Analyte
Stock solution
[Expected]
pH, Max UV wavelength
ε max × 10−3
Adenine
30 mg in 100 mL Buffer A
2.2 mM
pH 6–8, 260 nm
13.3
Xanthinea
12 mg in 1 mL 10 N NaOHa
0.79 mM
pH 2–6, 260 nm
8.85
Hypoxanthine
70 mg in 100 mL Buffer A
5.1 mM
pH 4–7, 260 nm
7.90
Guanosine
24 mg in 100 mL Buffer A
0.85 mM
pH 7, 260 nm
11.7
Inosine
120 mg in 100 mL Buffer A
4.47 mM
pH 3–6, 260 nm
7.10
Adenosine
120 mg in 100 mL Buffer A
4.49 mM
pH 7–12, 260 nm
14.90
Deoxyadenosine
55 mg in 100 mL Buffer A
2.04 mM
pH 2, 258 nm
14.1
2.
Standard curve mix: The single standard curve mix is made by adding each analyte to Buffer A for a final concentration of 100 μM for each analyte in a total volume of 50 mL. Vortex each stock analyte solution well prior to making the mix. Aliquot 500 μL into singly labeled microfuge tubes and store in −80 °C freezer.
3.
Prepare working calibrators according to Table 2.
Table 2
Standard curve construction for urine purines and succinyladenosine
Working sol’n | Int. std. | Sample buffer | Concentration (μM) |
|---|---|---|---|
5 | 10 | 95 | 5 |
10 | 10 | 90 | 10 |
25 | 10 | 75 | 25 |
50 | 10 | 50 | 50 |
75 | 10 | 25 | 75 |
100 | 10 | 0 | 100 |
4.
Prepare the following vials for the standard curve using the standard curve and internal standard mixes from the −80 °C freezer. The mixes are aliquoted into single use microfuge tubes. Thaw the standard curve and internal standard solutions to room temperature and sonicate for 2 min. Leave at room temperature during use and discard any remaining mixes as freezing/thawing degrades the analytes.
5.
Succinyladenosine (2.6 mM working solution): Add 12 mg of adenylosuccinic acid (Sigma, MO) to 10 mL of 40 mM Tris–HCl buffer (pH 8.0). Add 100 U of 5′-nucleotidase (Sigma, MO). Incubate at 37 ± 1 °C overnight. Check conversion using tandem mass. Substrate adenylosuccinate displays a 427 m/z peak and succinyladenosine 382 m/z. Add another 100 U enzyme if conversion is not complete.
6.
Quantitate the succinyladenosine concentration by diluting 200× and measuring its absorbance with the spectrophotometer. The millimolar extinction coefficient is 19.2 at 268 nm. Make an individual standard curve mix for succinyladenosine by diluting in buffer A to a final concentration of 100 μM. Aliquot 500 μL into singly labeled microfuge tubes and store in −80 °C freezer.
2.4 Quality Controls and Internal Standards
1.
Internal Standard Mix: 15N2-adenine (Sigma, MO), 13C5-adenosine , 13C10, 15N5-guanosine, 1,3-15N2 Xanthine , and 15N5 Deoxyadenosine (Cambridge Isotope Laboratories, MA). Prepare a 200 μM solution of each of the above solutions in running buffer A.
2.
Each run must contain two levels of spiked controls. Pool urines for a volume of about 100 mL. Put 50 mL each into two 50 mL conical tubes and centrifuge at 3000 × g for 10 min. Transfer urine supernatants into new 50 mL conical tubes. The level of endogenous adenosine in normal urine is very low and a small concentration spike is used to create the low control range. Aliquot 1 mL to labeled microfuge tubes and store in the −80 °C with the assay reagents. Establish control ranges by running ten control samples of each.
2.5 Supplies and Equipment
1.
Column: Atlantis T3, 5 μm particle, 2.1 × 100 mm (Waters, MA).
2.
Acquity UPLC coupled with TQD Tandem Mass Spectrometer (Waters, MA).
3 Methods
3.1 Stepwise Procedure
1.
Allow urine samples to thaw completely and mix thoroughly. Samples may be maintained at room temperature while processing.
2.
Prepare each urine sample as follows:
(a)
Sample volume: 50 μL
(b)
Internal standard mix: 10 μL
(c)
Sample buffer: 50 μL
Mix samples well and briefly centrifuge before loading to LCMS. Injection volume is 3 μL.
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