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Metabolites in Human Urine
Simple LCMS friendly method that is reproducible using (ANP) Aqueous Normal Phase LCMS
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printable Application Sheet
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Notes: For the screening of “enzyme defects” in humans, measuring the excretion of purines in urine is considered a good
diagnostic tool. High or low excretion levels of hypoxanthine was seen in cases of different types of leukemia. Betaine is
an important nutrient and due to its many metabolic roles there is an increasing demand for its measurement. Choline
appears to be a good tracer for the detection of a prostate lesion, however, further well-organized studies are needed
before it can be used clinically. The use of Cogent Diamond Hydride™ columns in ANP combined with the powerful MS
Agilent system prove useful for carrying out these studies as well as for the routine screening and monitoring of
metabolites in patients.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 0.1% formic acid
B: Acetonitrile + 0.1% formic acid |
| Mobile Phase |
Gradient t0 = 1.44 min
| Time |
%B |
Time |
%B |
| 0.0 |
95 |
35.00 |
50 |
| 0.2 |
95 |
35.1 |
95 |
| 30.0 |
50 |
40.0 |
95 |
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| Flow Rate |
0.4 mL/min. |
| Peaks |
- Hypoxanthine; 137.04580 m/z (M+H)+, RT = 4.98 min
- Chenodeoxycholic acid; 393.29990 m/z (M+H)+, RT = 6.23 min
- Betaine; 118.08680 m/z (M+H)+, RT = 15.27 min
- Choline; 104.10754 m/z (M+H)+, RT = 16.82 min
Figure: EIC – extracted ion chromatogram of selected compounds (1,2,3,4) |
| Sample |
Human Urine – after simple extraction
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| Detection |
ESI – pos - Agilent 6210 MSD TOF mass spectrometer. |
Discussion
This method can be used for routine assays of urinary purines (hypoxanthine), bile acids (chenodeoxycholic acid) and nutrients
(betaine, choline) in biological fluids. The method is very sensitive (due to the high content of organic component (acetonitrile)
in the MP and the use of “MS friendly” (formic acid), reproducible (% RSD for gradient analysis is below 0.5%) and accurate (MW to
3-4 decimal points).
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