999
Anal. Chern. 1980, 52, 999-1001 CONDUCTOMETER T3LNTER
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Flgure 1. Conductometer-potentiostat hook-up AUX
R,
R~
cdl
WORK 0
Dummy cell: R,, compensated resistance; R, uncompensated resistance; R, Faradaic resistance; C, doublelayer capacitor Figure 2.
Several approaches have been suggested for compensating the “uncompensated” cell resistance; among them are the well known “positive feedback” a n d t h e more sophisticated “dynamic compensation’‘ method. T h e positive feedback method is frequently used in commercial potentiostats and requires manual adjustment. Although the three-electrode system is used frequently. it is quite impossible to estimate which part of the cell resistance is compensated by positive feedback. Inevitable overcompensation results in high-frequency oscillations and high currents, causing the solution to heat up a n d destroying the working electrode. In spite of these reservations, whenever simple cell geometry allows the resistance between the tip of the reference electrode and the working electrode to be estimated, proper adjustment of the positive feedback is possible: the uncompensated resistance can be then calculated a n d the adjustment carried out in accordance with the instructions of the manufacturer. We suggest adjusting the positive feedback after measuring
the uncompensated resistance with a simple conductometer in a three-electrode system. This approach I S universally applicable and independent of cell geometry. Direct reading conductometers have two output leads, one virtually grounded, the other connected to the alternating voltage generator; the resistance between the two is very low. T h e counter and reference electrodes are connected to the potentiostat in the usual manner, potential in the vicinity of the tip of the reference electrode being kept constant; this electrode may now be considered virtually grounded (as far as ac currents are concerned). T h e working electrode is next connected to one lead of the conductometer; the second lead is connected t o the ground of the potentiostat (Figure 1). Clearly, the conductometer reading obtained is the reciprocal of the uncompensated resistance. After t h e resistance has been determined, the working electrode is disconnected from the conductometer and reconnected to the appropriate potentiostat input. T h e positive feedback potentiometer is then adjusted appropriately. The operation of the combination described in Figure 1 may be checked by substituting a dummy cell consisting of a capacitor and two resistors in series (Figure %), all having equivalent values close to those of the electrochemical cell under inspection. Certain kinds of conductometers cannot tolerate direct currents flowing through them a n d therefore potential adjustment is required to minimize cell currents. To test conductometer performance, a resistor i s connected in parallel to the capacitor of the dummy cell a n d a potential of 1 V imposed on it through the potentiostat. T h e conductometer reading should remain unchanged.
RECEIVED for review August 9, 1979. Accepted November 12, 1979.
Determination of Metabolites of Thymoxamine in Plasma by High Performance Liquid Chromatography A. E. Geahchan and P. L. Chambon” Laboratoire de Toxicologie et Hygisne Industrielle, Facult6 de Pharmacie, 8 a venue Rockefeller, 69373 Lyon, France
Thymoxamine 4-(2-dimethylaminoethoxy)-5-isopropyl-2methylphenyl acetate (Scheme I) is a specific competitive a-adrenoreceptor blocking agent (1, 2 ) . It is used as a peripheral vasodilatator in the treatment of hypertensive and vascular disorders ( 3 ) . Thymoxamine is rapidly a n d completely hydrolyzed by plasma (in vitro) within 5 min after administration to give the desacetyl derivative (Scheme I) which is later demethylated to give demethyldesacetylthymoxamine (Scheme I). T h e quantitative determination of desacetylthymoxamine, the major metabolite of thymoxamine, is important both for monitoring and optimizing blood levels of patients receiving therapeutic doses, as well as for the study of pharmacokinetic properties. T h e existing spectrophotometric method ( 4 ) requires fairly large amounts of serum (2.5-4 mL) to achieve the desired sensitivity a n d precision. Furthermore, interferences from endogenous compounds may occur, and the separation of the two metabolites is not achieved. This paper describes a high performance liquid chromatographic method for the rapid, accurate, a n d sensitive determination of desacetylthymoxamine and demethyldesacetylthymoxamine in plasma in the therapeutic range with the capability of separating the drug 0003-2700/80/0352-0999$01 OO/O
Scheme I ‘CH3
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from interferents normally troublesome with direct spectrophotometric methods. T h e method is simple and sensitive and it does not require the formation of volatile derivatives.
EXPERIMENTAL Apparatus. A Chromatem M 38 pumping system (Touzart & Matignon, France) coupled to a Jobin Yvon J Y 3 variable wavelength spectrofluorimeter equipped with a 20-pL flow cell, was used for this study. The detector was operated at 295 and 320 nm as excitation and emission wavelengths, respectively. For plasma samples taken after intravenous administration of thym1980 American Chemical Society
1000
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980 1
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Flow dependence of column efficiency H (calculated from
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",V V i O O 1 ° W V A M M O N I U M C A R B O N A T E A O ) IN
METHANOL
Effect of the percentage v/v of 0.01 YO ammonium carbonate aqueous in methanol on efficiency (upper) resolution (lower) Figure 1.
oxamine (30 mg/kg), a sensitivity of 0.1 (arbitrary unit) was used; but for the lower levels normally present in plasma, a sensitivity of 0.03 (arbitrary unit) (full scale deflection) was required. A reverse phase system consisting of a 22-cm-4.6-mm column packed with Partisill0 ODS 2 (Reeve Angel, USA) was utilized with methanol-0.017'~ w/v water ammonium carbonate (97.5:2.5 by vol) as eluent a t a flow rate of 2 mL/min. The column was packed by a slurry technique using hexane-chloroform (1:3 v/v) as a slurry liquid with a packing pressure of 500 bars. Injections were achieved with a high-pressure sample injection valve fitted with a 20-pL sample loop. Reagents and Chemicals. Thymoxamine, desacetylthg moxamine, and demethyldesacetylthymoxamine were purchased from Laboratoire Dedieu, France. Working standards were prepared by diluting the stock solutions with methanol to give 2.5, 5 , 10, and 20 mg/L. T o prepare sodium carbonate solution, dissolve 20 g of sodium carbonate in 100 mL of bidistilled water. T o prepare ammonium carbonate solution, dissolve 0.1 g of ammonium carbonate in 1L of bidistilled water. All other chemicals were reagent grade (Merck-Darmstadt). Extraction Procedure. Transfer 0.5 to 1.0 mL of plasma to a 10-mL stoppered test tube. Add 50 pL of sodium carbonate buffer (pH 9) and stir on a Vortex mixer (30 s). Add 5 mL of chloroform and shake the tube for 5 min on a mechanical shaker. Centrifuge the tube for 5 min. The organic layer is then transferred by disposable pipet to a dry clean 5-mL test tube and evaporated to dryness in a 50 "C water bath using a stream of nitrogen. The extraction and evaporating steps are repeated. The dry residue is redissolved by adding 200 p L of methanol, quickly capping each tube and vortexing. The methanol mixture of 20 ~ L is L injected into the liquid chromatograph. The plasma concentrations are determined from a standard curve established by plotting the peak heights of the standards against the standard concentrations.
RESULTS AND DISCUSSION A simple binary solvent mixture consisting of methanol and 0.170water ammonium carbonate proved t o be useful. This mixture was chosen after testing solvents containing increasing amounts of water ammonium carbonate. At a flow rate of 2 mL/min, the maximum efficiency for all solutes in the mixture and t h e best resolution between the two metabolites of thymoxamine occur a t 2.5% of 0.1% w / v aqueous ammonium carbonate in methanol (Figure 1). A methanol-0.1 % w / v aqueous ammonium carbonate (97.5:2.5 v/v) mixture was used as the mobile phase. Optimization of flow rate is readily deduced from t h e flow dependence of t h e height equivalent t o a theoretical plate ( H E T P ) (Figure 2).
Figure 3.
Chromatogram of desacetylthymoxamine ( I ) and de-
methyidesacetylthymoxamine(11). Concentration of each metabolite:
2 mg/L
Figure 4. Chromatogram of plasma extract: 1 mL of plasma is spiked with desacetylthymoxamine (I) and demethyldesacetytthymoxamine (11) (0.4 mg/L of each metabolite). Residue is taken up in 200 pL of methanol
Obviously, maximum efficiency for the two solutes in t h e mixture occurs at a flow rate of about 0.9 mL. However, we must compromise between loss of efficiency and decrease in analysis time. At a flow rate of 2 mL/min, little resolution is lost, b u t increasing t h e flow rate results in a desirable
1001
Anal. Chem. 1980, 52, 1001-1003
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Figure 5. Arithmetic plot of plasma concentrations of desacetylthymoxamine after administration of 30 mg/kg of thymoxamine to the monkey
decrease in analysis time while still preserving adequate resolution. A typical chromatogram is shown in Figure 3. The linearity of the procedure was found t o be good in the range of 0-50 mg/L. Figure 4 illustrates a chromatogram of a plasma spiked with desacetylthymoxamine and demethyldesacetylthymoxamine
(0.4 mg/L of each metabolite). The mean extraction recoveries were determined by adding known amounts of the two metabolites t o samples of a plasma pool. Desacetylthymoxamine and demethyldesacetylthymoxamine metabolites added to plasma over the range of 500 to 2000 ng/mL were l,ecoverable to the extent of 96.5% f 2.3 ( n = 6) and 81% h 3.1 ( n = 6), respectively. Control samples of plasma showed no interference peaks. We estimated the detection limit a t 2 ng and 4 ng for desacetylthymoxamine and demethyldesacetylthymoxamine, respectively (total injected amount). Application of the method to the administration of desacetylthymoxamine in plasma after intravenous administration of 30 mg/kg of thymoxamine to a monkey is demonstrated in Figure 5 . After being hydrolyzed, the drug is rapidly eliminated from the plasma pool.
LITERATURE CITED (1) (2) (3) (4)
Mercier, J.; Canellas, J.; Roquebot, J. Thdrapie 1971, 26, 785-794. Corbett, J. L.; Eidelman, 6. H. Lancet 1972, 7775, 461-463. Cristol, R. Gaz. Med. 1971, 78, 4270-4274. Arbab, A. G.:Turner, P. J . Pbarm. Pharmacol. 1971, 23. 719-721.
RECEIVED for review October 9,1979. Accepted December 18, 1979.
Syringe Micropyrolyter for Gas Chromatographic Determination of Acetylcholine, Choline, and Other Quaternary Ammonium Salts Frantigek Mikes, Geraldine Boshart, Karl Wuthrich, and Peter G. Waser Pharmacological Institute, University of Zurich, 8006 Zurich, Switzerland
Analytical pyrolysis is finding an ever-increasing number of applications in the biomedical field, including the analysis of drugs and biomolecules (1-3). One of the most important applications has been the pyrolytic demethylation of the quaternary amine neurotransmitter acetylcholine (ACh) and its precursor choline (Ch) followed by gas chromatography (GC) and quantitative determinations with either flame ionization detection (FID) ( 4 , 5 ) or mass spectrometry (MS)multiple ion detection (MID) (6-9). Pyrolysis-GC (Pyr-GC) is easier and faster to perform than the alternative chemical demethylation-GC method for choline esters ( I O ) , but it requires a larger initial economic investment for pyrolysis equipment. There are three types of pyrolyzers commercially available: flash filament, Curie point, furnace type, and in development a laser type of pyrolyzer (1-3). Most of these pyrolyzers have been designed to fit one type of gas chromatograph and require alterations in transferring them to another. Switching from Pyr-GC to direct injection GC is also a time consuming alteration with most commercial pyrolyzers. This paper describes the construction of a syringe, flash filament type of pyrolyzer, designed especially for the rapid quantitative analysis of nonvolatile molecules with appropriate standards, which can easily and inexpensively be built in any analytical laboratory. It may be quickly and easily manipulated and used with any gas chromatograph without alteration. One may also alternate between normal on-column injections and the pyrolyzer application without any loss of time or change in the system.
EXPERIMENTAL Syringe Micropyrolyzer Construction. Figure 1 shows a 0003-2700/80/0352-1001$01 OO/O
schematic diagram of the new syringe micropyrolyzer. The pyrolyzer body is a stainless steel Swagelok reducing union (J),1 / 4 inch X 1/16 inch (No. 400-1-SS,Crawford Fitting Co., Cleveland, Ohio) bored out to a U shape (Q) allowing efficient wash out of thermal degradation products. The pyrolysis filament (R) is a platinum (Pt) wire (Mbteaux Precibux, Neuchatel, Switzerland) 0.3 mm 0.d. X 10-18 mm long (total length including clamped ends 2@28 mm), clamped between two brass nipples (No. 220-PO2, Amphenol, Chicago, Ill.) with the use of a crimper. The nipples are inserted into leads (D) (No. 220-P02, Amphenol) embedded in a Teflon plug (F) above the body. The carrier gas stainless steel inlet tube (C), 3 cm X 1.5-mm o.d. X 0.2-mm i.d., is also embedded in the Teflon plug, which is in turn encased in a brass cylinder (E) to protect it from deforming by heat or manipulation. In order to minimize memory effects by adsorption of pyrolysis products on the Teflon, a stainless steel disk (G)with three holes for the leads and carrier gas is inserted in the top of the pyrolyzer chamber, directly below the Teflon plug. An air tight pyrolyzer chamber is achieved by placing a Teflon O-ring (HI, 10-mm 0.d. X 7-mm i.d. X 1.2 mm thick, under the disk and wrapping the outer threads of the Swagelok union body with a double layer of Teflon seal tape (S)(maximum temperature 280 "C, No. 121, Dodge Fibers Co., Hoosick Falls, N.Y.) before closing the chamber. inch) is equipped with The lower part of the reducing union an exchangeable needle (N) (0.46-mm 0.d. x 0.25-1nm i.d.) with soldered disk (L) (Gauge 26, Hamilton Bonaduz AG, Bonaduz, Switzerland). When using the syringe pyrolyzer, the carrier gas flow is diverted from the normal GC line by closing one brass toggle valve (B,) (No. B-OGS2, Whitey Co., Oakland, Calif.) and opening the other valve (B2)to the pyrolyzer before inserting the syringe into the injector (M). For the carrier gas line from the second valve (B2)to the pyrolyzer, polytetrafluorethylene tubing is used. The rapid heating of the Pt wire is achieved by connecting the leads (D) to a filament transformer (No. F-21A, ll5V/6.3 V-lOA, 1980 American Chemical Society
