AMPK Methods and Protocols

3.3 Preparation
of the HPLC System

The HPLC system used (DIONEX UltiMate 3000, Thermo Sci-

entific) consists of a column module with binary pumps, a

temperature-controlled autosampler (96-well plate) with a 50μl

injection valve, a temperature-controlled column compartment

module, and a fluorescent (UV) detector equipped with a 11μl

flow cell (Fig.1a). For peak analysis and quantification, the Chro-

meleon 7 software (version 7.1.2, Thermo Scientific) is used.

HPLC chromatograms are obtained at a wavelength of 254 nm

(Fig.1c and d).

1. Sonicate the pyrophosphate buffer during ~10 min (seeNote 3).


2. Fix the SUPELCOSIL™reversed-phase column (LC-18-DB,
   25 cm4.6 mm, 5μm, Sigma-Aldrich 58,355-U SUPELCO;
   Fig. 1b) to the system in the temperature-controlled column

0.0 5.0 10.0 15.0 20.0

-2.0

5.0

10.0

15.0

22.0 mAU

1 - 6.327

2 - 7.867

3 - 9.7204 - 10.100

5 - 12.273

6 - 14.753

7 - 15.747

min

0.0 5.0 10.0 15.0 20.0

-5.0

0.0

10.0

20.0

30.0

35.0 mAU

min

1 - 6.347

2 - 7.873

ATP 3 - 14.740

ADP

AMP

ADP

AMP

ATP

Standard

Hepatocytes

A C

D

B

Fig. 1HPLC system and examples of chromatogram. The HPLC system used (a; DIONEX UltiMate 3000,
Thermo Scientific) consists of a RS column module with binary pumps, a temperature-controlled RS
autosampler with a 50μl injection valve, a temperature-controlled RS column compartment module contain-
ing a SUPELCOSIL™reversed-phase LC-18 column (b), and a RS fluorescent (UV) detector equipped with a
11 μl flow cell. HPLC chromatograms obtained at a wavelength of 254 nm for standard (c) or test (d) samples
are shown as examples

Determination of Adenine Nucleotides by HPLC 233