Quantitative Analysis Of Estrogen In Human Serum By Chemical Derivatization Assisted High Performance Liquid Chromatography-Tandem Mass Spectrometry

Estrone(E1),estradiol(E2)and estriol(E3)are the most common human endogenous estrogens,and they have important effects on human physiological metabolism.However,they are present at low levels in humans and there is a large matrix interference in the serum.These factors make it difficult to detect estrogen.With the advantages of high sensitivity and accuracy,mass spectrometry technology has become the preferred method for detecting estrogen.However,estrogens have weak acid nature,poor ionization efficiency,result in a poor mass spectrometric response.Therefore,derivatization assisted high performance liquid chromatography-tandem mass spectrometry method is needed for quantitative analysis.This paper compared and exploresed 5 derivatization reagents,selected the best derivatization reagent 3-methyl-8-quinoline sulfonyl chloride to establish a chemical derivatization assisted high performance liquid chromatography-tandem mass spectrometry method for detecting human serum estrogen.It is a new quantitative method of estrogens,which has not been reported in the literature.This method has the characteristics of simple derivation process,high detection sensitivity and accurate quantitative results.Finally,the method applied to clinical detection of estrogen in human serum,and can provide a new method for clinical mass spectrometry detection of endocrine diseases.The main research contents were as follows:Part 1:Comparison and exploration of estrogen derivatization methodsA variety of derivatized reagents have been reported for estrogen detection,but there is a wide range of options create confusion for investigators.It can be selected an optimal reagent for particular experiment,by systematically comparing the advantages and disadvantages of these derivatization reagents the best reagent can be selected for a specific experiment.This thesis compared the derivatization reagent dansyl chloride(DNSCl),1,2-methylimidazole-5-sulfonyl chloride(DMISCl),which have been reported,and the derivatization reagent 3-methyl-8-quinolinesulfonyl chloride(MQSCl),quinoline-8-sulfonyl chloride(QSCl),1-methyl-1H-pyrazole-4-sulfonyl chloride(MPy SCl)that have not been reported for estrogens detection yet.Then calculated the potential ionization efficiency of these 5 derivatization reagents,which were in good agreement with the experimental results.In the experiment,derivatization reaction conditions were optimized,including p H,buffer solution acetone/water volume ratio,reaction temperature,reaction time,derivatization reagent concentration and mass spectrometry parameters.The derivatization efficiency of the five derivatization reagents>98.5%.The derivatized product had good stability within24 hours.With the analysis of the secondary mass spectra,the fragmentation mode of each derivative was explored.After comparing the peak area and sensitivity of the five derivative products,it was found that 3-methyl-8-quinolinesulfonyl chloride has the highest signal intensity and sensitivity.Part Ⅱ:Establishment of a method for quantitative analysis of estrogen by chemical derivatization assisted high performance liquid chromatography-tandem mass spectrometryThis work established a 3-methyl-8-quinolinesulfonyl chloride derivatization assisted high performance liquid chromatography-tandem mass spectrometry method for the quantitative analysis of estrogen,and the prescription was verified.After the derivatization reaction,the extraction solvent and chromatographic conditions were optimized,ethyl acetate was selected as the extraction solvent,and then the standard curve was established by the isotope internal standard method,r~2 were in 0.9919-0.9939,the quantification limits of E1,E2 and E3 were 2.7,4.6 and 5.1 pg/m L,respectively.The intraday precision of the derived accuracy and the inter-day precision were both within 13.2%,the recovery rates of the standard addition were between92.5-114.3%,and the matrix effect were between 91.5–122.9%,indicated that the method had a good accuracy and high precision.The serum samples were repeatedly frozen and thawed,and the stability of the derived products during the 7-day freeze-thaw cycle was lower than 14.4%,which demonstratesd estrogens in biological samples and the estrogens detection method were good stability.The chemical derivatization high performance liquid chromatography-tandem mass spectrometry estrogen analysis method established above was applied to clinical serum samples,serum samples were from 12 healthy adult women,7 healthy girls and 10 precocious girls.The serum estrogen concentrations of 12 adult women were estrone(E1)0.059-0.199 ng/m L,estradiol(E2)0.013-0.130 ng/m L,and estriol(E3)0.023-0.069 ng/m L.The content of detected E2 was close to the reference range reported in the literature,and the contents of detected E1 and E3 were within the reference range provided by Quest Diagnostics.For prepuberty(6-8 years old)compared estrogen in healthy girls with estradiol(E2)in samples with confirmed precocious puberty.The concentration of E2 in samples of healthy girls was 0.038±0.010 ng/m L,while the concentration of E2 in precocious puberty was 0.140±0.066 ng/m L.The E2 concentration of the two groups of samples showed significant differences,indicating that there was a relationship between E2 and disease,and it provided basic experimental data for children with precocious puberty to monitor the estrogens content in their bodies.