Simultaneous liquid-chromatographic determination of 12 common sedatives and hypnotics in serum

We present a method for simultaneously determining 12 hypnotics and sedatives (primidone, methyprylon, phenobarbital, butabarbital, butalbital, ethchlorvynol, pentobarbital, amobarbital, phenytoin, glutethimide, secobarbital and methaqualone) in 200 microliter of serum. Serum proteins are precipitated with an acetonitrile solution containing 5-(4-methylphenyl)-5-phenylhydantoin, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer, at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm; their quantities are estimated from their peak heights. Each analysis requires no longer than 30 min at the optimum column temperature of 50 degrees C. The lower limit of detection for all of these drugs is less than 10 ng/sample for drug standard. A sensitivity of 1.0 mg/liter of serum is attained routinely for each of the drugs. Analytical recoveries for the 12 drugs varied from 94 to 112%, with good day-to-day precision (CV between 3.8 and 10.4%). Of more than 35 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.