(3) R. S. Juvett, Jr., and R. L. Fisher, Anal. Chem., 37, 1752 (1965). (4) W. C. Butts and W. T. Rainey. Jr., Anal. Chem., 43, 1538 (1971). (5) G. Schwedt and H. A. Russel, Chromatographia, 5, 242 (1972). (6) M. G. Lai and H. V. Weiss, Anal. Chem., 34, 1012 (1962). (7) J. E. Portmann and J. P. Riley, Anal. Chim. Acta. 31, 509 (1964). (8)A. J. McCormack, S.C. Tong, and W. D. Cooke, Anal. Chem., 37, 1470 (1965). (9) C. A. Bache and D. J. Lisk, Anal. Chem., 38, 1757 (1966). (10) H. Kawaguchi, T. Sakamoto, and A. Mizuike. Talanta, 20, 321 (1973). (11) Y. Talmi and A. W. Andren, Anal. Chem., 46, 2122 (1974). (12) Y. Talmi, Anal. Chim. Acta, 74, 107 (1975). (13) S. L. Eck, Kalarnazoo College, Kalarnazoo, Mich., 1974, private cornmunication. (14) F. E. Lichte and R. K., Skogerboe, Anal. Chem., 44, 1480 (1972).
(15) R. M. Dagnal, T. S.West, and P. Whitehead, Analyst, (London),08, 647 (1973). (16) A. R. Byrne, Anal. Chim. Acta, 59, 91 (1972).
RECEIVEDfor review February 27,1975. Accepted April 22, 1975. Research supported by the National Science Foundation-RANN Program under NSF Interagency Agreement No. 389 with the U.S.Energy Research and Development Administration. Oak Ridge National Laboratory is operated for the US.Energy Research and Development Administration by the Union Carbide Corporation.
Determination of Amitriptyline at Nanogram Levels in Serum by Electron Capture Gas-Liquid Chromatography Jack E. Wallace,’ Horace E. Hamilton,’ Linda K. Goggin,’ and Kenneth Blum2 University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
An analytical method for the quantitative determlnation of amitriptyllne/nortriptyline in small amounts of biologlc fluids at the nanogram level Is described. The procedure involves the oxidation of the drugs to a polyaromatic carbonyl derlvatlve, anthraquinone, that has the intrinsic capability to capture electrons with an efflciency comparable to that of polyhalogenated compounds. The electrophilic property of the product provides the basis for the extreme sensitivity of the procedure. Oxidation is accomplished by employment of a solution of cerlc sulfate-sulfuric acid that selectively oxidlres only the ethylene group of the lO,ll-dlhydro-5-dlbenzo[ a,d]cycloheptene moiety of the molecule. The product is measured by electron capture gas-liquid chromatography. Quantitation of the method is extensively enhanced by utilization of ethylanthraquinone as the internal standard.
Amitriptyline and its monomethylamino analog, nortriptyline, are among the therapeutic agents most commonly administered for the amelioration of depression. I t is established that the tricyclic antidepressants demonstrate marked pharmacokinetic heterogeneity which promotes differences in plasma levels for subjects receiving equivalent dosage regimens (1-6). Identical dosages may result in subtherapeutic levels in certain subjects and toxic levels in others (1, 4 , 5 ) . Most investigators, therefore, consider the measurement of circulating blood levels to be of significant clinical importance during tricyclic antidepressant therapy (5, 6). It has been observed ( 5 ) that combined plasma amitriptyline and nortriptyline ( a principal metabolite of amitriptyline and a commercially available therapeutic) levels exhibit greater correlation with the clinical management of depression than do plasma levels of either amitriptyline or nortriptyline alone. Earlier spectrophotometric methods (7-9) for the determination of the tricyclic antidepressants lacked a certain level of sensitivity for application to the determination of the drugs in serum or plasma. The authors of this report (10) recently described a spectrophotometric procedure for the determination of amitriptyline and nortriptyline that D e p a r t m e n t of Pathology. D e p a r t m e n t of Pharmacology.
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
provided sufficient sensitivity for the measurement of blood levels if both amitriptyline and nortriptyline were analyzed simultaneously a t high therapeutic and toxic levels. Sensitive methods for the specific determination of nortriptyline are available utilizing isotopic labeling with subsequent scintillation spectrometry ( 1 1 ) or polyfluoroacetylation with subsequent quantitation by electron capture gas-liquid chromatography (12). However, these latter techniques are not applicable to the analysis of amitriptyline which has a tertiary amine group a t the functional nitrogen. A number of gas-liquid chromatographic (GLC) methods for the determination of amitriptyline and nortriptyline have been reported in recent years. Hucker and Miller (13) described a technique which, by application of the Hoffman reaction, converts both of these tricyclics to a common derivative prior to GLC determination. Their report did not contain any biologic data, and the data presented were for a concentration range greater than that encountered in plasma specimens. Braithwaite and Whatley (14) reported a GLC procedure for amitriptyline in urine, and Norheim (15) described a GLC method for the determination of amitriptyline and nortriptyline in biologic specimens. The latter procedure provided a limit of sensitivity of 1 pg/ml for a 5-ml specimen. GLC methods with a 20 ng/ml limit of detection for a 5-ml plasma specimen have recently been described for the simultaneous and separate determination of amitriptyline and nortriptyline (16, 1 7 ) . These procedures require a unique chromatographic packing and a special treatment of the packed column (16) or are somewhat tedious and time consuming, requiring additional solvent wash, back-extraction, and evaporative techniques not required in the method proposed in this report ( 1 7 ) . Currently available GLC procedures for the tricyclic antidepressants, with the exception of Walle and Ehrsson’s procedure (12) for the analysis of nortriptyline, utilize flame ionization detectors. This report depicts a rapid electron capture gas-liquid chromatographic procedure capable of detecting 1 ng/ml or reliably quantitating 5 ng/ml of amitriptyline/nortriptyline in 0.5-ml plasma or serum specimen within one hour of the time the sample is received a t the laboratory. The procedure is dependent upon the oxidation of the parent compounds to anthraquinone which is subsequently chromatographed.
EXPERIMENTAL Apparatus. A Hewlett-Packard Model 5713A gas chromatograph equipped with a three-foot 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 63Ni-electroncapture detector was utilized for gas chromatographic analysis. The packed column was silanized with a commercial silanizing agent (Silyl-8, Pierce Chemical Co., Rockford, Ill.) and conditioned overnight at a column temperature of 260 "C prior to use. Chromatography was performed at column and detector temperatures of 210 and 350 OC, respectively, and a carrier gas (5% methane in argon) flow rate of 45 ml/min. Reagents. A 5.5M sulfuric acid solution containing 25 mg of ceric sulfate per milliliter is prepared by adding 76 ml of concentrated sulfuric acid, very slowly and with constant stirring, to a large beaker containing 6.25 g ceric sulfate and 174 ml water. The solution is stable for two months at room temperature. The n-hexane and n- heptane utilized are of spectroanalytical quality. Procedure. One-half milliliter of plasma or serum, one drop of 1N sodium hydroxide, and 2.5 ml of n-hexane are placed in a 15-ml tube fitted with a glass stopper or Teflon-lined screw cap and shaken vigorously for one minute. Following a brief centrifugation (1-2 minutes at 2000 rpm), 2.3 ml of the hexane layer is transferred to a second tube and similarly extracted into 2.0 ml of the ceric sulfate-sulfuric acid solution. Then 1.8 ml of the ceric sulfate solution is placed in a 10- X 75-mm glass culture tube and placed in a 95 "C bath for ten minutes. The tube is allowed to cool to ambient temperature, and the tube contents vortexed (Vortex Genie Mixer) for one minute with 0.1 ml of heptane containing 100 ng/ml of ethylanthraquinone (Aldrich Chemical Co., Milwaukee, Wis.) as an internal standard. The heptane layer is drawn off with a disposable Pasteur pipet and placed in a 6- X 50-mm disposable glass culture tube to await chromatographic analysis. Five-microliter aliquots are injected into the column of the gas-liquid chromatograph. Quantitation is based upon the peak height ratios of the oxidized amitriptyline and/or nortriptyline (anthraquinone) to ethylanthraquinone. RESULTS Previous reports have established t h a t a common oxidation product of amitriptyline and nortriptyline is anthraquinone, a polyaromatic carbonyl compound, (9, IO, 18) and that ceric sulfate-sulfuric acid serves as a model oxidation system for its production (10). The oxidation of amitriptyline and subsequent detection of anthraquinone by electron capture provides the analyst with a reliable and sensitive gas chromatographic method capable of determining amitriptylinehortriptyline at low nanogram levels in biologic fluids (Figure 1). T h e reproducibility of t h e method was demonstrated by performing quadruplet determinations of solutions of amitriptyline HCl at concentrations from 25 t o 1000 ng/ml in ceric sulfate-sulfuric acid oxidant in which a mean coefficient of variation of 2.3%was observed. Application of Procedure. Plasma samples containing amitriptyline HC1,5 t o 1500 ng/ml, were determined by the method described (Table I). Near-quantitative recoveries were achieved a t concentrations of 200 ng/ml or less, but decreasing recoveries were observed a t higher concentrations, with a 81% mean recovery at 1500 ng/ml. In the plotting of a standard curve of actual concentration vs. amount determined, the data can provide for two straight line relationships in which a n intercept between t h e two lines occurs a t 200 ng/ml. T h e deviation from quantitative recoveries at higher concentration, nevertheless, does not significantly influence data in the normal therapeutic range. Plasma from eight depressed patients receiving amitriptyline therapy were examined, and the plasma levels calculated by three techniques; plotted on a calibration curve, calculated on the basis of a single 200 ng/ml standard, and calculated on the basis of the mean of three standards, respectively, 50, 200, and 400 ng/ml (Table 11).T h e levels of drug observed, encompassing the subtherapeutic and therapeutic range, were not significantly altered regardless of t h e
II
I
E E
C
2
4
6
NI N u T E S
Flgure 1. Gas-liquid chromatograms of amitriptyline extracted from plasma spiked at concentrations of 0, 25, 50, and 100 ng/ml (I, 11, 111, IV)
Ethylanthraquinone was added as internal standard at a final concentration of 100 nglml. Anthraquinone (oxidizedamitriptyline) and ethylanthraquinone are identified on the chromatograms as A and E, respectively
number of standard values used t o establish the calibration curve. For greatest accuracy when examining clinical specimens, it is suggested that at least two plasma standards be analyzed-one in the therapeutic range, 200 ng/ml, and one in t h e toxic range, Le., 1000 ng/ml. Calculation of the observed specimen levels could be conveniently based upon the standard most closely related t o the level in the biologic specimen. It is of significance t h a t plasma specimens t o which 1 ng/ml amitriptyline HCI have been added are routinely differentiated from corresponding plasma specimens containing no amitriptyline. However, duplicate determinations of plasma of twelve healthy adult subjects not receiving tricyclic therapy indicates a mean plasma blank of 1.5 f 0.6 ng/ml. Consequently, observed plasma concentrations of 2 ng/ml or less may be considered as indicative of the ab-
Table I. P l a s m a Amitriptyline Determinations Plasma
Amount
concn,
determined,
ngiml
ng/ml
5 25 50 100 2 00 400 600 1000 1500 a
5 26 50 98 187 341 500 810 1220
*1 1 i 1 i 2
+
3 29 7 48 i 8 i
* * *
Mean % recovery
95 103 102 92 94 85 83 81 81
*
11 4 f 2 i 2 2 i
*
i i i i
7 1 5 1
Mean of 8 determinations f standard deviation.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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I
E
\
m
z
: :\ IO.
Table 11. Plasma Amitriptyline/Nortriptyline Levels of Patients Receiving Amitriptyline Therapy dosage,
Duration since
Patient
mglday
dosage, hr
A B
250 100 150 100 150 150 150 25
C
D E
5.
F G
H
Both spectrophotometric (0-0-0) (0-0-0)are presented
18 16
Uncertain 5 17 16 16' 16
Single Plotted
standard
3 standards
164 233 154 615 206 172 7 10
167 234 157 622 208 176
164 228 153 605 203 172 8 11
7 11
and gas-liquid chromatographic data
sence of amitriptyline and/or nortriptyline. Chromatograms of derivatized extracts of plasma containing 0,25,50, and 100 ng/ml are shown in Figure 1. Six serum specimens were obtained over a ten-day interval from an adult female who upon arrival a t the hospital was semi-comatose following an overdose of an undetermined amount of amitriptyline HCl (Elavil). The first two specimens from that patient were analyzed individually either by a spectrophotometric procedure (IO) or the procedure described in this report. The remaining four specimens were determined in duplicate by both the spectrophotometric procedure and the electron capture gas-liquid chromatographic technique. Since the serum levels were relatively elevated, excellent agreement was observed between the two methods (Figure 2). Critical Evaluation of Procedure. A number of nonpharmacologic compounds were examined for possible use as an internal standard. Retention times relative to anthraquinone were observed for anthrone (1.00), methylanthraquinone (1.09), ethylanthraquinone (1.58), chloroanthraquinone (1.84), butylanthraquinone (2.00), and dichloroanthraquinone (3.37). Ethylanthraquinone was chosen as the internal standard of choice on the basis of relative retention time, sensitivity, and peak symmetry. The reproducibility of the described method is such that reliable results may be obtained in the absence of an internal standard. Quadruplet determinations were performed on solutions of amitriptyline HCl in ceric sulfate-sulfuric acid a t concentrations of 25, 100, and 200 ng/ml; the coefficient of variation for the anthraquinone peak height and the anthraquinone/ethylanthraquinone peak height ratio, both relative to concentration, was 7.0 and 3.2%, respectively. A number of structurally related compounds were examined for possible interference. Plasma extracts containing 250 ng/ml of imipramine, desipramine, chlorpromazaine, trifluoperazine, neocuproine, and phenanthroline were indistinguishable from similar plasma extracts containing no drugs. Nortriptyline, protriptyline, and cyproheptadine are converted to anthraquinone by the oxidation conditions employed in the procedure. Proper adjustment of pH during the initial extraction of the biologic specimen provides the analyst with a certain amount of selectivity in the analysis of individual tricyclics (Table 111). Extractions performed a t alkaline conditions (pH S-10) afford equivalent sensitivities for amitriptyline and nortriptyline whereas protriptyline yields a recovery and resultant sensitivity less than half that achievable with amitriptyline or nortriptyline. Extractions performed a t physiologic pH (7.0-7.4) 1518
Plasma concentration,a n g l m l b
Amitriptyline
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
Table 111. Influence of pH of Extraction upon Relative Recovery Achieved with Various Tricyclicsa pH of extraction
Amitriptyline
NortripQline
6 7.3 9-10
0 .8gb 1.oo 1.oo
0.22 0.77 1.oo
Protriptyline
0.11 0.25 0.49
a Mean of duplicate analysis of plasma containing 100 ng/ml of the respective tricyclic antidepressants. Recovery (sensitivity) relative to t h a t achieved a t pH9-10 with amitriptyline and nortriptyline.
provide a similar sensitivity for amitriptyline but diminished recoveries for nortriptyline and protriptyline. Extractions performed a t slightly acidic conditions (pH 6) afford a slightly diminished recovery of amitriptyline but a significant reduction in recovery for other tricyclics, that results in a sensitivity for amitriptyline several times that achieved with the other tricyclics. At a more acidic p H (3.0-4.0), the extraction efficiency for the three tricyclics examined is greatly diminished, with a mean relative recovery for amitriptyline of 45% compared to that achieved a t extractions using a pH greater than 9.0. Cyproheptadine recovery is essentially independent of p H over the range 6-10, with a recovery and sensitivity approximately one-fifth that achieved with amitriptyline and nortriptyline a t pH 9-10. The ceric sulfate-acid oxidation of amitriptyline to anthraquinone was examined a t various incubation temperatures and durations. The relative yield of anthraquinone for a 10-minute incubation a t 85, 90, 95, 100, and 105 "C temperature was 0.90 f 0.01, 0.96 f 0.02, 1.00 f 0.02, 0.95 f 0.02, and 0.77 f 0.03, respectively (mean of triplicate determinations, f standard deviation). The relative yield a t 95 "C for 5 , 10, and 15 minutes was 0.92 & 0.09, 1.00 f 0.04, and 0.90 f 0.14, respectively (mean of triplicate determinations, f standard deviation). Although the optimum time condition for the volumes and concentration of reactants was 10 minutes a t 95 "C, it is apparent that a 5-minute incubation may be utilized if required, During an emergency or stat situation, the interval between receipt of specimen and data reporting may be shortened to under twenty minutes by utilization of a 5 minute incubation, rapid cooling of the reaction tube by immersion in an ice bath, and shorter extraction times. Each of the three extractions (plasma to hexane, hexane to sulfuric acid, sulfuric acid to heptane) may be reduced to one-half minute with only a slight decrease in sensitivity; however the coefficient of variation utilizing the shorter extraction times is approximately doubled.
The anthraquinone/ethylanthraquinone peak height ratio is generally extremely stable from day to day, such that in a situation of acute toxicity, approximate quantitation may be based upon standards previously analyzed. For routine analyses, concurrent standards are recommended, thus resulting in an analysis time of slightly over one hour. Aqueous solutions of amitriptyline and nortriptyline and heptane solutions of ethylanthraquinone are stable for a t least one month, factors further enhancing the applicability of the described procedure to the analysis of specimens from the emergency room. The determination of nanogram amounts of drugs in biologic materials necessitates rigid control of possible sources of contamination. Reliable determinations of amitriptyline in amounts less than 100 ng/ml require special attention to the cleanliness of glassware and the laboratory environment in general. Glassware utilized in this study was subjected to both chromic acid-sulfuric acid and ultrasonic cleaning prior to cycling through a commercial laboratory dishwasher.
DISCUSSION The proposed method is, to our knowledge, the most sensitive available for the determination of amitriptyline and nortriptyline in biologic extracts. The procedure is rapid, offering a typical analysis time of approximately forty to sixty minutes. The inability of the method to differentiate between amitriptyline and nortriptyline limits its applicability to pharmacokinetic studies, but does not detract from its usefulness to clinicians and toxicologists. Previous investigations have indicated that plasma levels of total amitriptyline and nortriptyline are more clinically significant than levels representing the concentration of each compound ( 5 ) . Protriptyline and cyproheptadine are potential sources of interference, but these tricyclics are detected with a sensitivity approximately one half and one fifth, respectively, of that achieved with amitriptyline and nortriptyline. In addition, these compounds are currently administered far less frequently than amitriptyline and would not likely be administered concurrently with amitriptyline or nortriptyline. Flame ionization detection of amitriptyline and nortriptyline provides, under ideal conditions, a sensitivity limit of approximately 20 ng/ml in a 5-ml specimen (0.1 microgram) (16, 1 7 ) whereas the present method, utilizing electron capture detection of anthraquinone, provides a sensitivity of approximately 2 ng/ml in a 0 5 m l specimen (0.001 microgram).
Previously utilized electron capture detectors (ECD), which contained a thin foil of tritium or nickel-63 as the radioactive source and used non-pulsed electronics, provided a very narrow range of linear response to concentration and were quite susceptible to contamination. The gas chromatographic system employed in the present study, which has the 63Ni plated on the wall of the cylindrical cavity of the detector, significantly extended the linear response of electron capture detection and reduced the detector's susceptibility to contamination and sample overload, problems which have hindered the successful application of previous ECD's to the analysis of biologic extracts. The expanded range of linear response is primarily due to the mode of operation in that the electron population is sampled by frequency modulated pulses to produce a constant detector current.
ACKNOWLEDGMENT The authors are grateful to David King, Diana Bason, Laurence Early, Jr., John Sulak, and Michael E. Wallace for their excellent technical assistance.
LITERATURE CITED (1) W. Hammer, C. M. Idestrom, and F. Sjoquist, Excerpta Med. lnt. Congr. Ser., 122, 301 (1967). (2) F. Sjoquist, W. Hammer, C. M. Idestrom, M. Lind, D. Tuck, and M. Asberg, Excerpta Med. h t . Congr. Ser., 145, 246 (1967). (3) W. Hammer, S. Martens, and F. Sjoquist, Clin. Pharmacol. Tber., I O , 44 (1969). (4) M. B. Asberg, F. Cronholm, and F. Sjoquist, Brit. Med. J., 4, 18 (1970). (5) R. A. Braithwaite, R. Goulding. G. Theans, J. Bailey, and A. Coppen. Lancet, 1, 7764 (1972). (6) V. Marks, W. E. Lindup. and E. M. Baylis, Adv. Clin. Cbem., 18, 87 (1973). (7) I. Huss, "Detection of Amitriptyline in Urine and Body Tissues", Merck, Sharp and Dohme Research Laboratories, 19 April, 1963. (8) I. Sunshine and J. Baumler, Nature(London), 199, 1103 (1963). (9) J. E. Wallace and E. V. Dahl. J. Forensic Sci., 12, 484 (1967). (10) H. E. Hamilton, J. E. Wallace, and K. Blum, Anal, Cbem., 47, 1139 (1975). (11) W. M. Hammer and 8. B. Brodie, J. Pharmacol. Exp. Tber., 157, 503 (1967). (12) T. Walle and H. Ehrsson, Acta Pharm. Sci., 8, 27 (1971). (13) H. B. Hucker and J. K. Miller, J. Cbromatogr., 32, 406 (1968). (14) R. A . Braithwaite and J. A. Whatley. J. Cbromatogr., 49, 303 (1970). (15) G. Norheim, J. Cbromatogr., 88, 403 (1974). (16) R. A. Braithwaite and B. Widdop, Clin. Cbem. Acta, 35, 461 (1971). (17) H. B. Hucker and S. C. Stauffer. J. Pbarm. Sci, 83,296 (1974). (18) R. Banche, J. Pharm. Sci., 81, 986 (1972).
RECEIVEDfor review March 14, 1975. Accepted April 25, 1975. The research was supported in part by Grant R0100729-01 from the National Institute on Drug Abuse, Rockville, Md., and in part by Grant 5SOlRR95654-05 from the National Institute of Health (General Research Support).
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