(17) M. Novotny, . L. Lee, and K. D. Bartle, J. Chromatogr. Sci., 12, 606 (1974) (18) B. B. Chakraborty and R. Long, Environ. Soi. Technol., 1, 829 (1967). (19) G. M. Janini, K. Johnston, and W. L. Zielinski, Jr., Anal. Chem., 47, 670 (1975) (20) T. W. Stanley, J. W. Meeker, and M. J. Morgan, Environ. Sci. Technol., 1, 927 (1967). (21) D. Broceo, V. Cantuti, and G. P. Carton!, J. Chromatogr., 49, 66 (1969). E. J. Ciar, “Polycyclic Hydrocarbons”, 2 vols, Academic Press, New (22) York, 1964. (23) “UV Atlas of Organic Compounds”, 5 vols, Plenum Press, New York, .
.
1967-68. (24) R. A. Friedel and M. Orchln, "UV Spectra of Organic Compounds”, John Wiley and Sons, New York, 1967. (25) T. J. Porro, R. E. Anacreon, P. S. Flandreau, and I. S. Fagerson, J. Assoc. Off. Anal. Chem., 56, 607 (1973). (26) R. Jeltes, J. Chromatogr. Sci., 12, 599 (1974). (27) R. L. Cooper, Analyst (London), 79, 573 (1954).
Received for review June 11,1975. Accepted November
7,
1975.
Determination of Theophylline in Plasma Ultrafiltrate by Reversed Phase High Pressure Liquid Chromatography L. C. Franconi and G. L.
Hawk*
Waters Associates, Maple Street, Milford, Mass. 01757
B. J. Sandmann and W. G. Haney University of Missouri-Kansas City, School of Pharmacy, 5100 RockhiH Road, Kansas City, Mo. 64110
A procedure for the determination of theophylline In plasma by reversed phase high pressure liquid chromatography Is presented. This method uses molecular filtration to remove plasma proteins prior to chromatographic analysis. It permits the accurate measurement of plasma levels of theophylline without interference from the dietary xanthines and their metabolites, caffeine, and 3,7-dimethylxanthine. No Interference from commonly used drugs or their metabolites was found from 75 randomly collected plasma samples. In 55 comparative determinations, the LC method was found to be comparable to the GLC and spectrophotometrlc methods currently being employed.
drugs (1) Theophylline appears to be one of numerous for which monitoring drug plasma concentration is often necessary to ensure effective therapy. Significant variations in theophylline plasma concentrations resulting from a given daily dose have been noted (2), and response to the drug relates to the drug plasma concentration. Therefore, rapid and accurate procedures for the analysis of theophylline in plasma are essential. Because of the strong ultraviolet absorption characteristics of the drug, most procedures designed for its determination in plasma have been based on spectrophotometric analysis (3, 4). Typically, theophylline contained in a volume of plasma is extracted into an organic solvent from which it is back-extracted into an aqueous basic solution. The aqueous solution is neutralized, and the ultraviolet spectrum is determined. Such procedures have two intrinsic deficiencies. First, the partition coefficient of theophylline between organic solvents and water is low, and the initial extraction is, therefore, inefficient. Second, the class of compounds of which theophylline is a member, is often encountered in plasma as a consequence of the metabolism of endogenous biochemicals and of exogenously administered substances, e.g., the ubiquitious caffeine. The currently available ultraviolet procedures are not sufficiently selective to distinguish between members of this class of compounds (5). In addition, a number of drugs and their me372
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tabolites strongly absorb at the analytical wavelength, and those present in the final extract (for example, weak acids such as the barbiturates) offer additional complications. This lack of selectivity has led to an interest in chromatographic procedures. Gas-liquid chromatography (GLC) has been used for this determination (5), but derivatives of the drug must be formed prior to analysis. In addition, the chromatographic internal standard used in this GLC method is known (6) to be unstable in the methylating reagent. A GLC procedure offering several improvements has also been recently proposed (7). High speed liquid chromatography has enjoyed some acceptance, but the column packings used thus far have been unstable (8) and retention volumes of metabolites and related substances have been high (9). In addition, these procedures continue to rely on the inefficient extraction of theophylline from plasma or After this study was completed, a procedure for the serum. determination of theophylline by direct injection of plasma was reported (10). In light of the above, a non-extractive procedure for the analysis of theophylline in patient plasma by reversed phase HPLC has been developed and evaluated. Results of this procedure have been compared with results from an ultraviolet and a GLC analysis.
EXPERIMENTAL Apparatus and Operating Conditions. A liquid chromatograph equipped with a 6000 psi pump and high pressure injector
(ALC Model 202 with Model 6000 pump and U6K injector, Waters was used for the analyses, and eluent was monitored continuously at 254 nm. The peak areas were determined by triangulation. The mobile phase was 0.01 M sodium acetate (adjusted to pH 4.0 with acetic acid) and acetonitrile in a ratio by volume of 9:1, and the flow rate was 2.0 ml/min. Plasma samples were filtered at ambient temperature using a multiple unit system (Pellicon Carrousel and Pellicon PSED 01310, Millipore Corporation, Bedford, Mass.) with 3.0-ml, 13-mm stirred polycarbonate cells. The pressure was maintained at 50 psi with nitrogen. Membranes. A membrane (Membrane A) consisting of a continuous polymer film supported on a microporous substrate of mixed esters of cellulose and with a nominal molecular weight limit of 25 000 was evaluated in this study. In addition, a molecular Associates, Milford, Mass.)
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
VOLUME OF ULTRAFILTRATE (MICROLITERS)
Figure 2. Theophylline recovery from consecutive aliquots of ultrafiltrate Solutions containing 5.0 ( ), 10.0 (D), 17.5 (O), and 22.5 () pg/ml filtered through Membrane A. Solutions containing 5.0 ( ), 10.0 ( ), 17.5 (·), and 22.5 ( ) pg/ml filtered through Membrane B
o
Figure
1.
2
4
6
8
10
Chromatogram of plasma ultrafiltrate containing theophyl-
line
chromatographic system. After six to ten injections, however, the pressure required to maintain a given flow rate increased substantially, and a period of backflushing was required. While this procedure generally resulted in restora-
tion of the original chromatographic characteristics, it appeared that a preliminary ultrafiltration to remove plasma proteins would make the procedure more amenable to routine application. A chromatographic trace of plasma ultrafiltrate from a patient taking theophylline is presented in filter (Membrane B) composed of a proprietary, (Millipore Corporation) noncellulosic material was used. Each membrane was 13 Figure 1. mm in diameter and used as obtained from the supplier. Two membranes were selected for evaluation in this Column. The column packing material consisted of porous silica Initial work using aqueous solutions (Figure 2) demstudy. beads having an average particle size of 10 microns and a chemicalonstrated that each membrane has a tendency to bind the ly bonded, monomoiecular layer of octadecyltrichloriosilane (µ drug. However, the volume of filtrate at which the theoBondapak Cig, Waters Associates). The plate count for the 30 cm X 4 mm i.d. stainless steel column was 1250 with respect to theophylline concentration in the filtrate attains that in the filtered solution is independent of the concentration of the phylline. Calibration Curves. Solutions of theophylline were prepared in filtered solution. Therefore, the binding sites are nonselecthe mobile phase to contain 2.0, 5.0, 7.5, 10.0, 12.5, 17.5, and 22.5 tive and readily saturated. On the basis of these studies on pg/ml. An aliquot (20.0 pi) of each solution was injected into the samples, Membrane B was used in the remaining aqueous chromatographic system, and the area of the theophylline peak work. was determined. Studies of the filtration efficiency of theophylline in Membrane Selection. Solutions (2.0 ml) of the above concentrations of theophylline in distilled water were filtered through one plasma samples are presented in Figure 3. Direct filtration of the membranes chosen for the study, and consecutive 100-pl of spiked plasma resulted in an average recovery of 83.6%. portions were collected to a total of 800 pi, A quantity (20.0 pi) of a small quantity of alkaline buffer added to plasHowever, each 100-pi portion was injected into the chromatography system, ma prior to filtration through Membrane B results in virtuand theophylline concentration in the filtrate was determined by ally 100% recovery of theophylline in the fourth 100-pl alireference to the previously derived calibration curves. Determination of Filtration Efficiency. Human plasma was quot. This procedure was used for the remaining determispiked with quantities of theophylline to yield final concentration nations, and use of the filtration device allowed routine of 5.0, 10.0, and 20.0 pg/ml, and incubated overnight at 37 °C. Pornon-extractive analysis without subsequent interference tions (1.0 ml) of this plasma were filtered; the filtrate was collected with the chromatographic characteristics of the system. and analyzed according to the above procedures. Alternately, the Calibration curves using the developed procedure were 1.0-ml plasma volume was mixed with 0.1 ml of borate buffer pH 9.4 (prepared according to (11)) prior to filtration, and the deterprepared on each of ten consecutive days. Values (and % mined concentration of theophylline was corrected for the dilustd dev) obtained for the correlation coefficient, slope and tion. 7-intercept were 0.998, 0.509 (±2.1%), and —0,031 (±1.9%). Analysis of Patient Plasma. Plasma (1.0 ml) was mixed with In the final evaluative step, results of the developed pro0.1 ml borate buffer and filtered. The first 300 pi of filtrate was cedure were compared with results of other established discarded, and 20 pi of the next 100 pi was injected. The theophylprocedures. Two procedures widely used for the analysis of line concentration in the filtrate was determined by reference to the calibration curves after correction for the volume dilution. theophylline in plasma were selected for these comparative studies. The ultraviolet assay (12) appears to be the most RESULTS AND DISCUSSION selective of the numerous UV procedures available, and reIn initial studies, plasma was injected directly into the sults of the HPLC assay were compared with results of this Separation of theophylline from 20 pi of plasma ultrafiltrate. Eluent; 0.01 M sodium acetate (pH 4.0) and acetonitrile 90:10 (v/v), µ Bondapak C18, 4 mm X 30 cm: chart speed 12 in./hr
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
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Table I. Recovery of Theophylline from Plasma determined by Three Methods
as
Theophylline concentration, mg/ml Method
Added
Found
Figure 3. Theophylline recovery from plasma ultrafiltrates as determined by HPLC
HPLC GLC UV HPLC GLC UV HPLC GLC UV HPLC GLC UV HPLC GLC UV
2.00 2.00 2.00 5.00 5.00 5.00 10.00 10.00 10.00 15.00 15.00 15.00 25.00 25.00 25.00
2.01 1.68 1.74 4.84 4.42 4.69 9.70 8.03 8.76 14.1 13.2 13.7 24.9 21.7 22.1
Plasma containing 5.0 ( ), 10.0 ( ), and 20.0 (O) µ /ml filtered directly. Plasma containing 5.0 ( ), 10.0 ( ), and 20.0 (·) Mg/ml filtered after addition of buffer. Concentration is corrected for dilution
HPLC GLC UV
VOLUME OF ULTRAFILTRATE (MICROLITERS!
aN
method to emphasize their relative precision and accuracy. A GLC procedure (5), previously shown to be sensitive and selective, was also included in the study in order to solidify conclusions regarding the selectivity of the HPLC assay. Table I presents results of the studies on spiked plasma designed to determine the accuracy and precision of the three procedures. Generally, it appears that, in the absence of interference, the ultraviolet procedure is more accurate and precise than the GLC procedure. The relatively large standard deviation of results of the two methods is likely due to erratic extraction of the drug from plasma. Thus, when the extraction step is bypassed as in the HPLC procedure, both precision and accuracy are improved. However, in such a non-extractive procedure where one is not afforded the additional selectivity of an extraction, a major concern is the possibility of interferences in the procedure. Therefore, the following metabolites and related substances were examined and determined not to interfere with the results: caffeine, 7-methylxanthine, 1-methylxanthine, xanthine, 3-methylxanthine, 1-methyluric acid, hypoxanthine, and uric acid. Furthermore, to establish that commonly used drugs and their metabolites did not interfere with the results, plasma samples from patients known not to be taking theophylline were assayed for theophylline by the HPLC procedure. No interferences were encountered in 75 such plasma samples. Table II presents a comparison of selected results of the three procedures in determining theophylline in patient plasma. Since 55 such comparative determinations were made, only the most representative are shown. It will be noted that the ultraviolet procedure ordinarily gives results higher than the two chromatographic procedures. This is to be expected on the basis of previous statements regarding the relative selectivity of the three procedures. While it is not always possible to elucidate the nature of the interference in the UV analysis, it would appear that the closely related substances, theobromine and caffeine, give rise to problems in samples 6 and 8, respectively. It is also interesting to note that, in the one case where results of the two chromatographic procedure are at variance (sample 3), the HPLC result is supported by the UV result. As a general rule, however, the GLC and HPLC results were comparable considering the standard deviation or results of the two procedures. 374
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
=
Std dev, %'
Recovery,%
100.5 84.0 87.0 96.8 88.4 93.5 97.0 80.3 87.6 94.0 88.0 91.3 99.4 86.8 88.4
Av
3.56 9.42 6.31 2.74 6.82 6.74 2.58 4.74 5.60 2.47 5.63 5.24 2.26 7.87 4.84
97.54 85.5 89.56
Av Av
2.72 6.89 5.74
10.
Table II. Selected Results of the Analysis of Theophylline in Patient Plasma Sampie 1
2 3
4 5
6 7
8
Other drugs in patient regimen
Codeine, ASA, Ephedrine Papaverine, Phenobarbital, Glyceryl Guaiacolate Ephedrine, Butabarbital
Hydroxyzine, Ephedrine Isoproterenol, Phenobarbital, Ephedrine, Chlordiazepoxide Nitrofurantoin, Glutethimide,
Theobromine Diphenhydramine, Erythromycin, Dioctylsodium Sulfosuccinate Codeine, Aspirin, Phenacetin, Caffeine, Methyldopa
Theophylline concentration, Mg/ml -
GLC
UV
HPLC
8.7
11.2
8.4
3.4 24.2 7.6
3.1 21.7 12.1
3.1 20.4 8.1
15.7
19.3
14.0
9.1
15.4
8.6
1.6
1.1
1.0
9.1
15.0
8.7
Since there are substances present in the plasma filtrate (e.g., cholesterol) which might be expected to have a high affinity for the lipophilic column packing material, it was anticipated that these substances might ultimately build up on the stationary phase to the point where the chromatographic characteristics of the system were compromised. To prevent this, the system was flushed first with acetonitrile, then tetrahydrofuran, then chloroform, then hexane, and then through the reverse system back to the original mobile phase. Thus far, filtrates from over 300 plasma samples have been injected into the system with no apparent change in chromatographic characteristics of the system. In terms of technician time, one technician can process eight samples per hour with a minimum of active involvement using the HPLC procedure. For the ultraviolet procedure, four samples can be processed in the same time interval with considerable more activity. The GLC procedure is very complex and only 10 samples per day could be processed. It is also worthy to note that the non-extractive HPLC technique is simple and easily mastered. In drug analysis, there are three reasons for the extraction step. The first is to concentrate the sample so that it will allow detection in those systems which lack sufficient
sensitivity for direct analysis. With sufficiently sensitive detectors, the concentrating function of extraction should not be necessary. Second, a properly designed extraction procedure prevents introduction into the analytical system of many substances that might interfere with the analysis. As a matter of philosophy, however, a high efficiency chromatographic column should be capable of serving in this capacity. Last, extraction precludes introduction into the system those materials (e.g., plasma proteins) that might subsequently interfere with the efficiency of the analytical system. A properly selected molecular filtration system It would apserves effectively to prevent this occurrence. such a as described here would that therefore, system pear, be of use in the non-extractive analysis of a number of drugs.
LITERATURE
CITED
(1) E. S. Vessell and G. T. Passananti, Clin. Chem., 17, 851 (1971).
(2) J. W. Jeme, E. Wyze, F. S. Rood, and F. M. MacDonald, Clin. Pharmacol. Ther., 13, 349 (1972). (3) J. A. Schack and S. H. Waxier, J. Pharmacol. Exp. Ther., 97, 283
(1949). (4) R. C. Gupta and G. D. Lundberg, Anal. Chem., 45, 2403 (1973). (5) V. P. Shah and S. Riegelman, J. Pharm. Sci., 63, 1283 (1974). (6) R. Osiewlcz, V. Aggarwal, R. M. Young and I. Sunshine, J. Chromatogr. Sci., 88, 157 (1974). (7) G. F. Johnson, W. A. Dechtlaruk, and . M. Soloman, Clin. Chem., 21,
144(1975).
. T. Nagasawa, J. Lab. Clin. Med., 84, 584 (1974). C. V. Manlon, D. W. Shoeman, and D. L Azarnoff, J. Chromatogr., 101, 169 (1974). M. Weinberger and C. Chldsey, Clin. Chem., 21/7, 834 (1975). "The United States Pharmacopeia", 19th Rev., Mack Publishing Co., Easton, Pa., 1975, p 654. P, A. Mitenko and R. I. Ogilvie, Clin. Pharmacol. Ther., 13, 329 (1972).
(8) R. D. Thompson and (9)
(10) (11)
(12)
Received for review August 22, 1975. Accepted October 24,1975.
Application of Inexpensive Equipment for High Pressure Liquid Chromatography to Assays for Taurine, 7-Amino Butyric Acid, and 5-Hydroxytryptophan James L. Meek Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032
An inexpensive pump is described lor high pressure liquid chromatography which can be assembled from readily available parts. This pressurized tank type apparatus can be used with conventional liquid chromatography detectors,
with the spectrophotometers or fluorometers available in most laboratories. The system has been applied to measurement of specific amino acids in brain tissue: taurine, yaminobutyric acid and 5-hydroxytryptophan. The assays, which can measure less than 50 pmol, require only 3-7 min per sample, and require no sample preparation, other than precipitation of proteins. The apparatus can perform complex separations for analytical work, but its simplicity, high speed, and ease of sample preparation make it also suitable for enzymatic and clinical studies. or
The technique of high pressure liquid chromatography (LC) offers many advantages in the assay of certain classes of compounds: speed, sensitivity, selectivity, and ease of sample preparation (1). Excellent instruments are available commercially which can deliver a single solvent to a chromatographic column at pressures up to 3-6000 psi and monitor the effluent at a single wavelength in a low dead volume cell. For many purposes, operation at slightly lower pressures (up to 1500 psi) allows the use of a much less expensive pump of the pressurized tank type. If a recorder and detector are available, only columns, a flow cell, and one of these pumps would be required for a simple LC system. Other “home made” devices for LC have been described (2-8) but they are either specialized or require extensive machining. This pump is constructed of stainless
steel parts available as stock items from valve and fitting retailers, and requires less than an afternoon to assemble. The apparatus has been used in this laboratory for the trace analysis in tissues of several amino acids of neurochemical interest: 5-hydroxytryptophan, taurine, and yaminobutyric acid (GABA). These compounds illustrate the advantages of LC: they can be assayed in tissue with a 10-100 times increase in sensitivity and speed over conventional fluorimetric or absorbance methods, and the assays of these compounds in biological material require no sample preparations other than precipitation of proteins.
EXPERIMENTAL Pumping System. The pump in its simplest form (Figure 1) consists of a pressurized solvent tank with the necessary valves and fittings. A parts list is given in the appendix for this design, as well as for a more sophisticated 2-tank version (Figure 2). A regulator provides nitrogen to the top of the solvent reservoir. A 3-way valve at the bottom either stops the flow of solvent or directs it to a drain or to a sample injection device. The injectors are available commercially, or can be made from a Swagelok Tee with a Vi-inch o.d. chromatographic column in one branch of the Tee, and a Kel F insert in the other branch to serve as a needle guide and septum retainer (Figure 3a). A Teflon insert inside the Tee accepts the syringe needle, and restricts the dead volume to 5 µ\ (or less, if desired). The length of the needle guide determines whether delivery of the sample is off-column or directly into the packing. If glass columns are used they are connected with 32ga Teflon tubing (0.010-inch i.d., 0.027-inch o.d.). A short piece of Vi-inch diameter Teflon rod drilled out with a No. 72 drill (0.025 inch) holds the tubing in the injector Tee. The tubing is stretched to reduce its diameter, cut, and then the thin portion is inserted into the rod, and pulled through until the unstretched tubing emerges. Excess tubing is cut off flush with the end of the rod. This type of friction-fit adapter (which is leak proof to 500 psi) is also used in the flow cell ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
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