Ag1 x8 resin

Thirty drops of 1 M NaOH were added to 1.8
mL of 0.5 M PLP and cooled to 0 °C in an ice bath. The mixture
of sodium borohydride (5.8 mg, 0.15 mmol) and [³H]-sodium
borohydride (25 mCi) in 450 μL of 0.1 M NaOH was added to the
PLP solution and stirred for 1 h at 0 °C. After being stirred,
120 μL of concd HCl was added to the solution slowly (became
pH 4). To the solution at pH 4, 20 equiv of ground MnO₂ was added, and the mixture was stirred at room temperature for a
total of 2 h. The mixture was brought to pH 8 with 1 M NaOH, and the
solution was centrifuged. The supernatants were collected and were
loaded onto a gel filtration column packed with Bio-Rad AG1-X8 resin
(hydroxide form). The mobile phase used was water and 5 M acetic acid
(gradient of 90% water to 0% water), flowing at 1.5 mL/min for 300
min. Fractions were collected every 10 mL and tested for its UV absorption
and radioactivity. The fractions with desired product were lyophilized.
The lyophilized product was redissolved in 100 μL of 1 mM PLP
and loaded onto an HPLC with an Econosil C18 column (10 mm ×
250 mm, 10 μm). The mobile phase used was 0.1% aqueous TFA with
5% acetonitrile flowing at 0.5 mL/min for 40 min. Under these conditions,
PLP eluted at 15 min. Fractions running with the PLP peak were collected
and counted for radioactivity using liquid scintillation counting.
The product was collected and lyophilized.