Establishment And Application Of Candidate Reference Method For Glycocholic Acid

Bile acids(BAs)are a series of water-soluble steriods,which are divided into free bile acids and conjugated bile acids,making a difference on keeping human health through enterohepatic circulatory system.As a conjugate of cholic acid and glycine,glycocholic acid(GCA)is of great significance in the clinical diagnosis and disease observation for some diseases,such as cholangiocarcinoma(CCA),liver cirrhosis(LC),benign biliary tract disease(BBD),and pancreatic cancer(PC).Currrently,many methods were used for GCA determination.Regrettably,differences caused by different detection methods or different laboratories may be found of the same specimen,which brings certain obstacles for the accurate,reliable and comparable results,and then directly affecting the diagnosis and treatment of the disease.Hence,in order to achieve the comparability and accuracy test results of GCA,it is imperative to carry out standardization research.The core of standardization research is to realize the traceability of measurement results,and the establishment of reference system is the necessary condition to carry out the traceability chain of metrological traceability.Tracing to the international system of units(SI)is an ideal situation of metrological traceability,many means were usd for metrological traceability:(1)Sending to qualified identification or calibration institutions for verification and calibration directly.(2)The establishment of corresponding reference methods in interlaboratory.(3)The use of certified reference material(CRM).(4)The traceability of recognized physical standard or proficiency testing should be carried out when tracing to the national standard is impossible or not applicable.However,there is no internationally agreed GCA reference measurement procedure(reference method)and certified reference material,temporatily.Therefore,the establishment of accurate and reliable reference method is an effective way to achieve traceability.In view of this phenomenon,an advanced isotope dilution liquid chromatography-tandem mass spectrometry(ID-LC-MS/MS)technique was applied to develop a CRM for GCA.The mass spectrometry conditions,chromatographic conditions and pretreatment conditions were optimized.The performance evaluation of the method was performed.At the same time,the established routine reference method was used to compare and evaluate the current routine detection methods,aiming to provide basis and reference for the standardization of GCA test results.ObjectiveTo optimize the liquid chromatography(LC)conditions,mass spectrometry(MS)conditions and sample preparation conditions of the determination of serum GCA using the ID-LC-MS/MS method,for the establishment of the accurate and precious candidate reference method for GCA.In addition,to evaluate the analysis performance of the established method whether meet the requirement of reference method.MethodsSpecial amount of GCA and GCA-d4 were added to the charcoal stripped serum.The Waters TQ-S triple quadrupole mass spectrometer was used to establish the candidate reference method of GCA,using the ID-LC-MS/MS.The liquid chromatography(LC)conditions,mass spectrometry(MS)conditions and sample preparation conditions were evealuated and optimized.Performance evaluation for GCA candidate reference method:precision,linearity and sensitivity,sepecificity,matrix effect,carryover,stability and uncertainty,according to the CLIS documents.Finally,comparation was carried out between the established method and conventional detection methods.ResultsMS condition:The tests were analyzed by ID-LC-MS/MS system in the negative ions and detected in the multiple-reaction monitoring(MRM)mode,using the specific transitions m/z 464.4?74.0 and 468.4?74.0,respectively.LC condition:C18 column(ACQUITY UPLC(?)BEH,1.7 ?m,100 mm× 2.1 mm)was used in this study.The mobile phase used in the chromatographic separation consisted of a binary mixture of eluent A(0.2 mmol/L ammonium acetate containing 0.5%formic acid in water)and eluent B(acetonitrile containing 0.5%formic acid).The total run time of this test was 5min.Pretreat condition:Serum samples were extracted with protein precipitation before analysis.The mean extraction efficiency for the method was determined to be 76.1±2.3%.No obvious ion expression,interference,matrix effect and carry-over were observed.Method performance:Excellent linearity coefficients(R2=1.000)were obtained in the wide concentration range from 0.039 to 40 ? g/mL.The lowest limit of detection and(LLOD)and lowest limit of quantification(LLOQ)for developed RMP was 0.01 ng/mL and 0.05 ng/mL,respectively.The intraassay CVs were 1.39%,1.01%,and 0.46%for low,medium,and high level concentration samples,respectively;and the interassay CVs were determined to be 2.01%,1.24%,and 0.84%,respectively.The total imprecisions were below 2.14%for three levels of samples.Good recoveries were achieved at five spiked levels(98.0-100.9%).Samples can be stored at differernt storage conditions over different periods,suggesting the samples were relative satable.The uncertainty of the candidate reference method was within the acceptable range,which meets the evaluation requirements.Method application:The test results between the current conventional method and the candidate reference method are less comparable,and both have positive bias.The establishment of the reference method can increase the credibility of the GCA test result to some extent.Conclus ionThe well characterized method displayed good accuracy and definitive uncertainty,which linearity,sensitivity,precision,and specificity of the method were fully validated.No interference,matrix effect,and carry-over were observed.Altogether,the described method can be used as a candidate reference method for GCA measurement to provide serum value assignments for calibration and verification of method performance.