Commutability Of Reference Materials And Performance Of Methodsfor Aminotransferase Measurement Feasibility Of Measuring Aminotransferase Activity Byliquid Chromatography Mass Spectrometry

Objectives:We aimed toevaluate the commutability of several materials including external quality assessment scheme(EQAS)materials,RELA-IFCC materials,human serum pools(HSPs),bovine serum albumin(BSA)solution,swine sera,commercial calibrators and controls.Meanwhile,weestimated the biases of 2 routine assays withpyridoxal-5-phosphate(P-5'-P)from IFCC measurement reference procedures(RMPs)and 11 assays without P-5'-Pfroman average value of routine assays for serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activity measurements.Methods:The commutability assessment and method comparison were performed according to the clinical and laboratory standards institute(CLSI)EP14-A3 protocol and the CLSI EP9-A3,respectively.We analyzedpatient samples(PS)andprocessed materials foraminotransferase activity measurement byIFCC RMPs,11 routine assays withoutP-5'-Pand 2 assays with.Measurement results without P-5'-P of the patient samples were pairwise analyzed by Deming regression for slopes and intercepts,and 95%prediction interval(PI)were calculated to evaluate the commutability of processed materials.And the ordinary linear regression(OLR)and 95%PI were used to identify the commutability of processed materials on the measurement results with P-5'-P.The bias estimated on PS from IFCC RMPs or average values were analyzed for 13 routine assays.The criterions used toevaluate the accuracy of 13 routine assays were the specifications derived from biological variation.Results:The Deming slopes ranged from 0.879 to 1.058 and intercepts from-1.95 to 3.30 for ALT activity measurement;the OLR slopes were 0.905 and 0.994 as well as intercepts were-6.71 and 8.00.For AST activity measurement,the Deming slopes varied from 0.817 to 1.136 and intercepts from-2.80 to 2.02;the OLR slopes were 0,891 and 0.855 as well as intercepts were 1.54 and 2.23.The EQAS materials,RELA materials,commercial calibrators and controls showed commutability on 10%to 84%pairs,0 to 33%pairs and 19%to 81%pairs,respectively.The customized HSPs,processed HSPs1,processed HSPs2,processed HSPs3,BSA solution and swine sera were commutable on 0 to 76%pairs,24%to 100%pairs,5%to 95%,14%to 95%,19%to 76%,and 37%to 75%pairs,respectively.For ALT measurement results,Ortho,Abbott,Beckman,Biosino,BSBE,MACCURA and Siemens Advia shouwed positive bias;Siemens Dimension,KHB,Leadman and Roche showed negative bias.For AST measurement results,Beckman,Biosino,DiasSys,Leadman and Siemens Adviashouwed positive bias;Ortho,Siemens Dimension,Abbott,BSBE,KHB,Roche and Wako showed negative bias.Eleven assays for ALT measurement could meet the desirable specification(11.5%),and 10 assays for AST measurement could meet the minimum specification(9.9%).Conclusions:The comparability of most routine assays is well.Poor commutability of EQAS materials,commercial calibrators and controls adopted in China has been observed among current ALT and AST assays.Human serum base supplemented with human original recombinase is considered as a potential source of EQAS materials with desirable commutability.Objectives:We aim to explore a method measuringserumglutamic acid(Glu)inaminotransferase(ALT and AST)substrate mixture using liquid chromatography mass spectrometry(LC-MS/MS)for developing a new procedure of measurement of serum aminotransferase activity.Methods:We combined LC fraction collection and LC-MS/MSdetection to establish the method.Glu-13C5was used as the isotope internal standardand the mass of samples,standards and isotope internal standard were weight accurately.Serum combined with aminotransferase substrate mixturewas extracted with methanol containing 0.1%formic acid and Hexane,then reconstituted with the mobile phase.The reconstituted samples were purified by the LC fraction collection and analyzed by the LC-MS/MS.Linearity,limits of detection,limits of quantification,accuracy,imprecision and sample recovery were evaluated for method validation.Results:The LC fraction collection was performed with a Hypercarbcolumn.The mobile phase consisted of an watert/methanol(98:2.V/V)mixtureincluding 0.05%trifluoroacetic acid.The flow rate was run at 1000?L/minand the injection volume was 20?L.The optimal fraction timetable for ALT mixture was 3.3-3.7 min and for AST mixture was 3.6-4.0 min.Then,the fractionwasinjected onto a RP18 column and eluted with a mobile phase of an water/methanol(98:2,V/V)mixtureincluding 0.02%acetic acid at a flow rate of 150?L/min.The electrospray ionizationmass spectrometer was operated in the positive ionmodeon the API 4000in the multiple reaction monitoringmode.Thetotal chromatographic run time was 4.5 min for ALT fraction and 6 min for AST fraction.The regression coefficient of calibration curve for ALT mixture varied 0.9990-0.9998.and for AST mixture varied 0.9530-0.9942.The bias of middle and high level Sigma standardswas below 5%for ALT mixture,but varied dramatically for AST mixture.The within-runCVs was less than3%,and the inter-day CVswas less than10%for ALT mixture;the both CVs also varied dramatically for AST mixture.Theanalytical recoveries of added Glu ranged from 89.0%to 111.9%for ALT mixture and from 77.9%to 183.1%for AST mixture.Conclusions:Wedeveloped a methodmeasuring glutamic acid in human serum combined with aminotransferase substrate mixture using LC-MS/MS.The methodvalidation has beencomplemented.The results for ALT mixture was satisified but it remained problems for AST mixture.Subsequent research for establishing the new procedure of measurement of serum aminotransferase activity should follow on.