MP George*1; D Presser1; A Szczesniewski1; C Yu2, K McCann 2, Jinglan Zhang2
(*All correspondence to m-p_george@agilent.com)
1Agilent Technologies, Santa Clara, CA;
2Mount Sinai School of Medicine, New York, New York. |
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Tandem mass spectrometry offers an efficient and cost effective identification of inborn errors of metabolism to a group of disorders such as amino acid academia, fatty acid oxidation, and organic acidurias. Chase, DH and coworkers published numerous methods ( 1,2) for the MS/MS newborn screening and this methods are adopted worldwide by newborn screening labs. All these methods use three modes of MS/MS namely, precursor ion scan, neutral loss scan, and multiple reaction monitoring (MRM) and the concentration of the compounds are calculated in spectral domain using spectral intensity. We developed a method using the same MS/MS acquisition methods in different time segments using an injector program and use integrated peak height for the extracted ion chromatogram in precursor ion scan, neutral loss scan and MRM modes. We compared the data both in spectral domain technique and integrated peak height technique and found the later technique reduced the coefficient of variation (%CV) for some of the compounds from 35% to under 20%. In addition, our method has the option to recheck the abnormal results with scan results and MRM results.
Blood spot specimens (3.2 mm) were extracted with 100uL methanol containing isotopic internal standards at known concentrations. The extract is filtered through 4.2 micron filter. The filtered extract is derivetized with 3.0 N HCL butanol. The extract is evaporated and re-dissolved with 80:20 meoh:water. Samples are injected in the MS/MS using an auto sampler without any HPLC column. The auto sampler is programmed to make three repeat injections in 0.1 minute interval as part of the method. Total run time for all the three injections is 1.6 minutes which gave a cycle time of less than 2 minutes for each sample. The method in the MS/MS software was set up to acquire the first segment of precursor ion scan of 85 m/z , second segment of neutral loss scan of 102, 56, 119, and 161, and the third segment for MRM transitions for all free carantine, acylcarnites, and amino acids. We extracted and integrated each ion peak and used the peak height for the calculation to achieve concentrations. We also used slow flow injection and acquired the data using the spectral peak intensity to calculate the concentrations with slow flow. Accuracy and precisions were established using the Central for Disease Control (CDC) quality control samples. Further we analyzed 1000 newborn blood spots using both methods. We present the data and the conclusions from these methods.
References:
1. Chase DH, Hillman SL, Millington DS, Kahler SG, Roe CR, Naylor EW. Clin Chem. 1995;41:62-68
2. Chase DH, Hillman SL, Van Hove JL, Naylor EW. Clin Chem 1997;43:2106-2113 |
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