Interference of theophylline and caffeine in the gas-chromatographic estimation of phenobarbital.

To the Editor: We report our experience with 5-ethyl5-p-tolyl barbituric acid (EPTB) as an internal standard for estimating serum phenobarbital and the problem we encountered with sera containing theophylline or caffeine, or both. Phenobarbital decomposes quite erratically in the presence of water and alkaline methylating agenth during oncolumn gas-liquid chromatographic methylation (1-3). Because EPTB and phenobarbital decompose in a reproducible manner under identical conditions (4), Pippenger et a!. (5) suggest the routine use of EPTB in estimating phenobarbital; we introduced it as the internal standard in our assay procedure for serum phenobarbital, which is a modification of the method of O’Callaghan et a!. (6). To 1 mL of serum was added 0.5 mL ofO.5 molIL HC1, followed by the internal standards 5-(p-methylphenyl)-5-phenylhydantoin (MPPH; 1000 zmolfL, 0.1 mL in ethanol) and EPTB (1000 mo1/L, 0.1 mL in ethanol). The mixture was extracted with 6 mL of chloroform for 20 mm by rotation at 60 turns/mm. After centrifugation and removal of the aqueous/protein layer, the chloroform layer was evaporated and the residue dissolved in 0.5 mL of ethanol/water (90/10 by vol). After extraction of this solution with 3 mL of n-hexane by rotation for 5 mm, the n-hexane (upper) phase was discarded and the ethanol was evaporated under nitrogen. The residue was dissolved in 25 &L of a 0.2 mol/L solution of trimethyiphenylammonium hydroxide in methanol and 1 iL was injected into the chromatograph. The glass column, 1.8 m long and 2 mm i.d., was packed with 3% OV-17 on Gas Chrom Q, 100/ 120 mesh. The carrier gas was helium (flow rate 25 mL/min), injection port and detector temperatures were 260 #{176}C, and the column was operated isothermally at 230 #{176}C. Our initial use of EPTB as the internal standard resulted in surprisingly low phenobarbital values for the control serum (Ortho Diagnostics Inc.), but other sera with phenobarbital added gave the expected result. By comparing the ratio of peak heights of EPTB/ MPPH for all the samples, we consistently obtained a ratio of 2.80 ± 0.16, except for the Ortho control serum, for which the ratio was 5.60. This suggested that a second compound was co-eluting with the internal standard EPTB. After gas chromatography of the Ortho control serum without added internal standard EPTB, a large peak appeared at 1.9mm. From previous experience we knew that caffeine had a retention time of 1.9 mm. As theophylline (one of the constituents of the Ortho control serum) is methylated on-column to form caffeine, this drug was clearly the cause of our low phenobarbital values, because of its co-elution with EPTB. We proved this by demonstrating the coelution of caffeine (250 tmoIfL) and the on-column methylated derivatives of theophylline (250 tmol/L) with EPTB (100 tmo1fL), at 1.9 mm. Thus erroneous results for phenobarbital can be obtained in samples containing theophylline or caffeine (or both) when EPTB is used a internal standard. For this reason, we are still using MPPH as the internal standard for phenobarbital, but we are currently investigating other chromatographic systems with the view of using EPTB. We caution others who are using similar chromatographic systems for phenobarbital of this co-elution problem.