血浆中甲基丙二酸的定量分析检测方案(液质联用仪)

2 3 Quantitative Analysis of MethylmalonicAcid in Plasma Using SOLAu Plates forSample Preparation and a TSQ EnduraTriple Quadrupole Mass Spectrometerfor Clinical Research Mindy Gao， Jon Bardsley， James Goldberg， Marta KozakThermo Fisher Scientific， San Jose， CA MMA， plasma， triple quadrupole mass spectrometer， TSQ Endura， SOLAp To demonstrate a sensitive， robust， reliable quantitative analysis ofmethylmalonic acid in plasma for clinical research. Application Benefits ·Wide quantitative analytical range from 25 to 100，000 nM Simple， economical， easily automated sample preparation method · Confident analyte identification with ion ratio confirmation · Robust method without matrix effects ·Can be implemented on dual- and four-channel LC systems Introduction Liquid chromatography-mass spectrometry (LC-MS)analytical methods are widely used for quantitation ofmethylmalonic acid (MMA) in clinical researchlaboratories. The quantitation of MMA poses severalchallenges， including (i) the need for a simple samplepreparation method； (ii) achievement of low limits ofquantitation； and (iii) separation from the naturallyoccurring structural isomer succinic acid. Here we presentan analytical method that successfully addresses all ofthese requirements. Methods Calibrators and Quality Controls Because it is difficult to obtain MMA-free plasma，calibration standards in the range of 25 to 100，000 nMwere prepared in 2% acetonitrile. Pooled plasmacontaining MMA at 110 nM was used as Level 0 QualityControl. Level 1 (260nM) and Level 2 (583 nM) QualityControls were purchased from RECIPEChemicals andInstruments GmbH (ClinChekcontrol kit P/N MS5082). Sample Preparation Plasma samples， calibrators， and QCs (all 100 pL aliquots)spiked with internal standard (d，-MMA) were processedby solid phase extraction using Thermo ScientificSOLAp WAX 96-well plates (P/N 60209-005).Analytes were eluted from the extraction plate with70 pL of 1% ammonium hydroxide， and the eluent wasneutralized with 30 pL of 10% formic acid. A 15 pLaliquot of eluent was analyzed by LC-MS. Liquid Chromatography A 3.5-minute chromatographic method with aThermo Scientific"Accucore"RP-MS column (2.6 pm，100 x2.1 mm， P/N 17626-102130) at room temperaturewas performed using a Thermo Scientific"DionexUltiMate""3000RS liquid chromatography pump withOAS autosampler.Mobile phases consisted of 0.4%formic acid in water and 0.1% formic acid in methanol(Fisher Chemical Optimagrade) for phases A and B，respectively. This chromatographic method separatedMMA from its endogenous structural isomer， succinic acid. Mass Spectrometry Compounds were detected on a Thermo ScientificTSQ Enduratriple quadrupole mass spectrometerequipped with an Ion Max"source and a heatedelectrospray (HESI) sprayer operated in negative ionizationmode. Two SRM transitions each for MMA and itsinternal standard were monitored for quantitation andconfirmation (Table 1). Table 1. SRM transitions acquisition parameters. Analyte Precursor(m/z) Product(m/Z) CollisionEnergy (V) RFLens () lonFunction MMA 117.1 73.3 10 79 Quantifier MMA 117.1 55.3 25 79 Qualifier MMA-D. 120.1 76.3 10 79 Quantifier MMA-D_ 120.1 58.3 25 79 Qualifier Method Performance Evaluation The limits of quantitation (LOQ) and linearity rangeswere determined by collecting calibration curve data.Method precision was evaluated by running replicatecalibration curve standards and QCs on three differentdays. Method accuracy was assessed by calculating%recovery for QC samples and for ten donor plasmasamples spiked with 200 nM of MMA. Matrix effectswere evaluated by spiking internal standard intoSPE-processed plasma samples from eight different donorsaQYnd calculating recovery against the same internal standardamount spiked into SPE-processed water. Data Analysis Data were acquired and processed usingThermo Scientific"TraceFindersoftware. The averageion ratios calculated for analyte confirmation were about4% and were within the allowed 50% (relative) ofaverage calculated from the calibrators. Results and Discussion Limits of quantitation were defined as the lowestconcentrations that had back-calculated values within20% and ion ratio within the specified range. Using thesecriteria， the limit of quantitation for MMA was 25 nM.The upper limit of the calibration curve was 100，000 nM.Figure 1 shows a representative calibration curve， alongwith quantifying and qualifying ion chromatograms forthe lowest calibration standard. Quadruplicate calibrationstandards'precision was better than 5%， and accuracywas within ±9%. Figure 1. Representative calibration curve. Method accuracy calculated as % recovery of QC andspiked donor samples ranged from 90% to 104% andfrom 96.8% to 107% for QCs and spiked donor samples，respectively. This shows that the use of the surrogatematrix for the calibration curve produces accurate resultsfor samples in plasma matrix. Intra-assay precision for all C levels was better than5.1%(Table2)， and inter-assay precision was better than6.4% (Table 3). Figure 2 presents chromatograms ofquantifying and qualifying ions in plasma QC samplesand also shows the chromatographic separation of thestructural isomer， succinic acid. Table 2. Intra-assay precision and accuracy. 0C Level 0 QC Level 1 QC Level2 Concentration (nM) 110 260 583 Precision (%RSD) <5.1 <2.0 <0.9 Accuracy (average %recovery) 89.7-101 91.8-95.3 96.9-104 Table 3. Inter-assay precision and accuracy. QC Level 0 QC Level 1 QC Level 2 Concentration (nM) 110 260 583 Precision (%RSD) 6.4 2.2 3.5 Accuracy (average %recovery) 96.7 93.1 99.7 Matrix effects were not observed. Recovery in eight donorsamples， calculated as the ratio between internal standardpeak area in matrix and internal standard peak area inneat solution， was within method analytical error andranged from 89.7% to 107%. Conclusion We demonstrated a sensitive and robust method forquantifying MMA in plasma that provides a wide dynamicrange， while employing simple and economical samplepreparation. With the short run time and data acquisitionwindow， this analytical method can be put into use on a4-channel Thermo ScientificTranscend"II LC system toaddress high-throughput analysis (50 samples/hour)requirement in clinical research. For research use only. Not for use in diagnostic procedures. To find a local representative， visit：thermofisher.com ( C2016 Th e rmo Fisher Sci e ntif i c In c . A ll r ights reserved. ClinCh e k i s a trademark of RECIP E C hemica l s + I n s truments GmbH.All other t rademarks are the pr o perty o f Thermo Fish e r Scie n tif i c In c . and its subsidiaries. Thi s info r mation is pr e s e nted as an example o f the capabilities of Thermo Fisher Scientific Inc. products. I t is n o t intended t o encourage use o f these products in a ny manners that might in f ringe the int e llectual property rig h ts of o th e rs. Spe c ifications， terms and p rici n g are s u bje c t to chang e . N ot a l l p roducts ar e av a ilable in a ll c ou ntries. Pl ea se cons u lt your l ocal s al e s repres e n tative for d etails. ) Figure . Quantifying and qualifying ions chromatographic peaks in QC samples. AThermo Fisher Scientific Brand To demonstrate a sensitive, robust, reliable quantitative analysis of methylmalonic acid in plasma for clinical research.Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for quantitation of methylmalonic acid (MMA) in clinical research laboratories. The quantitation of MMA poses several challenges, including (i) the need for a simple sample preparation method; (ii) achievement of low limits of quantitation; and (iii) separation from the naturally occurring structural isomer succinic acid. Here we present an analytical method that successfully addresses all of these requirements.We demonstrated a sensitive and robust method for quantifying MMA in plasma that provides a wide dynamic range, while employing simple and economical sample preparation. With the short run time and data acquisition window, this analytical method can be put into use on a 4-channel Thermo Scientific™ Transcend™ II LC system to address high-throughput analysis (50 samples/hour) requirement in clinical research.