Methylmalonic Acid in Serum / Plasma / Urine
Contents
1. A a B m m t f ii 4 _ adh aque E r4 a J f R pa ITI RI i 3 J fi pe 3 1 ESCEA P Fi i N f An i Y ha UJ zu L 1 g 1 zl T T 1 Instruction Manual ClinMass LC MS MS Complete Kit advanced Methylmalonic Acid in Serum Plasma Urine MS5100 5 w For in vitro diagnostic use C IVDD 98 79 EC gt COSMO BIO CO LTD Inspiration for Life Science Contents 1 INTRODUCTION 1 1 Intended use 1 1 1 IVD symbols 1 2 Clinical background 1 3 General description of the analytical procedure COMPONENTS OF THE COMPLETE KIT AND ACCESSORIES 2 1 Ordering information 2 1 1 Safety information 2 1 2 Storage conditions and lifetime of kit components 2 1 3 Disposal of laboratory waste REQUIRED INSTRUMENTS OPERATION OF THE ANALYTICAL SYSTEM 4 1 Flushing of the LC system 4 2 Equilibration of the LC system 4 3 Starting the analytical system 4 3 1 LC parameters 4 3 2 MS MS parameters 4 4 Standby mode IMPLEMENTATION OF THE ANALYTICAL PROCEDURE 5 1 Collection and storage of samples 5 1 1 Serum and plasma 5 1 2 Urine 5 2 Sample preparation 5 2 1 Reconstitution of the lyophilised serum calibrators controls 5 2 2 Serum and plasma 5 2 2 1 Work flow 5 2 3 Urine 5 2 3 1 Work flow 5 3 LC MS MS analysis 5 3 1 Compound optimisation MS MS 5 3 2 Equilibration of the analytical system and test run 5 3 3 Calibration run 5 3 4 Accuracy control 5 3 5 Examp2. Acid in Serum Plasma Urine advanced Required instruments Using this test kit requires a LC system with tandem mass spectrometer LC MS MS and evaluation software Requirements for the tandem mass spectrometer The tandem mass spectrometer should be of comparable or higher sensitivity as the instrument described in section 4 3 2 Required LC modules e Autosampler e Binary HPLC gradient pump e Column heater e Degasser For sample preparation the following laboratory instruments are required e Pipettes pipette tips e Tabletop centrifuge e Vortex mixer Methylmalonic Acid in Serum Plasma Urine advanced Page 9 4 4 1 4 2 4 3 Operation of the analytical system Flushing of the LC system Connect the LC modules excluding the column with the outlet capillary directed into a safe waste container Set the HPLC pump at a flow rate of 1 ml min and flush the LC system with 10 ml Mobile Phase A B Mobile Phase A B 50 50 Thereafter connect the analytical column within the column heater When connecting the analytical column please make sure the flow direction follows the arrow marking on the column Also take care that the fittings used are appropriate to the column These should be custom prepared with a new fitting and the column In case of questions please contact RECIPE for detailed installation instructions Equilibration of the LC system After flushing the system see section 4 1
3. Auflage Band 1 TH Books Verlagsgesellschaft Frankfurt Main 2012 page 714 11 Norman E J Urinary Methylmalonic Acid Test May Have Greater Value than the Total Homocysteine Assay for Screening Elderly Individuals for Cobalamin Deficiency Clinical Chemistry 2004 50 8 1482 1483 Methylmalonic Acid in Serum Plasma Urine advanced Page 23 Troubleshooting Problem Possible Cause Corrective Measure Gradient profile cannot be generated Defective HPLC pump Air within the system Check the pumps Degas the mobile phases and flush and purge the HPLC system thoroughly Fluctuation of the flow rate Check the pumps Interference signals Injection system contaminated e Rinse needle with acidified methanol or inject 10 x Mobile Phase B e Check flushport solvent level e Clean exchange needle seat assembly and or injection valve Sample vials contaminated Vial septum contaminated Use new vials Use another septum Mobile phase contaminated Change the mobile phases and flush the system Column s guard analytical column contaminated Change the guard analytical column Mass resolution too low Optimise mass resolution System not correctly configured Check all connections No signals Injector defect Check injector Defective HPLC pump Check the pumps MS MS system not ready for operation Check the MS MS system
4. The following coefficients of variation CV were obtained CV 96 Sample 1 5 76 Sample 2 6 08 Sample 3 3 47 7 2 Reference Ranges Plasma Serum 10 Urine 11 Normal range 73 271 nmol l 3 6 mmol mol creatinine The indicated reference ranges are taken from thoroughly selected and current scientific literature Their actuality corresponds to the printing date of this document Please note that these ranges do not reflect any recommendations by the manufacturer of this product but may be used as a guideline for the assessment of the reference range by the clinical laboratory Page 22 Methylmalonic Acid in Serum Plasma Urine advanced 8 References 1 2 3 4 5 6 7 8 9 W Herrmann R Obeid Ursachen und fr hzeitige Diagnostik von Vitamin B12 Mangel Deutsches rzteblatt 2008 108 40 680 685 Klee G G Cobalamin and Folate Evaluation Measurement of Methylmalonic Acid and Homocysteine vs Vitamin B12 and Folate Clinical Chemistry 2000 46 8 1277 1283 Refsum H Smith A D Ueland P M Nexo E Clarke R McPartlin J Johnston C Engbaek F Schneede J McPartlin C Scott J M Facts and Recommendations about Total Homocysteine Determinations An Expert Opinion Clinical Chemistry 2004 50 1 3 32 Rasmussen K Studies on Methylmalonic Acid in Humans Concentrations in Serum and Uri
5. lon Source Turbo ionspray TIS ESI Polarity negative Resolution Q1 and Q3 unit 0 7 amu Nebuliser Gas GS 1 60 GS 2 50 Curtain Gas CUR 35 Collision Gas CAD 5 lon Spray Voltage IS 1500 V Source Temperature TEM 550 C Interface Heater ihe ON Mass transitions see table 7 Table 7 Mass transitions API 4000 Precursor Product Dwell time DP CE CXP EP Substance amu amu ms V IV IV IV MMA Quantifier 116 9 73 100 35 12 3 10 MMA Qualifier 116 9 55 100 35 32 3 10 MMA d3 Quantifier 119 9 76 100 40 12 3 10 MMA d3 Qualifier 119 9 58 100 40 32 3 10 Methylmalonic Acid in Serum Plasma Urine advanced Page 13 4 4 Standby mode When the analytical system is not in use the HPLC pump should be switched off The mobile phases can be left within the LC system The vacuum pumps of the tandem mass spectrometer MS MS system should be in permanent operation In order to protect the ion source and multiplier the MS MS system should be switched into the standby mode If the system is not used for more than 2 days the analytical column should be disconnected and closed tightly Page 14 Methylmalonic Acid in Serum Plasma Urine advanced 5 Implementation of the analytical procedure 5 1 Collection and storage of samples 5 1 1 Serum and plasma The routine analysis of MMA is primarily performed from serum If serum is no
6. scale factor 21 Page 20 Methylmalonic Acid in Serum Plasma Urine advanced 7 Test data 7 1 Test performance The results were obtained with the API 4000 MS MS system 7 1 1 Linearity detection limit quantitation limit Serum Plasma Urine ug l nmol I ng 1 nmol I Linearity 3 8000 25 67745 77 168420 651 1426200 LLOD 1 8 15 46 391 LLOQ 3 0 25 77 651 LLOD Lower limit of detection LLOQ Lower limit of quantitation 7 1 2 Recovery For MMA mean recovery rates between 91 116 were obtained 7 1 3 Precision 7 1 3 1 Intraassay For the evaluation of the intraassay precision 3 samples with the following concentrations were used MMA nmol l Sample 1 148 Sample 2 275 Sample 3 614 The samples were measured in 3 analytical series each by 8 fold determination n 24 n number of values per sample The following coefficients of variation CV were obtained mean values CV 96 Sample 1 5 65 Sample 2 3 60 Sample 3 3 77 Methylmalonic Acid in Serum Plasma Urine advanced Page 21 7 1 3 2 Interassay For the evaluation of the interassay precision 3 samples with the following concentrations were used MMA nmol l Sample 1 186 Sample 2 479 Sample 3 686 The samples were measured in 8 analytical series each by 2 fold determination n 16 n number of values per sample
7. shortly on a vortex mixer Afterwards put the sample into the autosampler 5 2 3 1 3 LC MS MS analysis Depending on the sensitivity of the LC MS MS system in use inject 2 5 ul of the sample into the LC MS MS System 5 2 3 1 4 Stability of the prepared samples 5 3 5 3 1 The prepared samples can be stored at least 7 days at temperatures between 2 8 C and at least 3 months at temperatures below 18 C multiple freeze thaw cycles should be avoided LC MS MS analysis Independent from the analytical method the mass accuracy of the tandem mass spectrometer MS MS should be checked at regular intervals A mass calibration may be required For information regarding the check up of the MS MS system please refer to the documentation provided by the instrument manufacturer Compound optimisation MS MS For the optimisation of the MS MS system parameters the Optimisation Mix is provided compound optimisation The Optimisation Mix contains the analyte methylmalonic acid and the Internal Standard IS d3 Methylmalonic Acid The Optimisation Mix should be diluted with Diluting Solution D order no MS5021 according to the sensitivity of the MS MS system in use For the API 4000 MS MS system we recommend a 1 1 dilution of the Optimisation Mix with Diluting Solution D The compound optimisation procedure for the MS MS system in use should then be followed in order to optimise the ionisation source parameters and the compoun
8. ClinMass Optimisation Mix as well as the ClinCal Calibrators and ClinChek Controls lyophilised after reconstitution please also refer to the respective product data sheets Table 1 Storage conditions of kit components Order no Product description Storage conditions MS5005 Autosampler Washing Solution Store at 15 30 C MS5108 Mobile Phase A Store at 15 30 C MS5112 P Precipitant with Internal Standard Store below 18 C Serum Calibrator Set lyophil Level 0 3 ze MS5013 Store at2 8 C MS5114 Optimisation Mix Store below 18 C MS5109 Mobile Phase B d Store at 15 30 C MS5020 Sample Preparation Vials Store at ambient temperature 8 C MS5021 D Diluting Solution for Urine Store at 2 8 C 2 C a Methylmalonic Acid in Serum Plasma Urine advanced Page 7 2 1 3 MS5130 Analytical Column Nu Store at 15 30 C MS5080 Serum Controls lyophil e ii I Store at 2 8 C MS5082 Level I II IH Se Refers to the lyophilised product For storage conditions after reconstitution please refer to the product data sheet Disposal of laboratory waste For disposal laboratory waste should be collected separately according to the different chemical properties Recommendations for the disposal of product and packaging are indicated in section 13 of the respective Material Safety Data Sheet MSDS Page 8 Methylmalonic
9. Decrease of sensitivity lon source contaminated Mass spectrometer contaminated Leakage of injection valve Clean the ion source Clean the mass spectrometer Check the injector Shift of mass calibration Mass resolution too high low Recalibrate MS MS system Optimise the mass resolution Page 24 Methylmalonic Acid in Serum Plasma Urine advanced Problem High fluctuations of signals Possible Cause Spray instable Corrective Measure Check the spray needle capillary and clean or exchange if necessary Fluctuation of the flow rate Check the HPLC pumps Gas flow rate instable Check the gas lines No vacuum Defective vacuum pumps Check the pre and high vacuum pumps Leakage within the vacuum Check the vacuum tubes and system fittings No gas supply Defective nitrogen generator Check the nitrogen generator Defective compressor Gas bottle is empty Check the compressor Replace the gas bottle Inlet gas pressures are not within the specified range Regulate the inlet gas pressures Methylmalonic Acid in Serum Plasma Urine advanced Page 25 10 Appendix EC Declaration of Conformity Declaration of Conformity for in vitro diagnostic medical devices acc to article 9 1 of the directive 98 79 EC The company RECIPE Chemicals Instruments GmbH Dessauerstra e 3 D 80992 Munich Germany declares that the CE l
10. abelled product ClinMass Complete Kit advanced for Methylmalonic Acid order no MS5100 meets all applicable provisions of the directive on in vitro diagnostic medical devices 98 79 EC The conformity assessment was performed according to annex III The technical documentation is held according to annex III no 3 Munich 12 09 2013 RR Alfred Bauer General Manager LLETEDTRTI PL te li RECIPE CHEMICALS INSTRUMENTS GmbH DessauerstraBe 3 D 80992 M nchen Tel 49 89 54 70 81 0 Fax 11 info Qrecipe de Zertifiziert nach Certified acc to ISO 9001 ISO 13485 www recipe de
11. be determined in addition to total vitamin B12 and MMA Holo TC is the intracellularly utilised form of vitamin B12 and as a precursor of coenzyme B12 is required for the conversion of MMA and homocysteine A metabolically manifested vitamin B12 deficiency will thus be indicated by lowered levels of Holo TC and by increased levels of MMA and homocysteine 1 3 For the determination of homocysteine the following products available ClinMass LC MS MS Complete Kit for Homocysteine in Plasma Serum order no MS2000 ClinRep HPLC Complete Kit for Homocysteine in Plasma order no 23000 Methylmalonic Acid in Serum Plasma Urine advanced Page 3 The determination of MMA can be performed from serum plasma and urine Serum samples are generally used for MMA determination as this matrix is used for parallel cobalamin level tests The advantage of determination from serum therefore is the sample availability Furthermore nutrition seems to have less influence on the MMA serum level than is the case with urine 4 5 Additional measurement of creatinine is also necessary for the determination from urine as the MMA creatinine ratio is required for data interpretation 5 The advantage of determination from urine however lies in the significantly higher MMA levels which facilitate the analyses In cases of patients with impaired renal function serum MMA measurements may provide false positive results due to reduced urinary MMA excreti
12. d in Serum Plasma Urine advanced 2 1 Components of the complete kit and accessories Ordering information Order No MS5100 MS5005 MS5108 MS5109 MS5112 MS5013 MS5114 MS5020 MS5021 MS5130 MS5080 MS5081 MS5082 Please note Apart from the use in sample preparation Diluting Solution D for Urine order no MS5021 is also intended for the optimisation and test run of the analytical system see sections 5 3 1 and 5 3 2 Diluting Solution D for Urine is therefore also required for Description ClinMass Complete Kit advanced for Methylmalonic Acid in Serum Plasma Urine for 300 assays Contents Autosampler Washing Solution Mobile Phase A Mobile Phase B P Precipitant with Internal Standard Serum Calibrator Set lyophil Level 0 3 Sample Preparation Vials Manual Separately available components Autosampler Washing Solution Mobile Phase A Mobile Phase B P Precipitant with Internal Standard Serum Calibrator Set lyophil Level 0 3 Optimisation Mix Sample Preparation Vials D Diluting Solution for Urine Start Accessories Analytical Column with test chromatogram ClinChek Controls Serum Control Iyophil Level Serum Control lyophil Level Il Serum Control lyophil Level I Il analysis of serum and plasma samples Quantity 1 pce 1 x MS5005 1 x MS5108 1 x MS5109 3 x MS5112 1 x MS5013 3 x MS5020 1000 ml 1000 ml 200 ml 40 ml 4x1x2ml 2m
13. d specific mass transition parameters Methylmalonic Acid in Serum Plasma Urine advanced Page 17 5 3 2 5 3 3 5 3 4 Equilibration of the analytical system and test run Equilibrate the entire analytical system for at least 30 min before injecting samples Before each series of analyses perform a blank injection injection volume O ul or injection of Mobile Phase A This procedure provides reproducible results right from the first sample injection In order to confirm the performance of the analytical system repeatedly inject the Optimisation Mix until two consecutive chromatograms comparable in retention times and peak areas are obtained A dilution of the Optimisation Mix with Diluting Solution D will be required depending on the sensitivity of the MS MS system in use For the API 4000 MS MS system we recommend a 1419 dilution of the Optimisation Mix with Diluting Solution D Calibration run For calibration a ClinCal 4 Level Serum Calibrator Set level O 3 order no MS5013 is available The serum calibrators can also be reliably used for the accurate determination of MMA from plasma and urine samples Please note that a scale factor must be considered for the quantitation of urine samples see section 6 The calibrators are lyophilised and subsequent to reconstitution see section 5 2 1 must be prepared as described for the patient samples see section 5 2 For each analytical series freshly prepa
14. l 100 pcs 50 ml 1 pce 10x2ml 10x2ml 2x5x2 ml Page 6 Methylmalonic Acid in Serum Plasma Urine advanced 2 1 1 2 1 2 Safety information Several of the kit components e g mobile phases and reagents are chemical preparations and may contain hazardous substances For safety information please consult the Material Safety Data Sheet MSDS of each component The calibrator and control materials are prepared from human serum Although the products are tested for the absence of common infection markers they still should be considered as potentially infectious For this reason we recommend the product to be handled with the same precautions as patient samples Detailed safety information is indicated in the respective Material Safety Data Sheet MSDS Storage conditions and lifetime of kit components Please unpack the kit components from the transport packaging immediately upon receipt and follow the instructions for storage conditions indicated on the product labels and table 1 Unused components stored under appropriate conditions can be used until the expiry date indicated on the product label After use of ClinMass Reagents and ClinMass Mobile Phases the bottles must be closed tightly and stored immediately under the required conditions Provided proper use and storage procedures are followed the lifetime of the reagents is the same as for the unused products For storage conditions and lifetime of the
15. l of the serum or plasma sample calibrator control patient Mix for 30 sec on a vortex mixer and subsequently centrifuge for 5 min at 10000 x g 5 2 2 1 2 LC MS MS analysis Transfer the centrifuged supernatant to a sample vial which is suitable for the autosampler in use Depending on the sensitivity of the LC MS MS system inject 2 5 ul of the supernatant 5 2 2 1 3 Stability of the prepared samples The prepared samples can be stored at least 7 days at temperatures between 2 8 C and at least 3 months at temperatures below 18 C multiple freeze thaw cycles should be avoided 5 2 3 Urine 5 2 3 1 Work flow Sample preparation Dilution 1000 ul 50 ul D Diluting Solution urine for Urine patient Jl mix shortly vortex mixer Addition of IS 400 ul 100 ul P Precipitant diluted urine contains Internal Standard T mix shortly vortex mixer LC MS MS analysis Inject 2 5 ul sample 5 2 3 1 1 Dilution For dilution pipette 1000 ul Diluting Solution D into a sample preparation vial order no MS5020 and add 50 ul of the urine sample patient Subsequently mix shortly on a vortex mixer Page 16 Methylmalonic Acid in Serum Plasma Urine advanced 5 2 3 1 2 Addition of IS Pipette 400 ul Precipitant P contains Internal Standard IS into a sample vial suitable for the autosampler in use Subsequently add 100 ul of the diluted urine see section 5 2 3 1 1 and mix
16. le chromatogram O 00 u Ul aAa Ne h h O o 10 12 13 14 14 14 14 14 14 14 14 15 15 16 16 17 17 17 18 Contents 6 EVALUATION 19 7 TEST DATA 20 7 1 Test performance 20 7 1 1 Linearity detection limit quantitation limit 20 7 1 2 Recovery 20 7 1 3 Precision 20 7 1 3 1 Intraassay 20 7 1 3 2 Interassay 21 7 2 Reference Ranges 21 8 REFERENCES 22 9 TROUBLESHOOTING 23 10 APPENDIX EC DECLARATION OF CONFORMITY 25 Methylmalonic Acid in Serum Plasma Urine advanced Page 1 1 Introduction 1 1 Intended use This ClinMass Complete Kit is intended for the determination of methylmalonic acid from human serum plasma and urine with LC MS MS The kit components have to be used in accordance with this user manual The kit is not designed for combination with components from other manufacturers 1 1 1 IVD symbols Symbols according to EU directive 98 79 EC for in vitro diagnostic medical devices IVDD which are used on the product labels and in this user manual For in vitro diagnostic use Order number wl Manufacturer Lot number Upper temperature limit C Temperature limits C to C Expiry date See instructions for use Ej Ed a gt Page 2 Methylmalonic Acid in Serum Plasma Urine advanced 1 2 Clinical background Vitamin B12 cobalamin is an essential nutrient and plays an important role for the normal functioning of the human organism The coe
17. n Figure 2 Chromatogram of the ClinChek Serum Control level I order no MS5080 Methylmalonic Acid in Serum Plasma Urine advanced Page 19 6 Evaluation The analyte detection is achieved using compound specific mass transitions see section 4 3 2 The evaluation of the analyte concentration is performed by the internal standard method using the peak areas Calibration curves are achieved for the calibrators by plotting the ratio analyte peak area internal standard peak area against the ratio analyte concentration internal standard concentration The analyte concentrations for samples and controls are calculated from the calibration curve Please consult the software user manual of the MS MS manufacturer in order to ensure correct evaluation of the results For the calculation of mass concentrations ug l into molar concentrations nmol l and vice versa the analytical results should be multiplied with the factors shown in table 8 Table 8 Conversion factors Analyte Molecular Conversion factor Conversion factor weight g mol nmol l gt ug l ug l gt nmol l MMA 118 09 0 118 8 468 Urine samples In case of urine samples the creatinine level must be quantified and the results interpreted from the mol MMA mol creatinine ratio Due to the calibration with the ClinCal Serum Calibrator no dilution within sample preparation the urine MMA analytical results must be multiplied with the
18. nary Excretion in Normal Subjects after Feeding and during Fasting and after Loading with Protein Fat Sugar Isoleucine and Valine Clinical Chemistry 1989 35 12 2271 2276 Rasmussen K Moelby L Mogens Krogh J Studies on Methylmalonic Acid in Humans II Relationship between Concentrations in Serum and Urinary Excretion and the Correlation between Serum Cobalamin and Accumulation of Methylmalonic Acid Clinical Chemistry 1989 35 12 2277 2280 Rasmussen K Vyberg B Pedersen K Brochner Mgrtensen J Methylmalonic Acid in Renal Insufficiency Evidence of Accumulation and Implications for Diagnosis of Cobalamin Deficiency Clinical Chemistry 1990 36 8 1523 1524 Norman E J Morrison J A Screening Elderly Populations for Cobalamin Vitamin B12 Deficiency Using the Urinary Methylmalonic Acid Assay by Gas Chromatography Mass Spectrometry The American Journal of Medicine 1993 94 589 594 Carvalho V M Kok F Determination of serum methylmalonic acid by alkylative extraction and liquid chromatography coupled to tandem mass spectrometry 2008 Analytical Biochemistry 381 67 73 Magera M J Helgeson J K Matern D Rinaldo P Methylmalonic Acid Measured in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry Clinical Chemistry 2000 46 11 1804 1810 10 L Thomas Labor und Diagnose Indikation und Bewertung von Laborbefunden f r die medizinische Diagnostik 8
19. nstallation and storage of the analytical column Column heater 25 C Autosampler Use the recommended needle wash settings for minimum sample carry over from the autosampler supplier E g For the Agilent autosampler set a 6 second needle wash using the flushport Injection volume 2 5 ul Injection interval 3 0 min 2 5 min 2 0 min see table 3 table 4 and table 5 The Mobile Phase A B and flow rate gradients of the HPLC pump are programmed according to the tables below Please note The gradients indicated are based on the standard configurations of mixer and pulse damper Use of other configurations will affect retention times and performance of the analytical procedure The retention time of MMA should be between 1 9 2 3 min see table 3 1 4 1 8 min see table 4 and 1 3 1 7 min see table 5 respectively The parameters shown in table 3 refer to the Agilent 1100 1200 LC system with mixer Table 3 Mobile Phase A B gradient and flow rate for the LC system Agilent 1100 1200 with mixer Time Mobile Phase A Mobile Phase B Flow rate min 96 96 ml min 0 00 100 0 0 7 0 01 70 30 0 7 0 05 70 30 0 7 0 10 40 60 0 7 1 10 40 60 0 7 1 11 0 100 0 7 1 20 100 0 0 7 3 00 100 0 0 7 Methylmalonic Acid in Serum Plasma Urine advanced Page 11 The parameters shown in table 4 refer to the Agilent 1200 LC system without mixer and the Agile
20. nt 1200 SL LC system Table 4 Mobile Phase A B gradient and flow rate for the LC system Agilent 1200 without mixer and Agilent 1200 SL Time Mobile Phase A Mobile Phase B Flow rate min 96 96 ml min 0 00 100 0 0 7 0 20 100 0 0 7 0 21 70 30 0 7 0 25 70 30 0 7 0 30 40 60 0 7 0 90 40 60 0 7 1 00 0 100 0 7 1 01 100 0 0 7 2 50 100 0 0 7 The parameters shown in table 5 refer to the Agilent 1290 UHPLC system with the 35 ul JetWeaver mixer Table 5 Mobile Phase A B gradient and flow rate for the UHPLC system Agilent 1290 Time Mobile Phase A Mobile Phase B Flow rate min 96 96 ml min 0 00 100 0 0 7 0 50 100 0 0 7 0 51 70 30 0 7 1 00 70 30 0 7 1 10 40 60 0 7 1 30 40 60 0 7 1 31 0 100 0 7 1 40 100 0 0 7 2 00 100 0 0 7 Page 12 Methylmalonic Acid in Serum Plasma Urine advanced 4 3 2 MS MS parameters The MS MS parameters indicated in the following tables are recommended values only This particularly applies to the mass transition specific parameters The values should be regarded as starting points for optimisation The optima vary between different MS MS systems and therefore should be optimised for the system to be used Compound optimisation see section 5 3 1 The parameters shown in table 6 refer to the MS MS system API 4000 Table 6 MS MS parameters API 4000 API 4000
21. nzyme form of vitamin B12 coenzyme B12 participates in two metabolic key positions One of these reactions is the vitamin B12 dependent conversion of methylmalonyl coenzyme A CoA to succinyl CoA 1 In cases of Vitamin B12 deficiency methylmalonyl CoA accumulates and methylmalonic acid MMA is subsequently released see figure 1 1 2 Accordingly vitamin B12 deficiency results in quantitative accumulation of MMA in blood and urine This occurs already in the early stages of insufficiency i e when vitamin B12 levels still appear normal see below making MMA a sensitive early biomarker for intracellular functional vitamin B12 deficiency 2 _COOH Coenzym B12 o CH CH C CH CH COOH m S CoA CoA S O Methylmalonyl CoA Succinyl CoA Methylmalonic Acid Figure 1 Vitamin B12 deficiency and release of MMA In contrast the determination of vitamin B12 in serum as total vitamin B12 which is frequently used due to its cost efficiency does not show adequate selectivity and sensitivity at the lower reference level range below 400 pmol l 1 As such this can lead to potential false negative diagnosis in cases of intracellular functional vitamin B12 deficiency where vitamin B12 levels appear normal 156 pmol l In such cases however the serum MMA is already significantly increased 300 nmol l and clearly indicates the deficiency 1 In these particular cases holotranscobalamin Holo TC and homocysteine will
22. on 6 However calculation of the urine MMA creatinine ratio can compensate for this 7 Mass spectrometry based methods have been widely tested for the determination of MMA GC MS has been routinely applied to the quantitation of MMA however due to the requirement of derivatisation prior to analysis an alternative method with less time consuming sample preparation and hence faster turn around time is of continued interest The application of LC MS MS methods to MMA determination has received increased attention in the last few years which however still bears some challenges due to the low endogenous concentration of MMA the highly polar nature low molecular weight low pKa and dicarboxylic acid structure Furthermore chromatographic separation from the naturally occurring structural isomer succinic acid SA present in physiological concentrations approximately 50 times higher than MMA is critical and not elementary Many methods hence require lengthy sample preparation steps such as solid phase extraction derivatisation evaporation and or ultra filtration and can also show sub optimal resolution from succinic acid 8 9 This method was developed for the routine analysis of methylmalonic acid MMA in human serum plasma and urine samples Sample preparation is simple and rapid and analogous for the different biological matrices Calibration is performed using lyophilised serum calibrators at clinically relevant levels Lyophilised se
23. ransferred to the gas phase where they subsequently pass into the MS MS which is composed of two quadrupoles and connected through a collision cell In this analytical method the MS MS measurement of the analytes is performed in the MRM Multiple Reaction Monitoring mode In this mode only selected ions known as the precursor ions with a defined mass charge ratio m z are isolated in the first quadrupole and subsequently are transferred into the collision cell These ions are then fragmented by impact with an inert gas argon or nitrogen at defined voltage settings Among the fragments generated known as the product ions only those with a defined m z ratio are isolated in the final quadrupole for subsequent detection Thus measurement in MRM mode ensures identification and quantification with high selectivity and sensitivity with the analyte identification based on characteristic mass transitions for the compound of interest The ClinMass Optimisation Mix is provided for the optimisation of the MS MS parameters see section 5 3 1 and for the test run of the analytical system see section 5 3 2 The calibration of the analytical system is performed by use of ClinCal Serum Calibrators For this purpose a 4 Level Serum Calibrator Set is provided see section 5 3 3 Quality control is performed by use of ClinChek Serum Controls These controls are available in two different concentrations see section 5 3 4 Methylmalonic Aci
24. red calibrators should be used Accuracy control For the quality control of the analytical measurements ClinChek Serum Controls in two concentrations are available level order no MS5080 level Il order no MS5081 level I Il order no MS5082 The serum controls can also be reliably used for the accurate determination of MMA from plasma and urine samples These controls are lyophilised and subsequent to reconstitution see section 5 2 1 must be prepared as described for the patient samples see section 5 2 For each analytical series freshly prepared controls must be used In case of large analytical series we recommend to inject these controls additionally at the end of the series Page 18 Methylmalonic Acid in Serum Plasma Urine advanced 5 3 5 Example chromatogram Example chromatogram of the ClinChek Serum Control level order no MS5080 recorded with the Agilent 1200 LC system without mixer and the MS MS system API 4000 0 56 1 0224 Retention Time 100 4 Analyte min min 9500 00 aud Succinic Acid 0 56 RR Methylmalonic Acid 1 79 ous d3 Methylmalonic Acid 1 79 M Internal Standard IS 7000 00 6500 00 6000 00 e 5500 00 5000 00 1 79 4600 00 4000 00 3600 00 3000 00 4 2500 00 2000 00 1500004 1000 00 tt pr DENM Eri d RENS 0 00 Tr m nr r 0 1 02 03 04 05 0 6 0 7 os 09 10 1 4 12 13 14 15 15 17 18 19 20 2 1 22 23 2 4 Time mi
25. rum controls are also available for quality assurance An isotope labelled internal standard d3 methylmalonic acid is used in order to compensate for matrix effects and measurement variations Samples are analysed using negative ion electrospray in MRM mode for maximum sensitivity and selectivity Page 4 Methylmalonic Acid in Serum Plasma Urine advanced 1 3 General description of the analytical procedure In this analytical method MMA is determined from human serum plasma or urine by HPLC with electrospray tandem mass spectrometry LC MS MS The routine analysis of MMA is primarily performed from serum However the methodology presented here can also be applied to plasma citrate EDTA and heparin and urine matrices see collection and storage of samples section 5 1 In the case of urine samples required for patients with renal insufficiency see section 1 2 the creatinine level must also be quantified and results interpreted from the MMA creatinine ratio see evaluation section 6 Prior to the LC MS MS analysis a short sample clean up is performed in order to remove the sample matrix and to spike with the internal standard sample preparation see section 5 2 After the chromatographic separation on the analytical column within the HPLC system MMA is ionised by electrospray ionisation ESI and detected by the tandem mass spectrometer MS MS In electrospray ionisation the sample components are ionised and then t
26. t available plasma citrate EDTA and heparin plasma can also be used The samples can be stored at least 3 days at room temperature 15 30 C at least 7 days at temperatures between 2 8 C and at least 3 months at temperatures below 18 C multiple freeze thaw cycles should be avoided 5 1 2 Urine In the cases of patients with impaired renal function the analysis is performed from the second early morning urine The stability of urine samples is identical to those of serum and plasma samples for storage conditions see section 5 1 1 5 2 Sample preparation 5 2 1 Reconstitution of the lyophilised serum calibrators controls ClinCal Serum Calibrators and ClinChek Serum Controls see section 2 1 are lyophilised and must be reconstituted before use Information regarding reconstitution analyte concentrations storage and stability is indicated in the respective product data sheets 5 2 2 Serum and plasma 5 2 2 1 Work flow Sample preparation Precipitation 400 ul 100 ul P Precipitant serum plasma contains Internal Standard calibrator control patient 11 mix for 30 sec vortex mixer TE centrifuge 5 min 10000 x g LC MS MS analysis Inject 2 5 ul supernatant Methylmalonic Acid in Serum Plasma Urine advanced Page 15 5 2 2 1 1 Precipitation Pipette 400 ul Precipitant P contains Internal Standard IS into a sample preparation vial order no MS5020 and then add 100 u
27. the equilibration is performed as follows e Set the HPLC pump to a flow rate of 0 7 ml min set the column heater to 25 C and equilibrate the column with approximately 10 ml Mobile Phase A gradient starting condition e Subsequently stop the HPLC pump and connect the outlet capillary of the analytical column with the tandem mass spectrometer Starting the analytical system The following sections provide the parameters for the LC system see section 4 3 1 and the tandem mass spectrometer see section 4 3 2 For optimisation equilibration testing and calibration of the LC MS MS system please refer to section 5 3 Please consult the user manual of the tandem mass spectrometer to ensure appropriate usage User trainings provided by the instrument manufacturer may also be advisable Methylmalonic Acid in Serum Plasma Urine advanced LC parameters Table 2 LC parameters Binary HPLC gradient pump Flow rate 0 7 ml min see table 3 table 4 and table 5 Mobile Phases A and B Make sure that the bottles are closed well to avoid alteration of the retention times through evaporation of components of the mobile phases Column The analytical column is installed in the column heater 25 C At a flow rate of 0 7 ml min the backpressure of the analytical column should not exceed 300 bar For the complete HPLC system the backpressure should not exceed 400 bar Please see section 4 4 for appropriate dei
Download Pdf Manuals
Related Search