Therapeutic Drug Monitoring: Measurement of Antiepileptic and Barbiturate Drug Levels in Blood by Gas Chromatography with Nitrogen—Selective Detector DEREK P
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ANNALS O F C LIN IC A L AND LABORATORY SCIEN CE, Vol. 6, No. 5 Copyright © 1976, Institute for Clinical Science Therapeutic Drug Monitoring: Measurement of Antiepileptic and Barbiturate Drug Levels in Blood by Gas Chromatography with Nitrogen—Selective Detector DEREK P. LEHANE, Ph.D., PAUL MENYHARTH, B.S., GIFFORD LUM, M.D., and ARTHUR L. LEVY, Ph.D. Chemistry Laboratory St. Vincent’s Hospital and Medical Center of New York, New York, NY 10011 ABSTRACT The nitrogen-specific detector for gas chromatography consists of a non volatile rubidium silicate bead, around which nitrogen-containing com pounds are pyrolyzed. Speed, sensitivity, specificity, accuracy, small sam ple size and minimum sample handling are characteristics of the nitrogen detector that render it superior to conventional gas chromatographic detec tors. The detector has been utilized to effect a simple and rapid quantita tion of allobarbital, amobarbital, butabarbital, heptabarbital, pentabarbital, phénobarbital and secobarbital, plus the anticonvulsants diphenylhydan- toin and primidone. Extraction of the drugs from acidified serum into or ganic solvent containing internal standard is followed by oncolumn méthy lation with methanolic trimethylphenyl ammonium hydroxide. The drugs, separated on a column of 3 percent OV-lOl on Gas-Chrom Q, 100-120 mesh, are readily quantitated by simple calculations based on peak-height ratios. Therapeutic drug monitoring is discussed in relation to recent con cepts of drug-protein binding, drug-drug interactions, drug biotransforma tion and problems of multiple drug therapy. Introduction chromatographic analysis of phosphorus- and nitrogen-containing compounds by Therapeutic drug monitoring is techni introduction of a specific alkali flame de cally demanding but extremely useful tector. Various commercial modifications because it correlates patient status di of this thermionic detector have been rectly with blood levels. Currently, gas- applied to toxicologic assays; recent clin chromatographic procedures are being ical applications of the detector include widely applied to the routine monitoring quantitation of blood levels of tricyclic of drug levels in blood. Karmen and Giuf- antidepressants,3 analgesics6 and drugs of frida7 increased the sensitivity of gas- abuse.15 4 0 4 THERAPEUTIC DRUG MONITORING 405 Since barbiturates and the anticonvul sants, diphenylhydantoin and primidone, CN* CN each contain two nitrogen atoms, they are ideally suited to gas-chromatographic analysis using the nitrogen-specific de Rb Rb* tector. As the detector signal is propor surface tional to the nitrogen content of the mol of the ecule, the presence of two nitrogen atoms L-------JL. bead favors high sensitivity, increased approx Rb++e" Rb* imately ten-fold over that of conventional \ flame ionization detectors.15 Recently, the nitrogen detector has been success FIGURE 1. Proposed mode of action of the fully applied to the analysis of blood nitrogen-selective detector.8 levels of both barbiturates2 and an tiepileptic drugs.5,16 Based on these pro cyan radical takes up one electron from cedures, a rapid and reliable assay has the alkali, resulting in a symmetrical been developed for diphenylhydantoin, cyanide ion and a positive alkali which is primidone and eight barbiturate drugs. captured by the bead (figure 1). The cyanide ion migrates to the collector elec The Nitrogen-specific Detector trode, but presumably liberates one elec tron either by oxidation, resulting in The nitrogen-specific detector is a neutral products, or by reaction with hy modification of the standard flame ioniza drogen atoms, forming HCN. The elec tion detector. A glass bead containing al tron thus is collected and is responsible kali as a primarily non-volatile rubidium for the specific nitrogen response. silicate is positioned between the jet and The thermionic nitrogen detector dis the collector electrode. The bead is al plays significant advantages over conven ways at a negative potential of 130v tional flame-ionization or electrón- against the collector electrode. Kolb and capture detectors, not the least being its Bischoff8 have presented a concise ac speed, sensitivity, specificity, accuracy, count of the theoretical aspects of the ni small sample size, minimum sample trogen detector. The specific sensitivity handling and detection limits indepen toward nitrogen-containing compounds dent of the matrix.15 The detection limit is facilitated by a low hydrogen flow rate of the nitrogen detector is 0.1 Pg N, with of 10 ml per minute together with an air a 105- fold linearity. flow of approximately 100 ml per minute. The heat generated from such a low Principle volume of hydrogen is insufficient for Internal standard (5-(p-methyl- achieving the necessary temperature for phenyl)-5-phenylhydantoin) in organic alkali emission by the bead; an additional solvent is added to serum. The drugs pass electrical heater is therefore employed. into the organic phase, which is removed The hydrogen is burned around the hot and evaporated to dryness. The residue is bead, thus forming a diluted and cold dissolved in methanolic trimethylphenyl flame zone. If an organic nitrogen- ammonium hydroxide; injection of this containing compound enters this flame solution into the injection port heater of zone around the bead, it is pyrolyzed the gas-chromatograph results in quan rather than oxidized, resulting in the pro titative methylation of the antiepileptic duction of cyan (C = N) radicals. Such a and barbiturate drugs. 4 0 6 LEHANE, MENYHARTH, LUM AND LEVY Reagents Special Apparatus Included are: Necessary reagents include: Reactivials, 1.0 ml.** Extraction solvent: chloroform- Rolded filter paper, 12.5 cm diame isopropanol-benzene: 94-4-1, v/v/v. ter. tf Hydrochloric acid, 1.0M. Gas-chromatograph model #900, with Methanolic trimethylphenyl am heated nitrogen detector. $$ monium hydroxide, 0.1M.* Column: single glass column, 1.83 m x 5-(p-methylphenyl)-5-phenylhydantoin.f 2 mm internal diameter, packed with 3 Diphenylhydantoin. j percent OV-lOl on Gas-Chrom Q, 100- Allobarbital, amobarbital, butabarbital, 120 mesh.§§ pentabarbital, phenobarbital and seco Column oven: programmed one min barbital.§ ute at 175°, then 24° per minute to 285°, Heptabarbital.11 then four minutes at 285°. Ion-free serum.H Injection port heater: 300° Detector oven: 285° Stock Standard Solutions Gas flow rates: helium 30 ml per min ute; hydrogen 10 ml per minute; and air Each drug is dissolved in methanol to 100 ml per minute. give a final concentration ofone g per liter. Before initial use, the column is con ditioned overnight at 300°, with a helium Working Standard Solutions flow rate of 30 ml per minute. Aliquots of each of the stock standard Procedure solutions are mixed and diluted with To a teflon-lined screw-capped tube methanol to give a level for each drug of are added, in sequence, 1 ml serum or 100 mg per 1. From this solution aliquots standard, 0.1 ml 1.0M hydrochloric acid are diluted in ion-free serum to give and 12 ml extraction solvent containing working standard mixtures containing 5 internal standard. The mixture is then mg per 1, 10 mg per 1, 20 mg per 1, 30 mg shaken vigorously for one minute. The per 1, 40 mg per 1, and 50 mg per 1, respec organic phase is filtered into a second tively, of each drug. tube and evaporated to dryness at 50° under a stream of nitrogen. The dried res Internal Standard Working Solution idues can be stored in stoppered tubes at room temperature prior to chromatog The internal standard, 5-(p-methyl- raphy. phenyl)-5-phenylhydantoin, is dissolved The sides of the tube are washed with in extraction solvent to give a concentra 0.8 ml extraction solvent. The solution is tion of 1.5 mg per 1. transferred to a Reactivial and is evapo rated to dryness under nitrogen. * Eastman Organic Chemicals, Rochester, NY 14650. f Aldrich Chemical Company, Milwaukee, ** Pierce Chemical Company, Rockford, IL WI 53233. 61105. $ Sigma Chemical Company, St. Louis, ff Schleicherand Schuell, Keene, NH 03431. MO 63178. £{ Perkin-Elmer Corporation, Norwalk, CT § Gane’s Chemical Works, Carlstadt, NJ 07072. 06856. 11 Geigy Pharmaceuticals, Ardsley, NY 10502. §§ Applied Science Laboratories, State College, 11 Clinton Laboratories, Santa Monica, CA 90404. PA 16801. THERAPEUTIC DRUG MONITORING 4 0 7 Methanolic trimethylphenyl am monium hydroxide (50 ¡A) is added to the vial. The contents are mixed and an aliquot (0.7 /xl)is injected into the gas- chromatograph. Calculations Peak heights are measured for drugs, standards and internal standards. A height ratio for each standard concentra tion is calculated: Peak height (standard) _ Peak height (internal standard) R (height ratio) and values of R are plotted graphically against the respective standard concen tration. Values of R are similarly calculated for each peak of an analytical sample, and the drug concentration read from the re spective standard curve. Results A typical chromatogram of the separa tion of the various barbiturate and an tiepileptic drugs is shown in figure 2. Application of temperature programming in conjunction with the nitrogen- selective detector achieves maximal sep aration of the drugs in the shortest time span, each sample cycle being 12 m in utes duration. Calibration of drug levels is performed rapidly and simply by plot ting peak height ratios; in this regard, the nitrogen detector is of particular value since the internal standard (peak 10 in figure 2) appears as a sharp spike, while FlGURE 2. Gas chromatogram illustrating the temperature-programmed separation of the barbitu with conventional flame ionization detec rate and anticonvulsant drugs in ion-free serum, tion the internal standard appears as a with relative retention ratios (internal standard = low, broad peak. As shown in figure 2, 1.00). OV-lOl, 3 percent, programmed one minute peaks are well separated from the solvent at 175°, then 24° per minute to 285°, then four min utes at 285°. Included are (1) allobarbital, 0.18; (2) front, while peak tailing and overlap are butabarbital, 0.24; (3) amobarbital, 0.29; (4) pen minimal.