30 Lijuan Zhang1 Biqi Lu1 Chao Lu1 Jin-Ming Lin2 1 State
Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, China 2 Department of Chemistry, Tsinghua University, Beijing, China
Received September 10, 2013 Revised October 28, 2013 Accepted October 29, 2013
J. Sep. Sci. 2014, 37, 30–36
Research Article
Determination of cysteine, homocysteine, cystine, and homocystine in biological fluids by HPLC using fluorosurfactant-capped gold nanoparticles as postcolumn colorimetric reagents We have demonstrated for the first time the suitability of fluorosurfactant-capped spherical gold nanoparticles as HPLC postcolumn colorimetric reagents for the direct assay of cysteine, homocysteine, cystine, and homocystine. The success of this work was based on the use of an on-line tris(2-carboxyethyl)phosphine reduction column for cystine and homocystine. Several parameters affecting the separation efficiency and the postcolumn colorimetric detection were thoroughly investigated. Under the optimized conditions, cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples were determined. Detection limits for cysteine, homocysteine, cystine, and homocystine ranged from 0.16– 0.49 M. The accuracy in terms of recoveries ranged between 94.0–102.1%. This proposed method was rapid, inexpensive, and simple. Keywords: Colorimetric assays / Cysteine-cystine / Homocysteine-homocystine / On-line reduction / Postcolumn detection DOI 10.1002/jssc.201300998
1 Introduction Cysteine, homocysteine, cystine, and homocystine occur widely in biological tissues and fluids [1–3]. The determination of cysteine, homocysteine, cystine, and homocystine has gained high interest within the biomedical community over recent years as the ratio of cysteine to cystine or homocysteine to homocystine serves as a sensitive indicator of oxidative stress, an important biomarker for a wide range of diseases [4, 5]. Consequently, it is vital for researchers in biochemistry and biomedicine to detect cysteine, homocysteine, cystine, and homocystine [6,7]. The quantification of cysteine, homocysteine, cystine, and homocystine is usually done sequentially. In brief, the determination of cysteine and homocysteine is commonly based on pre-/postcolumn derivatization with chromophores/fluorophores in conjunction with HPLC, and then the procedure is repeated on a second sample after cystine and homocystine are reduced to their respective thiol species using an off-line approach. The concentrations of cystine and homocystine are then indirectly calculated from the difference between the measurements obtained in the Correspondence: Professor Chao Lu, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15 East Road Beisanhuan, Box 79, Beijing 100029, China E-mail: [email protected] Fax: +86-10-64411957
Abbreviations: CL, chemiluminescence; FSN, fluorosurfactant; TCEP, tris(2-carboxyethyl)phosphine hydrochloride C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
two separate steps [8–10]. The sequential detection of thiols and disulfides is not only time consuming but also vulnerable to the autoxidation of thiols during the entire procedure. Only a few reports exist for the direct determination of thiols and disulfides, which are mostly electrochemical and MS detection procedures [11–15]. Although electrochemical detection offers some advantages over derivatization techniques in terms of procedural simplicity, its application is limited as a result of their high overpotentials for oxidation. MS is a well-established and routine analytical technique for the direct determination of thiols and disulfides [16]. In 2010, Kasicka et al. reviewed the wide applications of functionalized gold nanoparticles in separation sciences [17]. As a promising functionalizing reagent for gold nanoparticles, nonionic fluorosurfactant (FSN) has been used for capping spherical gold nanoparticles in aqueous solution to sense cysteine and homocysteine [18]. FSN-capped gold nanoparticles have received a great deal of attention in biosensors, including colorimetry [19–24], fluorescence [25, 26], resonance light scattering [27,28], and chemiluminescence (CL) [29–34]. However, to the best of our knowledge, FSN-capped spherical gold nanoparticles have not been utilized to detect disulfides, mainly due to the lack of a suitable on-line reduction column for disulfides. Therefore, it is still necessary to design new optical assays based on FSN-capped spherical gold nanoparticles for sensing disulfides owing to the advantages of FSN ligands over other ligands by their virtues of good biocompatibility and stability [35]. Colour Online: See the article online to view Fig. 1 in colour. www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 30–36
We recently used FSN-prepared triangular gold nanoparticles as the HPLC postcolumn specific CL reagents to detect thiols and disulfides directly by incorporating an online tris(2-carboxyethyl)phosphine (TCEP) reduction column for disulfides in a single chromatographic separation [36]. We reasoned that the combination of on-line reduction for disulfides and gold nanoparticles based HPLC postcolumn CL detection was a promising method for sensing thiols and disulfides in real samples. However, this strategy has three limitations: (i) like other previously reported CL-based postcolumn detection systems, an additional CL system (e.g. luminol/H2 O2 ) accompanied with complex procedures is required to generate the CL signals [37–39]; (ii) the synthesis of the uniform triangular gold nanoparticles as the HPLC postcolumn CL reagents requires cautious processing conditions [31]; (iii) the pH value of the HPLC mobile phase, the on-line reduction of disulfides and the luminol/H2 O2 CL reaction need to be prepared carefully to make them compatible. In this work, we set out to develop a simple method to simultaneously detect underivatized cysteine, homocysteine, cystine, and homocystine by using an on-line TCEP column, FSN-capped spherical gold nanoparticles as postcolumn colorimetric reagents, and HPLC as the separation technique. The new method presented here is simple, highly specific, and convenient. Furthermore, the practicality of this method is further demonstrated through the detection of cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples.
2 Materials and methods 2.1 Reagents and materials All reagents were of analytical grade and used without further purification. All solutions were prepared with deionized water (Milli Q, Millipore, Barnstead, CA, USA). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 ·3H2 O, 99.99%) and trisodium citrate (99%) were purchased from Acros (Geel, Belgium). Zonyl FSN-100 (F(CF2 CF2 )1–7 CH2 CH2 O(CH2 CH2 O)0–15 H), cysteine (≥99%), homocysteine (>90%), cystine (>99%), homocystine (>99%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl, ≥98%), and ethylenediaminetetraacetic acid (>99%) were purchased from Sigma-Aldrich (St. Louis, USA). Methanol, HClO4 (70–72%), ZnO, NH3 . H2 O NaOH, HCl, and trifluoroacetic acid ( ≥99.5%) were purchased from Beijing Chemical Reagent Company (Beijing, China). The pH of phosphate buffer solution was adjusted with NaOH or HCl. 10 mM stock solutions of cysteine, homocysteine and their disulfides were freshly prepared by dissolving an appropriate amount of thiols in deionized water or disulfides in acidic solution, respectively, and stored at 4⬚C until further use. Their corresponding working solutions were freshly diluted with deionized water. The HPLC mobile phase containing 0.05% m/v TFA was fresh daily prepared, filtered through 0.22 m membranes, and then degassed prior to use. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.2 Apparatus The HPLC system consisted of a 1525 Binary HPLC pump (Waters Corporation, USA), a Shimadzu LC-10AT pump (Shimadzu, Japan), a 2707 Auto Sampler (Waters Corporation, USA), a 2489 UV/Vis detector set at = 650 nm (Waters Corporation, USA), a SunFire C18 guard column (4.6 × 20 mm, 5 m particle size), a SunFire C18 analytical column (4.6 × 150 mm, 5 m particle size), and a Zn(II)-TCEP reduction column (3.0 cm in length and 5.0 mm id). Isocratic elution of a mobile phase containing 0.05% m/v TFA was performed with a 1525 Binary HPLC pump. The postcolumn reaction module included a Shimadzu LC-10AT pump (Shimadzu, Japan) to deliver the FSN-capped gold colloidal solution and a reaction coil (30 cm length, 0.5 mm id) immersed in a thermostatic water bath at 70⬚C.
2.3 Synthesis of gold nanoparticles All glassware for the preparation of gold nanoparticles was thoroughly washed with freshly prepared aqua regia (HNO3 /HCl = 1:3), rinsed extensively with deionized water, and then dried in an oven at 100⬚C for 2–3 h. Fourteen nanometer gold nanoparticles were prepared according to the literature [18]. Briefly, a 50 mL solution of 0.04% m/v trisodium citrate was brought to a vigorous boil with stirring in a round-bottomed flask fitted with a reflux condenser, and then 85 L of 5% m/v HAuCl4 was added to the above solution. The solution was maintained at boiling point with continuous stirring for 15 min and then was cooled to room temperature with continued stirring. Then 400 L of 5% FSN m/v was added. The suspension was stored at 4⬚C until further use.
2.4 Homemade on-line reduction column for disulfides A homemade on-line reduction column for disulfides was prepared as described in our previous paper [36]. First, Zn(ClO4 )2 ·6H2 O was prepared by the reaction of ZnO with HClO4 , recrystallizing the product from water, and drying the crystals in vacuum. Second, Zn(II)-TCEP complexes were synthesized by dissolving TCEP·HCl (0.2 mmol) and Zn(ClO4 )2 ·6H2 O (0.2 mmol) in 5.0 mL of H2 O/methanol (1:3), adding 1.5 mmol of NH3 aq, and leaving overnight at room temperature. The obtained white crystals of Zn(II)TCEP were washed with H2 O/MeOH (1:3), and then with deionized water. Finally, a stainless-steel column (3.0 cm in length and 5.0 mm id) was thoroughly washed with deionized water, and dried in an oven at 100⬚C for 2–3 h. Then 2.0 g of crystals of Zn(II)-TCEP were packed into the stainless-steel column through a rubber funnel. Both ends of the reduction column were furnished with glass wool in order to prevent the crystals from washing away. Finally, the as-prepared reduction column was washed with deionized water for 30 min. www.jss-journal.com
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Figure 1. Schematic representation of the on-line reduction–HPLC–colorimetric detection system. P1 : 1525 Binary HPLC pump; P2 : Shimadzu LC-10 AT pump; G: guard column; A: analytical column; R: reduction column; the injection volume was 20 L; the temperature of heater was set at 70⬚C; the length of the reaction coil was 30 cm; W: waste; the mobile phase of 0.05% m/v TFA was pumped by P1 at 0.6 mL/min; the 2.9 nM FSN-capped gold nanoparticles were pumped by P2 at 0.3 mL/min.
2.5 Detection procedures
3 Results and discussion
A schematic diagram of the HPLC separation, on-line reduction, and postcolumn colorimetric detection system is illustrated in Fig. 1. To summarize, the standard mixtures of the four amino acids or real samples were mixed with 0.05% m/v TFA by an automatic sampler. The flow rate of 0.05% m/v TFA was 0.6 mL/min. Then the four analytes were separated on a SunFire C18 RP column before cystine and homocystine were reduced by a homemade on-line reduction column. The separated solution was mixed with 2.9 nM FSN-capped gold nanoparticles containing 100 mM phosphate buffer solution, which could keep the suitable ionic strength and pH value (pH 6.0) for the interaction between the gold nanoparticles and aminothiols. The FSN-capped gold nanoparticles were pumped by using a Shimadzu LC-10 AT pump at 0.3 mL/min. Subsequently, the mixture was heated in a reaction coil to enhance the aggregation rate by immersing in a 70⬚C water bath. The UV/Vis absorbance intensity was monitored at 650 nm.
3.1 Principle of direct detection for cysteine, homocysteine, cystine, and homocystine
2.6 Pretreatment of real samples
3.2 Experimental parameters
Human urine and plasma samples were collected from three healthy volunteers, and the analysis was conducted immediately after the sample collection. In order to prevent the autoxidation of thiols by complexing potentially catalytic metal cations and acidifying the solution, 97 L of 1 mM ethylenediaminetetraacetic acid solution and 3 L of formic acid were added to 900 L of urine/plasma samples. Next, 20 L of 3.0 mM perchloric acid was added into a 100 L of the above solution with gentle vortex-mixing in a centrifuge tube, put aside at room temperature for 10 min, and then centrifuged at 13 000 rpm for 10 min. The clear supernatant was filtered through a 0.22 m filter and diluted before HPLC analysis. The standard addition method was carried out by spiking different concentrations of thiols and disulfides standard solutions to urine or plasma samples.
The flow rate of the mobile phase is crucial for HPLC systems. The strongest absorbance of gold nanoparticles was obtained at 650 nm and the four amino acids could be separated completely when the flow rate of TFA was 0.6 mL/min (Fig. 2A). When the flow rate of TFA was >0.6 mL/min, the absorbance of the gold colloidal solution decreased obviously as a result of inadequate aggregation of the FSN-capped gold nanoparticles induced by aminothiols. However, the lower flow rates of TFA led to chromatographic peak broadening. Therefore, 0.6 mL/min was selected as the optimal flow rate. The flow rate of the postcolumn gold nanoparticles was also optimized in the range of 0.2–0.8 mL/min (Fig. 2B). When the flow rate of gold nanoparticles was >0.3 mL/min, the absorbance of gold nanoparticles at 650 nm was decreased seriously due to the incomplete aggregation of the gold
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In this study, we fabricated one TCEP reduction column for cystine and homocystine using commercially available TCEP as an effective reducing agent according to the following two steps. The phosphorus atom of TCEP attacks the sulfur along the S–S bond axis to form a thiophosphonium salt. Subsequently, a rapid hydrolysis of the thiophosphonium salt releases the second thiol fragment and the phosphine oxide [36]. The reduction products of cystine and homocystine, that is, cysteine and homocysteine, can exchange the FSN ligands on the surface of the gold nanoparticles, resulting in the rapid aggregation of gold colloidal solution under highly saline conditions, along with the color change from wine red to blue. Note that 650 nm was selected in the absorption spectra of the gold nanoparticles in the presence of cysteine as the optimum absorbance wavelength in this study due to high detection sensitivity for cysteine and homocysteine.
www.jss-journal.com
J. Sep. Sci. 2014, 37, 30–36
Figure 2. Effects of the reaction conditions on on-line reductionHPLC-colorimetric detection in presence and absence of cysteine: (A) flow rate of TFA: the flow rate of the FSN-capped gold nanoparticles was 0.3 mL/min; the reaction temperature was 70⬚C; the length of the reaction coil was 30 cm; (B) flow rate of the FSNcapped gold nanoparticles: the flow rate of TFA solution was 0.6 mL/min; the reaction temperature was 70⬚C; the length of the reaction coil was 30 cm; (C) the length of the reaction coil: the flow rates of TFA and the FSN-capped gold nanoparticles were 0.6 and 0.3 mL/min, respectively; the reaction temperature was 70⬚; (D) the flow rates of TFA and FSN-capped gold nanoparticles were 0.6 and 0.3 mL/min, respectively; the length of the reaction coil was 30 cm.
nanoparticles. Accordingly, the optimal flow rate of gold nanoparticles was selected as 0.3 mL/min. Note that the flow rates of TFA and postcolumn gold nanoparticles were lower than those in the earlier published papers dealing with determination of the aminothiols in biological fluids [18, 36]. The effect of postcolumn colorimetric reaction coil length was optimized in the range of 30–100 cm. The absorbance of gold nanoparticles at 650 nm gradually decreased with an increase in the length of reaction coil from 50 cm due to the broadening and tailing of chromatographic peaks. The base line signal increased with an increase of the reaction coil length in the range of 30–100 cm as a result of the slight self-aggregation of gold nanoparticles (Fig. 2C). Considering the effect of the base line and the absorbance, a reaction coil of 30 cm was chosen throughout this study. The aggregation kinetics of FSN-capped gold nanoparticles induced by aminothiols was highly dependent on the incubation temperature. Herein, we optimized the incubation temperature in the range of 40–80⬚C (Fig. 2D). When the incubation temperature was 70⬚C, the base line was increased dramatically, which was ascribed to the serious self-aggregation of gold nanoparticles. These results indicated that FSN-capped gold nanoparticles were unstable at higher temperatures. Therefore, the suitable temperature for this system was 70⬚C because of the rapid reaction of the gold nanoparticles with aminothiols at this temperature [18]. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. Typical HPLC chromatograms of standard solution containing cystine, cysteine, homocysteine, and homocystine (A) without or (B) with Zn(II)-TCEP reduction column. (1) cystine; (2) cysteine; (3) homocysteine; (4) homocystine. The retention times were 2.45, 3.08, 4.03, and 5.25 min for cystine, cysteine, homocysteine, and homocystine, respectively. The concentrations of cysteine and homocysteine were 10 M while the concentrations of cystine and homocystine were 5 M. Experimental conditions: the mobile phase was 0.05% m/v TFA at 0.6 mL/min. The flow rate of 2.9 nM FSN-capped gold nanoparticles was 0.3 mL/min. The injection volume was 20 L. The reaction temperature was 70⬚C. The length of the reaction coil was 30 cm.
3.3 Zn(II)-TCEP reduction column for disulfides In our previous study [18], aminothiols, such as cysteine and homocysteine, could induce the aggregation of the FSN-capped spherical gold nanoparticles as a result of the ligand exchange between FSN molecules and cysteine/homocysteine. However, the FSN-capped spherical gold nanoparticles did not respond to their disulfides because these larger molecules might induce the steric effect. In this study, we utilized an on-line Zn(II)-TCEP column for the reduction of disulfides prior to their reaction with the FSNcapped gold nanoparticles. Note that the amount of Zn(II)TCEP was in excess in order to ensure complete reduction. In Fig. 3, the typical chromatograms of cysteine, homocysteine, cystine, and homocystine before and after on-line reduction are shown. The results revealed that cystine and homocystine could be detected in the presence of the on-line reduction column. In addition, the chromatographic peaks of 5 M cystine and homocystine were of the same high as those of the 10 M cysteine and homocysteine after the complete on-line reduction.
3.4 Method performance data The proposed method was validated by evaluating the linearity, recovery, and LODs. Figures 3–5 show that the complete separation of cysteine, homocysteine, cystine, and homocystine was obtained for the standard solutions, blank and spiked samples. Furthermore, the typical chromatograms of human urine and plasma samples are shown along with the samples spiked with standard aminothiols. It was obvious that cystine, cysteine, homocysteine, and homocystine in human urine and plasma samples were well separated by using the proposed system and their retention time was close to that www.jss-journal.com
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Figure 4. HPLC chromatograms of (A) a human urine sample and (B) a human urine sample spiked with 20 M homocysteine and 20 M homocystine. The blank or spiked urine samples solution were diluted five times with deionized water prior to HPLC injection.
Figure 5. HPLC chromatograms of (A) a human plasma sample and (B) a human plasma sample spiked with 20 M homocysteine and 20 M homocystine. The blank or spiked plasma samples solution were diluted five times with deionized water prior to HPLC injection.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2014, 37, 30–36
of the standard aminothiols. These results demonstrated that the present method would be fully validated and accurate for the determination of cysteine, homocysteine, cystine, and homocystine in real samples. The calibration curves of cysteine, homocysteine, cystine, and homocystine at six concentration levels showed good linearity in their respectively reported range as given in Table 1. The regression equation and correlation coefficient (R2 ) calculated from the calibration curves of standard solutions are shown in Table 1. Moreover, we used the F-test method to evaluate the fit goodness of the formulas [40]. The obtained P values by F-test for the calibration curves of cysteine, cysteine, homocysteine, homocystine were
Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, China 2 Department of Chemistry, Tsinghua University, Beijing, China
Received September 10, 2013 Revised October 28, 2013 Accepted October 29, 2013
J. Sep. Sci. 2014, 37, 30–36
Research Article
Determination of cysteine, homocysteine, cystine, and homocystine in biological fluids by HPLC using fluorosurfactant-capped gold nanoparticles as postcolumn colorimetric reagents We have demonstrated for the first time the suitability of fluorosurfactant-capped spherical gold nanoparticles as HPLC postcolumn colorimetric reagents for the direct assay of cysteine, homocysteine, cystine, and homocystine. The success of this work was based on the use of an on-line tris(2-carboxyethyl)phosphine reduction column for cystine and homocystine. Several parameters affecting the separation efficiency and the postcolumn colorimetric detection were thoroughly investigated. Under the optimized conditions, cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples were determined. Detection limits for cysteine, homocysteine, cystine, and homocystine ranged from 0.16– 0.49 M. The accuracy in terms of recoveries ranged between 94.0–102.1%. This proposed method was rapid, inexpensive, and simple. Keywords: Colorimetric assays / Cysteine-cystine / Homocysteine-homocystine / On-line reduction / Postcolumn detection DOI 10.1002/jssc.201300998
1 Introduction Cysteine, homocysteine, cystine, and homocystine occur widely in biological tissues and fluids [1–3]. The determination of cysteine, homocysteine, cystine, and homocystine has gained high interest within the biomedical community over recent years as the ratio of cysteine to cystine or homocysteine to homocystine serves as a sensitive indicator of oxidative stress, an important biomarker for a wide range of diseases [4, 5]. Consequently, it is vital for researchers in biochemistry and biomedicine to detect cysteine, homocysteine, cystine, and homocystine [6,7]. The quantification of cysteine, homocysteine, cystine, and homocystine is usually done sequentially. In brief, the determination of cysteine and homocysteine is commonly based on pre-/postcolumn derivatization with chromophores/fluorophores in conjunction with HPLC, and then the procedure is repeated on a second sample after cystine and homocystine are reduced to their respective thiol species using an off-line approach. The concentrations of cystine and homocystine are then indirectly calculated from the difference between the measurements obtained in the Correspondence: Professor Chao Lu, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15 East Road Beisanhuan, Box 79, Beijing 100029, China E-mail: [email protected] Fax: +86-10-64411957
Abbreviations: CL, chemiluminescence; FSN, fluorosurfactant; TCEP, tris(2-carboxyethyl)phosphine hydrochloride C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
two separate steps [8–10]. The sequential detection of thiols and disulfides is not only time consuming but also vulnerable to the autoxidation of thiols during the entire procedure. Only a few reports exist for the direct determination of thiols and disulfides, which are mostly electrochemical and MS detection procedures [11–15]. Although electrochemical detection offers some advantages over derivatization techniques in terms of procedural simplicity, its application is limited as a result of their high overpotentials for oxidation. MS is a well-established and routine analytical technique for the direct determination of thiols and disulfides [16]. In 2010, Kasicka et al. reviewed the wide applications of functionalized gold nanoparticles in separation sciences [17]. As a promising functionalizing reagent for gold nanoparticles, nonionic fluorosurfactant (FSN) has been used for capping spherical gold nanoparticles in aqueous solution to sense cysteine and homocysteine [18]. FSN-capped gold nanoparticles have received a great deal of attention in biosensors, including colorimetry [19–24], fluorescence [25, 26], resonance light scattering [27,28], and chemiluminescence (CL) [29–34]. However, to the best of our knowledge, FSN-capped spherical gold nanoparticles have not been utilized to detect disulfides, mainly due to the lack of a suitable on-line reduction column for disulfides. Therefore, it is still necessary to design new optical assays based on FSN-capped spherical gold nanoparticles for sensing disulfides owing to the advantages of FSN ligands over other ligands by their virtues of good biocompatibility and stability [35]. Colour Online: See the article online to view Fig. 1 in colour. www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 30–36
We recently used FSN-prepared triangular gold nanoparticles as the HPLC postcolumn specific CL reagents to detect thiols and disulfides directly by incorporating an online tris(2-carboxyethyl)phosphine (TCEP) reduction column for disulfides in a single chromatographic separation [36]. We reasoned that the combination of on-line reduction for disulfides and gold nanoparticles based HPLC postcolumn CL detection was a promising method for sensing thiols and disulfides in real samples. However, this strategy has three limitations: (i) like other previously reported CL-based postcolumn detection systems, an additional CL system (e.g. luminol/H2 O2 ) accompanied with complex procedures is required to generate the CL signals [37–39]; (ii) the synthesis of the uniform triangular gold nanoparticles as the HPLC postcolumn CL reagents requires cautious processing conditions [31]; (iii) the pH value of the HPLC mobile phase, the on-line reduction of disulfides and the luminol/H2 O2 CL reaction need to be prepared carefully to make them compatible. In this work, we set out to develop a simple method to simultaneously detect underivatized cysteine, homocysteine, cystine, and homocystine by using an on-line TCEP column, FSN-capped spherical gold nanoparticles as postcolumn colorimetric reagents, and HPLC as the separation technique. The new method presented here is simple, highly specific, and convenient. Furthermore, the practicality of this method is further demonstrated through the detection of cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples.
2 Materials and methods 2.1 Reagents and materials All reagents were of analytical grade and used without further purification. All solutions were prepared with deionized water (Milli Q, Millipore, Barnstead, CA, USA). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 ·3H2 O, 99.99%) and trisodium citrate (99%) were purchased from Acros (Geel, Belgium). Zonyl FSN-100 (F(CF2 CF2 )1–7 CH2 CH2 O(CH2 CH2 O)0–15 H), cysteine (≥99%), homocysteine (>90%), cystine (>99%), homocystine (>99%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl, ≥98%), and ethylenediaminetetraacetic acid (>99%) were purchased from Sigma-Aldrich (St. Louis, USA). Methanol, HClO4 (70–72%), ZnO, NH3 . H2 O NaOH, HCl, and trifluoroacetic acid ( ≥99.5%) were purchased from Beijing Chemical Reagent Company (Beijing, China). The pH of phosphate buffer solution was adjusted with NaOH or HCl. 10 mM stock solutions of cysteine, homocysteine and their disulfides were freshly prepared by dissolving an appropriate amount of thiols in deionized water or disulfides in acidic solution, respectively, and stored at 4⬚C until further use. Their corresponding working solutions were freshly diluted with deionized water. The HPLC mobile phase containing 0.05% m/v TFA was fresh daily prepared, filtered through 0.22 m membranes, and then degassed prior to use. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.2 Apparatus The HPLC system consisted of a 1525 Binary HPLC pump (Waters Corporation, USA), a Shimadzu LC-10AT pump (Shimadzu, Japan), a 2707 Auto Sampler (Waters Corporation, USA), a 2489 UV/Vis detector set at = 650 nm (Waters Corporation, USA), a SunFire C18 guard column (4.6 × 20 mm, 5 m particle size), a SunFire C18 analytical column (4.6 × 150 mm, 5 m particle size), and a Zn(II)-TCEP reduction column (3.0 cm in length and 5.0 mm id). Isocratic elution of a mobile phase containing 0.05% m/v TFA was performed with a 1525 Binary HPLC pump. The postcolumn reaction module included a Shimadzu LC-10AT pump (Shimadzu, Japan) to deliver the FSN-capped gold colloidal solution and a reaction coil (30 cm length, 0.5 mm id) immersed in a thermostatic water bath at 70⬚C.
2.3 Synthesis of gold nanoparticles All glassware for the preparation of gold nanoparticles was thoroughly washed with freshly prepared aqua regia (HNO3 /HCl = 1:3), rinsed extensively with deionized water, and then dried in an oven at 100⬚C for 2–3 h. Fourteen nanometer gold nanoparticles were prepared according to the literature [18]. Briefly, a 50 mL solution of 0.04% m/v trisodium citrate was brought to a vigorous boil with stirring in a round-bottomed flask fitted with a reflux condenser, and then 85 L of 5% m/v HAuCl4 was added to the above solution. The solution was maintained at boiling point with continuous stirring for 15 min and then was cooled to room temperature with continued stirring. Then 400 L of 5% FSN m/v was added. The suspension was stored at 4⬚C until further use.
2.4 Homemade on-line reduction column for disulfides A homemade on-line reduction column for disulfides was prepared as described in our previous paper [36]. First, Zn(ClO4 )2 ·6H2 O was prepared by the reaction of ZnO with HClO4 , recrystallizing the product from water, and drying the crystals in vacuum. Second, Zn(II)-TCEP complexes were synthesized by dissolving TCEP·HCl (0.2 mmol) and Zn(ClO4 )2 ·6H2 O (0.2 mmol) in 5.0 mL of H2 O/methanol (1:3), adding 1.5 mmol of NH3 aq, and leaving overnight at room temperature. The obtained white crystals of Zn(II)TCEP were washed with H2 O/MeOH (1:3), and then with deionized water. Finally, a stainless-steel column (3.0 cm in length and 5.0 mm id) was thoroughly washed with deionized water, and dried in an oven at 100⬚C for 2–3 h. Then 2.0 g of crystals of Zn(II)-TCEP were packed into the stainless-steel column through a rubber funnel. Both ends of the reduction column were furnished with glass wool in order to prevent the crystals from washing away. Finally, the as-prepared reduction column was washed with deionized water for 30 min. www.jss-journal.com
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Figure 1. Schematic representation of the on-line reduction–HPLC–colorimetric detection system. P1 : 1525 Binary HPLC pump; P2 : Shimadzu LC-10 AT pump; G: guard column; A: analytical column; R: reduction column; the injection volume was 20 L; the temperature of heater was set at 70⬚C; the length of the reaction coil was 30 cm; W: waste; the mobile phase of 0.05% m/v TFA was pumped by P1 at 0.6 mL/min; the 2.9 nM FSN-capped gold nanoparticles were pumped by P2 at 0.3 mL/min.
2.5 Detection procedures
3 Results and discussion
A schematic diagram of the HPLC separation, on-line reduction, and postcolumn colorimetric detection system is illustrated in Fig. 1. To summarize, the standard mixtures of the four amino acids or real samples were mixed with 0.05% m/v TFA by an automatic sampler. The flow rate of 0.05% m/v TFA was 0.6 mL/min. Then the four analytes were separated on a SunFire C18 RP column before cystine and homocystine were reduced by a homemade on-line reduction column. The separated solution was mixed with 2.9 nM FSN-capped gold nanoparticles containing 100 mM phosphate buffer solution, which could keep the suitable ionic strength and pH value (pH 6.0) for the interaction between the gold nanoparticles and aminothiols. The FSN-capped gold nanoparticles were pumped by using a Shimadzu LC-10 AT pump at 0.3 mL/min. Subsequently, the mixture was heated in a reaction coil to enhance the aggregation rate by immersing in a 70⬚C water bath. The UV/Vis absorbance intensity was monitored at 650 nm.
3.1 Principle of direct detection for cysteine, homocysteine, cystine, and homocystine
2.6 Pretreatment of real samples
3.2 Experimental parameters
Human urine and plasma samples were collected from three healthy volunteers, and the analysis was conducted immediately after the sample collection. In order to prevent the autoxidation of thiols by complexing potentially catalytic metal cations and acidifying the solution, 97 L of 1 mM ethylenediaminetetraacetic acid solution and 3 L of formic acid were added to 900 L of urine/plasma samples. Next, 20 L of 3.0 mM perchloric acid was added into a 100 L of the above solution with gentle vortex-mixing in a centrifuge tube, put aside at room temperature for 10 min, and then centrifuged at 13 000 rpm for 10 min. The clear supernatant was filtered through a 0.22 m filter and diluted before HPLC analysis. The standard addition method was carried out by spiking different concentrations of thiols and disulfides standard solutions to urine or plasma samples.
The flow rate of the mobile phase is crucial for HPLC systems. The strongest absorbance of gold nanoparticles was obtained at 650 nm and the four amino acids could be separated completely when the flow rate of TFA was 0.6 mL/min (Fig. 2A). When the flow rate of TFA was >0.6 mL/min, the absorbance of the gold colloidal solution decreased obviously as a result of inadequate aggregation of the FSN-capped gold nanoparticles induced by aminothiols. However, the lower flow rates of TFA led to chromatographic peak broadening. Therefore, 0.6 mL/min was selected as the optimal flow rate. The flow rate of the postcolumn gold nanoparticles was also optimized in the range of 0.2–0.8 mL/min (Fig. 2B). When the flow rate of gold nanoparticles was >0.3 mL/min, the absorbance of gold nanoparticles at 650 nm was decreased seriously due to the incomplete aggregation of the gold
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In this study, we fabricated one TCEP reduction column for cystine and homocystine using commercially available TCEP as an effective reducing agent according to the following two steps. The phosphorus atom of TCEP attacks the sulfur along the S–S bond axis to form a thiophosphonium salt. Subsequently, a rapid hydrolysis of the thiophosphonium salt releases the second thiol fragment and the phosphine oxide [36]. The reduction products of cystine and homocystine, that is, cysteine and homocysteine, can exchange the FSN ligands on the surface of the gold nanoparticles, resulting in the rapid aggregation of gold colloidal solution under highly saline conditions, along with the color change from wine red to blue. Note that 650 nm was selected in the absorption spectra of the gold nanoparticles in the presence of cysteine as the optimum absorbance wavelength in this study due to high detection sensitivity for cysteine and homocysteine.
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Figure 2. Effects of the reaction conditions on on-line reductionHPLC-colorimetric detection in presence and absence of cysteine: (A) flow rate of TFA: the flow rate of the FSN-capped gold nanoparticles was 0.3 mL/min; the reaction temperature was 70⬚C; the length of the reaction coil was 30 cm; (B) flow rate of the FSNcapped gold nanoparticles: the flow rate of TFA solution was 0.6 mL/min; the reaction temperature was 70⬚C; the length of the reaction coil was 30 cm; (C) the length of the reaction coil: the flow rates of TFA and the FSN-capped gold nanoparticles were 0.6 and 0.3 mL/min, respectively; the reaction temperature was 70⬚; (D) the flow rates of TFA and FSN-capped gold nanoparticles were 0.6 and 0.3 mL/min, respectively; the length of the reaction coil was 30 cm.
nanoparticles. Accordingly, the optimal flow rate of gold nanoparticles was selected as 0.3 mL/min. Note that the flow rates of TFA and postcolumn gold nanoparticles were lower than those in the earlier published papers dealing with determination of the aminothiols in biological fluids [18, 36]. The effect of postcolumn colorimetric reaction coil length was optimized in the range of 30–100 cm. The absorbance of gold nanoparticles at 650 nm gradually decreased with an increase in the length of reaction coil from 50 cm due to the broadening and tailing of chromatographic peaks. The base line signal increased with an increase of the reaction coil length in the range of 30–100 cm as a result of the slight self-aggregation of gold nanoparticles (Fig. 2C). Considering the effect of the base line and the absorbance, a reaction coil of 30 cm was chosen throughout this study. The aggregation kinetics of FSN-capped gold nanoparticles induced by aminothiols was highly dependent on the incubation temperature. Herein, we optimized the incubation temperature in the range of 40–80⬚C (Fig. 2D). When the incubation temperature was 70⬚C, the base line was increased dramatically, which was ascribed to the serious self-aggregation of gold nanoparticles. These results indicated that FSN-capped gold nanoparticles were unstable at higher temperatures. Therefore, the suitable temperature for this system was 70⬚C because of the rapid reaction of the gold nanoparticles with aminothiols at this temperature [18]. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography
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Figure 3. Typical HPLC chromatograms of standard solution containing cystine, cysteine, homocysteine, and homocystine (A) without or (B) with Zn(II)-TCEP reduction column. (1) cystine; (2) cysteine; (3) homocysteine; (4) homocystine. The retention times were 2.45, 3.08, 4.03, and 5.25 min for cystine, cysteine, homocysteine, and homocystine, respectively. The concentrations of cysteine and homocysteine were 10 M while the concentrations of cystine and homocystine were 5 M. Experimental conditions: the mobile phase was 0.05% m/v TFA at 0.6 mL/min. The flow rate of 2.9 nM FSN-capped gold nanoparticles was 0.3 mL/min. The injection volume was 20 L. The reaction temperature was 70⬚C. The length of the reaction coil was 30 cm.
3.3 Zn(II)-TCEP reduction column for disulfides In our previous study [18], aminothiols, such as cysteine and homocysteine, could induce the aggregation of the FSN-capped spherical gold nanoparticles as a result of the ligand exchange between FSN molecules and cysteine/homocysteine. However, the FSN-capped spherical gold nanoparticles did not respond to their disulfides because these larger molecules might induce the steric effect. In this study, we utilized an on-line Zn(II)-TCEP column for the reduction of disulfides prior to their reaction with the FSNcapped gold nanoparticles. Note that the amount of Zn(II)TCEP was in excess in order to ensure complete reduction. In Fig. 3, the typical chromatograms of cysteine, homocysteine, cystine, and homocystine before and after on-line reduction are shown. The results revealed that cystine and homocystine could be detected in the presence of the on-line reduction column. In addition, the chromatographic peaks of 5 M cystine and homocystine were of the same high as those of the 10 M cysteine and homocysteine after the complete on-line reduction.
3.4 Method performance data The proposed method was validated by evaluating the linearity, recovery, and LODs. Figures 3–5 show that the complete separation of cysteine, homocysteine, cystine, and homocystine was obtained for the standard solutions, blank and spiked samples. Furthermore, the typical chromatograms of human urine and plasma samples are shown along with the samples spiked with standard aminothiols. It was obvious that cystine, cysteine, homocysteine, and homocystine in human urine and plasma samples were well separated by using the proposed system and their retention time was close to that www.jss-journal.com
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Figure 4. HPLC chromatograms of (A) a human urine sample and (B) a human urine sample spiked with 20 M homocysteine and 20 M homocystine. The blank or spiked urine samples solution were diluted five times with deionized water prior to HPLC injection.
Figure 5. HPLC chromatograms of (A) a human plasma sample and (B) a human plasma sample spiked with 20 M homocysteine and 20 M homocystine. The blank or spiked plasma samples solution were diluted five times with deionized water prior to HPLC injection.
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of the standard aminothiols. These results demonstrated that the present method would be fully validated and accurate for the determination of cysteine, homocysteine, cystine, and homocystine in real samples. The calibration curves of cysteine, homocysteine, cystine, and homocystine at six concentration levels showed good linearity in their respectively reported range as given in Table 1. The regression equation and correlation coefficient (R2 ) calculated from the calibration curves of standard solutions are shown in Table 1. Moreover, we used the F-test method to evaluate the fit goodness of the formulas [40]. The obtained P values by F-test for the calibration curves of cysteine, cysteine, homocysteine, homocystine were
