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Departments of 1Chemistry, 2Biological Sciences, and 3Computing Science, University of Alberta, Edmonton, Alberta, Canada


 
  


 

 

= Metabolites were separated by 1D HPLC (250 x 10 mm Six independent HPLC experiments were performed to increase In this work , MS data were collected for all the pooled fractions at both positive and

Overall, 0 fractions were collected from each


C18 column) using 0.1% TFA in ACN the concentration of the metabolites for NMR analysis. The negative ion mode to determine the molecular mass of the metabolites which assists the
experiment and subjected to speed vac to remove
= To identify and quantify metabolites from human extracted urine sample was injected (5 mL each) onto a C18 interpretation of NMR spectra for metabolite identification.
= The fractions were subjected to speed vac concentrator to acetonitrile and pooled into 13 fractions based on
biofluids using MS and NMR column at a flow rate of 5 mL/min using 0.1% TFA in acetonitrile
remove acetonitrile and then lyophilized. retention time and then freeze-dried and lyophilized


! and the UV-chromatograms are shown in 6
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overnight. The dried pooled fractions were dissolved  
*MSD  SPC, time=0.2 :0. 43 of M  \2 SAMP \05 2402.D
  
API- S, Pos, Scan ¬ . Example of MS Search Results
= The flow chart of the extraction is shown in 6
" in solvents compatible with NMR and internal 00
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= 1D-HPLC coupled to MS and NMR 
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standards were added according to the standard

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operating protocol and analyzed by 500 MHz NMR as
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= A total of 48 metabolites were identified and $

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quantified from a normal human urine sample 0 r
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ACN

m AU

m AU
r
  
 
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Centrifuge 1


1


50 00 50 200 250 300 350 m/z

(A) P2

Urine 1 1

6
*+ (A) MS spectrum (positive mode) of pooled fraction 2 obtained from the single




p

 quadrupole mass spectrometer by flow injection of 2 dL solution. (B) MS search results for
(1:1)


a single m/z value (136.1) against a Human Metabolome Database ¬ www.hmdb.ca)

 1
1
 
 
 
 
 
 7

in u t e s

The determination of all possible metabolites from urine is  

 

important in the study of human diseases. GC and GC-MS 




-
p. Partial list of identified and quantified human urine metabolites obtained using N R Chenomx
Remove eclipsed software. The reported concentration is from the Human etabolome Database
have been widely used for the analysis of specific  

 



metabolites in urine. However, the sample preparation for Supernatant ACN








Compound Name

W

umol mol creatinine umol mol creatinine

m AU

m AU

       





GC and GC-MS is time consuming, because derivatization 1 1
Adenine 1 . . . 1 1.7 (> years) 

(B) P3       






N Acetylaspartatic acid

17 . 7 . < (> 1 years)
procedure is often required. Sample preparation for HPLC 1 1

 
   
Original 



Acetoacetic acid 1 . 1 9 . <
is relatively simple. In this work we have developed a volume Xanthine

1 .




. 7 . .7
 




 
    
method to isolate, purify, identify and quantify metabolites Ferulic acid 19 . 791
  
.

.1 .



   
  
 
in urine by HPLC-MS and NMR.

1
1 

in u t e s


7

Homovanillic acid 1 . 791 .1 < .7
Fractionation by  

 

Pyruvic acid
 
. 1
   
. 1 .





      

HPLC 




anillic acid 1 . 7
  
.17
 
. 7



Succinic acid 11 . 1 . . 7 .7 

 

 


      
lutaric acid 1 . 7 . 1. 

 


 






(C ) P4

Hydroxybutyric acid


1 . 7

 
. .


 

           
m AU

m AU
1


1



lycolic acid 7 . 1 . 7 1 .7 . 

       

1


1


aleic acid 11 . 1 9 . 1 <

       
Hippuric acid 179. . 17 . 

p 



 




 

 
Phenol 9 . 1 . .97 

= Single quadrupole mass spectrometer (Agilent HP


 
 
 
 
 


,    6

 (&
1 1 7

1100 MSD )  

in u t e s

 

= 500 MHz NMR (Varian Unity Inova) 




Ô The MS data were used to generate a list of possible metabolites based on the
 

 

= HPLC system (Beckman Courier) (D) P5 molecular mass match with those listed in a Human Metabolome Database.





Ô NMR was used to identify the metabolites. A total of 48 compounds were identified by
m AU

m AU



   

 1


1


1


1


HPLC-NMR.

= Urine sample was collected in a sterile container




from a healthy volunteer Ô Most of the metabolites identified in this work are high abundance. In the future, 2D-
Pictures of some urine fractions


HPLC will be used to enrich low abundance metabolites.
= Acetonitrile (ACN) and aqueous methanol were
 1
1
 
 
 
 
 
 7
%
in u t e s
Ô e will also focus on identifying novel metabolites using 2D-HPLC-FT-MS and NMR.
used to extract the metabolites
6
"+ Flow chart of metabolite extraction protocol
6
$+ The first four UV-chromatograms of the reversed 6
)+ Typical examples of the NMR spectra for '& 




= The supernatant were subjected to rotary (only with ACN), fractionation by HPLC and picture of phase HPLC separation of the extracted metabolites from pooled fractions 2(A), 3(B), 4(C) and 5(D).
 ater and This work was funded by Genome Alberta and Genome Canada through the Human
evaporation at room temp. urine fractions collected from reversed phase HPLC. normal human urine. DSS (at 0.0 ppm) peaks are removed from the spectra. Metabolomics Project.