Bin Rui1*, Xiaotao Wei2, Long Chen1, Yun An3 and Han Wen1*

1School of Life Science, Anhui Agricultural University, 230026, Hefei, China.
2School of Information and Computer, Anhui Agricultural University, 230026, Hefei, China.
3School of Tea and Food Technology, Anhui Agricultural University, 230026, Hefei, China.



Mass spectrometry is an analytical technique for molecules detection after separating the particle motion (charged molecules, atoms and fragment ions) according to their mass-to-charge ratio, under the action of an electric field and a magnetic field. In this study, ten kinds of fragment ions of metabolites in Escherichia coli were detected by ion trap liquid mass instrument and triple quadrupole mass chromatograph. Two different detection methods were then compared, to provide a foundation for later metabolomic research. This experiment detects metabolites with multistage mass spectrometry by electrospray ion trap mass spectrometry and triple quadrupole mass spectrometry, when scanning at anion mode using Agilent SB-C18 (250mm×4.6mm×5ìm) chromatographic column and using ammonium acetate and acetonitrile as the mobile phase. The result showed that both methods can act as multistage mass spectrometry, while triple quadrupole mass spectrometry enables to produce finer ions under low collision energy. Triple quadrupole mass spectrometry is a superior method to acquire a clear peak in detecting fragment ions of metabolites.

Keywords: Ion trap mass spectrometry; triple quadrupole mass spectrometry;
metabolites; mass-to-charge ratio.


In studies of metabolic processes in microorganisms, it’s very important to detect target metabolite, both qualitatively and quantitatively. Detecting target metabolite precisely and determining its content are important goals in modern metabolomics. Such studies have progressed significantly in recent years as a result of new and improved methodologies1-4.
The aim of this study is to analyse Escherichia coli carbon center metabolites by High Performance Liquid Chromatography, coupled with Tandem Mass Spectrometry. Escherichia coli is commonly used for experimental purposes, and particularly in genetic engineering. There have been 904 different Escherichia coli metabolites mentioned in research papers, involving in 932 different reactions [5-6], the number of which seems to be rapidly increasing as more research follows. Carbon central metabolism are common to many reactions in Escherichia coli and are therefore important to study to further understand the metabolomics of this commonly used bacterium. This study is to establish the experimental conditions of High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry for Escherichia coli carbon center metabolites. In addition, we hope to provide some references with regards to cell extraction methods and detection, for future studies.
There have been many studies describing extraction methods of mesostate in TCA and modern analysis techniques for phosphorylation products in most of the central metabolic pathways. Gas chromatography coupled with mass spectrometry is very common in metabolite detection, but its disadvantage is that heat-sensitive compounds have denatured decomposition more easily during the detection. Both triple quadrupole mass spectrometry and ion trap mass spectrometry are multi-stage mass spectrometry. Triple quadrupole mass spectrometry is mainly used for quantitative and qualitative analysis of target compounds and is often referred to as space tandem mass spectrometry. Ion trap mass spectrometry is time tandem mass spectrometry which is generally used for qualitative analysis and structural analysis of unknown compounds7-9.
Recently, with the development of MS and interface technology of LC/MS, it is now possible to determine conjugated metabolites directly. It has been reported that glucuronide conjugates can be determined directly by LC/MS/MS. The basis for the structure of this conjugate is that the new fragment ions are found when a molecular ion peak draws off 176u in the secondary mass [10]. In this study, we set out to explore and establish analysis of Escherichia coli carbon center metabolites using high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). It is one of the most important methods for standard sample detection of these kinds of metabolites.

Materials and methods

Main Instruments
Triple quadrupole mass spectrometer; ion trap mass spectrometer; sonicator; electronic analytical balance

Experimental Methods
Chromatographic conditions
Chromatographic column: 250mm × 4.6mm × 5um; column temperature: 35°C capillary temperature: 380°C; molecular weight range: 50-400°C mobile phase A: 10mmol/L ammonium acetate solution; mobile phase B: acetonitrile
Mass spectrometry conditions
negative ion scan; electrospray ionization voltage (ESI): 4.5 kV ion source temperature: 480°C; injection volume: 5 ml
Mass spectrometry conditions
Negative ion scan; electrospray ionization voltage (ESI): 4.5 kVion source temperature: 480!; injection volume: 5 ml
Preparation standard solution of samples
We prepared ten kinds of metabolites, which are pyruvic acid; glucose- 6-phosphate dipotassium salt; fructose-6-phosphate dipotassium salt; succinic acid; aspartic acid; isocitric acid trisodium salt; citric acid; glutamate acid; ±-ketoglutarate acid; fumaric acid acid respectively. To make all the standard solutions, use an electronic analytical balance to enable highly accurate weighing.

Table 1. Gradient elution parameters

Time Mobile phase A Mobile phase B
0 0 100
3 10 90
15 50 50
25 50 50
27 90 10
35 100 0


Table 2. The parameters of ten kinds of metabolites detected by ion trap mass spectrometry

Metabolites parent ion ion CE
Citric acid 191 87 25eV
D-Frutose-6-phosphate dipotassium salt 259 97 27eV
Dipotassium D-Glucose-6-phosphate 259 97、78.9 27eV
2-ketoglutaric acid 145 101 18eV
Glutamic acid 146 128 20eV
Fumaric acid 115 71 20eV
Pyruvic acid 87 87 10eV
Aspartic acid 132 88 27eV
Succitric acid 117 73 27eV
Isocitric acid trisodium salt hydrate 191 111、173.1 27eV


Table 3. The parameters of ten kinds of metabolites detected by triple quadrupole mass spectrometry

metabolites CE parent ion  (m/z) ion(m/z)
Dipotassium D-Glucose-6-phosphate    135    259 139, 169, 199
Aspartic acid    135    132 71.2, 88.1
Citric acid    135    191 111.0
D-Frutose-6-phosphate dipotassium salt    135    259 139, 169
Pyruvic acid    135    87 43.0
Succitric acid    135    117 73.1
Isocitric acid trisodium salt hydrate    135    191 73.1, 167.1
Glutamic acid    135    146 84.0, 102.1
2-ketoglutaric acid    135    145 57.1, 145.1
Fumaric  acid    135    115 71.0


Results and Discussion

Results of ion trap LC
X-axis represents ion response; Y-axis represents the time respectively; ESI: electrospray ionization voltage; MRM-multistage reaction detection; Frag-collision voltage; CID-collisional dissociation voltage


Fig. 1. Mixing spectrum of ten different metabolites.


The collision order of ten metabolites: glutamate acid→aspartic acid→glucose- 6-phosphate dipotassium salt→fructose-6-phosphate dipotassium salt→succinic acid→isocitrate→pyruvic acid→fumaric acid→citric acid→α-ketoglutarate acid


Fig. 2. Spectrum of glutamate acid with ion trap LC


Fig. 3. Spectrum of citric acid with ion trap LC


Fig. 4.  Spectrum of pyruvic acid with ion trap LC


Fig. 5. Chromatogram of glutamate acid with triple quadrupole LC

X-axis represents ion response; Y-axis represents the time respectively; ESI: electrospray ionization voltage; MRM-multistage reaction detection; Frag-collision voltage; CID-collisional dissociation voltage

Fig. 6. Spectrum of glutamate acid with triple quadrupole LC

X-axis represents mass to charge ratio; Y-axis represents ion response

Fig. 7. Chromatogram of citric acid with triple quadrupole LC

X-axis represents ion response; Y-axis represents time;

Fig. 8. Spectrum. of citric acid with triple quadrupole LC

Fig. 9. Chromatogram of pyruvic acid with triple quadrupole LC

Fig. 10. Spectrum. of pyruvic acid with triple quadrupole LC

Compared with Fig. 5 and Fig. 6, Fig. 1 and Fig. 2 show that only under a collision-induced voltage of -20 eV can fluid glutamate – prospective parent ion 146.1, be broken down for Sub ion and detected by an ion trap mass spectrometer. However, glutamate – prospective parent ion 146 detected by liquid chromatography triple quadrupole mass spectrometer could be broken down for Sub ion if the collision-induced voltage was -15 eV. So, the required collision-induced voltage for a Gas chromatography-ion trap mass spectrometry instrument is lower. The chromatomap illustrates the differences in the peak time. Fig. 1 (MS/MS) shows that glutamic acid appears at 4.18 min, whereas in Fig. 5 (GC-ITMS) the time to appearance is 3.76 min. This suggests that the Gas chromatography-ion trap mass spectrometry instrument can make the glutamic acid peak time earlier. From Fig. 2 it can be seen that the sub ion is 128.1 and in Fig. 6 it is 84.0 and 102.1. This suggests that MS/MS and GC-ITMS can both be used in multistage mass spectrometry, and that GC-ITMS can obtain smaller sub ions at a lower collision energy.
Comparing Fig. 1 and Fig. 3 with Fig. 7 and Fig. 8, we can learn that the collision energy of breaking citrate prospective parent ion 191 in MS/MS is -25 eV and in GC-ITMS is only -15eV. Comparing the chromatograms in Fig. 1 and Fig. 7, the peak of citrate in Fig. 7 is sharper and fuller than it is in Fig. 1. Sub ion in Fig. 3 is 87.3 and in Fig. 8 is 111.0. So, the two mass spectrometry methods can both be used in a multi-stage mass spectrometry, but in contrast, GC-ITMS can get an improved peak for Citric acid detection.
Comparing Fig. 1 and Fig. 4 with Fig. 9 and Fig. 10, collision energy of breaking pyruvate parent ion 87.0 is -10eV in MS/MS and is only -7eV in GC-ITMS. The pyruvate peak in Fig. 9 is sharper and more beautiful than it is in Fig. 1. and there is no broken parent ion 87.1 in Fig. 4, but in Fig. 10 sub ion we can see that 43.0 appears in the spectrum. The minimum mass set by the ion trap LC-MS instrument is 50, which means that any fragment ions below 50 cannot be detected. It is possible that the collision energy produced by Ion Trap is too small to detect pyruvate fragment ions by GC-ITMS. In that case, GC-ITMS would be a better way to detect pyruvate.


In conclusion, using GC-ITMS we can obtain smaller particle size fragment ions. Relatively speaking, detecting metabolite fragment ions by GC-ITMS is an improved method to obtain a clearer peak.


This work was supported by the Chinese National Natural Science Foundation [grants 31200626 and 31460233, 31372402); the Biology Key Subject Construction of Anhui (2014SKQJ017).


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