Briefly, to 360 μL EDTA plasma in a microfuge tube was added 180 μL distilled water, and after mixing and closure with a screw cap boiled for 30 min. After cooling on ice, the tubes were fast frozen in liquid nitrogen and thawed quickly in an ambient temperature water bath to allow for greater compaction of the pellet during subsequent centrifugation at 25,000 × g for 15 min without cooling. The supernatant was then removed, its volume measured, 0.1 volumes of 6 M trichloroacetic acid added with mixing, and then centrifuged for 25,000 × g for 10 min at 4°C. The supernatant was stored at -80°C until analysis. Standards were generated by this same procedure after spiking pooled plasma with a known amount of folic acid (determined using ε281 nm = 27,600 at pH 7.0). The level of the standard was corrected for the measured value of endogenous unmetabolized folic acid in the pooled plasma prior to spiking. The use of spiked plasma not only takes into account the recovery during workup (~87%), but also prevents adsorption of folic acid to the walls of the autosampler vials during analysis.
For the first stage of the HPLC analysis 50 μL of sample or standard was injected with a Varian ProStar 420 autosampler into a Zorbax C8-SB column (1.8 μ, 50 × 4.6 mm, Agilent) protected with a Hypersil C8 BDS (3 μ, 4.0 × 3.0 mm SecurityGuard cartridge, Phenomenex), both placed in a water jacket maintained at 40°C. This was eluted at 0.75 ml/min with 20 mM ammonium hydroxide plus acetic acid to pH 3.6/acetonitrile (93:7). Just prior to the elution of the folic acid from the above Zorbax -C8 column, the mobile phase was automatically redirected by a switching valve (Vici-Cheminert model C72) from waste to the head of two coupled Luna phenyl-hexyl columns (3 μ, each 150 × 4.6, Phenomenex) placed in a water jacket maintained at 40°C. After complete transfer of the folic acid peak from the Zorbax-C8 to the phenyl-hexyl columns, the switching valve was returned to the initial state causing the remainder of the eluate from the first column to again go to waste. The phenyl-hexyl columns were developed with 40 mM ammonium hydroxide plus phosphoric acid to pH 2.1/acetonitrile (9:1) at a flow of 0.8 ml/min. The pressures of the two pumps were equalized by attaching the appropriate length of 65 μ PEEK capillary tubing (Upchurch) to the waste port of the switching valve. The timing of elution from the Zorbax-C8 column was determined by connecting this capillary directly to a photodiode array (Waters model 996) and injection of 50 μL of 400 nm folic acid in 0.55 M trichloroacetic acid without switching. The above combination of columns and mobile phases was specially optimized for the more challenging chromatography of non-fasted samples.
Fluorescence detection of folic acid was greatly enhanced by coulometric electrochemical oxidation of the mobile phase from the phenyl-hexyl columns by passage first through an ESA 1010 dual cell with both electrodes set to the potential of maximal response (ESA model 5100A controller, typically between 0.45 V and 0.6 V). The output of the electrochemical cell, primarily the folic acid cleavage products pterin-6-carboxylic acid and p-aminobenzoyl glutamate, was fed into a Waters model 2476 fluorometer set to 362 nm excitation and 455 nm emission. After analysis of the spiked plasma standards, 50 μL of 0.55 M trichloroacetic acid was injected to establish that the system was free of residual folic acid. In the event of detection of a folic acid peak, several injections of 0.1 M Tris-Cl, pH 8.0/acetonitrile (1:1) were made without switching, to clear the autosampler of any carryover. All samples were injected twice (from a cooled and dark sample tray), the second time at an oxidation potential 0.3 V lower than the first. At this lower voltage specifically the folic acid peak decreases by about 95%, thus permitting not only its definitive identification, but also facilitation of placement of baselines for quantification of peak heights using an Empower (Waters) data system. The limit of detection (S/N = 3) was 0.15 nM in plasma, and the intra assay CV = 3%.