Determination of Metapramine in Plasma by Gas Chromatography with Nitrogen-Selective and Electron-Capture Detection Alaln R. Vlala,” Jean-Paul Cano, and Alain G. Durand Laboratoire de Toxlcologie G6n6rale et Biotoxicologie, Facult6 de Pharmacie, 27 Boulevard Jean Moulin, 13005 Marseille, France
The0 Erlenmaier Hewlett-Packard GmbH, Boblingen, West Germany
Roger M. Garreau Hewlett-Packard France, Lyon, France
Metapramine is determined in plasma by gas chromatography wlth deslpramlne as an Internal standard, a nitrogen-phosphorus selective detector (N-P D) for the detectlon of the underivatized compounds and an electron capture detector (ECD) for the N-heptafluorobutyrlc derlvatlves. The detection limits are respectlvely 15 ng/mL and 35 ng/mL plasma and the quantltatlve repeatablllties acceptable (relative standard deviation 3.85 % and 6.64 % ). The N-P D method Is the more rapld, the more sensitlve, and the more reproduclble. However, the ECD method Is the more selectlve with regard to the possible metabolltes of metapramine. The two methods are complementary and may be used for pharmacokinetlc lnvestlgatlons as well as for toxicological purposes.
advantages of these two detection methods are discussed.
EXPERIMENTAL Apparatus and Operating Conditions. Two HewlettPackard Model 5710 Gas Chromatographs were used. One was equipped with a recently introduced nitrogen-specific detector (Model 18789A “Dual N-P FID”) and a glass column, 1.5 m long, 2 mm i.d., packed with 3% of OV-1/OV-17 mixture (1/4-3/4) on Gas Chrom Q, 100/120 mesh. Temperatures were regulated at: column 230 “C, injection port 200 “C, detector 300 “C. Gas flow rates were: nitrogen carrier gas 30 mL/min; hydrogen 5.8 mL/min; air 50 mL/min. The second chromatograph was equipped with a 63Nielectron-capture detector (Model 18724 “EC”) and a glass column, 1.2 m long, 4 mm i.d., with the same packing as described above. Temperatures were regulated at: column 220 “C; injection port 250 “C; detector 300 “C. The carrier gas (10% methane in argon) flow rate was 25 mL/min. A sustained program of investigations involving the tricyclic The columns were conditioned for at least 48 h at 270 “C before drugs (1-5) had focused attention on metapramine (“19560 use. If they had not previously been in continuous use, they were RP”), which is 5-methyl-10-methylamino-10,1l-dihydrodi- treated before an analysis by several injections of a plasma blank extract with metapramine and internal standard, derivatized or benzo[b, flazepine. This psychoactive agent differs from not (depending on the detector type), to ensure maximal perimipramine derivatives in that its molecule contains only one formance. These precautionary measures would be essential. methyl group on the nitrogen atom to the central ring in Reagents. All solvents and reagents (ethyl ether, cyclohexane, addition to a methylamino group on carbon 10 (Figure 1). N hydrochloric acid, 1.0 N sodium hydroxide) were analytical 0.1 Experimental studies have demonstrated that pharmacograde. All aqueous solutions were prepared with bi-distilled water. logically as well as biochemically, by its action on cerebral Ethanol was purified by distillation from potassium hydroxide amine metabolism, this compound produces a central activity and zinc. The buffer solution, pH 9.8 was prepared by adding pattern which is different from that of other available psy34.9 mL of 0.1 N sodium hydroxide to 65.1 mL of a solution choactive agents (6). It is administered in tablets as the containing 12.37 g boric acid and 100 mL of 1.0 N sodium hydroxide per liter. The purity of the internal standard desipramine fumarate and in solution for injection as the chlorohydrate. chlorohydrate was checked by thin-layer chromatography on silica Metapramine, like the other tricyclic compounds, has two gel, using ether-acetone-diethylamine (9O:lO:l) or benzenecharacteristics which profoundly affect its study with regard acetone (100:20) treated with 10 mL of a 570 ammonia solution to pharmacokinetics and analytical toxicology. Its distribution ( I , 4 , s ) as developing solvent. A diluted solution of iodoplatinate through the human body is very rapid, so that only low was used as locating reagent. The heptafluorobutyric anhydride concentrations are found in blood serum samples. In addition, “Puriss. p.a.” (obtained from Fluka AG, Buchs SG, Switzerland) it undergoes considerable metabolism in the liver, so that it was required as the derivatizing agent for the ECD analysis, and is present in urine samples only in low concentrations and was kept in sealed vials to prevent the ingress of water from the heavily masked by metabolites. The high sensitivity of gas atmosphere. chromatography, plus its specificity to the appropriate Standard Solutions. The stock solution of metapramine chlorohydrate in ethanol contained 0.1 mg of metapramine base compounds, led to its adoption for this study, using blood per mL. By diluting it successively in the same solvent, standard serum as the preferred samples. solutions with concentrations between 25 ng and 200 ng per 100 A number of gas chromatographic procedures have been pL were produced. The stock solution of the internal standard, proposed for the determination of antidepressants in plasma; desipramine chlorohydrate in ethanol, contained 0.1 mg of dethe most recent tend to utilize nitrogen-selective detectors sipramine base per mL and was diluted with the same solvent (7-IO), but none of these involves metapramine. In this paper, to obtain a standard solution of 400 ng per 100 pL. It was found a procedure for metapramine is described which utilizes both that both solutions remained stable for several weeks when stored a detector which is selective to nitrogen-containing molecules, in a refrigerator. and which requires no derivatization, and an electron-capture Glassware. After rinsing in distilled water, all glassware was soaked for 4 h in a hot solution of 3% “RBS 25 Biodegradable” detector which demands a derivatization. The respective 2354
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Table I. Retention Times (Minutes) of Metapramine, Desipramine, and Possible Metabolites of Metapramine in Different Detection Modes
"2
N-P D detection ME TAP R A M I NE ("19560 RP")
COMPOUND A ("19148 RP")
Metapramine
ECD detection
4 40
A B D G H I
4.40
2.67 3.87 3.20 5.47
5..33
Desipramine
8.67
Heptafluorobutyric derivatives of: Me tapramine
CH3 COMPOUND B ("26443 RP")
F?
H
COMPOUND G ( " 9 9 8 9 RP")
H
k
COMPOUND D ("19422 R P " )
k COMPOUND H ("19749 RP")
C $J H ~ - C H ~ - C H ~ - N < ~ CH3
COMPOUND I ("23669 R P " )
D E S l P R A M l NE (Internal standard)
Figure 1. Metapramlne, possible metabolltes, and desipramine
(obtained from T.C.S., 59130 Lambersart, France). After rinsing again in distilled water, it was left overnight in a dichromatosulfuric mixture. It was finally rinsed in distilled water and ethanol, and dried at 110 "C. Procedure. A known quantity of the internal standard solution (containing 400 ng desipramine) was inserted in a centrifuge tube, fitted with a glass stopper. The solvent was allowed to vaporize completely, then 1 to 3 mL of plasma sample was added. After vibrating for 15 s, 2 mL of buffer pH 9.8 was added. An extraction of this aqueous solution was performed by adding 3 mL of diethyl ether, mechanically shaking for 10 min and centrifuging to separate the two phases. The ether phase was removed and the extraction procedures repeated with another 3 mL of diethyl ether. The two ether extracts were combined and concentrated to a volume of about 1 mL. Then 3 mL of 0.1 N hydrochloric acid were added and this mixture shaken for 10 min, and centrifuged. The aqueous phase was neutralized with 1 N sodium hydroxide and 1 mL of buffer pH 9.8 added. Then the solution was again extracted twice with 3 mL diethyl ether as described above. The two ether phases were combined, and the solution evaporated to dryness under vacuum in a nitrogen atmosphere. Finally the residue was dissolved in 100 p L of ethanol. Between 1 and 2 pL of this solution was injected into the gas chromatograph equipped with the nitrogen-specific detector. The ratio of the peak areas E / E l ( E = metapramine, EI = internal standard) was calculated and referred to a calibration curve (actually a straight line). This calibration had been obtained by performing identical analyses of plasma standard samples to which
3.80 2.90 1.50
A G H
I Desipramine Compounds B and D: no derivatives are formed
3.20 4.67
9.80
known quantities of metapramine and a constant quantity of internal standard had been added. After the analysis of the sample on the N-detector instrument, the ethanolic solution was evaporated to dryness and the residue dissolved in 100 pL cyclohexane and 1 pL heptafluorobutyric anhydride. Vibration for 30 s was followed by heating to 122 "C for 1 min in the closed tube. The sample was then evaporated t o dryness under vacuum in a nitrogen atmosphere. The residue was dissolved in 100 pL cyclohexane. Between 1 and 3 pL of this solution was injected into the gas chromatograph equipped with the electron-capture detector. The ratio of the peak areas E / E I ( E = derivative of metapramine, E1 = derivative of internal standard) was calculated and referred t o a calibration curve (again a straight line), which was defined by performing identical analyses of plasma reference samples to which known quantities of metapramine and a constant quantity (around 400 ng) of internal standard had been added. To increase the reliability of the results, the calibration curves for both detector systems were checked for each series of samples. This was done by performing the complete analysis procedure with two different standard plasma samples containing exactly known quantities of metapramine (e.g., 7 5 and 125 ng) together with 400 ng internal standard. Each analysis on plasma samples and each injection into the chromatographs was repeated at least twice.
RESULTS Internal Standard. Several compatible compounds which could be used as an internal standard were studied. T h e compounds A, G, H, I, and desipramine provoked particular interest (Figure 1). T h e realization t h a t some of the tested compounds might eventually interfere with peaks of metapramine metabolites, led to the definite conclusion that desipramine was the best choice. The chromatographic separations of the unmodified compounds and of their N heptafluorobutyric derivatives were all shown to be satisfactory (Figure 2 and Table I). Derivatizing Technique. Several derivatization techniques had been tested to form derivatives of metapramine with a high response for the electron-capture detector. N-Heptafluorobutyric imidazole and heptafluorobutyric anhydride were tested under varying operational conditions-temperatures, periods of heating, solvents, etc. T h e best results, with respect t o sensitivity and reproducibility were produced by a reaction with heptafluorobutyric anhydride at 122 "C for 1 min, as already described under Procedure. ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Table 11. Recovery of Metapramine Added to Human Plasma Metapramine
Metapramine
added,
found
Recovery,
Re1 std
WmL
ng/mL
%
dev., %a
N-P D Detection 48 72 96 126 168 189 252
47.8 71.8 96.3 126.7 167.8 189.0 252.2
99.5 99.7 100.3 100.5 99.8 100.0 100.1
7.9 2.7 3.2 2.5 3.3 2.7 4.7
Average: 3.85
ECD Detection 48 72 96 126 168 189 252
48.0 71.4 96.0 126.0 167.8 188.5 250.8
100.0 99.1 100.0 100.0 99.8 99.7 99.5
8.3 5.0 9.3 4.9 5.8 4.5 8.7
Average: 6.64 a
N-P D "blank"
ECD "blank"
N-P D sample
ECD sample
Figure 2. Chromatograms obtained from the plasma extract of a patient who received metapramine fumarate in tablets
The linearity of responses provided proof of good derivatization, subsequently confirmed by thin-layer chromatography. The reaction conditions for the formation of N-heptafluorobutyric derivatives applied also to compounds A, G, H and I (Figure 1 and Table I). E x t r a c t i o n P r o c e d u r e . The tests on standard plasma samples led to the conclusion that diethyl ether (6 mL in two fractions of 3 mL each) at pH 9.8 ensured a complete extraction of metapramine and desipramine-as well as of the compounds A, B, D, G, H, and I. Additionally, their purification by re-extraction into 0.1 N hydrochloric acid was carried out without measurable loss. Sensitivity, Reproducibility, a n d Accuracy. The limits of sensitivity for the quantitative determination of meta2356
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Seven determinations,
pramine by the two detection systems were established after the extraction of 2 mL of plasma, solution of the residue in 100 pL solvent, and injection of 1.0 to 1.5 pL. These limits had been found for 15 ng/mL plasma with the N - P D (attenuation S = 4) and 35 ng/mL plasma (attenuation S = 3) after derivatization. These limits could, if needed, be lowered substantially by increasing the volume of the plasma sample, reducing the amount of solvent added to the residue, and increasing the injected quantity. The calibration curves for the two analyses were established under the analytical conditions already described using plasma samples which contained a constant quantity of internal standard (500 ng) and increasing quantities of metapramine (from 48 to 252 ng)-made by dilution from the two stock solutions containing 48 and 126 bg/mL, respectively. The determinations were repeated 7 times for each quantity. The accuracy of the results obtained is presented in Table 11. Relative standard deviations of the results for concentrations of metapramine in the range 48-252 ng/mL were found to be 2.5-7.9% and 4.5-9.370, respectively, by N-P D and ECD detection. The deviation was then smaller for the N-P D than for the ECD (which required derivatization). Specificity. The chromatographic analysis of the plasma "blanks" showed that no compound extracted under the conditions described had interfered with the metapramine, its possible metabolites, and the internal standard desipramine, derivatized or not. Concerning the analysis of plasmas taken from patients under treatment with metapramine, the double detection (N-P D and ECD) procedure allowed the accuracy of determination of the drug concentration to be increased and also the reliability of results (Figure 2 and Table I). It also revealed the nature and quantity of metabolites of the metapramine which were present in the sample. The specificity of this procedure was demonstrated by the table of the retention times for the different compounds studied (Table I). Any doubts relating to interferences between compound A and metapramine and between compounds I and H, which could remain after the N-P D procedure, were eliminated after EC detection of the derivatized products. Applications. The procedure was evolved to establish the plasmatic kinetics of metapramine in human subjects. An example is given in Figure 3, where the curve related to a patient who had received oral administration of 150 mg of
1
12
24 time (hours)
Figure 3. Plasma levels of metapramine after oral administration of a single dose of 150 mg (as fumarate). (A-A-A) N-P D detection. (0--0--0)ECD detection of heptafluorobutyryl derivatives
metapramine fumarate in tablet form. The plasma concentrations were determined not only for single doses but also after repeated dosages of the drug and also after intravenous injection of metapramine chlorohydrate. T h e urinary excretion was also investigated and the results will be published a t a later date. It is important to point out that in plasma the half-life of the compound is 5 to 6 h, that a large disproportion exists between the administered dose and the unchanged drug content in the plasma, and that the biotransformations of the metapramine are fast and intensive.
DISCUSSION A comparison of the two chromatographic detection methods clearly showed that the N - P D method was faster and easier because the disadvantages of derivatization were avoided. This method also provided excellent reproducibility and was considerably more sensitive than the ECD method which required derivatization. In this particular application however, its selectivity could cause some inconvenience be-
cause there was unfortunate mergence of chromatographic peaks from compound A and metapramine on the one hand and from compounds I and H on the other. Where single doses of metapramine were concerned, this caused no problem because compound A did not appear a t all in the human plasma and compounds I and H appeared only several hours after administration and then only in negligible quantities compared with the metapramine. Consequently the N - P D method of detection was perfectly applicable for the establishment of the plasmatic kinetics of unchanged metapramine after single doses of the drug. After repeated doses it was no longer possible, a t the beginning of the second day, to assure the absence of measurable quantities of compound A, formed by metabolism of metapramine. T h e ECD method was slower and more demanding as indicated by the “derivatizing technique” described earlier. The sensitivity was lower than that achieved by the N - P D. On the other hand, in this particular application, the specificity of the method was better because no interference between the main metabolites was observed, including compound A. This method therefore allowed the results of the kinetic studies of metapramine to be refined and it provided better recognition of the metabolites which could accompany the unchanged compound in the plasma. Simultaneous quantitative determinations of both the unchanged drug and its main metabolites could even be considered. The two detection methods therefore were complementary to each other in this work. Thus the analytical procedure described is available for pharmacokinetic and bioavailability studies involving metapramine, also for its therapeutic control. It can also be used for diagnostic or prognostic purposes in possible cases of accidental or deliberate poisoning.
ACKNOWLEDGMENT Suzanne Monjanel has provided invaluable assistance with the mathematical calculations involved.
LITERATURE CITED A. Viala, F. Gouezo, and C. Gola, J . Chromatogr., 45, 94 (1969). A. Viala. J. P. Cano. F. Gouezo. C. Gola. and A. Anaeletti. Sci. Med.. 2, 409 (1971) A Viala. J P Cano. C Gola, and F Gouezo, J Chromatogr , 59, 297 I
11971) \ . - . ./.
A. Viala, C. Gola, J. P. Cano, and F. Gouezo, Ann. Pharm. Fr., 30, 445 (1972). C. Goh, Etudes toxicologiques sur les m6dicarnent.s antid6pressurs d6riv6s de I’imipramine, Pharm. Thesis. Marseilles, 1971. Specia Laboratories, Paris, personal communication. L. A. Gifford, P. Turner, and C. M. B. Pare, J . Chromatogr., 105, 107 ( 1975). A . Jorgensen, Acta Pharmacol. Toxicol. 36,79 (1975). J. Vasiliades and K . C. Bush, Anal. Chern.. 48, 1708 (1976). D. N. Bailey and R. I . Jatlow. Clin. Chem. ( Winston-Sa/em, N . C . ) , 22, 777, 1697 (1976).
RECEIVED for review July 11, 1977. Accepted September 20, 1977. Specia Laboratories of Paris have provided considerable support of this work, and also provided us with the required reference compounds.
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