| Objective:To establish an analytical method for methylene diphenyl diisocyanate(MDI)hemoglobin adducts in blood by using ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Methods:1.Blood sample collection and globin isolation according to the method in Gries W’study.2.4,4’-MDA-Hb adduct,AcMDA-Hb adduct and ABP-Val-Hyd-Hb adduct were hydrolyzed as follows respectively.(1)4,4’-MDA Hydrolysis:The 20mg hemoglobin sample was weighed into a 15mL centrifuge tube,hydrolyzed with 1.0mL 2M hydrochloric acid solution at 100℃ for 12 hours.(2)AcMDA Hydrolysis:The 20mg hemoglobin sample was weighed into a 15mL centrifuge tube,adding 20μg/L internal standard solution AcMDA-D8 10μL,then hydrolyzed with 1mL 0.1M sodium hydroxide solution at 37℃ for 0.5 hours.(3)ABP-Val-Hyd Hydrolysis:The 20mg hemoglobin sample was weighed into a 15mL centrifuge tube,adding 20μg/L internal standard solution ABP-Val-Hyd-D8 10μL,then hydrolyzed with 1.0mL 2M hydrochloric acid solution at 100℃ for 2 hours.3.The above hydrolysate was extracted twice with dichloromethane and concentrated by vacuum concentrator.The residue was dissolved in acetonitrile and separated by liquid chromatography,detected under the conditions of electrospray ionization positive ion mode(ESI+)and multi reaction monitoring(MRM).4.The working curve method was used for 4,4’-MDA quantitative detection,Isotope internal standard curve method was used for AcMDA and ABP-Val-Hyd quantitative detection.Results:1.This study established a method for the three hemoglobin adducts of MDI determination in blood by UPLC-MS/MS.(1)Method performance of 4,4’-MDA:The linearity range of 4,4’-MDA is 0.01 μg/L～25.00μg/L with a correlation coefficient of 0.9998,the LOD and LOQ were 0.13ng/gHb(0.656pmol/g)and 0.44ng/gHb(2.219pmol/g),respectively.The recovery was ranged from 97.0%～105.2%;the RSD of intra-and inter-batch precision were 6.1%-8.4%and 6.8%～9.2%,respectively.The 4,4’-MDA-Hb adduct can be stored for 1 month at 4～6℃.(2)Method performance of AcMDA:The linearity range of AcMDA is 0.01μg/L～25.00μg/L with a correlation coefficient of 0.9996,the LOD and LOQ were 0.07ng/gHb(0.291pmol/g)and 0.23ng/gHb(0.957pmol/g),respectively.The recovery was ranged from 95.3%～97.6%;the RSD of intra-and inter-batch precision were 6.2%～7.0%and 7.9%～8.0%,respectively.The AcMDA-Hb adduct can be stored for 2 months at 4～6℃.(3)Method performance of ABP-Val-Hyd:The linearity range of ABP-Val-Hyd is 0.05μg/L～20.00μg/L with a correlation coefficient of 0.9995,the LOD and LOQ were 0.39ng/gHb(1.205pmol/g)and 1.29ng/gHb(3.989pmol/g),respectively.The recovery was ranged from 94.9%～97.8%;the RSD of intra-and inter-batch precision were 4.6%～6.2%and 4.2%～5.8%,respectively.The ABP-Val-Hyd-Hb adduct can be stored for 4 months at 4～6℃.The coexisting interferences in the field and the matrix components in the sample do not interfere with the three hemoglobin adducts determination under the selected experimental conditions.2.Using the detection method to test 16 blood samples from general population over 45 years old.The concentration of 4,4’-MDA in five people was between(<0.13ng/gHb)～0.63ng/gHb,one of which detected AcMDA was 0.09ng/gHb.ABP-Val-Hyd was not detected(<0.39ng/gHb)in 16 blood samplesConclusion:This study established a method for the three hemoglobin adducts of MDI determination in blood by UPLC-MS/MS.Each performance index of the method can meet the requirements of the Guide for establishing occupational health standards-part 5:Determination methods of chemicals in biological materials(GBZ/T 210.5-2008).This method is suitable for the determination of three kinds of MDI hemoglobin adducts by application in general population. |
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