Article pubs.acs.org/JAFC
Simultaneous Determination of Glutathione, Cysteine, Homocysteine, and Cysteinylglycine in Biological Fluids by IonPairing High-Performance Liquid Chromatography Coupled with Precolumn Derivatization Wenbing Zhang,† Pingliang Li,† Qianqian Geng, Yongheng Duan, Mingcheng Guo, and Yongsong Cao* College of Agriculture and Biotechnology, China Agricultural University, Beijing, China, 100193 ABSTRACT: Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 μM with the coefficient regression range of 0.9987−0.9994. Detection limits ranged from 0.04 to 0.08 μM (S/N = 3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50−124.25 μM, 0−72.81 μM, and 0−4.25 μM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva. KEYWORDS: cysteine, glutathione, homocysteine, high performance liquid chromatography, precolumn derivatization, 4-chloro-3,5-dinitrobenzotrifluoride bimane (mBrB),26,27 ammonium-7-fluoro-2,1,3-benzoxadiazole4-sulfonate (SBD-F),28 and 4-(aminosulfonyl)-7-fluoro-2,1,3benzoxadiazole (ABD-F).29,30 Although these reagents have been well adapted in many fields, new labeling reagents are still needed to improve the detection properties. Ion-pairing chromatography is a mature and valuable analytical and separation strategy in biological fluids. Its used has been reported in a very wide range of applications concerning organic and inorganic ions, neutrals, and zwitterionic compounds.13−18 Therefore, the ion-pairing chromatography would be a better approach in separation and analysis of GSH, Cys, Hcy, and Cys-Gly in biological fluids. In our previous studies, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) has been demonstrated to be a feasible and superior derivatization reagent for amino compounds.43−45 CNBF has one activated halide leaving group which can be easily displaced by the thiol group (−SH), leading to formation of the stable thioether with well-defined absorbance at 230 nm. The aim of the present work is to develop and validate a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva. Then the levels of thiols in three biological fluids collected from five healthy donors were determined with this novel method.
1. INTRODUCTION Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids and important biomarkers for a wide range of human diseases.1,2 GSH is the most abundant nonprotein thiol, and its altered levels occur in plasma of patients with leukemia, diabetes, or AIDS.3 Cys is involved in a variety of important cellular functions, and its levels in urine have been recognized as an important indicator for kidney stones and cystinuria.4,5 The elevated level of Hcy in plasma is considered as a risk factor for cardiovascular disease.1,6 Increased concentration of Cys-Gly in plasma or urine has been observed to be linked to rheumatoid arthritis.1 Therefore, quantification of these thiols in biological fluids is important for monitoring some special pathological conditions of human. There have been many analytical methods for these thiols, which mainly involved fluorescent assays,7 electrochemical assays,8 capillary electrophoresis,9,10 gas chromatography,11,12 high performance liquid chromatography (HPLC),13−37 gas chromatography with mass spectrometry,38,39 and liquid chromatography with mass spectrometry.40,41 Recently, the derivatization followed by HPLC determination of the resulting derivatives attracted more attention because it is relatively simple and can simultaneously determine several thiols with good sensitivity.33,42 The derivatization reagent is very important for the method sensitivity and recovery because it can provide chromophore or fluorophores and stabilize the thiol molecules. There have been a number of commonly used derivatization reagents, such as 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT),16−18 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),19 o-phthalaldheyde (OPA),24,25 monobromo© 2014 American Chemical Society
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Glutathione, L-cysteine, DLhomocysteine, L-cysteinylglycine, trichloroacetic acid (TCA), 5,5′Received: Revised: Accepted: Published: 5845
March 22, 2014 June 9, 2014 June 10, 2014 June 10, 2014 dx.doi.org/10.1021/jf5014007 | J. Agric. Food Chem. 2014, 62, 5845−5852
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Figure 1. General reaction of thiols with 4-chloro-3,5-dinitrobenzotrifluoride.
Figure 2. Effects of CNBF/thiol molar ratio (A), pH (B), reaction temperature (C), and reaction time (D) on the resulting peak areas of the thiol derivatives. (A) Constant conditions: thiol standard 100 μM, pH 8.0, 25 °C, 20 min. (B) Constant conditions: thiol standard 100 μM, CNBF 500 μM, 25 °C, 20 min. (C) Constant conditions: thiol standard 100 μM, CNBF 500 μM, pH 8.0, 20 min. (D) Constant conditions: thiol standard 100 μM, CNBF 500 μM, pH 8.0, 25 °C. 2.3. Sample Preparation. In the present study, venous blood samples (about 2.0 mL), urine samples (about 5.0 mL), and saliva samples (about 1.0 mL) were collected from five healthy donors (male:female = 3:2, 23−30 years of age, Beijing, China) on April 27th, 2012. Blood samples were placed in precooled ethylenediaminetetraacetic acid (EDTA) Vacutainer tubes and immediately centrifuged at 2500g for 10 min at 4 °C. The supernatant liquid (plasma) was collected and stored at −80 °C for the analysis. The urine samples were collected and stored in a 5 mL tube at −80 °C until analysis. Five minutes after the gargling with distilled water, donors kept tongue in touch with palate and made the saliva naturally flow down into a 5 mL tube. Then the saliva samples were stored at −80 °C for analysis. The pretreatment method was referenced to the report by Cevasco et al.33 with some modifications. After thawing, 100 μL of sample (plasma, urine, or saliva) was mixed with 10 μL of a solution of TCEP (50 mM in borate buffer, pH 7.4) in a vial and incubated at 25 °C for 10 min. Then 90 μL of a solution containing 1% TCA and 1 mM EDTA was added and the total solution was centrifuged for 10 min at 10000g. The obtained supernatant was collected for the subsequent derivatization.
dithiobis(2-nitrobenzoic acid), and trifluoroacetic acid (TFA) were analytical grade reagent and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). CNBF was provided by Alfa Aesar (Ward Hill, MA, USA). Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Fluka (Germany). Acetonitrile and methanol (HPLC grade) was purchased from J.T. Baker (Phillipsburg, NJ, USA). The ultrapure water was obtained by a Milli-Q water purification system (Millipore, Billerica, MA, USA). The standard solutions of 5.0 mM GSH, Cys, Hcy, and Cys-Gly were prepared in ultrapure water. The CNBF standard solution of 40.0 mM was prepared in methanol. The stock solutions were stored in the dark at 4 °C when not in use. H3BO3−Na2B4O7 buffer was prepared by mixing 0.2 mol/L H3BO3 solution with 0.05 mol/L Na2B4O7 solution to the required pH value. 2.2. Instruments. A HPLC system, consisting of two LC-20ATvp pumps and an SPD-20Avp ultraviolet detector (Shimadzu, Japan), was applied for the separation and analysis. A reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm, 5 μm) and Chromatograph Solution Light Chemstation for LC system were employed to obtain and process chromatographic data. 5846
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2.4. Derivatization Method. After the pretreatment step, 100 μL of the supernatant, 20 μL of 10 mM CNBF solution, 20 μL of methanol, and 50 μL of borate buffer (0.2 M, pH 8.0) were mixed and incubated at 25 °C for 20 min. Then the derivatization was terminated with 10 μL of 2 M HCl. The resulting solution was filtered through a 0.22 μm filter membrane (Millipore Corporation, Belfast, MA, USA) and injected into the chromatographic system. 2.5. Chromatographic Method. Before the analysis, the C18 column equipped with a C18 guard column (4 mm × 3 mm i.d.) was pre-equilibrated with the mobile phase for 20 min. HPLC separation of the thiol derivatives was carried out on a reversed-phase Kromasil ODS C18 column. The mobile phase was composed of acetonitrile (eluent A) and water with 0.1% (v/v) TFA (eluent B). Optimum separation for plasma samples was achieved in gradient condition as follows (expressed as the proportion of eluent B): 0−9.0 min, a linear gradient starting from 70% to 20%; 9.0−10.0 min, 20%; 10.0−15.0 min, linear gradient from 20% to 70%. Optimum separation for urine and saliva samples (proportion of eluent B): 0−5.0 min, a linear gradient starting from 70% to 20%; 5.0−8.0 min, 20%; 8.0−11.0 min, linear gradient from 20% to 70%. The analysis was executed with injection volume of 20 μL, flow rate of 1.0 mL/min and UV detection wavelength at 230 nm. Each sample was injected in triplicate, and the analysis was carried out at 25 °C. 2.6. Comparison with a Reference Method. To validate this method, the thiols in the plasma samples from the five donors were also determined by a reference method19 and the data from the two methods were compared by the Independent Sample t-Test using SPSS (version, 17.0). The plasma sample was derivatized with 10 mM DTNB at room temperature in pH 7.2 for 5 min, filtered, and injected into HPLC. The mobile phase was composed of methanol (A) and 100 mM KH2PO4 (B) with the ratio of 12:88 (A/B, v/v). The analysis was executed with injection volume of 20 μL, flow rate of 1.2 mL/min, and UV detection wavelength at 230 nm.
The reaction time was also an essential factor, and its effects on derivatization were studied (Figure 2D). It can be seen that when the time ≥ 20 min, the peak areas of four thiol derivatives reached the most. Therefore, the derivatization time was 20 min. CNBF has relatively poor water solubility, thus organic solvent should be added to the reaction medium to avoid the precipitation of the reagent and derivative. The tests showed the content of methanol in the total solutions should be more than 15%, especially for the derivatization of Cys. 3.2. Optimization of Separation Conditions. Test showed that, without ion-pairing reagents in the mobile phase, the thiol derivatives were too polar to be well retained on the reversed-phase column, which resulted in difficulty in the separation. In the present study, TFA was used as the ionpairing agent to achieve fast and optimum separation of four thiol derivatives, CNBF, and matrix interferences. The effects of TFA concentration (range of 0.01−0.20%) on separation of the derivatives were also studied. It was found that high concentration may cause damage to the C18 column. Test also indicated 0.1% (v/v) TFA was the optimum adding concentration. To obtain better separation within shorter analysis time, the separation conditions for plasma, urine, and saliva were investigated respectively as previously mentioned in section 2.5. In the present work, a wavelength of 230 nm was selected in the HPLC-UV analysis to obtain good absorption and minimize the interferences. Typical chromatograms of reaction of thiols with CNBF and plasma sample with spiking thiols are shown in Figure 3, in which the retention times of GSH, Hcy, Cys-Gly, and Cys were 4.05, 5.45, 7.50, and 11.80 min respectively and the separation can be completed within 15 min. Typical chromatograms of reaction of thiols with CNBF, urine sample with spiking thiols, and saliva sample with spiking thiols are shown in Figure 4, in which the retention times of GSH, Hcy, Cys-Gly, and Cys were 3.85, 4.95, 6.50, and 9.00 min respectively and the separation can be completed within 11 min. 3.3. Stability of the Derivative. The stability of thiolCNBF in methanol−water (20:80, v/v) at 4 °C was investigated over 5 days without light irradiation. Results showed there was no significant change in the peak areas of GSH-CNBF, Cys-CNBF, Hcy-CNBF, and Cys-Gly-CNBF. When sample was illuminated by ordinary light of 100 W bulb for 24 h, there was also no significant change in the peak areas of the four thiol derivatives. Seven days later, the average degradation rates at room temperature were 4.20%, 5.05%, 5.40%, and 4.85% for GSH-CNBF, Cys-CNBF, Hcy-CNBF, and Cys-Gly-CNBF respectively. It appeared that the thiol derivatives were very stable. 3.4. Validation of the Method and Application to Sample Analysis. Text mixtures of different concentrations of thiols were prepared and analyzed by using the optimized derivatization procedure and separation conditions. Sensitivity of the method was determined by the limit of detection (LOD) at a signal-to-noise ratio of 3 and the limit of quantification (LOQ) at a signal-to-noise ratio of 10.29,30 The usual numerical value used is the relative standard deviation (RSD) for reproducibility. The reproducibility of this analytical method was evaluated both intraday and interday with thiol concentration of 50 μM. The linear calibration range, regression equation, R2, LOD, LOQ, and RSD of four thiols with this new method were calculated, and the results are listed in Table 1. Results showed that the correlation coefficients for
3. RESULTS AND DISCUSSION 3.1. Optimization of Derivatization Conditions. The general reaction of CNBF with thiols is shown in Figure 1. CNBF can react with thiols in low concentration to form stable thioether derivatives under alkaline conditions. The main factors affecting the derivatization yields were concentration ratio of CNBF to thiol, pH value, reaction temperature, and reaction time. To determine the optimum derivatization conditions, the effects of the four factors on the peak areas (yields) were investigated, and the results are shown in Figure 2. The optimum concentration ratio of CNBF to thiol was of primary importance. Its effects on the peak areas are shown in Figure 2A. The thiol (100 μM) was reacted with various concentrations of CNBF (300, 400, 500, 600, and 700 μM, CNBF/thiol = 3−7:1). At concentration ratio ≥5:1, the peak areas of four thiols reached the most and kept constant. The levels of GSH, Cys, Hcy, and Cys-Gly in human fluids were reported to be in the range of 2.07−17.80, 72.0−312.70, 1.30− 9.20, and 0.49−11.30 μM.14,22,33 In the real analysis, 2.0 mM was the adequate concentration for CNBF to keep the concentration ratio ≥5:1. The optimum reaction pH value was determined by derivatizing thiols in H3BO3−Na2B4O7 buffer with the pH values ranging from 7.5 to 9.0. Figure 2B shows that the peak areas of thiol-CNBF are the most at pH range of 8.0−9.0. Because high pH could lead to the hydrolysis of CNBF, pH 8.0 was selected for all experiments. A temperature range of 25−60 °C was used to study the optimum temperature. Figure 2C shows when other conditions are kept constant, temperature has no significant influence on the peak areas of four thiols. Therefore, 25 °C (room temperature) was selected for the derivatization temperature. 5847
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Figure 3. Chromatograms obtained from the reaction of CNBF with thiols (A, 40 μM of GSH, Cys, Hcy, and Cys-Gly; B, plasma samples; C, plasma samples with spiking 40 μM of GSH, Cys, Hcy, and CysGly). Chromatographic conditions: column, reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm i.d., particle size 5 μm); UV detection, λ = 230 nm; mobile phase, acetonitrile (A) and water with 0.1% (v/v) TFA (B). Gradient conditions: B, 0−9.0 min, a linear gradient starting from 70% to 20%; B, 9.0−10.0 min, 20%; B, 10.0− 15.0 min, linear gradient from 20% to 70%; flow rate, 1.0 mL/min; 25 °C.
these thiols were from 0.9987 to 0.9994. The RSDs for the thiol-CNBF are from 2.26% to 2.92% for within-day determination (n = 6) and from 2.65% to 3.55% for between-day determination (n = 6). The detection limits for four thiols ranged from 0.04 μM to 0.08 μM. In the present work, the applicability of the proposed method was evaluated in plasma, urine, and saliva. Four thiol peaks were identified by spiking thiol standards to the fluid sample. Before adding the reducing reagent TCEP, the fluid sample was spiked with thiol standard 5 and 50 μM, respectively. Then the fluid samples with and without spiking were extracted and analyzed. Because the fluid contained thiols, the recovery should be calculated by the formula
Figure 4. Chromatograms obtained from the reaction of CNBF with thiols (A, 50 μM of GSH, Cys, Hcy, and Cys-Gly; B, urine samples; C, urine samples with spiking 50 μM of GSH, Cys, Hcy, and Cys-Gly; D, saliva samples; E, saliva samples with spiking 50 μM of GSH, Cys, Hcy, and Cys-Gly). Chromatographic conditions: column, reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm i.d., particle size 5 μm); UV detection, λ = 230 nm; mobile phase, acetonitrile (A) and water with 0.1% (v/v) TFA (B). Gradient conditions: B, 0−5.0 min, a linear gradient starting from 70% to 20%; B, 5.0−8.0 min, 20%; B, 8.0−11.0 min, linear gradient from 20% to 70%; flow rate, 1.0 mL/ min; 25 °C.
recovery = (C − Cd)/Cs × 100%
where C represents the total found concentration of thiol after spiking, Cd represents the initially measured concentration before spiking, and Cs represents the spiked concentration. The recoveries of thiols from plasma, urine, and saliva were investigated. Table 2 shows the recoveries of thiols are in the range of 82.5% to 91.6% for plasma, 86.8% to 95.0% for urine, and 80.5% to 88.5% for saliva, depending on the sample
investigated. RSDs are from 1.75% to 3.62% for plasma, 1.85% to 3.84% for urine, and 2.00% to 4.08% for saliva. 5848
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Table 1. Linear Calibration Range, Regression Equation, R2, LOD, LOQ, and RSD of Thiol-CNBFa parameters calibration range (μM) regression eq, y coeff regression, R2 RSD, n = 6 within-day between-day LOD (μM)b LOQ (μM)c a
GSH-CNBF
Cys-CNBF
Hcy-CNBF
Cys-Gly-CNBF
0.15−500.0 71698x + 561.9 0.9994
0.15−500.0 90251x − 1263 0.9987
0.15−500.0 68920x + 865 0.9992
0.15−500.0 73505x + 1106.5 0.9994
2.46 2.90 0.06 0.15
2.85 2.70 0.04 0.12
2.92 3.55 0.08 0.15
2.26 2.65 0.06 0.15
x: concentration of thiol-CNBF (μM). y: peak area of thiol-CNBF. bS/N = 3, per 20 μL injection volume. cS/N = 10, per 20 μL injection volume.
Table 2. Average Recoveries of Thiols from Plasma, Urine, and Saliva Samples by Using Proposed Method fluid plasma
initiala (μM)
Spiked (μM)
totalb (μM)
foundc (μM)
recovery (%)
RSDd (%)
GSH
26.50
Cys
122.40
Hcy
5.70
5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00
30.93 72.30 126.65 165.50 9.83 47.20 23.70 64.50 10.02 52.90 77.24 117.91 5.54 45.20 13.40 55.75 6.95 45.95 7.57 47.44 5.31 43.71 4.26 44.25
4.43 45.80 4.25 43.10 4.13 41.50 4.45 45.25 4.62 47.50 4.43 45.10 4.34 44.00 4.55 46.90 4.15 43.15 4.03 43.90 4.10 42.50 4.26 44.25
88.6 91.6 85.0 86.2 82.5 83.0 89.0 90.5 92.3 95.0 88.5 90.2 86.8 88.0 91.0 93.8 83.0 86.3 80.5 87.8 82.0 85.0 85.2 88.5
3.62 2.05 3.24 2.05 2.58 2.10 2.90 1.75 3.02 1.85 3.84 2.73 3.68 3.50 2.55 2.32 3.62 2.15 4.08 2.25 2.96 2.05 2.50 2.00
thiols
Cys-Gly urine
saliva
19.25
GSH
5.40
Cys
72.81
Hcy
1.20
Cys-Gly
8.85
GSH
2.80
Cys
3.54
Hcy
1.21
Cys-Gly
0
Mean value of six initial measured concentrations in biological fluid before spiking. bMean value of six initial measured concentrations in biological fluid after spiking. cThe total measured concentration minus the initial measured concentration. dMean value of six determinations. a
3.5. Comparison of CNBF with Other Derivatizing Reagents. The comparison of CNBF with these reported derivatizing reagents is given in Table 3. In comparison to the ultraviolet derivatizing reagents, CNBF was superior to CMQT and BCPB in the method sensitivity but inferior to them in the derivatization velocity. Although the methods with AuNPs (Au nanoparticles) and TCDI (1,1′-thiocarbonyldiimidazole) as the derivatizing reagent were more sensitive than the present method, the derivatization times with them were much longer (2 h). In comparison to several fluorescent reagents, the present method was more sensitive than the method with NDA or 5-BMF as the derivatizing reagent but showed no superiority to that with OPA, mBrB, SBD-F, SBD-BF, ABD-F, MIPBO, and TMPAB-o-M. However, derivatization with SBD-F, SBDBF, ABD-F, MIPBO, and TMPAB-o-M required more time or higher temperature than that with CNBF. mBrB has the one drawback that the reagent itself, the hydrolysis products, and the impurities are fluorescent, which may interfere with analysis.46 Apart from thiols, OPA can also very easily react with primary amino compounds in biological samples, resulting
in an increase of interferences in the form of amino−OPA adducts.42 CNBF can easily react with thiols (25 °C, pH 8.0), but its reaction with amines needed higher temperature (60 °C) and higher pH (9.0),43−45 indicating that CNBF has good selectivity for the analysis of thiols. In conclusion, this CNBFderivatization based method showed superiority in relative sensitivity and selectivity. Besides, HPLC with a UV detector, known for its stability, universality, and low demand in terms of maintenance, belongs to the standard instrumentation of an analytical laboratory for clinical samples.2,23 Therefore, the proposed method is an attractive choice for the determination of thiols in human biological fluids. 3.6. Analysis of Fluid Samples and the Validation by the Reference Method. The developed method was applied to quantification of GSH, Cys, Hcy, and Cys-Gly in plasma, urine, and saliva collected from five donors. The detected concentrations of GSH, Cys, Hcy, and Cys-Gly are given in Table 4. In the plasma samples, the concentrations were in the range of 8.75 to 26.50 μM, 86.45 to 124.25 μM, 2.50 to 10.57 μM, and 11.20−27.00 μM for GSH, Cys, Hcy, and Cys-Gly 5849
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Table 3. Comparison of the Derivatization Conditions and Detection Limit of the Derivatizing Reagents Reported for Determination of Thiols in Different Matrixa anal. meth
thiol analytes
derivatizing reagent
derivatization conditions
CE-LIF CE-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-UV
GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly Cys, Hcy Cys Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH Hcy GSH, Cys, Hcy, GSH, Cys GSH, Cys, Hcy, Cys-Gly GSH, Cys GSH, Cys, Hcy, Cys-Gly GSH, Cys GSH, Cys, Hcy GSH, Cys, Hcy, Cys-Gly GSH GSH, Cys, GSH, Cys, Hcy, Cys-Gly
5-BMF AuNPs CMQT CMQT CMQT DTNB CMPI TCDI TCDI BCPB OPA OPA mBrB mBrB SBD-F ABD-F ABD-F MIPBO MIAC SBD-BF NDA TMPAB-o-M CNBF
RT, 10 min RT, 2 h RT, 1 min RT RT RT, 5 min RT, 10 min RT, 2 h RT, 2 h RT, 10 min RT, 1 min Post column RT, 5 min RT, 30 min 60 °C, 1 h 60 °C, 10 min 50 °C, 10 min 40 °C, 35 min RT, 1 min RT, 30 min RT, 4 min 37 °C, 6 min 25 °C, 20 min
matrix human plasma, human plasma human plasma human saliva human plasma human plasma human plasma ND human plasma, human plasma ND human cell human plasma human muscle human plasma human plasma human plasma, human urine human plasma human plasma must and wine pig liver human plasma,
urine
urine
urine, saliva
urine, saliva
detection limit
ref
0.061−0.156 μM 10−65 nM 0.1−0.3 μM 0.5 μM 0.5 μM ND 4 pmolb 2 pmolb ND 0.2 μM 25 fmolc 100 fmolc 2 pmolb 30, 50 nM ND 20, 5 nM 0.1−0.5 μM 3.5−15.0 fmolc 1.2−2.0 pmolb 0.01−0.1 μM 0.1 μM 0.13−0.25nM 0.04−0.08 μM
9 10 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 this work
CE-LIF, capillary electrophoresis with laser-induced fluorescent detection; CE-UV, capillary electrophoresis with ultraviolet detection; HPLC-UV, high performance liquid chromatography with ultraviolet detection; HPLC-FL, high performance liquid chromatography with fluorescent detection; 5-BMF, 5-(bromomethyl) fluorescein; CMQT, 2-chloro-1-methylquinolinium tetrafluoroborate; AuNPs, the synthesized triangular gold nanoparticles; CMPI, 2-chloro-1-methylpyridinium iodide; DTNB, 5,5-dithiobis (2-nitrobenzoic acid); TCDI, 1,1′-thiocarbonyldiimidazole; BCPB, 1-benzyl-2-chloropyridinium bromide; OPA, o-phthalaldheyde; mBrB, monobromobimane; SBD-F, ammonium-7-fluoro-2,1,3-benzoxadiazole-4-sulfonate; MIPBO, 5-methyl-(2-(m-iodoacetylaminophenyl) benzoxazole; MIAC, N-(2-acridonyl) maleimide; SBD-BF, 5-bromo-7fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; NDA, 2,3-naphthalenedialdehyde; TMPAB-o-M, 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene; ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3 -benzoxadiazole; RT, room temperature; ND, not described. bThe full unit: pmol per injection (20 μL). cThe full unit: fmol per injection (20 μL). a
Table 4. Detected Concentrations of GSH, Cys, Hcy, and Cys-Gly in the Plasma, Urine, and Saliva Collected from Five Donors samplea
donor
plasma
01 02 03 04 05 01 02 03 04 05 01 02 03 04 05
urine
saliva
a
GSH (μM) 12.2 8.75 18.5 26.2 15.6 5.40 12.1 1.43 6.57 9.35 1.43 2.80 3.20 1.25 0.90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.85 0.60 1.40 1.52 0.95 0.07 0.35 0.08 0.12 0.08 0.35 0.50 0.33 0.20 0.11
12.0 9.00 17.9 25.9 15.5
Cys (μM) ± ± ± ± ±
1.15b 0.64b 2.15b 1.93b 0.80b
124 ± 5.50 105 ± 7.25 86.5 ± 5.03 122 ± 10.20 90.8 ± 6.80 72.8 ± 1.25 61.0 ± 0.87 56.8 ± 1.04 59.0 ± 3.25 66.0 ± 1.95 4.25 ± 0.60 3.54 ± 0.41 1.95 ± 0.05 3.55 ± 0.70 1.24 ± 0.20
Hcy (μM)
130 ± 8.20b 105 ± 11.5b 82.2 ± 2.55b 120 ± 7.83b 91.5 ± 6.40b
4.02 ± 10.6 ± 2.53 ± 5.70 ± 2.50 ± 1.20 ± 2.45 ± 0.82 ± 1.10 ± NDc 0.84 ± 1.21 ± 0.75 ± 0.98 ± 1.33 ±
0.12 0.97 0.22 0.10 0.20 0.02 0.46 0.03 0.02 0.21 0.06 0.08 0.08 0.12
4.35 10.0 2.12 5.50 2.24
Cys-Gly (μM) ± ± ± ± ±
0.40b 0.46b 0.50b 0.50b 0.31b
11.2 ± 0.75 25.0 ± 1.68 27.0 ± 2.85 19.2 ± 1.02 23.0 ± 1.55 8.85 ± 0.24 12.00 ± 1.22 12.1 ± 0.53 11.0 ± 0.88 3.24 ± 0.09 NDc NDc 0.35 ± 0.04 NDc 0.55 ± 0.08
10.5 25.0 25.9 20.0 22.5
± ± ± ± ±
0.95b 1.22b 0.70b 1.50b 2.40b
Each fluid sample was determined in triplicates. bThe data was obtained with a reference method described in section 2.6. cNot detected.
respectively, with highest average concentration for Cys and lowest for Hcy. For urine samples, the concentration ranges were 1.43−12.11 μM, 56.75−72.81 μM, 0−2.45 μM, and 3.24− 12.09 μM for GSH, Cys, Hcy, and Cys-Gly, respectively. As in saliva, the range values were 0.90−3.20 μM, 1.24−3.25 μM, 0.75−1.33 μM, and 0−0.55 μM for GSH, Cys, Hcy, and CysGly, respectively. It can be concluded that the concentrations of
total thiols in saliva were far lower than those in plasma or urine. The data obtained with the reference method19 is also given in Table 4. The data with two methods was compared, and significant differences were considered at the P < 0.05 level. The results showed that there were no significant differences 5850
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of total aminothiols in plasma and urine. Electrophoresis 2003, 24, 1200−1207. (10) Chang, C. W.; Tseng, W. L. Gold nanoparticle extraction followed by capillary electrophoresis to determine the total, free, and protein-bound aminothiols in plasma. Anal. Chem. 2010, 82, 2696− 2702. (11) Kataoka, H.; Takagi, K.; Makita, M. Determination of total plasma homocysteine and related aminothiols by gas chromatography with flame photometric detection. J. Chromatogr. B 1995, 664, 421− 425. (12) Küster, A.; Tea, I.; Sweeten, S.; Rozé, J. C.; Robins, R. J.; Darmaun, D. Simultaneous determination of glutathione and cysteine concentrations and 2H enrichments in microvolumes of neonatal blood using gas chromatography−mass spectrometry. Anal. Bioanal. Chem. 2008, 390, 1403−1412. (13) Khan, M. I.; Iqbal, Z. Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ionpairing RP-HPLC coupled with electrochemical detector. J. Chromatogr. B 2011, 879, 2567−2575. (14) Khan, A.; Khan, M. I.; Iqbal, Z.; Shah, Y.; Ahmad, L.; Nazir, S.; Watson, D. G.; Khan, J. A.; Nasir, F.; Khan, A.; Ismail. A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection. Talanta 2011, 84, 789−801. (15) Bronowicka-Adamska, P.; Zagajewski, J.; Czubak, J.; Wróbel, M. RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain. J. Chromatogr. B 2011, 879, 2005−2009. (16) Bald, E.; Chwatko, G.; Głowacki, R.; Kuśmierek, K. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. A 2004, 1032, 109−115. (17) Bald, E.; Głowacki, R. Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection. Amino Acids 2005, 28, 431−433. (18) Głowacki, R.; Bald, E. Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection. J. Chromatogr. B 2009, 877, 3400−3404. (19) Katrusiak, A. E.; Paterson, P. G.; Kamencic, H.; Shoker, A.; Lyon, A. W. Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl− glycine, homocysteine and glutathione in plasma and cell extracts. J. Chromatogr. B 2001, 758, 207−212. (20) Kaniowska, E.; Chwatko, G.; Głowacki, R.; Kubalczyk, P.; Bald, E. Urinary excretion measurement of cysteine and homocysteine in the form of their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. A 1998, 798, 27−35. (21) Amarnath, V.; Amarnath, K. Specific determination of cysteine and penicillamine through cyclization to 2-thioxothiazolidine-4carboxylic acids. Talanta 2002, 56, 745−751. (22) Amarnath, K.; Amarnath, V.; Amarnath, K.; Valentine, H. L.; Valentine, W. M. A specific HPLC-UV method for the determination of cysteine and related aminothiols in biological samples. Talanta 2003, 60, 1229−1238. (23) Kuśmierek, K.; Bald, E. Reversed-phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1-benzyl-2-chloropyridinium bromide. Biomed. Chromatogr. 2009, 23, 770−775. (24) Mopper, K.; Delmas, D. Trace determination of biological thiols by liquid chromatography and precolumn fluorometric labeling with ophthalaldehyde. Anal. Chem. 1984, 56, 2557−2560. (25) Mukai, Y.; Togawa, T.; Suzuki, T.; Ohata, K.; Tanabe, S. Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection. J. Chromatogr. B 2002, 767, 263−268. (26) Ivanov, A. R.; Nazimov, I. V.; Baratova, L. Determination of biologically active low-molecular-mass thiols in human blood: I. Fast
between the data (P > 0.05). Therefore, the data with the proposed method can be validated by the reference method. Based on the reaction of CNBF with thiols, we developed a novel method for the determination of four physiologically important thiols, GSH, Cys, Hcy, and Cys-Gly, in plasma, urine, and saliva. The application of CNBF as a derivatization reagent seemed to be an attractive choice for the determination of thiols with TFA in mobile phase, which showed good separation of derivatized thiols from the interferences. The reaction of CNBF with thiols was rapid, required moderate reaction conditions, and led to stable products. In comparison to other derivatizing reagents for thiols, application of CNBF showed superiority in relative sensitivity, selectivity, and efficient derivatization procedure. The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 0−124.25 μM. This study provides a feasible HPLC method for the routine analysis of thiols required for clinical evaluation.
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AUTHOR INFORMATION
Corresponding Author
*No. 2 Yuanmingyuan West Road, China Agricultural University, Beijing, P.R. China, 100193. Tel: 86-10-62734302. Fax: 86-10-62734302. E-mail: [email protected]. Author Contributions †
These authors contributed equally to this work.
Funding
This work was supported by National Department Public Benefit Research Foundation of China (201003065) and Research Fund for the Doctoral Program of Higher Education of China (20120008130006). Notes
The authors declare no competing financial interest.
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REFERENCES
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Simultaneous Determination of Glutathione, Cysteine, Homocysteine, and Cysteinylglycine in Biological Fluids by IonPairing High-Performance Liquid Chromatography Coupled with Precolumn Derivatization Wenbing Zhang,† Pingliang Li,† Qianqian Geng, Yongheng Duan, Mingcheng Guo, and Yongsong Cao* College of Agriculture and Biotechnology, China Agricultural University, Beijing, China, 100193 ABSTRACT: Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 μM with the coefficient regression range of 0.9987−0.9994. Detection limits ranged from 0.04 to 0.08 μM (S/N = 3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50−124.25 μM, 0−72.81 μM, and 0−4.25 μM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva. KEYWORDS: cysteine, glutathione, homocysteine, high performance liquid chromatography, precolumn derivatization, 4-chloro-3,5-dinitrobenzotrifluoride bimane (mBrB),26,27 ammonium-7-fluoro-2,1,3-benzoxadiazole4-sulfonate (SBD-F),28 and 4-(aminosulfonyl)-7-fluoro-2,1,3benzoxadiazole (ABD-F).29,30 Although these reagents have been well adapted in many fields, new labeling reagents are still needed to improve the detection properties. Ion-pairing chromatography is a mature and valuable analytical and separation strategy in biological fluids. Its used has been reported in a very wide range of applications concerning organic and inorganic ions, neutrals, and zwitterionic compounds.13−18 Therefore, the ion-pairing chromatography would be a better approach in separation and analysis of GSH, Cys, Hcy, and Cys-Gly in biological fluids. In our previous studies, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) has been demonstrated to be a feasible and superior derivatization reagent for amino compounds.43−45 CNBF has one activated halide leaving group which can be easily displaced by the thiol group (−SH), leading to formation of the stable thioether with well-defined absorbance at 230 nm. The aim of the present work is to develop and validate a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva. Then the levels of thiols in three biological fluids collected from five healthy donors were determined with this novel method.
1. INTRODUCTION Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids and important biomarkers for a wide range of human diseases.1,2 GSH is the most abundant nonprotein thiol, and its altered levels occur in plasma of patients with leukemia, diabetes, or AIDS.3 Cys is involved in a variety of important cellular functions, and its levels in urine have been recognized as an important indicator for kidney stones and cystinuria.4,5 The elevated level of Hcy in plasma is considered as a risk factor for cardiovascular disease.1,6 Increased concentration of Cys-Gly in plasma or urine has been observed to be linked to rheumatoid arthritis.1 Therefore, quantification of these thiols in biological fluids is important for monitoring some special pathological conditions of human. There have been many analytical methods for these thiols, which mainly involved fluorescent assays,7 electrochemical assays,8 capillary electrophoresis,9,10 gas chromatography,11,12 high performance liquid chromatography (HPLC),13−37 gas chromatography with mass spectrometry,38,39 and liquid chromatography with mass spectrometry.40,41 Recently, the derivatization followed by HPLC determination of the resulting derivatives attracted more attention because it is relatively simple and can simultaneously determine several thiols with good sensitivity.33,42 The derivatization reagent is very important for the method sensitivity and recovery because it can provide chromophore or fluorophores and stabilize the thiol molecules. There have been a number of commonly used derivatization reagents, such as 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT),16−18 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),19 o-phthalaldheyde (OPA),24,25 monobromo© 2014 American Chemical Society
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Glutathione, L-cysteine, DLhomocysteine, L-cysteinylglycine, trichloroacetic acid (TCA), 5,5′Received: Revised: Accepted: Published: 5845
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Figure 1. General reaction of thiols with 4-chloro-3,5-dinitrobenzotrifluoride.
Figure 2. Effects of CNBF/thiol molar ratio (A), pH (B), reaction temperature (C), and reaction time (D) on the resulting peak areas of the thiol derivatives. (A) Constant conditions: thiol standard 100 μM, pH 8.0, 25 °C, 20 min. (B) Constant conditions: thiol standard 100 μM, CNBF 500 μM, 25 °C, 20 min. (C) Constant conditions: thiol standard 100 μM, CNBF 500 μM, pH 8.0, 20 min. (D) Constant conditions: thiol standard 100 μM, CNBF 500 μM, pH 8.0, 25 °C. 2.3. Sample Preparation. In the present study, venous blood samples (about 2.0 mL), urine samples (about 5.0 mL), and saliva samples (about 1.0 mL) were collected from five healthy donors (male:female = 3:2, 23−30 years of age, Beijing, China) on April 27th, 2012. Blood samples were placed in precooled ethylenediaminetetraacetic acid (EDTA) Vacutainer tubes and immediately centrifuged at 2500g for 10 min at 4 °C. The supernatant liquid (plasma) was collected and stored at −80 °C for the analysis. The urine samples were collected and stored in a 5 mL tube at −80 °C until analysis. Five minutes after the gargling with distilled water, donors kept tongue in touch with palate and made the saliva naturally flow down into a 5 mL tube. Then the saliva samples were stored at −80 °C for analysis. The pretreatment method was referenced to the report by Cevasco et al.33 with some modifications. After thawing, 100 μL of sample (plasma, urine, or saliva) was mixed with 10 μL of a solution of TCEP (50 mM in borate buffer, pH 7.4) in a vial and incubated at 25 °C for 10 min. Then 90 μL of a solution containing 1% TCA and 1 mM EDTA was added and the total solution was centrifuged for 10 min at 10000g. The obtained supernatant was collected for the subsequent derivatization.
dithiobis(2-nitrobenzoic acid), and trifluoroacetic acid (TFA) were analytical grade reagent and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). CNBF was provided by Alfa Aesar (Ward Hill, MA, USA). Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Fluka (Germany). Acetonitrile and methanol (HPLC grade) was purchased from J.T. Baker (Phillipsburg, NJ, USA). The ultrapure water was obtained by a Milli-Q water purification system (Millipore, Billerica, MA, USA). The standard solutions of 5.0 mM GSH, Cys, Hcy, and Cys-Gly were prepared in ultrapure water. The CNBF standard solution of 40.0 mM was prepared in methanol. The stock solutions were stored in the dark at 4 °C when not in use. H3BO3−Na2B4O7 buffer was prepared by mixing 0.2 mol/L H3BO3 solution with 0.05 mol/L Na2B4O7 solution to the required pH value. 2.2. Instruments. A HPLC system, consisting of two LC-20ATvp pumps and an SPD-20Avp ultraviolet detector (Shimadzu, Japan), was applied for the separation and analysis. A reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm, 5 μm) and Chromatograph Solution Light Chemstation for LC system were employed to obtain and process chromatographic data. 5846
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2.4. Derivatization Method. After the pretreatment step, 100 μL of the supernatant, 20 μL of 10 mM CNBF solution, 20 μL of methanol, and 50 μL of borate buffer (0.2 M, pH 8.0) were mixed and incubated at 25 °C for 20 min. Then the derivatization was terminated with 10 μL of 2 M HCl. The resulting solution was filtered through a 0.22 μm filter membrane (Millipore Corporation, Belfast, MA, USA) and injected into the chromatographic system. 2.5. Chromatographic Method. Before the analysis, the C18 column equipped with a C18 guard column (4 mm × 3 mm i.d.) was pre-equilibrated with the mobile phase for 20 min. HPLC separation of the thiol derivatives was carried out on a reversed-phase Kromasil ODS C18 column. The mobile phase was composed of acetonitrile (eluent A) and water with 0.1% (v/v) TFA (eluent B). Optimum separation for plasma samples was achieved in gradient condition as follows (expressed as the proportion of eluent B): 0−9.0 min, a linear gradient starting from 70% to 20%; 9.0−10.0 min, 20%; 10.0−15.0 min, linear gradient from 20% to 70%. Optimum separation for urine and saliva samples (proportion of eluent B): 0−5.0 min, a linear gradient starting from 70% to 20%; 5.0−8.0 min, 20%; 8.0−11.0 min, linear gradient from 20% to 70%. The analysis was executed with injection volume of 20 μL, flow rate of 1.0 mL/min and UV detection wavelength at 230 nm. Each sample was injected in triplicate, and the analysis was carried out at 25 °C. 2.6. Comparison with a Reference Method. To validate this method, the thiols in the plasma samples from the five donors were also determined by a reference method19 and the data from the two methods were compared by the Independent Sample t-Test using SPSS (version, 17.0). The plasma sample was derivatized with 10 mM DTNB at room temperature in pH 7.2 for 5 min, filtered, and injected into HPLC. The mobile phase was composed of methanol (A) and 100 mM KH2PO4 (B) with the ratio of 12:88 (A/B, v/v). The analysis was executed with injection volume of 20 μL, flow rate of 1.2 mL/min, and UV detection wavelength at 230 nm.
The reaction time was also an essential factor, and its effects on derivatization were studied (Figure 2D). It can be seen that when the time ≥ 20 min, the peak areas of four thiol derivatives reached the most. Therefore, the derivatization time was 20 min. CNBF has relatively poor water solubility, thus organic solvent should be added to the reaction medium to avoid the precipitation of the reagent and derivative. The tests showed the content of methanol in the total solutions should be more than 15%, especially for the derivatization of Cys. 3.2. Optimization of Separation Conditions. Test showed that, without ion-pairing reagents in the mobile phase, the thiol derivatives were too polar to be well retained on the reversed-phase column, which resulted in difficulty in the separation. In the present study, TFA was used as the ionpairing agent to achieve fast and optimum separation of four thiol derivatives, CNBF, and matrix interferences. The effects of TFA concentration (range of 0.01−0.20%) on separation of the derivatives were also studied. It was found that high concentration may cause damage to the C18 column. Test also indicated 0.1% (v/v) TFA was the optimum adding concentration. To obtain better separation within shorter analysis time, the separation conditions for plasma, urine, and saliva were investigated respectively as previously mentioned in section 2.5. In the present work, a wavelength of 230 nm was selected in the HPLC-UV analysis to obtain good absorption and minimize the interferences. Typical chromatograms of reaction of thiols with CNBF and plasma sample with spiking thiols are shown in Figure 3, in which the retention times of GSH, Hcy, Cys-Gly, and Cys were 4.05, 5.45, 7.50, and 11.80 min respectively and the separation can be completed within 15 min. Typical chromatograms of reaction of thiols with CNBF, urine sample with spiking thiols, and saliva sample with spiking thiols are shown in Figure 4, in which the retention times of GSH, Hcy, Cys-Gly, and Cys were 3.85, 4.95, 6.50, and 9.00 min respectively and the separation can be completed within 11 min. 3.3. Stability of the Derivative. The stability of thiolCNBF in methanol−water (20:80, v/v) at 4 °C was investigated over 5 days without light irradiation. Results showed there was no significant change in the peak areas of GSH-CNBF, Cys-CNBF, Hcy-CNBF, and Cys-Gly-CNBF. When sample was illuminated by ordinary light of 100 W bulb for 24 h, there was also no significant change in the peak areas of the four thiol derivatives. Seven days later, the average degradation rates at room temperature were 4.20%, 5.05%, 5.40%, and 4.85% for GSH-CNBF, Cys-CNBF, Hcy-CNBF, and Cys-Gly-CNBF respectively. It appeared that the thiol derivatives were very stable. 3.4. Validation of the Method and Application to Sample Analysis. Text mixtures of different concentrations of thiols were prepared and analyzed by using the optimized derivatization procedure and separation conditions. Sensitivity of the method was determined by the limit of detection (LOD) at a signal-to-noise ratio of 3 and the limit of quantification (LOQ) at a signal-to-noise ratio of 10.29,30 The usual numerical value used is the relative standard deviation (RSD) for reproducibility. The reproducibility of this analytical method was evaluated both intraday and interday with thiol concentration of 50 μM. The linear calibration range, regression equation, R2, LOD, LOQ, and RSD of four thiols with this new method were calculated, and the results are listed in Table 1. Results showed that the correlation coefficients for
3. RESULTS AND DISCUSSION 3.1. Optimization of Derivatization Conditions. The general reaction of CNBF with thiols is shown in Figure 1. CNBF can react with thiols in low concentration to form stable thioether derivatives under alkaline conditions. The main factors affecting the derivatization yields were concentration ratio of CNBF to thiol, pH value, reaction temperature, and reaction time. To determine the optimum derivatization conditions, the effects of the four factors on the peak areas (yields) were investigated, and the results are shown in Figure 2. The optimum concentration ratio of CNBF to thiol was of primary importance. Its effects on the peak areas are shown in Figure 2A. The thiol (100 μM) was reacted with various concentrations of CNBF (300, 400, 500, 600, and 700 μM, CNBF/thiol = 3−7:1). At concentration ratio ≥5:1, the peak areas of four thiols reached the most and kept constant. The levels of GSH, Cys, Hcy, and Cys-Gly in human fluids were reported to be in the range of 2.07−17.80, 72.0−312.70, 1.30− 9.20, and 0.49−11.30 μM.14,22,33 In the real analysis, 2.0 mM was the adequate concentration for CNBF to keep the concentration ratio ≥5:1. The optimum reaction pH value was determined by derivatizing thiols in H3BO3−Na2B4O7 buffer with the pH values ranging from 7.5 to 9.0. Figure 2B shows that the peak areas of thiol-CNBF are the most at pH range of 8.0−9.0. Because high pH could lead to the hydrolysis of CNBF, pH 8.0 was selected for all experiments. A temperature range of 25−60 °C was used to study the optimum temperature. Figure 2C shows when other conditions are kept constant, temperature has no significant influence on the peak areas of four thiols. Therefore, 25 °C (room temperature) was selected for the derivatization temperature. 5847
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Figure 3. Chromatograms obtained from the reaction of CNBF with thiols (A, 40 μM of GSH, Cys, Hcy, and Cys-Gly; B, plasma samples; C, plasma samples with spiking 40 μM of GSH, Cys, Hcy, and CysGly). Chromatographic conditions: column, reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm i.d., particle size 5 μm); UV detection, λ = 230 nm; mobile phase, acetonitrile (A) and water with 0.1% (v/v) TFA (B). Gradient conditions: B, 0−9.0 min, a linear gradient starting from 70% to 20%; B, 9.0−10.0 min, 20%; B, 10.0− 15.0 min, linear gradient from 20% to 70%; flow rate, 1.0 mL/min; 25 °C.
these thiols were from 0.9987 to 0.9994. The RSDs for the thiol-CNBF are from 2.26% to 2.92% for within-day determination (n = 6) and from 2.65% to 3.55% for between-day determination (n = 6). The detection limits for four thiols ranged from 0.04 μM to 0.08 μM. In the present work, the applicability of the proposed method was evaluated in plasma, urine, and saliva. Four thiol peaks were identified by spiking thiol standards to the fluid sample. Before adding the reducing reagent TCEP, the fluid sample was spiked with thiol standard 5 and 50 μM, respectively. Then the fluid samples with and without spiking were extracted and analyzed. Because the fluid contained thiols, the recovery should be calculated by the formula
Figure 4. Chromatograms obtained from the reaction of CNBF with thiols (A, 50 μM of GSH, Cys, Hcy, and Cys-Gly; B, urine samples; C, urine samples with spiking 50 μM of GSH, Cys, Hcy, and Cys-Gly; D, saliva samples; E, saliva samples with spiking 50 μM of GSH, Cys, Hcy, and Cys-Gly). Chromatographic conditions: column, reversed-phase Kromasil ODS C18 column (250 mm × 4.6 mm i.d., particle size 5 μm); UV detection, λ = 230 nm; mobile phase, acetonitrile (A) and water with 0.1% (v/v) TFA (B). Gradient conditions: B, 0−5.0 min, a linear gradient starting from 70% to 20%; B, 5.0−8.0 min, 20%; B, 8.0−11.0 min, linear gradient from 20% to 70%; flow rate, 1.0 mL/ min; 25 °C.
recovery = (C − Cd)/Cs × 100%
where C represents the total found concentration of thiol after spiking, Cd represents the initially measured concentration before spiking, and Cs represents the spiked concentration. The recoveries of thiols from plasma, urine, and saliva were investigated. Table 2 shows the recoveries of thiols are in the range of 82.5% to 91.6% for plasma, 86.8% to 95.0% for urine, and 80.5% to 88.5% for saliva, depending on the sample
investigated. RSDs are from 1.75% to 3.62% for plasma, 1.85% to 3.84% for urine, and 2.00% to 4.08% for saliva. 5848
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Table 1. Linear Calibration Range, Regression Equation, R2, LOD, LOQ, and RSD of Thiol-CNBFa parameters calibration range (μM) regression eq, y coeff regression, R2 RSD, n = 6 within-day between-day LOD (μM)b LOQ (μM)c a
GSH-CNBF
Cys-CNBF
Hcy-CNBF
Cys-Gly-CNBF
0.15−500.0 71698x + 561.9 0.9994
0.15−500.0 90251x − 1263 0.9987
0.15−500.0 68920x + 865 0.9992
0.15−500.0 73505x + 1106.5 0.9994
2.46 2.90 0.06 0.15
2.85 2.70 0.04 0.12
2.92 3.55 0.08 0.15
2.26 2.65 0.06 0.15
x: concentration of thiol-CNBF (μM). y: peak area of thiol-CNBF. bS/N = 3, per 20 μL injection volume. cS/N = 10, per 20 μL injection volume.
Table 2. Average Recoveries of Thiols from Plasma, Urine, and Saliva Samples by Using Proposed Method fluid plasma
initiala (μM)
Spiked (μM)
totalb (μM)
foundc (μM)
recovery (%)
RSDd (%)
GSH
26.50
Cys
122.40
Hcy
5.70
5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00 5.00 50.00
30.93 72.30 126.65 165.50 9.83 47.20 23.70 64.50 10.02 52.90 77.24 117.91 5.54 45.20 13.40 55.75 6.95 45.95 7.57 47.44 5.31 43.71 4.26 44.25
4.43 45.80 4.25 43.10 4.13 41.50 4.45 45.25 4.62 47.50 4.43 45.10 4.34 44.00 4.55 46.90 4.15 43.15 4.03 43.90 4.10 42.50 4.26 44.25
88.6 91.6 85.0 86.2 82.5 83.0 89.0 90.5 92.3 95.0 88.5 90.2 86.8 88.0 91.0 93.8 83.0 86.3 80.5 87.8 82.0 85.0 85.2 88.5
3.62 2.05 3.24 2.05 2.58 2.10 2.90 1.75 3.02 1.85 3.84 2.73 3.68 3.50 2.55 2.32 3.62 2.15 4.08 2.25 2.96 2.05 2.50 2.00
thiols
Cys-Gly urine
saliva
19.25
GSH
5.40
Cys
72.81
Hcy
1.20
Cys-Gly
8.85
GSH
2.80
Cys
3.54
Hcy
1.21
Cys-Gly
0
Mean value of six initial measured concentrations in biological fluid before spiking. bMean value of six initial measured concentrations in biological fluid after spiking. cThe total measured concentration minus the initial measured concentration. dMean value of six determinations. a
3.5. Comparison of CNBF with Other Derivatizing Reagents. The comparison of CNBF with these reported derivatizing reagents is given in Table 3. In comparison to the ultraviolet derivatizing reagents, CNBF was superior to CMQT and BCPB in the method sensitivity but inferior to them in the derivatization velocity. Although the methods with AuNPs (Au nanoparticles) and TCDI (1,1′-thiocarbonyldiimidazole) as the derivatizing reagent were more sensitive than the present method, the derivatization times with them were much longer (2 h). In comparison to several fluorescent reagents, the present method was more sensitive than the method with NDA or 5-BMF as the derivatizing reagent but showed no superiority to that with OPA, mBrB, SBD-F, SBD-BF, ABD-F, MIPBO, and TMPAB-o-M. However, derivatization with SBD-F, SBDBF, ABD-F, MIPBO, and TMPAB-o-M required more time or higher temperature than that with CNBF. mBrB has the one drawback that the reagent itself, the hydrolysis products, and the impurities are fluorescent, which may interfere with analysis.46 Apart from thiols, OPA can also very easily react with primary amino compounds in biological samples, resulting
in an increase of interferences in the form of amino−OPA adducts.42 CNBF can easily react with thiols (25 °C, pH 8.0), but its reaction with amines needed higher temperature (60 °C) and higher pH (9.0),43−45 indicating that CNBF has good selectivity for the analysis of thiols. In conclusion, this CNBFderivatization based method showed superiority in relative sensitivity and selectivity. Besides, HPLC with a UV detector, known for its stability, universality, and low demand in terms of maintenance, belongs to the standard instrumentation of an analytical laboratory for clinical samples.2,23 Therefore, the proposed method is an attractive choice for the determination of thiols in human biological fluids. 3.6. Analysis of Fluid Samples and the Validation by the Reference Method. The developed method was applied to quantification of GSH, Cys, Hcy, and Cys-Gly in plasma, urine, and saliva collected from five donors. The detected concentrations of GSH, Cys, Hcy, and Cys-Gly are given in Table 4. In the plasma samples, the concentrations were in the range of 8.75 to 26.50 μM, 86.45 to 124.25 μM, 2.50 to 10.57 μM, and 11.20−27.00 μM for GSH, Cys, Hcy, and Cys-Gly 5849
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Table 3. Comparison of the Derivatization Conditions and Detection Limit of the Derivatizing Reagents Reported for Determination of Thiols in Different Matrixa anal. meth
thiol analytes
derivatizing reagent
derivatization conditions
CE-LIF CE-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-UV HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-FL HPLC-UV
GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly Cys, Hcy Cys Cys, Hcy, Cys-Gly GSH, Cys, Hcy, Cys-Gly GSH Hcy GSH, Cys, Hcy, GSH, Cys GSH, Cys, Hcy, Cys-Gly GSH, Cys GSH, Cys, Hcy, Cys-Gly GSH, Cys GSH, Cys, Hcy GSH, Cys, Hcy, Cys-Gly GSH GSH, Cys, GSH, Cys, Hcy, Cys-Gly
5-BMF AuNPs CMQT CMQT CMQT DTNB CMPI TCDI TCDI BCPB OPA OPA mBrB mBrB SBD-F ABD-F ABD-F MIPBO MIAC SBD-BF NDA TMPAB-o-M CNBF
RT, 10 min RT, 2 h RT, 1 min RT RT RT, 5 min RT, 10 min RT, 2 h RT, 2 h RT, 10 min RT, 1 min Post column RT, 5 min RT, 30 min 60 °C, 1 h 60 °C, 10 min 50 °C, 10 min 40 °C, 35 min RT, 1 min RT, 30 min RT, 4 min 37 °C, 6 min 25 °C, 20 min
matrix human plasma, human plasma human plasma human saliva human plasma human plasma human plasma ND human plasma, human plasma ND human cell human plasma human muscle human plasma human plasma human plasma, human urine human plasma human plasma must and wine pig liver human plasma,
urine
urine
urine, saliva
urine, saliva
detection limit
ref
0.061−0.156 μM 10−65 nM 0.1−0.3 μM 0.5 μM 0.5 μM ND 4 pmolb 2 pmolb ND 0.2 μM 25 fmolc 100 fmolc 2 pmolb 30, 50 nM ND 20, 5 nM 0.1−0.5 μM 3.5−15.0 fmolc 1.2−2.0 pmolb 0.01−0.1 μM 0.1 μM 0.13−0.25nM 0.04−0.08 μM
9 10 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 this work
CE-LIF, capillary electrophoresis with laser-induced fluorescent detection; CE-UV, capillary electrophoresis with ultraviolet detection; HPLC-UV, high performance liquid chromatography with ultraviolet detection; HPLC-FL, high performance liquid chromatography with fluorescent detection; 5-BMF, 5-(bromomethyl) fluorescein; CMQT, 2-chloro-1-methylquinolinium tetrafluoroborate; AuNPs, the synthesized triangular gold nanoparticles; CMPI, 2-chloro-1-methylpyridinium iodide; DTNB, 5,5-dithiobis (2-nitrobenzoic acid); TCDI, 1,1′-thiocarbonyldiimidazole; BCPB, 1-benzyl-2-chloropyridinium bromide; OPA, o-phthalaldheyde; mBrB, monobromobimane; SBD-F, ammonium-7-fluoro-2,1,3-benzoxadiazole-4-sulfonate; MIPBO, 5-methyl-(2-(m-iodoacetylaminophenyl) benzoxazole; MIAC, N-(2-acridonyl) maleimide; SBD-BF, 5-bromo-7fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; NDA, 2,3-naphthalenedialdehyde; TMPAB-o-M, 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene; ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3 -benzoxadiazole; RT, room temperature; ND, not described. bThe full unit: pmol per injection (20 μL). cThe full unit: fmol per injection (20 μL). a
Table 4. Detected Concentrations of GSH, Cys, Hcy, and Cys-Gly in the Plasma, Urine, and Saliva Collected from Five Donors samplea
donor
plasma
01 02 03 04 05 01 02 03 04 05 01 02 03 04 05
urine
saliva
a
GSH (μM) 12.2 8.75 18.5 26.2 15.6 5.40 12.1 1.43 6.57 9.35 1.43 2.80 3.20 1.25 0.90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.85 0.60 1.40 1.52 0.95 0.07 0.35 0.08 0.12 0.08 0.35 0.50 0.33 0.20 0.11
12.0 9.00 17.9 25.9 15.5
Cys (μM) ± ± ± ± ±
1.15b 0.64b 2.15b 1.93b 0.80b
124 ± 5.50 105 ± 7.25 86.5 ± 5.03 122 ± 10.20 90.8 ± 6.80 72.8 ± 1.25 61.0 ± 0.87 56.8 ± 1.04 59.0 ± 3.25 66.0 ± 1.95 4.25 ± 0.60 3.54 ± 0.41 1.95 ± 0.05 3.55 ± 0.70 1.24 ± 0.20
Hcy (μM)
130 ± 8.20b 105 ± 11.5b 82.2 ± 2.55b 120 ± 7.83b 91.5 ± 6.40b
4.02 ± 10.6 ± 2.53 ± 5.70 ± 2.50 ± 1.20 ± 2.45 ± 0.82 ± 1.10 ± NDc 0.84 ± 1.21 ± 0.75 ± 0.98 ± 1.33 ±
0.12 0.97 0.22 0.10 0.20 0.02 0.46 0.03 0.02 0.21 0.06 0.08 0.08 0.12
4.35 10.0 2.12 5.50 2.24
Cys-Gly (μM) ± ± ± ± ±
0.40b 0.46b 0.50b 0.50b 0.31b
11.2 ± 0.75 25.0 ± 1.68 27.0 ± 2.85 19.2 ± 1.02 23.0 ± 1.55 8.85 ± 0.24 12.00 ± 1.22 12.1 ± 0.53 11.0 ± 0.88 3.24 ± 0.09 NDc NDc 0.35 ± 0.04 NDc 0.55 ± 0.08
10.5 25.0 25.9 20.0 22.5
± ± ± ± ±
0.95b 1.22b 0.70b 1.50b 2.40b
Each fluid sample was determined in triplicates. bThe data was obtained with a reference method described in section 2.6. cNot detected.
respectively, with highest average concentration for Cys and lowest for Hcy. For urine samples, the concentration ranges were 1.43−12.11 μM, 56.75−72.81 μM, 0−2.45 μM, and 3.24− 12.09 μM for GSH, Cys, Hcy, and Cys-Gly, respectively. As in saliva, the range values were 0.90−3.20 μM, 1.24−3.25 μM, 0.75−1.33 μM, and 0−0.55 μM for GSH, Cys, Hcy, and CysGly, respectively. It can be concluded that the concentrations of
total thiols in saliva were far lower than those in plasma or urine. The data obtained with the reference method19 is also given in Table 4. The data with two methods was compared, and significant differences were considered at the P < 0.05 level. The results showed that there were no significant differences 5850
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between the data (P > 0.05). Therefore, the data with the proposed method can be validated by the reference method. Based on the reaction of CNBF with thiols, we developed a novel method for the determination of four physiologically important thiols, GSH, Cys, Hcy, and Cys-Gly, in plasma, urine, and saliva. The application of CNBF as a derivatization reagent seemed to be an attractive choice for the determination of thiols with TFA in mobile phase, which showed good separation of derivatized thiols from the interferences. The reaction of CNBF with thiols was rapid, required moderate reaction conditions, and led to stable products. In comparison to other derivatizing reagents for thiols, application of CNBF showed superiority in relative sensitivity, selectivity, and efficient derivatization procedure. The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 0−124.25 μM. This study provides a feasible HPLC method for the routine analysis of thiols required for clinical evaluation.
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AUTHOR INFORMATION
Corresponding Author
*No. 2 Yuanmingyuan West Road, China Agricultural University, Beijing, P.R. China, 100193. Tel: 86-10-62734302. Fax: 86-10-62734302. E-mail: [email protected]. Author Contributions †
These authors contributed equally to this work.
Funding
This work was supported by National Department Public Benefit Research Foundation of China (201003065) and Research Fund for the Doctoral Program of Higher Education of China (20120008130006). Notes
The authors declare no competing financial interest.
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REFERENCES
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