Amino Acids

LC Conditions

Column

Luna 5 µm C18(2) 100 Å, LC Column 250 x 4.6 mm, Ea

Brand

Luna

Part No

00G-4252-E0

Phase Name

C18(2)

Elution Type

Gradient

Mobile Phase

A: Water

B: Acetonitrile

Gradient Profile

Step No.

Time(min)

%A

%B

1

20

20

80

1

0

100

0

Flow Rate

1.00 mL/min

Temperature

22°C

Detection

UV-Vis Abs.-Variable Wave.(UV) @ (22°C)

Detection Info

Sample Notes

Description of Sample Prep and HPLC: Do not use pipette tips or microcentrifuge tubes that have been autoclaved. The water that may collect from the steam and then dry may contaminate the samples. Collection: - Collect sample (3.5-4 ml unheparizied hematocrit tubes of peripheral blood or minimum of 100 mL culture media) - Centrifuge @ 3000 rpm, 4°C, 10 min - Transfer 75mL* of serum or culture media to a new microcentrifuge tube - Sample can now be stored @ -20 °C until ready to perform extraction Extraction: - Add 1.4 mL (2 x 0.7 mL) HPLC-grade methanol, vortex - Spin @ 15,800 g, 0 °C, 15 min - Transfer 1.2mL (2 x 0.6 mL) of supernatant to a new microcentrifuge tube - Dry completely in Speedvac on low heat (2-3 hr) - Store @ -20 °C until ready to run HPLC or proceed directly to next step HPLC: - Warm samples to room temp; resuspend dry extract in 100 mL distilled-deionized water (ddH2O), let sit at RT for about 30 min then vortex - Transfer 95 mL to HPLC sample vials (we use inserts inside vials); no bubbles - HPLC - run on a Luna C18(2) column (Phenomenex, 250mm x 4.6 mm) - 20mL injection volume with a linear water:acetonitrile gradient from 100:0 to 20:80 over 20 min; 1 mL/min - absorbance is read at 225 nm and integrated peaks are calculated by computer - Beckman System Gold 166 detector Standards: 75mL* of each triple standard is extracted as above. Each standard is run in duplicate with the average AUC used to construct a standard curve. Use distilled-deionized water (ddH2O) to make up standards. - Prepare standard stock solutions of kynuerinine, L-tryptophan and 1-methyl-DL-tryptophan (each = 1mg/ml). Protect from light and store @ -70 °C. - Triple standard is a mix of 50ml each of kynuerinine, L-tryptophan and 1-methyl-DL-tryptophan. When brought to a volume of 1ml, the concentration of each = 50 mg/mL - this dilution is used as the most concentrated standard - From the above dilution, make 4 additional serial dilutions (25, 12.5, 6.25 and 1.25 mg/mL) *A 150 mL sample will give a much more readable result if you suspect low amounts of kynuerinine. Directions for dissolving 1-methyl-DL-tryptophan (1 mg/mlL ~4.6mM) total volume 100mL 1-MeTrp 100mg 1N NaOH 1mL distilled H2O 98ml HCl ~1mL Weigh 1-MeTrp and place in bottle. Add 1N NaOH and mix until majority of powder is dissolved. Add water and stir for ~ 1hr. Titrate to pH=7 using 1N HCl. Aliquot and wrap in foil and store @ -70°C.