| Uric acid (UA) is a kind of endogenous antioxidant, mainly by the cell metabolism decomposition of nucleic acid and the decomposition of purine in food. Recently, hyperuricemia and gout prevalence rate increased year by year. Because there is no obvious symptoms in gout early stage, so it cannot be found early. And an increase of the uric acid concentration in human blood can lead to cardiovascular and other metabolic diseases. So it is urgent to develop a simple, cheap, highly sensitive method for early diagnosis. Noninvasive fingernail samples are easy to collect, no risk of infection and easily stored relative to the blood, there has not been applied to literature reports for the diagnosis of gout.In this study we using high performance liquid (HPLC)-ultraviolet (UV) detector to establish a method for the quantification of UA in human fingernails for the first time. Using Hitachi HPLC system, separated on an ODS-3 column (250 mm x 4.6 mm I.D.,4.0 μm) by isocratic elution using methanol:74 mM phosphate buffer (pH 2.2)=2:98 (v/v). An UV detector was used to monitor at 284 nm, the flow rate of 1.0 mL/min, analysis for 10 minutes. A good linearity was achieved from the calibration curves (r2≥0.9999) in the range of 1.0-10000 ng; the limit of detection (S/N= 3) was 2.0 pg, the inter-day and intra-day assay precisions were 1.68% to 8.02%. The mean recoveries (%) of the uric acid spiked in the human fingernail were 103.40%. The amounts of UA in the ten fingernails of 10 healthy volunteers (5 male and 5 female) were determined and successfully identified. The results show that uric acid concentration in men’s fingernails are all higher than women in each fingernail. And the thumb and little finger have an uneven concentration, while the forefinger, middle finger and third finger are relatively stable. So in the later study using the above three fingernails for uric acid quantitative analysis of the samples. Furthermore, we in order to further verify the validity nails in the diagnosis of gout, The amounts of UA in the 26 healthy volunteers (HV, age 22-60 years) fingernails and 22 gout patients (GP, age 26-65 years) fingernails were determined and compared. The results show that the mean amounts were 44.57 ± 9.37 and 144.70 ± 47.93 ng in 1.0 mg of fingernail from the HV (n = 26) and GP (n = 22), respectively. Uric acid content in the gout patient’s fingernails was obviously higher than that of healthy volunteers. A statistically significant correlation was observed with the UA (p< 0.01), so human fingernails quantitative analysis of the detection of uric acid for gout provides a new method for noninvasive diagnosis.Chirality is an important feature of endogenous metabolites, however, the mainstream of modern metabonomics research LC-MS technology also can’t identify chiral metabolites. But pre-column derivatization method can solve the problem of separation of chiral metabolites. Therefore, we have developed derivatization reagents by the L-Thiazolidine-4-carboxylic acid with chiral center and benzoyl chloride with isotope, synthesized having targeted amino functional groups and chiral metabolites isotope labeling mass spectrometry chiral reagents. And applied to the chiral metabolites 1-phenylethylamine (R, S-PEA) andl-(1-naphthyl) ethylamine (R, S-NEA) analysis, R, S-PEA resolution (Rs) is 1.90, R, S-NEA resolution is 2.21, achieve complete separation and has good separation efficiency. With best collision energy at 40 eV in mass spectrum detection, obtain high strength characteristic product ion of m/z for 77. This reagent can improve the sensitivity of chiral metabolites in mass spectrometry, still can use isotope ionization efficiency in the mass spectrum and the same m/z but the characteristics of different screening biomarkers of various diseases. This method will be for the chiral metabonomics analysis and chiral biomarkers screening provides a new analysis method. |
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