for Non-derivatized Samples - AB Sciex

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Chromsystems kit utlilized, but not provided, includes well plate samplers and extraction solutions, as well as buffers and internal standards for accurate analyte  ...

A Rapid iMethod™ Test for Research Into Inborn Errors of Metabolism (for non derivatized samples) iMethod™ Test for Research Into Inborn Errors of Metabolism Version 1.1 for Cliquid® Software LC/MS/MS is a powerful tool for the study of metabolic disorders. The simultaneous analysis of amino acids and acylcarnitines panels provides information on over 40 metabolic disorders such as Phenylketonuria (PKU) and Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD). The AB SCIEX LC/MS/MS iMethod™ Test for Inborn Errors in Metabolism Research is based upon flow-injection analysis. This method has been verified for use on both AB SCIEX 3200 and 4000 level LC/MS/MS systems. This method is for the screening of amino acids and acylcarnitines from dried blood spots. The use of MRM for the underivatized amino acids and acylcarnitines results in considerably faster sample preparation and workflow set up. Results are comparable to those obtained from derivatized blood spot filters. The

Chromsystems kit utlilized, but not provided, includes well plate samplers and extraction solutions, as well as buffers and internal standards for accurate analyte determinations. Sample preparation is performed via a simple extraction directly from the blood spot with injection of the supernatant.

Method performance This method was evaluated using a series of dried blood spot control samples. Five replicates of control samples at blank, low and high levels were processed according to the previously described conditions and each replicate was injected in triplicate. The %CV (n=15) values for the target analytes were obtained using the low level control replicates.

Table 1. Representative Analyte List Analyte Alanine Alanine (by MRM) Arginine Aspartic acid Citrulline Glutamic acid Glycine Leucine Methionine Methionine (by MRM) Ornithine Phenylalanine Tyrosine Valine Carnitine C2-Carnitine C3-Carnitine C4-Carnitine C5-Carnitine C5-DC-Carnitine C6-Carnitine C8-Carnitine C10-Carnitine C12-Carnitine C14-Carnitine C16-Carnitine C18-Carnitine

Level 1 control (Mean conc. µmol/L) 362.2 340.4 18.7 142.0 58.0 432.5 391.1 224.4 53.7 53.9 212.5 123.9 159.0 236.6 65.6 47.6 7.57 1.28 0.68 0.32 0.54 0.63 0.66 0.71 0.59 4.74 2.54

%CV 5.75 5.26 1.80 7.80 6.12 6.07 5.40 7.20 6.68 5.71 5.98 4.97 6.89 5.89 10.1 5.46 10.61 11.84 13.12 12.55 12.73 15.53 8.75 11.71 19.46 10.05 9.58

Level 2 control (Mean conc. µmol/L) 633.7 606.6 93.6 265.3 218.2 798.6 1125.8 458 195.6 200.5 602.7 482.1 438.9 427.8 128.7 87.9 15.07 4.15 2.38 2.49 2.08 2.14 2.05 2.23 2.08 11.4 7.3

%CV 4.10 3.32 5.24 4.82 3.87 4.39 3.16 4.29 4.25 3.42 4.41 4.10 4.64 5.03 9.87 5.45 8.13 8.44 10.92 13.27 8.72 6.86 6.29 8.41 8.40 6.70 8.37


Please note that the results presented above were obtained using a single instrument and single set of standards and samples. Prior to production use, the method should be fully validated with real samples, and the results here may not be typical for all instruments. Variations in LC column properties, chemicals, environment, instrument performance and sample preparation procedures will impact performance, thus these results should be considered as informative rather than representative.

While the information provided above outlines the instrument requirements and expected results obtainable from the AB SCIEX iMethod™ Test for Research into IEM, please note that the results obtained do require some experience with LC/MS/MS and sample preparation procedures. As such, webbased and on-site training are available to assist in the deployment of the iMethod™ Test and are recommended for inexperienced users. Please consult your local sales representative for more details.

System Requirements

Important Note

In order to run this method as outlined above, the following equipment and reagents are required:

The iMethod™ Test described above has been designed by AB SCIEX to provide the sample prep and instrument parameters required to accelerate the adoption of this method for routine testing. This method is provided for information purposes only. The performance of this method is not guaranteed due to many different potential variations, including instrument performance, tuning, and maintenance, chemical variability and procedures used, technical experience, sample matrices, and environmental conditions. It us up to the end user to make adjustments to this method to account for slight differences in equipment and/or materials from lab to lab as well as to determine and validate the performance of this method for a given instrument and sample type. Please note that a working knowledge of Analyst® Software may be required to do so.

• An AB SCIEX 3200 Series (3200 QTRAP® or API 3200™) or 4000 Series (4000 QTRAP® or API 4000TM) LC/MS/MS System • An Agilent 1100/1200 LC system with binary pump G1312A (without static mixer), well plate auto sampler, and thermostated column oven or a Shimadzu Prominence LC system with system controller, two LC-20AD isocratic pumps and SIL-20AC autosampler or either pump configuration used with a CTC autosampler. • Chromsystems Kits ( containing well plate samplers, extraction solutions, buffers and internal standards. • Chromsystems Mobile Phase • A microplate agitator able to run at 600 r.p.m • A 1/8" inch hole punch • Pipettes and standard laboratory glassware

Ordering Information Product Name

Part Number

iMethod™ Test for Research Into Inborn Errors of Metabolism Version 1.1 for Cliquid® Software


The purchase and use of certain of the chemicals listed below may require the end user to possess any necessary licenses, permits or approvals, if such are required in accordance with local laws and regulations. It is the responsibility of the end user to purchase these chemicals from a licensed supplier, if required in accordance with local laws and regulations. The suppliers and part numbers listed below are for illustrative purposes only and may or may not meet the aforementioned local requirements. AB SCIEX is not responsible for user’s compliance with any statute or regulation, or for any permit or approval required for user to implement any iMethod™ procedure. The information included in this product is intended for reference and research purposes only. AB SCIEX offers no guarantee as to the quality or suitability of this data and suitability of the information included in this (Library, database, etc.) for use with your specific application.

For Research Use Only. Not for use in diagnostic procedures. © 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Publication number: 1010410-01