| Background:Folate is a kind of B-vitamins and not active itself. Folate is catalytically converted to dihydrofolate and tetrahydrofolic acid by dihydrofolate reductase in vivo. Retrahydrofolic It acid is the active form of folate. As a carrier of one carbon unit, it involves in one-carbon metabolism and the synthesis of methyltetrahydrofolate, methylene tetrahydrofolate,anhydroleucovorin and leucovorin. Folate plays an important role in the progress of single nucleotide synthesis by the four kind of one carbon unit mentioned above and provides methyl for the methylation of protein nucleic acid and so on. Therefore, folate is closely related to nucleic acid metabolism and regulation, and energy metabolism and regulation.Epidemiological investigation and experimental researches show that folate deficiency and folate dysmetabolism contribute to multiple severe diseases including birth defects,neurological mental disease, cardiovascular disease, cancer, ect. At present, the correlation between folate and human health and the pathogenesis of folate deficiency remain major projects of basic science and clinical science.Establishing an accurate, efficient and convenient method for the determination of folate and its related metabolites is an important basic mean for studying folate. So far,there are a variety of folate detection methods with no unified standard such as colorimetric method, thin-layer chromatography(TLC), capillary electrophoresis(CE),microbiological test, radioactive isotopes immune analysis(RIA), gas chromatographytandem mass spectrometry(GC-MS/MS), high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS),etc. The disadvantages of these methods are time consuming, large sample volume, high cost, poor sensitivity, etc. So they can not meet the needs for scientific research and clinical application.Aiming at these defects of above methods, we try to established an efficient,convenient, sensitive and broad scope detection method for the determination of folate and its related metabolites by using ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Objectives:To investigate the feasibility and detecting condition of determining folate and its related metabolites in serum and tissue by UPLC-MS/MS. Further optimize and establish a new methods for the determination of folate and its related metabolites.Methods:1.Using standard substances of folate and its related metabolites to study and establish UPLC-MS/MS analysis conditions and parameters for these standard substances. The effect of three kinds of hydrophilic chromatographic column separating folate and its related metabolites were further compared. And the separating effect of folate and its related metabolites under different chromatographic conditions(mobile phase, elution mode,velocity and column temperature) and different mass spectrometer conditions(Ion source,positive and negative ions mode, test mode, the temperature of the ion source, ion injection voltage, collision and atomizing gas flow) were studied.2.Using normal human serum and tissue to investigate sample processing conditions.Comparing the advantages and disadvantages of proposed method and traditional method.Using different organic solvent(methanol, acetonitrile, ethyl acetate) to precipitate proteins in the sample and observing its effect of protein deposition. Observing the protective effect of different concentrations of antioxidants for folate and its related metabolites. The linear,detection and quantitative limit, RSD for the inter – day and intra – day and recovery rate stability of this method were verified by serum standard panel with different concentrations.3.Verification test were carried out using fifty normal cases and NTDs large samples to establish measurement standard and condition parameter of UPLC-MS/MS in ourresearch.Animal tissue was used to study the universality of this method.Results:1.Experimental result of the standard panel showed that: working fluid was made of acetonitrile and water in the proportion of 1:9. Mobile phase: A. 0.1% methanoic acid, B.acetonitrile. Gradient elution program: 0~1 min, 99.9%A~99.9%A; 1~3 min, 99.9%A~82%A; 3~4 min, 82%A~10%A; 4~5 min, 10%A~10%A; 5~6 min, 10%A~99.9 % A. Equilibration time: 1min, total analysis time: 7 min. Flow rate: 0.4 m L/min,column temperature: 40 ℃, the sample quantity: 5 μL, electrospray ion source, positive ion(ESI+) model, multiple reaction monitoring mode, capillary voltage: 3.0 KV, ion source temperature: 150 ℃, the temperature of the solvent. The effect of separating folate and its related metabolites were excellent under the above experiment condition and the sensitivity is 99.9%. The comparison results showed that Acquity UPLC BEH C18 column(Waters company, USA, 2.1 mm×50 mm,1.7 μm) had the best separating effect.2.Experimental result using standard substance with serum showed that samples treated with methanol had the best sedimentation effect. The antioxidant system made of100 μg/m L ascorbic acid, 100 μg/m L citric acid and 15 mg/m L DTT can effectively maintain the stability of folate and its related metabolites.3.Under the optimized condition of chromatography and mass spectrometry, the retention time of six kinds of compounds are within 3 min, with good peak shape and without the interference of impurity peak, smooth baseline. The linear range of six compounds are 10 ~ 500 ng/m L, 2 ~ 100 ng/m L, 0.5 ~ 50 ng/m L, 50-2000 ng/m L, 4 to 200ng/m L, 4 to 200 ng/m L respectively. The correlation coefficient(R2) was more than 0.998.The corresponding detection limit and the quantitative limit were 0.05 and 0.20 ng/m L,0.20 and 0.05 ng/m L, 0.20 and 0.05 ng/m L, 0.05 and 0.50 ng/m L, 0.20 and 0.50 ng/m L,0.20 and 0.50 ng/m L. The RSD of intraday precision and interday precision are less than5.84%. The standard addition recovery was 90.1% ~ 105.3% with good stability.4.Folate and its related six compounds in the pregnant women serum of normal group and NTDs group showed that, compared with normal group, serum SAM levels in NTDsgroup had no significant change(P > 0.05), 5- Me THF and 5- Fo THF levels were significantly decreased(P < 0.05), Hcy and SAH concentration significantly increased(P <0.05), while methylation index(SAM: SAH) reduced.Folate and its related metabolites in fetal brain tissue of two groups showed that compared with normal group, fetal brain tissue folate, 5-Me THF, 5-Fo THF, SAM levels of NTDs group were decreased(P < 0.05), while Hcy and SAH concentration were increased(P < 0.05).Conclusions:This research successfully established the UPLC-MS/MS for the determination of folate, 5-Me THF, 5- Fo THF, Hcy, SAM and SAH for the first time and verified this method using NTDs pregnancy serum. This method was characterized by less sample(5μL), time saving(7 min), low cost, high sensitivity and extensive application scope and can be used for testing large scientific research and clinical samples. |
PDF Full Text Request