| Objective:1.To establish a novel high performance liquid chromatography tandem mass spectrometric(HPLC-MS/MS)method and validated for the determination of four water-soluble vitamins(B1,B2,B6 and B9)in human serum.2.To evaluate the correlation and concordance of the results between the determinations of HPLC-MS/MS and Electrochemical(EC)method clinically used for routine vitamin determination in patients.Methods:1.HPLC-MS/MS method:Vitamin B1,B2,B6 and B9 in serum samples of patients from Qilu Hospital were extracted by liquid-liquid extraction with ethyl acetate.The chromatographic column was a Venusil XBP-C18 column(150×4.6 mm,3?m,100A),eluted with the mobile phase of 5 mM ammonium acetate:methanol(40:60,v/v)at a flow rate of 0.5 mL/min with a thermostated column oven 30?.The source parameters were as follows:spray gas(nitrogen)temperature 350?,spray gas flow 9 L/min,nebulizer pressure 40 psi,capillary voltage 4000 v.The fragment voltage of vitamin B1,B2,B6,B9 and IS were 100,140,100,110 and 130V,while the collision energy were 8,25,11,10 and 25 eV,respectively.Multiple reaction monitoring(MRM)mode was used for detect ion of vitamin B1,B2,B6,B9 and IS,with ion transition of m/z 265?122.1,377?244,170?152,442.2?295.8,195.1?137.8,respectively.The IS ion pairs were detected during 4-6 min,and vitamin B1,B2,B6,B9 ion pairs were detected during 0-4 min while applied with delta EMV 200.2.EC method:Determining vitamin B1,B2,B6 and B9 concentration of each serum sample also by EC method(LK3000V vitamin detector).The appropriate volume of serum was added into the sample processing solution,then were determined after the electrode processingand It was noted that the determination of vitamin B9 still needs electrode activation before electrode processing.3.A comparison between the HPLC-MS/MS and EC method was performed using patient serum samples obtained from those patients who were suffering from vitamin-related diseases.Statistics were performed utilizing SPSS 19 and MedCalc 16.2.0 software.HPLC-MS/MS results as X-axis and EC method results as Y-axis,a linear regression model was established and a regression analyses was performed with these data for the evaluation of correlation.Concordance was depicted by The Bland-Altman deviation graphs of vitamin B1,B2,B6 and B9.Results:1.HPLC-MS/MS was successfully established for the determinations of vitamin B1,B2,B6 and B9 in human serum.The calibration curves for vitamin B1,B2,B6,and B9 were linear within the range of 1-200ng/mL,5-40ng/mL 1-80ng/mL,5-40ng/mL(with r2=0.9916,0.9968,0.9922 and 0.991),the lower limits of quantitation were 1 ng/mL,5 ng/mL,1 ng/mL and 5 ng/mL,and the inter-or intra-day RSDs were less than 8.5%,respectively.The recoveries of vitamin B1,B2,B6,B9 and IS were acceptable and reproducible.No significant matrix effects were observed.The mean recoveries and matrix effects were 29.01%and 71.82%for vitamin B1,69.39%and 69.94%for vitamin B2,63.22%and 70.91%for vitamin B6,26.21%and 61.20%for vitamin B9 and 63.79%and 81.42%for IS,respectively.Stabilities were tested via three levels of QC in five replicates at different storing or processing conditions,i.e.storing at-20? for 7 days,freezing(-20?)and thawing(24±2?)for one cycle,and storing on the bench for 6h or in the auto-sampler for 20 h at room temperature.The accuracy was ranged from 89.8%to 107.7%and RSD was less than 10%.2.Vitamin B1,B2,B6 and B9 in 160 patient samples were determined by EC and HPLC-MS/MS methods.The mean concentrations of vitamins in patient serum obtained by EC were 21.44±5.26 for vitamin B1,6.50±0.80 for vitamin B2,12.36±3.54 for vitamin B6 and 5.59±1.49 for vitamin B9.3.A comparison between the HPLC-MS/MS and EC methods was performed using patient serum samples.Results of the Deming regression analysis demonstratedthat both methods were good correlated with square correlations(r2)of 0.965(B1:y=1.145x-2.741,n=160),0.965(B2:y=0.923x+1.091,n=160),0.956(B6:y=1.006x+0.391,n=160)and 0.901(B9:y=1.084x-2.241,n=160)respectively.Both methods were in good agreement,indicating the results were valid and repeatable.The Bland-Altman deviation graphs of vitamin B1,B2,B6 and B9.The 95%LOA for vitamin B1,B2,B6 and B9 were-2.00-2.70,-0.29?1.00,-1.70-2.60,-2.12?-1.15 ng/mL.No systematic bias for the differences and the magnitudes of vitamin B1,B2,B6 and B9 concentrations between the two measurements were observed.Conclusions:1.The established HPLC-MS/MS method was sensitive,accurate and suitable for determining water-soluble vitamins B1,B2,B6 and B9 in human serum2.EC method was rapid,convenient and sensitive for the determination of vitamins B1,B2,B6 and B9 in human serum.It was suitable for routine monitoring concentration of vitamins B1,B2,B6 and B9 in patient serum.3.It was a good correlation and a good agreemen with the data of vitamins B1,B2,B6 and B9 concentration results in human serum between HPLC-MS/MS method and EC method..In our study,a novel HPLC-MS/MS method was developed and validated for the determination of vitamin B1,B2,B6 and B9 in human serum and EC assay was also validated with the gold standard HPLC-MS/MS method.Therefore,the EC assay was successfully applied to our hospital for TDM of vitamin in patient serum. |
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