Development And Application Of Dried Spot Method Of Uric Acid In Human Saliva

In recent years,patients with hyperuricemia have continued to increase.However,hyperuricemia has no obvious symptoms in the early stages,so patients cannot be detected and treated in time,which will lead to the occurrence of gout,hypertension and kidney stones.The main cause of gout disease is long-term high concentration of uric acid,which will cause deposition of urate in the joints and kidneys.Gout disease can lead to joint deformity,skin ulceration,kidney damage and even threaten our life.Therefore,for early warning and diagnosis of gout and hyperuricemia,we have developed a cheap,fast,convenient storage and transportation method for quantitative analysis of uric acid in human saliva that allows users to collect samples outside the hospital.It is known as dried spot method of uric acid in human saliva(DSM-UHS).In this study,Shuang Quan filter paper was used,which was cut into a spot with diameter of 5 mm.The internal standard and saliva were dropped on the spot,which was dried at room temperature.The liquid extraction of the dried spot was carried out using lithium carbonate solution.The extraction was evaporated to dryness and reconstituted for injection into the HPLC-UV system.We used INERTAIL ODS-3(250 mm×4.6 mm,4 μm)column and the mobile phase was composed by methanol and 74 mM phosphate buffer solution(pH 2.2).And their radio was 2:98.The flow rate was 1 min/mL,and the analysis was performed within 7 minutes under the wavelength of 284 nm to detect.The linear regression equation of uric acid was y=0.1 129x+0.3872(R2≥0.9998),and a good linearity was achieved was in the range of 2-300 μg/mL.The limit of detection(S/N=3)was 5 μg/mL.The precision ranged was from 0.29 to 4.06%.The best collection time for saliva was 8:00-16:00,because the level of uric acid in human saliva was stable throughout the day.Dried spot of uric acid in human saliva was stored in 20℃ and 40℃ for 24 hours and 5 days.Their CV%was less than 8.94%.It shows that uric acid in dried saliva spots has good stability.The average recovery rate of uric acid in saliva from healthy volunteers and gout patients were 96.16%and 99.20%.The intra-day and inter-day precision of healthy volunteers and gout patients were 1.08-4.30%,1.06-3.78%and 1.25-4.66%,3.62-10.86%.We used DSM-UHS to detect the content of uric acid from 61 volunteers(46 health volunteers;15 gout patients).The average content of uric acid in saliva from health male was 22.28±12.59 μg/mL;the average content of uric acid in saliva from health female was 12.67±9.64 μg/mL,and the average concentration of uric acid in saliva from healthy volunteers and gout patients were 17.48±12.11 μg/mL and 47.08±14.57 μg/mL,respectively.The results showed that the content of uric acid in saliva was related to gender,and the content of uric acid in health male was higher than that in health female(p<0.01).And the content of uric acid in saliva from gout patients was significantly higher than that of healthy people,with a statistically significant difference(p<0.0001).We performed correlation between uric acid in saliva and serum from 61 volunteers,and we found positive correlation between content of uric acid in saliva and serum(r=0.6243,p<0.0001).Therefore,these results indicate that we can use the content of uric acid in human saliva to indirectly reflect the content of uric acid in serum.It provides powerful data for estimating concentrations of uric acid in serum from that in salivaThis study developed a new method for quantitative analysis of uric acid in human saliva,and provided effective basic data for the future development of a kit for detecting uric acid in saliva from humans.