The Method And Application Study Of Determination Of Free 3-Nitrotyrosine In Human Plasma By Solid Phase Extraction High Performance Liquid Chromatography

Objective: Studies in recent years have indicated that 3-nitrotyrosine is a kind of amino acid that is intrinsic and extremely few in vivo. In healthy human plasma, the level of free 3-nitrotyrosine in healthy human plasma is about 1 nmol/L. In a strengthened oxidative stress environment, the generation of reactive nitrogen species such as nitric oxide is increased. Through free radical reactions, they can nitrate free or protein-bound tyrosine in biological systems to increase the concentration of 3-nitrotyrosine, thereby damaging the chemical structures and biological function of normal amino acid and proteins and leading to pathological changes in tissues. This process has been implicated in more than 40 kinds of diseases. So, the detection of oxidative stress extent related to active substances such as reactive nitrogen species in vivo is of great importance for exploring the pathogenesis of diseases. Since reactive nitrogen species are very unstable in vivo and have very short half lives, it is very difficult to measure them in vivo directly. Therefore, their specific and stable product 3-nitrotyrosine has been frequently employed as a diagnostic marker of damage left by oxidative stress. A sensitive, accurate and reproducible solid phase extraction-high performance liquid chromatography assay is developed for the determination of free 3-nitrotyrosine in human plasma. It has been successfully applied to analyze the levels of 3-nitrotyrosine in plasma of healthy volunteers and patients with type 2 diabetes and patients with chronic hepatitis B in order to provide a sensitive and reliable determination method for studying the role of reactive nitrogen species in the course of type 2 diabetes and chronic hepatitis B.Methods: 1. Chromatographic conditions: Solid phase extraction-high performance liquid chromatography-fluorescence detection was performed with a Shimadzu model 10ATvp LC system, consisting of a controller (SCL-10Avp), two pumps (LC-10ATvp), a fluorescence detector (RF-10AXL), a column heater (HT-230A), and a 20-μL injection loop. The separation was performed on a Nova-Pak C₁₈(3.9×150 mm, 4μm) reversed-phase column protected by a Dikma SecurityGuard^TM guard cartridge (4×3.0 mm). A mobile phase consisting of a mixture of acetonitrile-0.02 mol/L phosphate buffer was filtered, degassed by sonication, and used at a column temperature of 35℃and a flow rate of 1.0 mL/min. The fluorescence detector was set at 470 and 530 nm for excitation and emission wavelengths, respectively. C₁₈ solid phase extraction cartridges (100 mg, 1 mL) were purchased from Waters.2. Major reagents: All solvents and reagents used were of chromatographic or analytical grade. Chromatographic grade acetonitrile was purchased from J. T. Baker. Analytical grade boric acid, potassium tetraborte, trifluoroacetic acid, sodium dihydrogen phosphate and dibasic sodium phosphate were all purchased from Shanghai sinopharm chemical reagent limited company. Ultrapure water was prepared using a Millipore Milli-Q water system. The standard of 3-nitrotyrosine andα-methyl-tyrosine (used as the internal standard) were obtained from Sigma-Aldrich. 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole, used as the fluorescent agent, was purchased from TCI in Tokyo. Blank human plasma, which was used for method validation experiments, was obtained from the Shandong Blood Center.3. Plasma sample preparation: Plasma protein was precipitated by proper organic solvent in a centrifuge tube. The suspension was centrifuged, then the precipitate was discarded and the supernatant was evaporated to dryness under a gentle stream of nitrogen. The sequence of solid phase extraction and fluorescence derivatization reaction was explored.4. Method optimization: The solid phase extraction conditions were optimized from the aspects of activation methods of solid phase extraction cartridge, washing reagent and eluting reagent; the derivatization conditions were optimized from the aspects of reaction temperature and time, buffer solution, concentration of acetonitrile and 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole; the chromatographic conditions were optimized from the aspects of column, the choice of organic phase in mobile phase and the ratio of organic phase to aqueous phase, as well as the volume of trifluoroacetic acid added to aqueous phase.5. Method investigation: External standard method and internal standard method were used for quantification. The working curves were calculated by unweighed least-squares linear regression analysis of the peak areas of 3-nitrotyrosine or the peak areas ratios of 3-nitrotyrosine toα-methyl-L-tyrosine against the corresponding plasma concentration of 3-nitrotyrosine. The detection limits were calculated. Quality control samples for determination of accuracy and precision of the method were independently prepared at low, medium, and high concentrations. Absolute and relative recoveries, intra-day and inter-day relative standard deviations and the stability of 3-nitrotyrosine derivative and 3-nitrotyrosine were calculated.6. Method application: The method was used to determined free 3-nitrotyrosine human plasma concentrations of healthy volunteers, patients with type 2 diabetes and patients with chronic hepatitis B. The results were analyzed by t test. The role of reactive nitrogen species in damaging amino acid and protein in the course of type 2 diabetes and chronic hepatitis B was confirmed through the results of statistics analysis.Results:1. In the sample preparation procedure step, Acetonitrile was used to precipitate plasma protein. The volume of plasma and acetonitrile was 500μL and 1 mL, respectively. Solid phase extraction was selected to use before fluorescence derivatization reaction. To 500μL of a plasma sample in a centrifuge tube, 1 mL of acetonitrile was added. The resulting suspension was vortex-mixed and centrifuged. The supernatant was collected and evaporated to dryness under a gentle stream of nitrogen. The residue was cleaned by solid phase extraction and the eluent was evaporated to dryness again before fluorescence derivatization reaction.2. The derivatization reaction was carried out at 60℃for 2 min under dark. The concentrations of potassium borate buffer (pH 9.5) and 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole were 0.05 mol/L and 4 mmol/L, respectively. The proportion of acetonitrile was 40%.3. Aliquot (20μL) of the reaction mixture is directly injected into an high performance liquid chromatography apparatus with acetonitrile-phosphate buffer (0.02 mol/L, plus 500μL/L TFA, pH 3.0) (36:64, V/V) as the mobile phase at the flow rate of 1.0 mL/min.4. When external standard method was used for quantification, the linear range was 0.50～50.0 nmol/L, coefficient correlation was 0.9999. The detection limit was 0.25 nmol/L(S/N= 5). The average absolute and relative recoveries in spiked sample were 89.3%～91.9% and 99.2%～107.9%. The intra-day and inter-day relative standard deviations were 1.5%～4.8% and 2.8%～9.2%, respectively. The average concentrations of 3-nitrotyrosine in normal human plasma and patients with type 2 diabetes were 1.18 nmol/L (SD = 0.43 nmol/L, n = 21) and 3.63 nmol/L (SD = 1.04 nmol/L, n = 23), respectively.5. When internal standard method was used for quantification, the calibration curve is linear over the concentration range of 0.5 nmol/L to 50.0 nmol/L in plasma, coefficient correlation was 0.9990. The limit of detection is 0.25 nmol/L (S/N= 5) . The average absolute and relative recoveries in spiked sample were 87.7%～91.5% and 96.9%—110.0%. The intra-day and inter-day relative standard deviations were 2.6%～6.4% and 3.1%～7.2%, respectively. The average concentrations of 3-nitrotyrosine in normal human plasma and patients with chronic hepatitis B were 1.19 nmol/L (SD = 0.41 nmol/L, n = 22) and 3.20 nmol/L (SD = 0.51 nmol/L, n = 20), respectively.Conclusions:1. In order to obtain accurate and reliable determination result of trace levels of 3-nitrotyrosine in human plasma, fluorometric method or chromatographic-mass spectrographic method which was extremely sensitive must be employed. However, the cost of chromatographic-mass spectrographic method was relatively higher, it was not easy for common analytical chemistry laboratory to equip and use in large sample study. The current method utilized high performance liquid chromatography instrument and fluorescence detector whose cost was lower and detected trace levels of 3-nitrotyrosine in human plasma. It is sensitive, accurate and reproducible, suitable for analysis of large sample numbers in clinic research.2. Significant differences of plasma free 3-nitrotyrosine concentration (P < 0.01) were found between healthy volunteers and patients with type 2 dependent diabetes and between healthy volunteers and patients with chronic hepatitis B, indicating that damage of amino acids and proteins left by reactive nitrogen species has occurred during pathogenesis of non-insulin dependent diabetes and chronic hepatitis B.