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doi.org/10.26434/chemrxiv.7771142.v1
A Fast and Sensitive Method Combining Reversed-PhaseChromatography with High Resolution Mass Spectrometry to Quantify2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite forDetermining Glucose UptakeAshley Williams, Deborah Muoio, Guofang Zhang
Submitted date: 26/02/2019 • Posted date: 27/02/2019Licence: CC BY-NC-ND 4.0Citation information: Williams, Ashley; Muoio, Deborah; Zhang, Guofang (2019): A Fast and Sensitive MethodCombining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake. ChemRxiv.Preprint.
Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite(2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the fieldhas long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues toassess glucose uptake, no analytical MS-based methods exist to detect trace amounts in complex biologicalsamples. In the present work, we show that 2DG is poorly suited for MS-based methods due to interferingmetabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-Orbitrap-MSmethod using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring thesodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The lowdetection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. Thenewly developed method was fully validated via calibration curves in the presence and absence of biologicalmatrix. The present work is the first successful LC-MS method that can quantify trace amounts of anonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method todetermine glucose uptake in biological samples.
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A fast and sensitive method combining reversed-phase chromatography with
high resolution mass spectrometry to quantify 2-fluoro-2-deoxyglucose and its
phosphorylated metabolite for determining glucose uptake
Ashley S. Williams1, Deborah M. Muoio1,2,3, and Guo-Fang Zhang1,2*
1Duke Molecular Physiology Institute and Sarah W. Stedman Nutrition and Metabolism Center, Duke University
Medical Center, Durham, NC 27701, USA
2Department of Medicine, Division of Endocrinology, Metabolism, and Nutrition, Duke University Medical Center,
Durham, NC 27710, USA
3Department of Pharmacology and Cancer Biology
*To whom correspondence should be addressed: Guo-Fang Zhang, guofang.zhang@duke.edu, Duke Molecular
Physiology Institute, 300 N Duke St, Durham, NC 27701
Lead contact: Guo-Fang Zhang
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ABSTRACT
Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite
(2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the field has
long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues to
assess glucose uptake, no analytical mass spectrometry (MS) - based methods exist to detect trace amounts in
complex biological samples. In the present work, we show that 2DG is poorly suited for MS-based methods due
to interfering metabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-
Orbitrap-MS method using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring
the sodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The low
detection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. The
newly developed method was fully validated via calibration curves in the presence and absence of biological
matrix. The present work is the first successful LC-MS method that can quantify trace amounts of a
nonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method to
determine glucose uptake in biological samples.
Keywords: 2-deoxyglucose, 2-deoxyglucose-6-phosphate, sodium adduct, glucose uptake, LC-MS
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INTRODUCTION
The gold standard for the determination of glucose uptake is the 2-deoxyglucose (2DG) method. 2DG is
a glucose analogue that lacks one hydroxyl group at C-2 (Scheme 1). Upon entering the cell, 2DG is rapidly
phosphorylated by hexokinase to form 2-deoxyglucose-6-phosphate (2DG-6-P), which is then trapped in the
tissue because the lack of a keto group on C-2 of 2DG prevents the entry of 2DG-6-P into glycolysis (1). A small
fraction of 2DG-6-P can be incorporated into glycogen or dephosphorylated back to 2DG, depending on tissue
type (1-3), although this appears negligible in tissues such as muscle and heart. Therefore, concentrations of
2DG-6-P in tissues or cells can be used as an index of glucose uptake in most of organs, except those with high
activity of glucose-6-phosphatase (G6Pase), such as the liver, kidney, and intestine.
For applications in rodent models, the 2DG method involves bolus administration of trace amounts of
2DG (13uCi 14C-2DG or ~227nmol) followed by serial collection of blood samples to determine rates of 2DG
disappearance from the plasma. The plasma 2DG decay curve is coupled with tissue measurement of 2DG-6-P
to calculate glucose uptake, or more specifically, the glucose metabolic index (Rg) (4). A critical, but often
overlooked, consideration for these assays is that high doses of tracer can inhibit hexokinase in the brain and
perturb whole body glucose metabolism (5). Therefore, determination of glucose uptake in vivo requires a
sensitive and reliable method to quantify trace (nanomolar) amounts of both 2DG and 2DG-6-P in plasma and
tissues.
Measurement of 2DG and 2DG-6-P can be achieved by a number of various techniques, such as NMR
(6), enzymatic assays (7-9), and scintillation counting of radiolabeled metabolites (2,10-14). While each of the
foregoing methods has advantages, notable drawbacks include the requirement for sophisticated
instrumentation and expertise, lack of sensitivity, and logistical and administrative constraints, respectively. By
contrast, a MS-based method would alleviate most of these concerns. However, to our knowledge, an MS-
based assay has not been developed, due in large part to the inherently difficulty of measuring both 2DG and
2DG-6-P using a single method. Moreover, each compound presents its own set of challenges. 2DG-6-P, when
compared to 2DG, is very difficult to quantify using GC-MS because derivatization with TMS or TBDMS causes
partial phosphoryl group hydrolysis and, once derivitized, 2DG-6-P is a relatively large molecule that may
degrade in the GC-MS column (15,16). Despite this challenge, a method to quantify 2DG and 2DG-6-P via GC-
MS has been described; however, this approach requires the conversion of 2DG-6-P to 2DG prior to analysis,
which is less than ideal (17). Second, 2DG-6-P readily forms a negative ion in the electrospray ionization (ESI)
source that can be fragmented in tandem mass spectrometry. Therefore, although 2DG-6-P is a very polar
compound, it is best suited for a LC-MS/MS method run in the negative mode. To this point, a previous study
successfully quantified 2DG-6-P by LC-MS/MS with a linear range from 8 to 30 µM (18), yet no LC-MS method
is available to quantify 2DG, presumably as a consequence of its polarity and poor ionization in the ESI source.
In the current study, we sought to develop and validate a MS-based method to detect trace amounts of
2DG and 2DG-6-P. To this end, we investigated 2DG and 2DG-6-P ionization in the ESI source and its potential
application for quantitation by LC-MS. We found that while 2DG and 2DG-6-P can be measured via LC-MS, the
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presence of interfering metabolites around the m/z for each compound decreases the accuracy and sensitivity
of the method. To circumvent this problem, we developed a novel LC-MS high resolution Orbitrap MS-based
method to measure 2FDG and 2FDG-6-P without derivatization or extensive sample preparation. The
replacement of 2DG and 2DG-6-P by 2FDG and 2FDG-6-P improved method selectivity and the newly
developed method was validated via calibration curves in distilled water and biological matrix.
MATERIALS AND METHODS
Chemicals and reagents
2DG was purchased from Chem-impex Int'l Inc (Wood Dale, IL). 2-DG-6-P was purchased from Santa Cruz
Biotechnology (Dallas, Texas). 2FDG-6-P was purchased from Omicron Biochemicals Inc. (Southbend, IN).
2FDG, 2ClDG, hexokinase from baker yeast, triethanolamine, formic acid, chloroform, adenosine triphosphate
(ATP), ethylenediaminetetraacetic acid disodium salt (EDTA), and other chemical reagents in analytical grade
or above were purchased from Sigma (St. Louis, MO).
Preparation of 2ClDG-6-P
To obtain 2ClDG-6-P, 150 ul 150 ul of 100 mM 2ClDG, 180 ul of 100 mM ATP, 180 ul of 100 mM MgCl2, 75 ul
of 100 mM EDTA, and 10 U hexokinase were added into 1 ml triethanolamine buffer (50 mM, pH=7.4). The
reaction was maintained at 37C for 30 minutes. The reaction was quenched by adding 1 ml of precooled
methanol. After centrifugation at 800 × g for 15 minutes, the supernatant was dried and dissolved in distilled
water. The concentration of 2ClDG-6-P was estimated based on 2DG-6-P. 2ClDG-6-P was used as the internal
standard (IS) for the external calibration curve, therefore, the absolute concentration was not required.
LC-QTOF-MS instrumentation and conditions
An Agilent 1200 HPLC connected to an Agilent 6520 QTOF mass spectrometer was employed for method
optimization including the LC method optimization, ionization, and fragmentation tests. HPLC 1200 was
configured with an isocratic pump, a binary pump, a thermostatted column compartment, and a temperature-
controlled autosampler. The isocratic pump delivered reference solution (5 µM purine and 1.5 µM HP-0921 in
methanol solution containing 5% water.) to the mass spectrometer at a flow rate of 0.5 ml/min with a split of
1/100. The binary pump was used to transport mobile phase (HPLC-MS grade water) at a flow rate of 0.5 ml/min
in isocratic elution mode. The column was a Microsorb-MV C18 column (100 × 4.6 mm, 3 µm) with C18 guard
column and was kept at 40 C in the oven compartment. The autosampler was maintained at 5C, and the
injection volume was 5 µl. The total running time is 7 minutes.
QTOF-MS was operated at electrospray mode. For 2-DG and glucose, QTOF was run in the positive and MS
scan mode with the following parameters: gas temperature, 350C; drying gas, 11 l/min; nebulizer, 50 psig; VCap,
3500V; fragmentor, 130V; skimmer, 65V; Oct 1 RF Vpp, 750 V; collision energy, 0V; mass scan range, 100 –
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1700. 2DG-6-P and G-6-P were detected in negative mode with same parameters except that ion polarity was
negative.
LC- Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer
LC-Q-Exactive+-Orbitrap-MS was used for final quantitation. The LC method was adopted from the previous LC-
QTOF-MS method. The parameters for Q-Exactive+-MS equipped with a HESI probe: heat temperature: 425 °C;
sheath gas: 30, auxiliary gas, 13; sweep gas, 3; spray voltage, 3.5 kV for positive mode; capillary temperature
was set at 320 °C, and S-lens was 45. A full scan range was set at 60 to 900 (m/z). The resolution was set at 70
000 (at m/z 200). The maximum injection time (max IT) was 200 ms. Automated gain control (AGC) was targeted
at 3 × 106 ions.
Sample preparation for blank plasma 2FDG calibration curves
Two ul of plasma were added to a tube prior to folch extraction using the following solvents: 200 µl methanol,
200 µl distilled H2O, and 200 µl chloroform. The sample mixture was vortexed and centrifuged for 20 minutes at
14,000g at 4°C. The upper phase (~350 µl) containing the extracted matrix from several preparations (~15) was
combined into a single tube. Approximately 350 µl of the upper phase, 50 µl of each 2FDG standard solution,
and 1 nmol 2ClDG (IS) were combined into a tube, vortexed, and dried completely by nitrogen gas at 37C. The
dried residue was resupended in 60 µl distilled water, vortexed, and placed in an autosampler vial for LC-MS
analysis.
Sample preparation for blank tissue 2FDG-6-P calibration curves
Unlabeled mouse skeletal muscle tissue was pulverized in liquid nitrogen. Approximately 10 mg powdered tissue
was weighed prior to folch extraction using the following solvents: 400 μl ice cold methanol, 400 μl chloroform,
and 400 μl distilled water using a Qiagen Tissuelyzer (30 Hz, 1 minute per solvent). After homogenization, the
sample mixture was centrifuged for 20 minutes at 14,000 ×g at 4°C. The upper phase (~750 µl) containing the
extracted matrix from several preparations (~15) was combined into a single tube. Approximately 750 µl of the
upper phase, 50 µl of each 2FDG-6-P standard solution, and 1 nmol 2ClDG (IS) were combined into a tube,
vortexed, and dried completely by nitrogen gas at 37C. The dried residue was resupended in 100 µl distilled
water, vortexed, and placed in an autosampler vial for LC-MS analysis.
Method validation
The linear range, limit of detection (LOD), limit of quantitation (LOQ) were assessed by a serial dilution of
standard stock solutions. The accuracy was assessed by measuring the recovery of low (81.4 fmol for 2FDG
and 48.8 fmol for 2FDG-6-P), middle (1.3 pmol for 2FDG and 1.6 pmol for 2FDG-6-P), and high amounts (10.4
pmol for 2FDG and 50 pmol for 2FDG6P) of standards spiked into blank plasma or blank skeletal muscle samples.
The recovery experiment was repeated over 3 different days.
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Data analysis
Mass spectrometric data was acquired by MassHunter Data Acquisition (B.06.01) software, MassHunter
Quantitative Analysis for QTOF (B.07.01) software, and Thermo Xcalibur software. Analyte concentrations were
calculated using a 1/x weighted linear regression analysis of the standard curve.
RESULTS AND DISCUSSION
Ionization and fragmentation of 2DG and 2DG-6-P
The goal of this study was to develop a LC-MS or LC-MS/MS method, therefore we started by injecting 2DG and
2DG-6-P into the QTOF to check the ionization. From the mass spectra of the two compounds (Figure 1), we
could not find the protonated 2DG (2DG-6-P) whose m/z is expected to be 165.0757 (C6H13O5) (Figure 1A).
Instead we identified a strong sodium adduct of 2DG at m/z 187.0580 (theoretical m/z of C6H12O5Na is
187.0577) (Figure 1A). As anticipated, the ionization of 2DG-6-P was a strong negative ion at m/z = 243.0310
(Figure 1B).
Next we investigated the fragmentation of 2DG and 2DG-6-P to determine if a MS/MS method could be
developed using the triple quadrupole (Q-TOF) mass spectrometer. In order to identify the fragments, we ran
the fragmentation with 2DG and 2DG-6-P with the increasing collision energy (CE) from 10 to 40 V. 2DG-6-P
was easily fragmented even with 10 V of CE (Figure 2A). Two fragments at m/z 96.709 and m/z 78.9607 were
likely to be [phosphoric acid]- and [phosphoric acid-H2O]-. With the increasing CE, [phosphoric acid-H2O]- became
the dominant fragment (Figures 2B-D). However, 2DG remained largely intact as a sodium adduct ion at
CE=10V (Figure 3A). Thus we increased the CE and found with increasing CE, 2DG was fragmented into
several small fragments (Figures 3B-D), particularly at CE=40V (Figure 3D). At high collision energy, 2DG was
fragmented at multiple locations and there was no a major fragment that could be used for tandem mass
spectrometry. This is supported by previous work on sodium adducts showing they are difficult to measure due
to insufficient fragmentation and poor reproducibility (19). Thus, for the remainder of the study, we focused our
efforts on the development of a LC-MS method to determine both [2DG-Na]+ and [2DG-6-P-H]-. For all
subsequent assays, mass spectrometry was run in the positive mode for the [2DG-Na]+ assay and in the negative
mode for the [2DG-6-P-H]- assay.
HPLC method development
Sugar and phosphorylated sugars are very polar compounds and this creates several challenges for LC-MS
method development including: (i) poor retention on reversed-phase columns which impedes method
development and reproducibility and (ii) peak tailing for phosphorylated sugars due to the metal tubing system.
Therefore, we tested different reversed-phase columns and mobile phases. A Microsorb-MV C18 column (100
× 4.6 mm, 3 µm) was found to have very good retention of both sugars and phosphorylated sugars. Organic
solvents (methanol and acetonitrile) in the mobile phase worsen peak tailing, thus we selected an isocratic mobile
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phase containing 98% H2O, 2% methanol and 0.01% formic acid based on optimization to ensure: (i) low peak
tailing, (ii) reproducible retention time of biological samples, and (iii) long term use of the column. Using these
conditions, 2DG and 2DG-6-P were well separated, and all of them came out before 3 minutes (data not shown)
and the total running time was 10 minutes per sample.
Sample preparation
Since the present work employed an isocratic LC method with aqueous as mobile phase, it was necessary to
remove all lipid during the sample preparation to avoid altering the LC column’s performance by lipid
accumulation. To this point, we employed a Folch wash approach for both tissue and plasma sample preparation.
In addition, a 1-µl injection volume was used to decrease the amount of biological matrix loaded onto the column.
The effect of sodium ion concentration on the relative abundance of [2DG-Na]+ in ESI
The lack of protonated 2DG in our Q-TOF extracted ion chromatograms and the presence of the sodium adduct
is supported by the phenomenon that neighboring hydroxy groups can form a stable triangular cyclic ion after
binding with one metal ion. In addition, glucose possesses a strong affinity to sodium and other metal ions in the
ESI source (20) and the formation efficiency of sodium adducts is higher for oxygen bases. Glucose and 2DG
have 6 and 5 oxygen atoms, respectively, and the strong sodium affinity indicated that the 2DG sodium adduct
could be used for quantitation since a glucose or 2DG sodium adduct would be more resistant to ionization
condition changes. Thus we hypothesized that a MS-based method for the quantitation of a sodium adduct is
required although it is highly unusual due to the difficulty of fragmentation and delicate ionization conditions (21).
To determine the effect of NaCl concentration on the ionization of 2DG and glucose, the relative abundance of
2DG and glucose was assessed in the presence of increasing concentrations of NaCl (Figure 4). Ionization of
2DG progressively increased with [NaCl] from 0 to 10 mM and slightly dropped at 100 mM NaCl. Notably,
intensity of the glucose sodium adduct remained constant from 0 to 10 mM NaCl and, similar to 2DG, 100 mM
NaCl suppressed the ionization of glucose presumably due to suppression of the ionization source. In sum, these
results show that NaCl concentration differently affects the relative abundance of 2DG and glucose using ESI. It
is plausible that differences between glucose and 2DG exist as (1) the loss of one –OH from 2DG could decrease
the sodium affinity compared to glucose and (2) formation of the glucose sodium adduct is maximized even at
trace amounts of sodium ion.
Endogenous interference in biological samples and the selection of a monohalogenated 2DG compound
Although the high resolution Orbitrap Q Exactive MS dramatically improves differentiation and separation of
several isobaric compounds, interference from complicated biological matrices remain a challenge due to the
presence of structural isomers and isotopologues. To assess whether unlabeled 2DG could be utilized as a
metabolic tracer in rodents, we sought to determine if there were any potential interfering metabolites around the
m/z of 2DG in untreated (a.k.a. blank) mouse plasma. As shown in Figure 5A, the extracted chromatogram of
[2DG-Na]+ (m/z = 187.0577) from a blank plasma sample demonstrates the existence of a highly expressed,
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interfering metabolite with a very similar m/z and retention time that would impede method sensitivity. Next we
investigated whether other monohalogenated glucose analogues with different m/z, such as 2FDG and 2ClDG,
could be used as a tracer and internal standard, respectively. Similar to 2DG, 2FDG (or [18F]FDG) is an
established glucose analogue which is often used to in humans and rodents to determine glucose uptake via
Positron Emission Tomography (PET) (22-25). We extracted the ion chromatograms of 2FDG and 2ClDG from
blank plasma samples (Figures 5B and 5D) and established that no interference was observed at a similar
retention time when the m/z of 2FDG or 2ClDG was extracted. 2FDG or 2ClDG were only present in the extracted
chromatogram when blank plasma was spiked with 2FDG (Figure 5C) or 2ClDG (Figure 5E). Notably, similar
interference was also observed for 2DG-6-P (m/z = 243.0264) in blank, untreated skeletal muscle (Figure 6A).
2FDG-6-P (m/z = 261.0180) or 2ClDG-6-P (m/z = 276.9885) was not found in the blank skeletal muscle sample
(Figures 6B and 6D) except when the blank skeletal muscle sample was spiked with 2FDG-6-P (Figure 6C) or
2ClDG-6-P (Figure 6E). While an ideal internal stand for quantitation is the stable isotope labeled compound,
we chose 2ClDG (and its phosphorylated metabolite, 2ClDG-6-P) as our internal standard(s) because stable
isotope labeled 2DG ([1-13C]2DG) and 2DG-6-P ([1-13C]2DG-6-P) are also subjected to the same interference
from the M+1 isotopologue of the endogenous compound at m/z 187.0577 and cannot be used as internal
standards. 2ClDG and 2ClDG-6-P do not have interfering metabolites in plasma and muscle tissue, therefore
they were chosen as internal standards for quantifying 2FDG and 2FDG-6-P, respectively.
Method validation and mass accuracy
After initial method optimization, all method validation was conducted using our UPLC-Q-Exactive-MS+ platform
which has the merit of high sensitivity and mass resolution. The high resolution of Q-Exactive-MS+ mass
spectrometry improves the selectivity of assay and decreases the baseline of mass scan. The measured m/z
values of analytes and internal standards in Q-Exactive-MS+ were compared to their theoretical values. The
detailed data is shown in Table 1. The mass accuracy were within 2.7 ppm among all analytes and internal
standards (see Table 1).
Linearity, detection and quantification limits for the method
Next we ran an external calibration curve to check the sensitivity and linearity of the method. Figure 7 shows
the chromatogram of 11 different concentrations of 2FDG (Figure 7A) and 12 different concentrations of 2FDG-
6-P (Figure 7C) with equal concentrations of the internal standards (2ClDG or 2ClDG-6-P) (Figures 7C and
7D). The peak shapes of 2FDG (Figure 7A), 2FDG-6-P (Figure 7C), and 2ClDG-6-P (Figure 7D) were sharp
(peak width < 0.2 minutes) and symmetric. Only 2ClDG (Figure 7B) showed a slight peak split which might be
caused by the spatial isomerism of Cl. However, the spatial isomerism of Cl in the relatively larger molecule,
2ClDG-6-P, is minimized and comes out as a single peak (Figure 7D).
To determine whether biological matrix affects the slope of the calibration curves, calibration curves of 2FDG
and 2FDG-6-P were prepared in distilled water and compared to those prepared in blank plasma/skeletal muscle
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extracts (Figure 8). Skeletal muscle extract had little effect on the calibration curve of 2FDG-6-P. In contrast,
plasma matrix increased the slope of calibration curve of 2FDG compared to the one generated in distilled water.
Although biological matrix had little effect on 2FDG-6-P calibration curve, subsequent calibration curves were
generated in corresponding biological matrix and the performance of the analytical method was further assessed
by investigating the linearity, slope, limit of detection (LOD), and limit of quantitation (LOQ) (Table 2). The
accuracy and reproducibility of the method was determined using three different concentrations of analytes from
the calibration curves (low, middle, and high) assayed over three different days (Table 3). Notably, no changes
were observed for either compound (2FDG and 2FDG-6-P) after storage for 2 weeks in the 4C LC sampler. As
demonstrated in Tables 2 and 3, the present method for 2FDG and 2FDG-6-P quantitation is sensitive, robust,
accurate, and reproducible.
CONCLUSIONS
The 2DG method is the gold-standard for the determination of glucose uptake. However, a single MS-
based method to measure both 2DG and 2DG-6-P does not exist. To fill this gap, we developed and validated a
simple, fast, and sensitive C18-based LC-Q-Exactive-Orbitrap-MS method to quantify both 2DG and 2DG-6-P
using a monohalogenated form of 2DG, 2FDG. Our method is unique because it leverages the inherent nature
of glucose analogues, such as 2FDG, to form sodium adducts in combination with the high resolution and
sensitivity of the Orbitrap Q-Exactive to reliably quantify trace amounts of 2FDG and 2FDG-6-P.
ACKNOWLEDGEMENTS
Financial support for this work was provided by the NIDDK Mouse Metabolic Phenotyping Centers (National
MMPC, RRID: SCR_008997, www.mmpc.org) under the MICROMouse program, grants DK076169 and National
Institutes of Health grants F32DK105922 (ASW) and R01DK089312 (DMM).
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Table 1. Mass accuracy of compounds measured by Q-Exactive-MS+
Compound Ion Formula Theoretical m/z Measured m/z Difference(ppm)
[2DG-Na]+ Positive C6H12O5Na 187.0576 187.0578 1.1
[2FDG-Na]+ Positive C6H11FO5Na 205.0482 205.0480 1.0
[2ClDG-Na]+ Positive C6H11ClO5Na 221.0187 221.0184 1.4
[2DG6P-H]- Negative C6H12O8P 243.0270 243.0274 1.6
[2FDG6P-H]- Negative C6H11FO8P 261.0170 261.0177 2.7
[2ClDG6P-H]- Negative C6H11ClO8P 276.9875 276.9881 2.2
Table 2. Calibration curve, linearity, LOD, and LOQ of the method
Compound Slope of calibration curve
for 1/x (10 points, n = 3) Linear range(fmol) R2 LOQ(fmol) LOD(fmol)
2FDG 5.70.2 81.4 - 10416 0.9955 81.4 40.7
2FDG-6-P 0.900.03 48.8-50000 0.9998 48.8 24.4
Table 3. Accuracy and reproducibility of the method at various concentrations of analytes
2FDG 2FDG-6-P
Low
(81.4fmol)
Medium
(1.3 pmol)
High
(10.4 pmol)
Low
(48.8fmol)
Medium
(1.6 pmol)
High
(50 pmol)
Replicate 1 – Recovery (%) 103.6 106 98 122 106 99
Intra deviation (SD) 4.8 1.6 3.2 6.7 5.6 4.5
Replicate 2 - Recovery (%) 119 110 100 117 107 99
Intra deviation (SD) 22.3 2.1 1.2 6.8 0.16 4.5
Replicate 3 -Recovery (%) 103 111 101.4 85 105 99
Intra deviation (SD) 5.9 2.2 3.9 105 2.7 1.4
Inter-day average 115 109 99 108 106 99
Inter-day deviation (SD) 10.4 2.5 1.2 20 1.2 0.04
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FIGURES
Scheme 1. Chemical structure of analytes and internal standards. The chemical structures of all compounds
utilized in this study including: 2DG, 2DG-6-P, 2FDG, 2FDG-6-P, 2ClDG, and 2ClDG-6-P.
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Figure 1. Ionization of 2DG and 2DG-6-P in the Q-TOF. Ionization of (A) 2DG and (B) 2DG-6-P in the Q-TOF.
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Figure 2. Fragmentation of [2DG-6-P-H]- in the Q-TOF. Fragmentation of M0 [2DG-6-P-H]- in the Q-TOF at
(A) CE = 10V, (B) CE = 20V, (C) CE = 30V, and (D) CE = 40V.
16
Figure 3. Fragmentation of [2DG-Na]+ in the Q-TOF. Fragmentation of the sodium adduct of 2DG, [2DG-Na]+,
in the Q TOF at (A) CE = 10V, (B) CE = 20V, (C) CE = 30V, and (D) CE = 40V.
17
Figure 4. Effect of sodium ion concentration on the relative abundance of 2DG and glucose using ESI.
Relative abundance of 2DG and glucose using ESI with varying concentrations of NaCl. Data represent mean
±SD. N=3 replicates.
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Figure 5. Endogenous plasma interferences to 2DG, 2FDG, and 2ClDG. Extracted ion chromatograms for
(A) 2DG m/z at 187.0577 in blank plasma, (B) 2FDG m/z at 205.0481 in blank plasma, (C) 2FDG m/z at 205.0481
in blank plasma spiked with 2.44pmol 2FDG, (D) 2ClDG m/z at 221.0185 in blank plasma, and (E) 2ClDG m/z
at 221.0185 in blank plasma spiked with 1nmol 2ClDG.
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Figure 6. Skeletal muscle interferences to 2DG-6-P, 2FDG-6-P, and 2ClDG-6-P. Extracted ion
chromatograms for (A) 2DG-6-P m/z at 243.0264 in blank skeletal muscle, (B) 2FDG-6-P m/z at 261.0180 in
blank skeletal muscle, (C) 2FDG-6-P m/z at 261.0180 in blank skeletal muscle spiked with 2.44 pmol 2FDG-6-
P, (D) 2ClDG-6-P m/z at 276.9885 in blank skeletal muscle, and (E) 2ClDG-6-P m/z at 276.9885 in blank skeletal
muscle spiked with 1 nmol 2ClDG-6-P.
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Figure 7. Chromatograms of 2FDG, 2ClDG, 2FDG-6-P, and 2ClDG-6-P from different concentrations of
analytes and equal concentrations of internal standard(s). Extracted ion chromatograms of 11-12 different
concentrations of (A) 2FDG and (C) 2FDG-6-P with equal concentrations of (B) 2ClDG (internal standard for
2FDG) and (D) 2ClDG-6-P (internal standard for 2FDG-6-P).
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Figure 8. Calibration curves in distilled water and biological matrices. Calibration curves for (A) 2FDG-6-P
in distilled water, (B) 2FDG in distilled water, (C) 2FDG-6-P calibration curve in skeletal muscle matrix and (D)
2FDG calibration curve in plasma matrix.
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