Determination of Theophylline in Plasma by Electron Capture Gas Chromatography Harvey A. Schwertner,* Thomas M. Ludden, and Jack E. Wallace Department of Pathology and Division of Clinical Pharmacology, Departments of Pharmacology and Medicine, The University of Texas Health Science Center, San Antonio, Texas 78284
A procedure for the determination of theophylllne in plasma by electron capture gas chromatography Is presented. The method Involves derivatization of theophylllne and the internal standard, 3-isobutyl-l-methylxanthine,with pentafluorobenroyl chloride. Measurement of the derivatives by means of the electron capture detector provides a detectlon llmlt of a1 least 10 ng/ml plasma for theophylllne. The slngle extraction method permits accurate measurement of plasma concentratlons (f0.22pg/ml) with little or no interference from theophylllne metabolites, caffelne metabolites, and other coextractable materlal. Mass spectra of theophylline extracted from the serum of patients on theophylline therapy were identical to those displayed by the reference theophylline.
Theophylline, 1,3-dimethylxanthine,is used for the treatment of bronchial asthma and other respiratory disorders. There is a pronounced intersubject variation in its biological half-life and its therapeutic response appears to be related to the plasma concentration ( 1 ). Consequently, knowledge of plasma concentrations is considered useful in management of theophylline therapy and accurate procedures for its determination in plasma are essential. Most procedures designed for the determination of theophylline have been based on relatively nonspecific spectrophotometric methods (2);however, a recent modification has been presented which largely eliminates interference from barbiturates ( 3 ) .The ultraviolet methods generally require 3 ml of plasma and large volumes of extraction solvents, and quantitation, based on external standards, is often complicated by variable recoveries. As with many spectrophotometric scanning methods, there are also problems with establishing an appropriate baseline and in eliminating background absorbance (3). Difficulties inherent in spectrophotometric methods have led to an increased interest in gas-liquid chromatographic procedures. Because of the polar nature of theophylline, derivatives must be formed prior to gas-liquid chromatographic analysis. Several alkylating agents, e.g., tetrapropyl ammonium hydroxide ( 4 ) , trimethylanilinium hydroxide (5, 6)) pentyl iodide (7), have been used for this purpose. The available gas chromatographic methods for determining plasma theophylline have several deficiencies, including low sensitivity and the requirement for one or more back-extraction steps (4,643).Temperature programming is required in several of the methods (6, 7) and a lack of a pr,oper internal standard has led, in many instances, to high relative standard deviations. A high pressure liquid chromatographic method offering several improvements over current methods has recently been published (9). A procedure for the determination of theophylline in plasma by electron capture gas chromatography is described in this report. The selectivity of the extraction method, coupled with the electron capturing properties of a new theophylline derivative, provides a procedure for the analysis of theophylline in biological fluids or tissue possessing high sensitivity, reasonable precision, and high specificity.
EXPERIMENTAL Apparatus and Operating Conditions. A Hewlett-Packard Model 5710 or a 5830A reporting gas chromatograph equipped with a 1-m glass coiled column, 4-mm i.d. (3%OV-17 on Gas Chrom Q, 100/120 mesh, Applied Science Laboratories, State College, Pa.) and a 63Nielectron capture detector were utilized for the gas chromatographic analysis. The packed column was silanized with a commercial silanizing reagent (Silyl-8, Pierce Chemical Co., Rockford, Ill.) and conditioned overnight at a column temperature of 250 OC, prior to use. Chromatography was performed a t column temperatures as indicated in the figures. The carrier gas (5%methane in argon) flow rate was 60 ml/min. If not used daily, the column requires several preliminary injections of the derivatized theophylline and internal standard to obtain maximum sensitivity and a constant and reproducible peak height or area ratio. Reagents. Pentafluorobenzoyl chloride was purchased from Pierce Chemical Company, Rockford, Ill. 3-Isobutyl-1-methylxanthine was obtained from Aldrich Chemical Company, Milwaukee, Wis. 1Methylxanthine, 3-methylxanthine, and 7-methylxanthine were purchased from Vega-Fox Biochemicals, Tucson, Ariz. Theophylline, anhydrous crystals, and theobromine were obtained from Sigma Chemical Company, St. Louis, Mo. Ammonium sulfate (granular) was obtained from Mallinckrodt Chemical Works, St. Louis, Mo. All solvents were of ACS reagent grade. Standard Curves. Theophylline standards were prepared in water in a concentration range of 2.5-25 pg/lOO pl. 3-Isobutyl-1-methylxanthine was dissolved in absolute ethanol a t a concentration of 12.5 pg/lOO p1 because of its insolubility in water. The solutions of theophylline standards and the internal standard were stored at 4 "C and were stable for a t least 3 months. Recoveries, standard curves, and other studies were performed with plasma obtained from outdated blood. Analysis of Patient Plasma. One ml of patient serum or plasma, 100 pl of internal standard, and a constant amount of granular ammonium sulfate (approximately 0.80 g in the spoon end of a porcelain spatula, Coors, 19:K) were added in sequence to 16 X 150 mm test tubes. Alternatively, 2 ml of a saturated ammonium sulfate solution can be used (see Results section). The samples were mixed continuously on a vortex-type mixer for 15 s. The extraction solvent consisted of 10 ml of a mixture of toluene and ethylacetate (8:2, v/v). The tubes were capped with Teflon-lined caps and placed in a horizontal position in an Eberbach shaker. A 15-min shaking period with the shaker operated a t full speed, 250 oscillations/min, was utilized for the extraction process. The phases were separated by a brief centrifugation at 2000 X g and the organic phase was transferred to 16 X 125 mm tubes with a forced-air transfer device made from stainless steel tubing. After evaporation of the organic solvent with the assistance of nitrogen from a suitable manifold (90 "C, dry temperature block), the tubes were removed from the temperature block and allowed to cool. Twenty-five pl of pentafluorobenzoyl chloride were added with an Eppendorf pipet, the tubes capped, and derivatization was performed a t 90 "C for 1 h. Excess derivatization reagent was removed a t room temperature under dry air or nitrogen, and the product was dissolved in 5 ml of ethylacetate. An aliquot of 2-5 pl of the resulting solution was injected into the chromatograph. The pentafluorobenzoyl derivatives in our laboratory were observed to be stable a t room temperature for a t least 48 h.
RESULTS Extraction Procedure. Because of the relatively low solubility of theophylline in toluene-ethylacetate, salting-out techniques were introduced to efficiently extract the drug from biologic specimens. In this context, it was determined that the amount of ammonium sulfate added to a given plasma volume is the most critical step in the proposed assay. The use
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
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Figure 1. Effects of adding saturating amounts of (NH4)*S04 or 2 mi of saturated (NH4)zSOd on results. Detector response is presented as the ratio of the peak areas
of a constant amount of salt ensuring complete saturation of the aqueous phase permits consistent recoveries from plasma in excess of 85% for both theophylline and 3-isobutyl-1methylxauthine. The fact that theophylline/internal standard peak area ratios of 0.97 and 1.94 are obtained for 12.5 pg/ml and 25 pg/ml theophylline, respectively, when compared to 12.5 pg/ml of internal standard, suggests that equal recoveries for both compounds a t varying concentrations are obtained (Figure 1).Excellent recovery of the drug can also be achieved with the addition of a saturated so1,utionof ammonium sulfate; however, the slope of the standard curve differs somewhat from that obtained with solid ammonium sulfate (Figure 1). The slope obtained using a saturated solution of ammonium sulfate depicts the importance of utilizing a sufficient amount of salt for, when the aqueous phase is not completely saturated, different extraction characteristics for theophylline and the internal standard are observed. In the recovery studies, relative standard deviations of 3%for theophylline measurements were observed for both methods of (",&SO4 addition. Mechanical shaking is recommended since it provides greater reproducibility in recovery than does hand shaking. The extraction solvent-salt combination utilized in this study excludes most lipid and other coextractable material. This is accomplished by the establishment of a lipid-protein interphase between the organic and the aqueous layer. The amount of coextractable material was determined by combining the extracts of 20 one-ml theophylline-free plasma samples and found to be 68 pg per ml of plasma. This amount constitutes less than 1%of the total lipid material and is reflected in the absence of other major chromatographic peaks and in the presence of a stable chromatographic recorder baseline. An extraction procedure employing chloroformisopropanol (95:5) ( 3 )was evaluated and was found to produce results similar to those obtained with toluene-ethylacetate (see Specificity section). Plasma samples of 100 p1 also could be effectively extracted with 2 ml of chloroform-isopropanol. The principal advantage of chloroform-isopropanol is that it can be evaporated more readily than toluene-ethylacetate. Since theophylline determinations are often performed on small volumes of plasma, e.g., samples obtained from finger or heel stick, the influence of plasma volume on analytical accuracy and reproducibility was evaluated. The influence of the salt concentration/plasma volume ratio was examined following the addition of 2 ml of saturated ("&SO4 to 1.0, 0.5, and 0.1 ml of plasma containing 5.0-25 pg/ml theophylline and 12.5pg/ml internal standard. A t each plasma theophylline concentration, the results of the assay obtained with 1.0,0.5, and 0.1 ml plasma were statistically different. The addition of normal saline or water to produce a constant total volume 1876
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Figure 2. Gas-liquid chromatogram of theophylline and the internal standard Concentrations of theophylline, from ieR to right, are 2.5, 5.0, 10.0, 15.0, 20.0, and 25.0 pg/mi. 3-Isobutyl-1-methyixanthine ( I S ) is 12.5 pg/mi. Column temperature, 210 OC. Retention time of theophylline and the IS. are 1.8 and 2.3 min, respectively. Quantitation is based on peak height ratios
of 1ml did not fully achieve consistent values. Accurate values, Le., values corresponding to amounts added, were obtained either by adding sufficient ("&SO4 to saturate the plasma or by adding theophylline-free plasma to give a constant 1ml total plasma volume. Alternatively, standard curves obtained from a given plasma volume and 2 ml of saturated (NH4)2S04 can be used. Reaction Conditions. Optimalconditions for derivatization of theophylline and the internal standard were determined experimentally. The peak height ratio of theophyllinehnternal standard did not change after 15 min a t 90 "C, Le., ratios a t 15,30, and 60 min were found to be statistically the same. The yield of derivatization product, however, does increase slightly from 15 to 60 min, whereupon it reaches a plateau. A variety of different derivatizing reagents, pentafluoropropionic anhydride, trifluoroacetic anhydride, and pentafluoropropionyl imidazole, failed either to result in the production of a single product or to react. Analytical Sensitivity, Reproducibility, and Accuracy. The procedure has a sensitivity limit of 50 pg injected or 10 ng/ml plasma (10% full-scale deflection). Quantitations can be based either on peak area ratios or on peak height ratios (Figure 2). Relative standard deviations of 3% were obtained with either method of extraction and with either method of calculating ratios. Standard curves were routinely determined by the least squares method. The accuracy of the obtained results is presented in Table I. The percentage accuracy obtained by calculation of peak area ratios averages 3.0-3.5% with both methods of extraction and shows the greatest deviation at the 2.5 pg theophylline level. The average difference between the amount added and the amount determined is f0.22 to f0.30 pg/ml and is independent of the plasma concentration. Variations in the chromatographic portion of the analytical procedure appear to account for most of the relative standard deviation. The variability in reproducibility can be minimized by limiting the use of the gas chromatographic column to theophylline analysis. It takes only a few minutes to change columns and for the instrumental conditions to stabilize. The approach works well for other drug analyses as well and has led to a
