of electric current occurred sometimes during the experiments when the voltage was higher than 25 kV. Therefore, 20 kV was selected for a relatively good resolution of the four analytes with a shorter analysis time. The influence of the capillary temperature was also studied with an optimized electrophoretic medium between 15 C and 25 C. When the temperature was higher than 25 C, an electric current breakdown was sometimes observed. So 20 C was selected as the optimum. According to the above results, the optimum derivatization conditions are 20 mM ammonium acetate (methanol medium) and a reaction time of 70 min at 55 C. The optimum separation conditions were 60 mM ammonium acetate (methanol medium) with 10% ACN, a separation voltage of 20 kV and a capillary temperature of 20 C. Linearity, Reproducibility and Detection Limits The linear relationship between the concentration of the four analytes and the corresponding peak area was investigated under the optimum separation conditions. The results are shown in Table 1. The calibration curves exhibited a good linear relationship over the concentration range of 75–800 mg L1 for Val, 60–600 mg L1 for Ile, 70–730 mg L1 for Ala and 110–870 mg L1 for Phe, respectively. The limit of detection (LOD) was considered as the minimum analyte concentration yielding a signal-to-noise ratio equal to 3. The determined results are also given in Table 1. The precision and accuracy of the proposed method were determined by investigating the repeatability, which was evaluated by relative standard derivatization (R.S.D.). The regression equations and correlation coefficients are also given in Table 1. The R.S.D. (n ¼ 5) of the migration time
Fig. 4. Electropherograms of the standard solution and samples. (A) Standard solution; (B) Compound Amino Acid Injection (9 AA); (C) Compound Amino Acid Injection (18 AA). Derivatization and separation conditions as in Fig. 2. Other conditions as in Fig. 1
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Table 2. Determination results of the analytes in the samples (n ¼ 3) Sample
Compound
Contents (mg mL1 )
Labeled (mg mL1 )
Recovery (%)
Compound amino acid injection (18 AA)
Val Ile Ala Phe
3.8 3.9 1.8 5.5
3.6 3.5 2.0 5.3
114 110 88 74
Compound amino acid injection (9 AA)
Val Ile Phe
6.3 5.8 8.9
6.4 5.6 8.8
116 96 78
Application Two compound amino acid injections (9 AA and 18 AA) were analyzed under the optimum conditions. The peaks of the analytes were identified by comparing the migration times and spiking the standards to the sample solutions. The typical electropherograms for the separation of the standards and samples are illustrated in Fig. 4A–C, respectively. Each sample was measured by analyzing three times with the proposed method. The resulting contents of the four compounds in the sample are given in Table 2. The recovery of the method was determined with the standard addition for Val, Ile, Ala and Phe in the two sample solutions, respectively. The results are also shown in Table 2. As becomes clear from Table 2, fairly satisfactory results were obtained when the new method was applied to the analysis of the two real samples. This implies that the new method can be applied to the simultaneous determination of the four hydrophobic amino acids in complicated medical preparations. Conclusion A new method combining nonaqueous capillary electrophoresis with LIF detection has been developed for the separation and determination of Ala, Ile, Val and Phe after NBD-Cl derivatization in methanol. The results indicate that the method extends the application area of LIF in CE. The results also indicated that analytes that are insoluble in nonaqueous media can not be analyzed by the new method and that the interferences caused by other amino acids in the samples were reduced to minimum. However, the method’s sensitivity still needs improving and the derivatization time needs to be reduced. Also, the developed method using NBD-Cl derivatization is well suited for the commercial LIF instrument (excitation 488 nm= emission 520 nm).
Acknowledgements. The project was supported by the National Natural Science Foundation of China (No. 20275014).
References [1] Shen Z D, Sun Z M, Wu L, Wu K, Sun S Q, Huang Z B (2002) J Chromatogr A 979: 227 [2] Zhou A, Cha X (2002) Biochemistry, 5th edn., People’s Medical Publishing House, p 22 [3] Chan K C, Janini G M, Muschik G M, Issaq H J (1993) J Chromatogr A 653: 93 [4] Lee P L, Slocum R H (1988) Clin Chem 34: 719 [5] Coufal P, Zuska J, Van De Goor T, Smith V, Gas B (2003) Electrophoresis 24: 671 [6] Dong Q, Jin W, Shan J (2002) Electrophoresis 23: 559 [7] Ivano A R, Nazimov I V, Lobazov A P, Popkovich G B (2000) J Chromatogr A 894: 253 [8] Zunic G, Jelic-Ivanovic Z, Colic M, Spasic S (2002) J Chromatogr B 772: 19 [9] Goldsmith R F, Earl J W, Cunningham A M (1987) Clin Chem 33: 1736 [10] Oguri S, Uchida C, Mishina M (1996) J Chromatogr A 724: 69 [11] Woo K L, Lee S H (1994) J Chromatogr A 667: 105 [12] Haynes P A, Sheumack D, Kibby J, Redmond J W (1991) J Chromatogr 540: 177 [13] Dorresteijn R C, Berwald L G, Zomer G, de Gooijer C D, Wieten G, Beuvery E C (1996) J Chromatogr A 724: 159 [14] Zhang J Y, Xie J P, Chen X G, Hu Z D (2003) Analyst 128: 369 [15] Chen A J, Zhang J Y, Li C H, Chen X F, Hu Z D, Chen X G (2004) J Sep Sci 27: 568 [16] Suntornsuk L, Pipitharome O, Wilairat P (2003) J Pharm Biomed Anal 33: 441 [17] Riekkola M L (2002) Electrophoresis 23: 3865 [18] Ward V L, Khaledi M G (1998) J Chromatogr B 718: 15 [19] Boulat O, Mclaren D G, Arriaga E A, Chen D Y D (2001) J Chromatogr B 754: 217 [20] Zhao J Y, Chen D Y, Dovichi N J (1992) J Chromatogr 608: 117 [21] Ueda T, Mitchell R, Kitamura F, Metcalf T (1992) J Chromatogr 593: 265 [22] Fan L Y, Chen H L, Zhang J Y, Chen X G, Hu Z D (2004) Anal Chim Acta 501: 129 [23] Xie J P, Zhang J Y, Liu H X, Liu J Q, Tian J N, Chen X G, Hu Z D (2004) Biomed Chromatogr 18: 600 [24] Fillet M, Servais A, Crommen J (2003) Electrophoresis 24: 1499 [25] Cherkaoui S, Varesio E, Christen P, Veuthey J L (1998) Electrophoresis 19: 2900 [26] Li Y Q, Qi S D, Chen X G, Hu Z D (2004) Electrophoresis 25: 3003