Metabolomics deals with a diversity of small molecules that differ greatly in their physical and chemical properties of size, polarity/hydrophobicity, and charge. While no single chromatographic method is ideal for all classes of metabolites, we have found that two methods—one for positive ionization mode and one for negative ionization mode (described below), provide a reasonable breadth of coverage. The route by which we arrived at this approach exemplifies some of the tradeoffs in targeted metabolomics.
Our initial approach was a reversed-phase chromatography method, on a C18 Fusion-RP (Phenomenex) column with acidic mobile phase [42
]. This method worked adequately for ~100 compounds in positive ion mode. However, many polar compounds did not retain on this column, eluting near the void volume, and nucleotide triphosphate compounds like ATP did not elute as well defined peaks. Subsequently, we compared the performance of nine different chromatography approaches involving seven different column chemistries [10
]. This study identified hydrophilic interaction chromatography (HILIC) [43
] on an aminopropyl column as an effective method to separate a broad range of cellular metabolites including amino acids, nucleosides, nucleotides, coenzyme A derivatives, carboxylic acids, and sugar-phosphates. By separating compounds into a 40-min positive ion run and a 50-min negative ion run, a total of ~140 compounds can be readily measured with a total running time of 90 minutes, using only one mass spectrometer. The same column chemistry can also be used to analyze additional classes of molecules, such as folates, which themselves include > 50 chemical species [48
]. This method is a reasonable alternative for those occasions where limited mass spectrometry resources are available.
Recent reports have demonstrated that reversed-phase chromatography with an amine ion pairing agent is a useful method for separation of a broad range of negatively charged metabolites, including nucleotides, sugar phosphates, and carboxylic acids [49
]. These methods utilize a volatile cationic compound, such as tributylamine [51
] or hexylamine [50
], to form ion pairs with negatively charged analytes, improving retention and separation on a C18 column. We performed a systematic comparison between our HILIC method and a variant of the reversed-phase, ion-pairing method of Luo et al.
], using both compound standards and cellular extracts under identical mass spectrometry conditions. The ion pairing chromatography in general offered better separation and higher signal for negatively charged metabolites (). This improved sensitivity may in part be due to improved separation leading to reduced ion suppression by co-eluting compounds. We found that this method generally did not work well in positive ionization mode, due to poor retention of amine-containing compounds and ion suppression effects by tributylamine.
Figure 1 Chromatographic traces for selected metabolites in a cellular extract comparing the performance for the HILIC (top panel) and reversed-phase ion pairing chromatographic (bottom panel) methods in negative ionization mode, under identical mass spectrometry (more ...)
Based on this study, we arrived at the dual chromatography method approach of HILIC chromatography in conjunction with positive mode ionization, and reversed-phase chromatography with tributylamine as an ion-pairing agent in conjunction with negative mode ionization, running on two separate LC-MS systems. The LC conditions for the positive mode have been previously reported [10
]: an aminopropyl column with acetonitrile and pH 9.45 aqueous buffer as the mobile phases and a running time of 40 minutes. The negative mode LC method uses a Synergi Hydro column (4 µm particle size, 150×2 mm, from Phenomenex, Torrance, CA), with solvent A being 10 mM tributylamine + 15mM acetic acid in 97:3 water:methanol, and solvent B being 100% methanol. The flow rate is 200 µl/min and running time is 50 minutes. The gradient is t = 0 min, 0% B; t = 5 min, 0% B; t = 10 min, 20% B; t = 20 min, 20% B; t = 35 min, 65% B; t = 38 min, 95% B; t = 42 min, 95% B; t = 43 min, 0% B; t = 50 min, 0% B. This dual chromatography methodology enables quantitation of approximately 250 water-soluble metabolites of validated identity including amino acids and derivatives, sugar phosphates, nucleotides, coenzyme A and derivatives, and carboxylic acids, with the number of quantifiable compound largely limited by the availability of standards, which are used for confirmation of targeted compound identity based on retention time and (for MS/MS approaches) mass spectrometry fragmentation pattern. Due to recent acquisition of several hundred additional standards from the Human Metabolome Database [53
], we anticipate generating methods covering a yet greater number of validated known compounds.
In contrast to the success in coupling ion pairing chromatography with negative ionization mode, there have been a limited number of studies exploring the possibility of coupling ion pairing with positive ionization mode. Because amino ion pairing agents work well with only with negatively charged compounds, a different kind of ion pairing agent is needed for positively charged metabolites. Volatile perfluorinated acids such as trifluoroacetic acid (TFA) have been used in the separation of peptides [54
]. Recently Hsieh et al.
reported the use of two similar ion pairing agents, heptafluorobutyric acid and nonafluoropentanoic acid in the mobile phase to improve retention and separation on a reversed-phase column, using APCI for ionization [55
]. Unfortunately, these volatile perfluorinated acids may cause ion suppression in ESI [56
]. More work is needed to find a proper ion pairing agent for positively charged metabolites in ESI.
A variety of additional alternative analytical techniques for metabolomics have been reported in the literature. Many researchers used multiple analytical platforms in parallel in order to detect as many compounds as possible. For examples, Sabatine et al.
used three different chromatographic modes for the analysis of plasma samples [57
]. Amino acids and amines were separated on a reversed-phase column at mobile phase pH 4, sugars and nucleotides by normal phase chromatography at mobile phase pH 11, and organic acids by reversed-phase chromatography at pH 6. A total of 477 multiple reaction monitorings were performed through 6 analytical methods for each sample. More recently, van der Werf et al.
developed a comprehensive metabolic platform that utilizes three GC-MS methods and three LC-MS methods [58
]. When applying this platform to the analysis of the metabolome of E. coli
, more than 400 compounds were detected.
In addition to using multiple chromatographic methods in parallel, two-dimensional chromatography approaches have also been described. In these methods the two chromatographic separations occur serially, and the chromatographic modes are orthogonal—they separate based upon different characteristics, in a manner similar to a 2D-nanoLC method, now widely used in proteomics for the separation of peptides [59
]. Kennedy and colleagues developed a two-dimensional liquid chromatography (2D-LC) method for metabolomics [60
]. In this approach, the samples were first separated on a strong anion exchange column, with fractions released to the reversed-phase for further separation by pulses of incrementally higher ionic strength. When this method was applied to the analysis of islets of Langerhans, roughly 200 peaks were detected [61
]. The main disadvantage of this approach is the long analysis time, as each fraction from the ion exchange column must be separated sequentially via reversed-phase.
Other notable developments in LC include the use of monolithic capillary columns [11
], high temperature LC [62
], and ultra performance liquid chromatography (UPLC, i.e., pressure > 400 bar to drive flow through columns packed with < 2 µm diameter particles [13
]). Recently, Guillarme et al.
systematically evaluated each of these three approaches compared to conventional LC [13
]. They found that each of the approaches provided at least comparable quantitative precision and accuracy to conventional LC while also expediting analysis, with the greatest gains obtained with UPLC.
On occasions in which metabolites of interests are at very low abundance or have poor ionization, it is often possible to increase the signal by derivatizing the compound(s) of interest, prior to running the sample by LC-MS. Our laboratory routinely applies several derivatization procedures for this purpose. These include the derivatization of thiol compounds such as cysteine and homocysteine which usually do not ionize very well, using S-methyl methanethiosulfonate (MMTS) [65
]. The derivatized compounds give substantially improved signal using the HILIC method in positive ionization mode. Similarly, reaction of amino acids with the benzylchloroformate gives improved retention in reversed-phase mode and negative mode ionization. Many other derivatizations have been reported in the literature [29
] and could be used to enhance the signal for specific classes of compounds at the expense of potentially impairing quantitative reliability (e.g., due to incomplete reactions or side reactions). For enhancing classspecific signals, a particularly promising approach involves schemes that simultaneously derivatize and capture metabolites on the surface of a bead from which they can subsequently be released and analyzed [68