Whole blood assay:
1. Collect blood into two heparinized blood tubes (for unstimulated control and forskolin).
2. Add forskolin stock to one tube, 10 µM final concentration.
3. Incubate control and forskolin stimulated sample for 24 hrs at 37°C incubator or water bath.
4. Centrifuge in table top centrifuge at 3000 rpm for 10 min.
5. Store plasma at -80°C.

Tissue preparation:
1. Euthanize and debleed animals and rapidly excise DRGs and spinal cord tissue, immediately freeze on dry ice or liquid nitrogen, store at –80°C.

Tissue homogenization, acidic oxidation [1] and solid phase extraction:
1. Add 200 µl of 0.1 M hydrochloric acid containing 20 ng/ml of the internal standard rhamnopterin to the defrosted tissue (small pieces of about 2-3 mg) or plasma sample (50 µl) in a 1.5 ml microcentrifuge tube.
2. Homogenize tissue with reusable pellet pestles and motor (Kontes Glass Company, Vineland, New Jersey, USA) for 45 s with at least 10 strokes, remove 10 µl of the suspension for protein determination.
3. Add 100 µl of iodine solution (1% (w/v) iodine and 2% (w/v) potassium iodide in 1 M hydrochloric acid), mix and incubate for 30 min in the dark at room temperature.
CRITICAL STEP: Always prepare fresh iodine solution.
4. Stop the reaction by adding 200 µl of 5% (w/v) ascorbic acid in water and vortex up to decolorization.
CRITICAL STEP: Always prepare fresh ascorbic acid solution.
5. Activate the cation-exchange cartridge (Oasis MCX extraction cartridge) with 1 ml methanol and 1 ml water before loading of the sample.
6. Load the completely decolorized sample onto the cartridge, allow binding and vacuum elute unbound substances, wash with 1 ml 0.1 M hydrochloric acid and 1 ml methanol.
7. Elute bound substances with 1 ml of 8% (v/v) ammonia solution in 25% methanol.
CRITICAL STEPS Points 5-7: Use a maximum speed of 1 ml per min for vacuum elution
8. Evaporate the organic solvent to dryness by a gentle stream of nitrogen at 40°C.
9. Reconstitute the residue with 200 µl water/0.002% formic acid (v/v) and transfer the solution in a screw vial with a glass insert containing a Silikon/PTFE seals in the screw cap (e.g. Macherey-Nagel).

Chromatography
1. Use a Gemini C18 column (150 × 2 mm i.d., 5 µm particle size, 110 Å pore size) for chromatographic separation, connected to a pump and autosampler for injection of the samples (e.g. Agilent 1100 Series binary pump (G1312A) and degasser (G1379A) connected to an HTC PAL autosampler).
2. Use as mobile phase eluent A (water with 0.002% formic acid) and eluent B (methanol with 0.002% formic acid) and apply the following gradient:

0 - 0.5 min A/B 100:0
0.5 - 4 min a linear gradient from 100:0 to 5:95
4 - 7 min A/B 5:95
7 - 8 min a linear gradient from 5:95 to 100:0 and finally
8 - 13 min A/B 100:0

3. Inject 10 µl of the sample onto the LC-MS/MS.
4. Set the flow rate to 0.3 ml/min and the runtime to 13 min.
5. Biopterin, neopterin and rhamnopterin elute after 5.7, 4.1 and 5.5 min, respectively.

MS/MS analysis
For determination of total biopterin and neopterin concentrations use liquid chromatography coupled to tandem mass spectrometry employing a 4000 Q TRAP triple quadrupole mass spectrometer with a Turbo V source.

1. Choose the negative ion mode with an electrospray voltage of -4300 V at 400 °C and set the auxiliary and nebulizer gas to 50 psi, the curtain gas to 10 psi and the collision gas to 5 psi Use pure nitrogen as gases.
2. Use precursor-to-product ion transitions of m/z 236 to 192 for biopterin (collision energy -20 V), 252 to 192 for neopterin (-22 V) and 265 to 192 for rhamnopterin (-20 V) as quantifier for the multiple reaction monitoring (MRM) with a dwell time of 150 ms.
3. Evaluate concentrations of the samples with calibration standards (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 50 ng/ml) using Analyst software 1.4 (Applied Biosystems). Every tenth sample in each run should be a quality control (1, 10, 50 ng/ml).