1001
Anal. Chem. 1980, 52, 1001-1003
1.
\.-----+
_______~__ ~
hrr
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
1002
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
l-E:
Figure 1. Schematic diagram of the new syringe flash filament micropyrolyzer. (A) Carrier gas, (B,) toggle valve on line with GC, (B2) toggle valve to pyrolyzer, (C) carrier gas inlet tube, (D) electrical leads, (E) brass cylinder, (F) Teflon plug, (G) stainless steel disk, (H) Teflon O-ring, (I) '/,-inch nut, (J) '/,-inch X '/,,-inch Swagelok reducing union-body, (K) '/,,-inch nut, (L) needle disk, (M) injector, (N) needle, (0)silanized glass wool, (P) column bed, (0)pyrolysis chamber, (R)
platinum wire, (S) Teflon tape Traid Transformer Co., Los Angeles, Calif.) from which 0-10 A is available. The required amperage is preset and switched on for the desired pyrolysis time. Utilization. In a typical pyrolysis, 3 pL of a saturated solution (ca. 1.7 mg/mL) of tetramethylammonium iodide (TMAI) in acetonitrile and 1 pL of a sample is applied to the P t wire and the solvent is evaporated using 2.5 A of current. The pyrolysis body is closed (Le., connect I and J) and the carrier gas valve to the pyrolyzer is opened. The pyrolyzer is purged with nitrogen for 1min, during which time it may be checked for possible leaks by immersing the whole body in alcohol. After purging, the carrier gas valve on line with the GC is closed and the pyrolyzer needle is inserted through the septum into the GC column, where the needle tip is located 5-10 mm above the filling bed (PI. The entrance to the column should contain enough silanized glass wool (O), so that the column bed is not disturbed by the carrier gas flow streaming from the pyrolyzer needle. The pyrolyzer is allowed to equilibrate for 2 to 5 min, during which it should approach 100 "C, a temperature suitable for analyzing pyrolytically demethylated choline esters. (When performing Pyr-GC-MS, i t is sufficient to preheat the pyrolyzer for 15-30 s with an electric hot air pistol, Rotax Type HP-67, Balzer AG, Basel, Switzerland, maximum temperature 250 "C, and no further equilibration is necessary.) The sample is then pyrolyzed by passing amperage through the Pt wire for 6 s. With the beginning of filament heating, the recorder chart is turned on ( t o ) . A t the end of the chromatogram the pyrolysis needle is removed from the injector, the valve to the pyrolyzer is closed, and the valve on line with the GC is opened. The lower part of the pyrolyzer (J, K) combined with the needle is cleaned under vacuum with water, methanol, and methylene chloride. The upper part of the pyrolyzer (I) with the Pt wire is cleaned by applying higher electric current (e.g., 7 A) for 10 s with nitrogen flowing. Occasionally the stainless steel disk may be cleaned by wiping with cotton slightly moistened with water. Chromatographic Equipment. For Pyr-GC-FID, a Fractovap Model GI gas chromatograph (Carlo Erba, Milano, Italy) was used, and for Pyr-GC-MS,a Pye Unicam 104 gas chromatograph coupled with an LKB 2091-051 mass spectrometer. The GC glass columns, 1.5 or 2.3 m X 2 mm i.d., were filled with Gas-Chrom Q (100/120 mesh) coated with either 570 4-dodecyldiethylenetriamine succinamide (DDTS) ( 1 1 ) and 5% OV-101 or 4% DDTS, 4 % Carbowax 20M, and 2% OV-101. Isobutane was used as ionizing gas for chemical ionization ((21)-MS.
0
4
i
rnl"
Figure 2. GC-FID chromatogram of pyrolytically demethylated choline esters. Sample: 5 ng each of acetylcholine (ACh)and butyrylcholine (BCh), 100 ng each of propionylcholine (PCh) and butyrylmethylcholine (BMCh). Column: 4 % DDTS, 2 % OV-101, 4 % Carbowax 20M on Gas Chrom Q (100/120 mesh), 1.5 m X 2 mm i.d.; column temperature 110 "C; injector temperature 315 '. N, flow 40 mL/min, H, flow 20 mL/min, air flow 300 mL/min
RESULTS AND DISCUSSION The micropyrolyzer has been in use for about one year and we have analyzed more than 30 quaternary ammonium (QA) salts (12)with it. One example of the pyrolysis of four choline esters can be seen in Figure 2. In comparison with the chemical demethylation-GC method for choline esters (IO), Pyr-GC has the advantage, when using an FID detector, of giving no solvent peak. The early trimethylamine peak from copyrolyzed TMAI does not interfere with the demethylated ACh peak and 5 ng of ACh+ can easily be detected. On the other hand, when injecting 100 ng of demethylated ACh in 1 pL of acetonitrile onto this column, only a slight shoulder a t an attenuation X8 was observed. A detection limit of 0.5 nmol (73 ng QA+) of ACh by GC-FID, when injecting the demethylated form in chloroform, has been reported (13). For quantitative analysis of ACh we had chosen the propionyl homologue (PCh) as standard and for Ch its @-methyl homologue (MCh). Both Ch and MCh are converted to their butyryl esters (BCh, BMCh) before analysis. When pyrolyzing a mixture of 100 ng each of ACh, PCh, BCh, BMCh eight consecutive times, the peak height ratio of ACh/PCh was 1.32 with a standard deviation of 0.018 and for BCh/BMCh 0.570 0.016. T h e peak height ratios of ACh and BCh t o their standards increase linearly with their relative concentrations between 10 pg and 10 ng using CI-MID (12) and between 5 and 1000 ng using FID. In optimizing the method, we have found two pyrolytic factors which greatly influenced the sensitivity. First, when a choline ester is copyrolyzed with a large excess of another QA salt (TMAI) (14), its peak height is equal to that obtained when injecting an equimolar amount of the chemically demethylated choline ester. Without this cofactor the response for picomole samples is significantly lower. Secondly, for any Pt wire, there exists an optimum electrical current range for pyrolyzing choline esters to obtain maximum demethylated product peaks, as shown in Figure 3. The longer the Pt wire, the lower the current a t which maximum peak heights are obtained. Peak height ratios, on the other hand, remain
*
Anal. Chem. 1980, 52, 1003-1005
30
*0
50
63
-0
.io03
matograph, where the carrier gas may be directed on line and/or through the pyrolyzer with quick switching. Injections may be performed with either the micropyrolyzer or a normal syringe without adjustments to the system. Thus extracts (e.g., of biogenic amines, drugs) may be analyzed in rapid succession by either or both application methods, and reference compounds of suspected degradation products may be injected between pyrolyses. T h e small pyrolysis chamber (less t h a n 0.5 mL) minimizes peak broadening and t h e small stainless steel body provides relatively fast heat exchange (heating and cooling). Four analyses per hour of ACh and Ch (as its butyryl ester) using Pyr-GC-FID and eight analyses per hour using Pyr-GC-CI-MID can easily be performed. For the quantitative analysis of other nonvolatile molecules (e.g., drugs, small biomolecules) using appropriate internal standards (preferably homologues), this type of simple a n d inexpensive pyrolyzer should also be applicable.
I 80
diperes
Figure 3. (a) Influence of platinum wire length (+ 18 mm, A 15 mm, 0 10 mm) and current intensity on ACh response. (b) Influence of current intensity on peak height ratios ( 0 ,0 )using a 15-mm long wire
ACKNOWLEDGMENT We thank H. Dollenmeier for helpful discussions on t h e electronics of our system and F. Lamprecht (Memo AG, Wallisellen, Switzerland) for the use of a high temperature thermocouple.
constant (ACh/PCh) or vary only slightly (BCh/BMCh) over quite a wide current range (Figure 3). T h e temperature on t h e surface of t h e Pt wire, when applying optimum current for a given wire, was measured with a 0.5-mm 0.d. NiCr-Ni thermocouple (Tastotherm D 1200, BT-l202d, Gulton, Frankfurt, GFR) to be 330-370 "C. T h e thermal demethylation of choline esters begins a t about 200 "C, and temperatures of 220-250 "C have been used for direct inlet-MS (12) or slow pyrolysis on a glass probe before GC-MS (8). With t h e filament transformer used, the temperature rise time (TRT) was 5-7 s. A faster T R T could be attained by connecting a capacitor discharge circuit (15)to the filament, but for the determination of choline esters it is not required. T h e upper temperature limit for t h e Pt filaments was measured with t h e NiCr-Ni thermocouple to be about 1150 "C (7.5 A with a 15 mm X 0.3-mm 0.d. wire). By increasing the temperature (amperage) further, filaments broke. By optimal pyrolytic conditions, the detection limit for ACh and the butyryl derivative of Ch is 5 ng of the QA cation using a n FID detector and 10 pg using CI-MID (12) with packed glass columns. A further increase in sensitivity by the use of open tubular glass columns is in progress. T h e advantages of the syringe micropyrolyzer here described may be summarized as follows. T h e total micropyrolyzer assembly including electrical current source may be constructed a t a cost of about 10% of a commercial one. I t is easy to manipulate and may be used with any gas chro-
LITERATURE CITED (1) Irwin, W. J.; Slack, J. A. Analyst (London) 1978, 103, 673-704. (2) "Analytical Pyrolysis", Jones, C. E R., Cramers, C. A., Eds.; Elsevier: New York, 1977. (3) Irwin, W. J J . Anal. Appl. Fyrol. 1979, 7 , 3-25, 89-122. (4) A.: Green. J. P.: Brown. 0. M.: Maraolis, S. J . Murochem. . , Szilaavi. P. I. 197%- 19, 2555-2566. (5) Schmidt, D. E.; Speth, R. C. Anal. Biochem. 1975, 67, 353-357. (6) Fidone, S. J.; Weintraub, S. T.; Stavinoha, W. B. J . Neurochem. 1976, 26. 1047-1049. (7) Sziiagyi, P. I:A.;Green, J. P. I n "Analytical Pyrolysis", C. E. R., Jones, Cramers, C . A., Eds.; Elsevier: New York, 1977; pp 417-418. (8) Polak, R. L.; Moienaar, P. C. J . Neurochem. 1979, 32, 407-412. (9) Maruyama, Y.; Kusaka, M.; Mori, J.; Horikawa, A,; Hasegawa, Y. J . Chromatogr. 1979, 164, 121-127. (10) Jenden. D. J.; Hanin, I.; Lamb, S. I.Anal. Chem. 1968, 40, 125-128. (1 1) Jenden. D. J.; Roch, M.; Booth, R . J . Chromatogr. Scl. 1972, 10, 15 1-1 53. (12) Mikeg, F.; Boshart, G.; Waser. P. G. I n "Recent Developments in Mass Spectrometry in Biochemistry and Medicine-VI" (Proceedings of the 6th International Symposium on MS in Biochemistry and Medicine, Venice, June 1979). Frigerio, A., Ed.; Elsevier: Amsterdam, 1980, in press. (13) Kosh, J. W.; Smith, M. B.; Sowell, J. W.; Freeman, J. J. J , Chromatogr. 1979, 163, 206-211. (14) Polak, R. L.; Molenaar, P. C. J . Neurochem. 1974, 23. 1295-1297. (15) Levy. R. L.; Fanter, D. L.; Wolf, C. J. Anal. Chem. 1972, 4 4 , 38-42.
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RECEIVED for review November 1, 1979. Accepted January 21, 1980.
Stocastic Variability of Noise with the Hall Electrolytic Conductivity Detector for Gas Chromatography Reginald K. S. Goo,' H. Kanai, V. Inouye, and H. Wakatsuki Laboratories Branch, Hawaii State Department of Health, Honolulu, Hawaii 968 13
T h e use of an electrolytic conductivity system in conjunction with a combustion furnace for the detection of chlorine, nitrogen, and sulfur compounds was first described by Coulson ( I ) . H e has shown t h a t the controlled oxidative or reductive pyrolysis of gas chromatographic eluants produced simple inorganic gases which may be subsequently dissociated in water a n d detected conductometrically. Coulson's detector, besides being unwieldy, had one serious drawback for pesticide residue analysis, its sensitivity was low. Since then, there has been a steady stream of improvements 0003-2700/80/0352-1003$01 .OO/O
(2-5). Then Hall (6) developed a sensitive and selective microelectrolytic conductivity detector. Since then, this electrolytic conductivity detector has been used in pesticide residue analysis because of its selectivity for chlorine, nitrogen, and sulfur. Also its superior discrimination against polar co-extractives makes it a desirable detector for complex matrices. Many laboratories such as ours, doing daily routine pesticide residue analysis, require consistent signal-to-noise ratios from day to day. Some of the factors which affect noise and sen-
e
1980 American Chemical Society
