Metabolite Identification in Blood Plasma Using GC/MS and the Agilent Fiehn GC/MS Metabolomics RTL Library
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Metabolite Identification in Blood Plasma Using GC/MS and the Agilent Fiehn GC/MS Metabolomics RTL Library Application Note Authors Abstract Mine Palazoglu and Oliver Fiehn Gas chromatography / mass spectrometry (GC/MS) offers high separating power and high UC Davis Genome Center sensitivity for metabolomic research. The utility of metabolomic screens largely depends on Davis, CA 95618 the number of identified metabolites and links to their biological interpretation. Often, the challenging step is in the identification of these metabolites. The new Agilent Fiehn GC/MS Metabolomics RTL (retention time locked) Library has specifically been developed to help facilitate the identification of metabolites. Human blood plasma was used to demonstrate metabolite identification in a complex biologi- cal matrix. The identification routines were complemented by mass spectral deconvolution and matching of sample peak spectra to Agilent Fiehn library spectra via fast, flexible, high- throughput searching. One of the most important criteria for unambiguous identification was the sample retention times, which were locked to the absolute retention time of an internal standard, d27-myristic acid. Retention time locking, a feature of the Agilent Fiehn library, results in increased identification confidence. The average retention time deviations were found to be less than 0.15 min, increasing the reliability and confidence in metabolite annota- tions. Implementing identification procedures such as this will become increasingly important in standardizing the reporting of metabolomics results, such as what has recently been sug- gested by the NIH/NIDDK, and the Metabolomics Society. Introduction biopolymers such as glycogen or fat. The The following GC/MS conditions were relative abundance of these conserved used. An Agilent 6890 GC oven was ramped Comprehensive identification and quantifi- intermediary metabolites can subsequently by 10°C/min from 60°C (1 min initial time) cation of small molecule metabolites (30- be interpreted in relation to the activity of to 325°C (10 min final time), resulting in a 1,500 Da) from complex biological matrices, the underlying catabolic and biosynthetic 37.5 min run time with cooling down to called “Metabolomics”, is a challenging pathways. Therefore, the utility of 60°C. 1 µL was injected into the Agilent task for analytical chemistry, even with metabolomic screens largely depends on split/splitless injector at 250°C by a 10µL modern instrumentation. No single method the number of identified metabolites and syringe with 4 sample pumps, 1 pre-injection can achieve this goal. For example, highly links to their biological interpretation. This wash and 2 post-injection washes using volatile compounds like terpenes cannot be application note demonstrates the identifica- both solvent A and solvent B. No viscosity assessed with liquid chromatography-based tion of metabolites in human blood plasma, delay or dwell time was applied using a fast methods. Gas chromatography (GC) must be using a single solvent mixture for protein plunger speed. Samples were introduced in used. Similarly, liquid chromatography (LC) precipitation and extraction, followed by both splitless and split conditions. For split- is clearly better suited for separation of chemical derivatization, analysis by GC/MS, less conditions, a helium purge flow of polyphenols than capillary electrophoresis data processing with mass spectral decon- 10.5 mL/min was applied for 1 min (8.2 psi). (CE), whereas bis- and trisphosphates (as in volution, validation of lists of identified Gas saver was on with a saver flow rate of glycolysis and Calvin cycle pathways) might metabolites, and a way to use links to 20 mL/min for 3 min. A 29 m long DB5-MS be most suitable for CE-separations. external chemical and biological databases. column with 10 m Duragard precolumn was Nevertheless, each of these three major used with 0.25 mm diameter and 0.25 µm separation methods can detect and quantify Experimental film thickness. A constant flow rate of a range of different compound classes in a 1 mL/min helium was used as carrier gas. multiparallel way, especially when mass Blood was collected from volunteers The Mass Selective Detector (MSD) was set spectrometry is used for unambiguous and between the hours of 12:00 and 3:00 p.m. at a signal data rate of 20 Hz with the MSD sensitive detection. GC/MS offers high Platelet depleted plasma (based on additional transfer line set to 290°C. The quadrupole separating power and high sensitivity for centrifugation) was isolated immediately mass spectrometer was switched on after a metabolomic research. Often, a large overlap within 15 min after plasma withdrawal. 5.90 min solvent delay time, scanning from of identified compounds is observed when Samples were kept on ice during the proce- 50-600 u. The source temperature was set comparing results between the major sepa- dure and plasma was immediately frozen at to 230°C and the quadrupole temperature ration methods, despite the large differences -80°C. Samples were thawed once, aliquoted was 150°C. Prior to acquisition, the MSD in separation mechanisms. The underlying and prepared for GC/MS analysis. 30 µL of was autotuned using FC43 according to the reason for this high degree of overlap (even blood plasma was extracted using 1 mL of a instrument manual. When using split injec- when comparing to nuclear magnetic reso- single phase mixture of isopropanol:ace- tions, parameters used were identical as nance techniques) is the conserved nature of tonitrile:water (3:3:2, v/v) at 20°C for 5 min. given above but with a split ratio of 1:10 and the most abundant metabolic intermediates. After centrifugation, 0.5 mL of supernatant a split flow rate of 10.3 mL/min. Absolute was aliquoted into a tube, and then com- retention times were locked to the internal standard d27-myristic acid using the RTL Metabolites occur at very different concen- pletely dried in a SpeedVac concentrator. system provided in Agilent’s ChemStation trations in complex biological matrices, The tube was subsequently derivatized in software. Retention time locking reduces from which they must be extracted without two steps. First, carbonyl functional groups run-to-run retention time variation. The compromising the structural integrity and were protected by methoximation using Agilent Fiehn GC/MS Metabolomics RTL relative abundances. Yet, a range of central 10 µL of a 40 mg/mL solution of Library (version June 2008) was employed metabolism pathways are highly similar in methoxyamine hydrochloride in pyridine at for metabolite identifications. This library is design between different species (e.g. 30°C for 90 min. Next, to increase the the most comprehensive library of metabo- mouse, rat and human) and even beyond volatility of the compounds, the samples lite GC/MS spectra that is commercially (e.g. between mammals, birds and simple were derivatized using 90 µL of N-methyl- available. It contains searchable GC/MS EI eukaryotes like yeast). Such conserved N-trimethylsilyltrifluoroacetamide with 1% spectra and retention time indexes from pathways contribute to generic metabolic trimethylchlorosilane (MSTFA + 1% TMCS, approximately 700 common metabolites. needs like oxidizing carbon sources to obtain Pierce) at 37°C for 30 min. This procedure energy or, conversely, using carbohydrates, resulted in the detection of two peaks for amino acids and fatty acids for cellular derivatized aldehydes and ketones: the growth, providing the metabolic building syn- and anti- forms such as glucose1 and blocks for proteins, complex lipids and storage glucose2. 2 Results is glucose, sometimes called ‘blood sugar’, plasma withdrawals are not standardized as it is regulated in humans around a between different clinical laboratories and A 22 year old female was tested for her concentration of 5 mM to provide energy to can result in analytical variability. Some blood plasma metabolome in two samples all organs, notably to the brain. Other clinics prefer the use of EDTA to prevent that were withdrawn four weeks apart in an abundant metabolites expected are free clotting, whereas others use heparin or ongoing pharmacogenomics research pro- cholesterol, saturated free fatty acids and a citrate for this purpose. The anti-clotting gram. Plasma samples were analyzed using range of amino acids, particularly alanine, agent should be controlled for best results. splitless and split injection into the GC/MS which serves as a three-carbon carrier Efforts to produce ‘best practice’ documents system. The major component in plasma between skeletal muscle and liver. Blood to standardize and harmonize protocols are under way by the National Institutes of Health; however, no documents have been released yet. Using Agilent’s ChemStation software, several abundant peaks were detected for the 1:10 split injection. Peak detection with background subtraction and subsequent matching of the resulting mass spectra to the Agilent Fiehn GC/MS Metabolomics RTL Library using either NIST MS searches or ChemStation’s PBM queries confirmed that alanine, citrate, glucose and cholesterol were readily identified at retention times of 7.72, 16.59, 17.65 and 27.55 min, respectively (Fig. 1). Mass spectral match factors for these abundant compounds exceeded 90 in PBM search and 900 in NIST MS search even without further spectral purification. The high abundance of the citrate peak and lack of an EDTA peak confirmed that these samples had been subjected to plasma sta- bilization by addition of citric acid, which renders measurements for endogenous citrate impossible. Nevertheless, for detailed analysis of less abundant compounds, automatic mass spectral deconvolution (AMDIS) is required. AMDIS, an automated GC/MS identification program from the U.S. National Institute of Standards and Technology, is included with the Agilent Fiehn library. Mass spectral deconvolution automatically finds peaks, deconvolutes spectra from co-eluting compounds using model ion traces that best describe the presence of unique peaks, and subsequently match spectra against user-defined mass spectral libraries, making searches quick and simple.