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Metabolites in Urine
Isobaric compounds (creatine and 4-hydroxyproline) separated from other components of synthetic urine
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printable Application Sheet
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Notes: Cr is a compound that is made primarily in the liver and then transported to muscles where it is used as an
energy source. Once in the muscle, some of the Cr is spontaneously converted to Cm. It is important to monitor the
Cr/Cm ratio. An increased ratio may be due to several diseases: congestive heart failure or dehydration. It may also
be seen with gastrointestinal bleeding, or increased protein in the diet. The ratio may be decreased with liver
disease (due to decrease in the formation of urea) and malnutrition. Cr and Cr analogs such as cyclocreatine were
found to have antitumor, antiviral, and antidiabetic effects and to protect tissues from hypoxic, ischemic,
neurodegenerative, or muscle damage.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-7.5P |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water, 0.1% formic acid
B: Acetonitrile, 0.1% formic acid |
| Mobile Phase |
| Gradient |
| Time |
%B |
Time |
%B |
| 0.0 |
95 |
35.0 |
50 |
| 0.2 |
95 |
35.1 |
95 |
| 30.0 |
50 |
40.0 |
95 |
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| Flow Rate |
0.4 mL/min. |
| Sample |
Synthetic urine |
| Compounds |
- Creatinine - Cm
M+H 114.0662, RT = 11.98 min
- Creatine - Cr
M+H 132.0768, RT = 12.52 min
- 4-Hydroxyproline
M+H 132.0655, RT = 13.16 min
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| Detection |
ESI – pos - Agilent 6210 MSD TOF mass spectrometer |
Discussion
Creatine and Creatinine are typically very difficult to separate from each other. A simple gradient with an MS friendly mobile
phase was used to resolve these two compounds. In addition 4-hydroxyproline which has a very similar mass weight as creatine was
also easily separated. A powerful combination of an Agilent MSD-TOF instrument (4 digit mass accuracy) and a Cogent Diamond
Hydride™ LCMS column takes away guessing from the analysis of metabolites in a complex mixture as shown.
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