Detection of Human Urinary 5 Hydroxymethylcytosine by Stable Isotope Dilution HPLC-MS/MS Analysis
Detection of Human Urinary 5 Hydroxymethylcytosine by Stable Isotope Dilution HPLC-MS/MS Analysis
Detection of Human Urinary 5 Hydroxymethylcytosine by Stable Isotope Dilution HPLC-MS/MS Analysis
pubs.acs.org/ac
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, Beijing 100085, China
Figure 1. Eect of mobile-phase additives on the HPLC separation and MS detection sensitivity of 5mC, 5hmC, and 5fC. Left panel represents
typical chromatograms of 5mC, 5hmC, and 5fC that were obtained using four dierent additives to the mobile phase. The peak area ratios of the
target analytes obtained by ammonium salt vs HCOOH are summarized in the table to the right. The mobile phase consisted of solvents A and B
(pure methanol). Solvent A was 2.0 mM ammonium salt (HCOONH4, CH3COONH4, and NH4HCO3) or 0.1% formic acid in water. An isocratic
elution of 95% A and 5% B was used, and the ow was set at 0.25 mL/min. The nal concentrations of 5mC, 5hmC, and 5fC standards were 10 nM,
and the injection volume was 5.0 L.
water 1:9 (v/v) and 5.0 mL of methanol/water 3:7 (v/v). standards. The intraday and interday precision was estimated
When methanol was removed by nitrogen gas at room by triplicate quantication of 5hmC and 5mC in human urine
temperature, the eluted fractions from each step were frozen samples (nos. 1113) per day for three consecutive days.
at 80 C and then lyophilized at 50 C under a vacuum of Collection and Preparation of Human Urine Samples.
0.01 mbar. The residues were centrifuged at 12 000 rpm for 10 The urine samples were collected from 13 healthy and young
min and redissolved in 50 L of H2O. volunteers (age 2330, ve males and eight females). One
To evaluate the SPE protocol, the collected SPE fractions female volunteer (no. 1) was pregnant in the 25th week at the
were further analyzed using a Shimadzu LC-20AD HPLC time of urine collection. All volunteers were nonsmokers. All
system equipped with an SPD-20A UV detector. 5hmU, 5mC, collected urine samples were stored at 80 C before they were
and its oxidation products were separated with a Venusil MP subjected to any pretreatment and analysis. The samples were
C18 column (4.6 100 mm, 5.0 m, Agela Technologies, fully thawed and centrifuged at 12 000 rpm for 5.0 min. The
Tianjin, China) using an isocratic elution of methanol/water collected urine samples were pretreated using HLB cartridges
(1:9) at 0.8 mL/min. The detection wavelength was set at 280 (3.0 mL, 60 mg per cartridge). Each cartridge was
nm, and the injection volume was 5.0 L. preconditioned with 3.0 mL of methanol followed by 3.0 mL
To t to HPLC MS/MS analysis, the collected urine samples of water; then urine samples of 200 L, each mixed with
were also pretreated using solid-phase extraction. However, a [D3]5mC of 40 nM (nal concentration) and [D3]5hmC 80
smaller HLB cartridge (3.0 mL, 60 mg per cartridge) was nM (nal concentration), were loaded; the cartridges were
tailored for this application. washed with 1.0 mL of H2O, followed by the nal elution using
Linearity, Accuracy, and Precision. The standard 1.0 mL of methanol/water of 3:7 (v/v). The eluted fractions
solutions of 5hmC and 5mC standards of varying concen- were evaporated with nitrogen gas, lyophilized, and redissolved
trations (2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 nM) mixed with 80 in 200 L of pure H2O.
nM [D3]5hmC and 40 nM [D3]5mC (as internal standards)
were prepared and then analyzed using the HPLC-MS/MS
method described above. Calibration curves were constructed
RESULT AND DISCUSSION
Ammonium Bicarbonate Enhances the MS Detection
by linearly plotting the peak area ratios of 5hmC and 5mC to of 5mC, 5hmC, and 5fC. Our recent work demonstrates that
the corresponding stable isotopic standards against the ammonium bicarbonate can signicantly improve the ESI-MS/
concentration of the added nonisotopic 5hmC and 5mC. MS detection of acroleindeoxyguanine adducts by suppress-
To calculate the recovery of the method, known amounts of ing the formation of MS signal-deteriorating metal complexes.51
5hmC and 5mC (10 and 20 nM) were added to the urine However, we do not know whether ammonium bicarbonate
samples, respectively. After addition of 40 nM [D3]5mC and 80 enhances the LC-ESI-MS/MS detection of 5mC and DNA
nM [D3]5hmC, each sample was passed through a HLB demethylation intermediates. Here we examined the possibility
cartridge (3.0 mL, 60 mg per cartridge) and analyzed by to improve LC-ESI-MS/MS detection of 5mC and its oxidation
HPLC-MS/MS. The recovery (R) was determined according to products (5hmC and 5fC) using ammonium bicarbonate.
the formula shown below. First, we tested four additives to the mobile phase, including
ammonium bicarbonate, ammonium acetate, ammonium
Cmeasured C background
R= 100% formate, and formic acid. As shown in the bottom trace of
Cadded Figure 1, 5hmC migrates out at 1.7 min and is well separated
from 5mC (2.6 min) and 5fC (5.9 min) by the use of 0.1%
Cmeasured and Cbackground represent the measured concentrations HCOOH as the additive to the mobile phase. Evidently, for all
of 5hmC or 5mC in spiked and nonspiked urine samples, three tested ammonium salts (2.0 mM), the retentions of
respectively; Cadded is the added amounts of 5hmC and 5mC 5hmC (2.62.7 min), 5mC (4.45.1 min), and 5fC (7.6 min)
1848 DOI: 10.1021/ac5038895
Anal. Chem. 2015, 87, 18461852
Analytical Chemistry Article
Figure 2. Typical ESI-MS spectra of 5hmC when four additives were used. An isocratic elution of 95% A (2.0 mM ammonium salt or 0.1% formic
acid) and 5% B (pure methanol) was used, and the ow was set at 0.25 mL/min. A 10 M 5hmC standard solution (nal concentration) was used,
and the injection volume was 5.0 L.
Figure 3. HPLC-UV analysis of each SPE fraction obtained from the mixture of 5hmC, 5hmU, and 5fCs pretreated with C18 (A) and HLB
cartridges (B). A 1.0 mL premixed solution (nal concentration of each deoxynucleoside: 1.0 M) was loaded into the preconditioned cartridges.
Each SPE fraction was lyophilized and redissolved in 50 L of H2O. The concentrations of 5hmC, 5hmU, and 5fC standards were 20 M. The
injection volume was 5.0 L.
increase. More importantly, compared with other three as the mobile-phase additive promotes 5hmC to form abundant
additives (HCOOH, HCOONH 4 , and CH 3 COONH 4 ), [5hmC+Na]+ and [5hmC+K]+ complexes. Using normalizing
NH4HCO3 increased the MS/MS signals of 5mC, 5hmC, and intensity of [5hmC+H ]+ as 1.0, the relative abundance of
5fC by 1.814.3 times (right panel, Figure 1). However, the metal5hmC complexes is about 19.1% to 202.9% (Figure 2).
MS intensity of 5caC and 5hmU (data not shown) decreased Interestingly, when using NH4HCO3 as the additive to the
when using NH4HCO3 compared with HCOOH. They mobile phase, neither the [5hmC+Na]+ nor the [5hmC+K]+
probably prefer to deprotonate in basic mobile phase (pH 8). complex forms (Figure 2). These data suggest that NH4HCO3
Second, we compared the ionization complex distribution of partially improves the ionization eciency of 5hmC by
5hmC using four additives to the mobile phase. The HPLC suppressing the formation of metal5hmC complexes during
fractions of 5hmC were directly scanned from m/z 100 to 300 the ESI process.
under the positive ionization mode by an ESI-triple quadrupole Optimization of SPE Columns. There are more than 3000
mass spectrometer. HCOOH, HCOONH4, or CH3COONH4 chemicals found in human urine, and the 4 most abundant
1849 DOI: 10.1021/ac5038895
Anal. Chem. 2015, 87, 18461852
Analytical Chemistry Article
ingredients are Na+, Cl, K+, and urea, the levels of which range Table 2. Quantication of 5hmC and 5mC in Human Urine
from 4.6 to 22.5 mM/mM creatinine.52 To avoid possible Samples
ionization source contamination and ion suppression caused by
5hmC (nM) (mean 5mC (nM) (mean 5hmC vs
coexisting ingredients in urine, two types of SPE cartridges sample gender SD) SD) 5mC
(C18 and HLB) were tested for the cleanup and enrichment of
1 female 51.4 3.2 112.4 2.6 0.46
the target deoxynucleosides. We found that the C18 cartridges
2 male 17.8 0.4 19.0 0.2 0.94
were unable to eectively adsorb 5hmC during cartridge 3 female 36.9 1.7 41.0 1.4 0.90
washing by pure water, and approximately 95% of 5hmC and 4 female 12.9 1.6 41.4 2.1 0.31
5hmU were washed away (Figure 3A). In comparison, the HLB 5 male 16.0 1.9 18.8 0.4 0.85
cartridges, lled with a water-wettable stationary phase mixed 6 female 5.4 0.1 1.2 0.2 4.67
with the immobilized hydrophilic ligands and lipophilic ligands, 7 male 17.9 0.7 55.0 1.4 0.32
show a much better performance in the separation and 8 male 6.7 0.3 4.6 0.1 1.45
enrichment of all the targets from urine (Figure 3B). Less 9 male 35.7 1.8 161.5 1.0 0.22
than 20% 5hmC is lost during washing with water, and most of 10 female 33.9 2.0 97.0 1.4 0.35
5hmC, 5hmU, and 5fC are retained in the HLB cartridges until 11 female 20.6 1.8 100.0 2.9 0.21
they are eluted with 1030% methanol (Figure 3B). Of note, 12 female 10.9 0.7 4.8 0.5 2.27
we could observe a matrix eect even when we used HLB 13 female 28.2 1.6 24.5 1.0 1.15
cartridges to enrich 5hmC from urinary samples. The MS average 22.6 13.7 52.4 50.2 1.08
signals of 5hmC and 5mC enriched from urinary samples were
repressed over 17 times and 2.7 times (data not shown),
respectively. However, the accurate quantication of urinary detected in all the urine samples. This is reasonable since those
5hmC and 5mC is obtained by adding stable isotopic standards undetectable DNA intermediates display an abundance 1 or 2
of [D3]5mC and [D3]5hmC in known amounts to the urinary orders of magnitude lower than that of 5hmC.15,33,45
samples. As shown in Figure 4A, the peak of 5hmC from human urine
Calibration Curves, Recovery, and Precision. Calibra- (no. 2) (2.7 min, blue line) completely overlaps that of the
tion curves were obtained according to Experimental Section. added internal stable isotope standard ([D3]5hmC). Consistent
Excellent linearity for 5hmC (y (peak area ratio of 5hmC to with the nonisotope 5hmC standard (Figure 4C), urinary
[D3]5hmC) = 0.0152 (concn nM) 0.0025) and 5mC (y 5hmC also generates a transition with the second highest
(peak area ratio of 5mC to [D3]5mC) = 0.0347 (concn nM) abundance (m/z 258 124) (Figure 4B). Moreover, the signal
+ 0.1329) was achieved in the concentration range from 2.0 to ratio of the primary ion transition (m/z 258 142) to the
64.0 nM with the correlation coecient of R2 0.999. secondary transition (m/z 258 124) for urinary 5hmC is
The recovery was measured by spiking human urine samples almost the same as that of the nonisotope 5hmC standard
with known amounts of 5hmC and 5mC (10.0 and 20.0 nM, (3.2, evaluated by peak height). These results suggest that
nal concentration). The estimated recovery is about 101.3 urinary 5hmC has the same chromatographic retention and the
4.1% for 10 nM 5hmC, 103.5 2.4% for 20 nM 5hmC, 70.2 same MS fragmentation pattern as the 5hmC standard,
0.9% for 10 nM 5mC, and 89.9 0.6% for 20 nM 5mC. consistently conrming the presence of 5hmC in human urine.
Three urine samples (nos. 1113) were used for evaluating The measured concentration of 5hmC in human urine is
the precision of the HPLC-MS/MS method, and the results are about 5.451.4 nM, and the average concentration is about
summarized in Table 1. The interday precision values shown by 22.6 13.7 nM (n = 13). The average concentration of 5hmC
relative standard deviations (RSD) vary from 2.9% to 10.6%, in male and female urine samples is about 18.8 10.5 (n = 5)
and the intraday precision values vary from 1.4% to 7.7%. and 25.0 15.5 nM (n = 8), respectively. There is no
Given the presence of the matrix repression eect, the limits signicant dierence in 5hmC levels between male and female
of detection (LODs, S/N 3) are estimated to be 25 amol for samples (student t test, p = 0.45).
5mC and 250 amol for 5hmC, and the limits of quantication To evaluate the metabolism activity of 5hmC in genomic
(LOQs, S/N 10) are 75 amol for 5mC and 760 amol for DNA, 5mC as an intrinsic reference is detected and quantied
5hmC. The detection sensitivity of 5mC and 5hmC was in all urine samples. The average concentration is about 52.4
measured using the latest series of Agilent triple quadrupole 50.2 nM (n = 13). Considering the normal concentration of
mass spectrometers (G6495). creatinine in adult human urine (>18 years old) is about 10
Identication and Quantication of 5hmC in Human mM,52 the normalized value of 5mC in our urine samples is
Urine. By the developed o-line SPE-coupled stable isotope about 5.2 5.0 nmol/mmol creatinine, which is consistent with
dilution HPLC-MS/MS method, we further examined 5hmC the previous work.42
and the other DNA intermediates in human urine. We collected Previous work reported that the level of 5hmC is much less
morning urine samples from 13 healthy volunteers. 5mC and than that of its precursor 5mC in mammalian tissues.18,3234 In
5hmC could be detected in all the collected urine samples mouse tissues, the highest levels of 5hmC are found in genomic
(Table 2). However, 5fC, 5caC, and 5hmU could not be DNA from brain tissues, including cortex, brain stem, and
1850 DOI: 10.1021/ac5038895
Anal. Chem. 2015, 87, 18461852
Analytical Chemistry Article
Figure 4. Identication of 5hmC in no. 2 urine sample by HPLC-MS/MS. Chromatograms of urinary 5hmC and [D3]5hmC (internal standard) are
shown in A; the overlapped chromatograms for the two most intensive MRM transitions of 5hmC obtained from no. 2 urine samples and 5hmC
standards are shown in B and C, respectively. The concentration of 5hmC standard used in C was 5 nM. The optimized method was used, and the
details are described in Experimental Section.
spleen, thymus, muscle, bladder, and testes,33,34 the level of
5hmC is relatively low and accounts for 0.73.7% of 5mC. The ACKNOWLEDGMENTS
average ratios of 5hmC vs 5mC in human tissues are also
calculated: 2.9 0.8% for lung (n = 18) and 21.1 2.9% for This work was supported by grants from the Ministry of
brain (n = 6).18 Moreover, the ratios are much less in human Science and Technology of China (2011YQ060084 to H.W.),
lung (1.4 0.4%, n = 24) and brain tumors (5.5 4.3%, n = the Ministry of Environmental Protection of China (201309045
35).18 to H.W.), the Strategic Priority Research Program of the
The molar concentration ratio of 5hmC to 5mC (from Chinese Academy of Sciences (XBD14030200 and
urinary samples) is about 0.214.67 (Table 2). The level of YSW2013A01 to H.W.), and the National Natural Science
5hmC is even higher than that of 5mC in four samples (nos. 6, Foundation of China (21327006, 21435008, and 21125523 to
8, 12, and 13), and the molar concentration ratio of 5hmC to H.W.).
5mC is about 1.154.67. On average, the measured molar
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