careful selection of column materials, preparation of the column, use of the gases of uniform quality, and setting the electrometer response and the recorder amplification a t the same value each time the determinations are made. The question of ester interchange within the column while the mixtures of esters are injected was not as serious as expected, but the ester interchange can become serious if mixtures having large retention volumes are analyzed. In resetting the chromatographic conditions with new columns, one of the reference compounds, like methyl cyanoacetate, can be used as an external standard to adjust the response to the same original value found in the first calibration. The use of a suitable internal standard to obviate the effect of temporary fluctuations in any of the parameters involved is being investigated further. The secondary butyl derivatives of the above series did not conform to the linearity of the logarithm of the retention volume or response in area units per micromole to the carbon number as expected. This is shown clearly from the values of the same reported in Table I, which do not fit in the plots of Figures 1and 3. Accuracy of Determinations. The response in peak area of the hydrogen flame ionization detector ( I ) depends linearly on the mass flow rate of the sample (at low values) described by: (1)
where I = quantity of ion current in coulombs, g = quantity of carbon in grams flowing, t = time in seconds, and K = proportionality constant (coulombs/g carbon). In the present investigation, the only polarizing potential available on the instrument was 112.5 volts a t a constant value, and the relative value of the ion current was given by the peak area in the integrator counts. The proportionality of the above relationship was maintained by keeping the mass flow rate of each sample within certain limits, by adhering to the technique described in the ex-
perimental section above. In short, the samples were all 0.02 molar in solution in methylene chloride, giving approximately 1 to 2.8 pg total carbon per sample in solution per microliter, and the peak area was covered by each sample in an average time of about 20 seconds depending on the retention volume. By this method, the mass flow rate of carbon amounts to nearly 0.5 to 1.4 x lo-? gram per second per microliter injection. If the maximum volume injected each time is 10 microliters, the response as measured was within the 1-270 accuracy in the determination of the sample size from the area. However, if the sample size injected was larger than 30 pg in any compound, the linearity became uncertain and the determination was no longer reliable.
CONCLUSIONS A gas chromatograph equipped with a hydrogen flame ionization detector can be calibrated us. pure samples of normal monohydric alcohols, alkyl cyanoacetates, and alkyl 2-cyanoacrylates. Logarithms of retention volumes and of micromolar responses are linearly related to the number of carbons of the individual compound in each homologous series. The identity and the amount of any unknown compound in the same series can be evaluated by analysis of the compound on the precalibrated gas chromatograph. ACKNOWLEDGMENT The authors gratefully acknowledge the invaluable help in the analytical work by Ronald Harrison, the physicochemical assistant. Received for review July 16, 1973. Accepted December 28, 1973. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
Gas Chromatographic Determination of Thiopental in Plasma Using an Alkali Flame Ionization Detector Lawrence T. Sennello and Fred E. Kohn Division of Experimentai Therapy, Abbott Laborafories. North Chicago. //I. 60064
Thiopental, an ultra short-acting barbiturate, is often used as a general anesthetic in conjunction with inhalation or with another intravenously administered anesthetic. Thus, it is occasionally desirable to monitor the level of the barbiturate in the blood of patients during and/or after anesthesia. Although the technical literature contains numerous procedures for the determination of thiopental and other barbiturates in biological fluids, most of the methods are relatively insensitive or time-consuming. Grochowska ( 2 ) and Oroszlan and Maengwyn-Davies (2) reported spectrophotometric procedures with sensitivities of about 20-300 pg per ml. Scoppa ( 3 ) reported a spectrofluorometric de( 1 ) 2 . Grochowska, Mikrochirn Acta. 5, 1905 (1968). (2) S. I . Oroszlan and G . D Maengwyn-Davies, J. Arner. f h a r r n A s s 49, 507 (1960). (3) P . Scoppa, Biochirn. A p p l , 13, 274 (1966).
752
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
termination with a sensitivity of about 0.5 pg per ml of fluid. Braddock and Marec ( 4 ) published a gas chromatographic procedure for the determination of thiopental and pentobarbital in biological fluids with lower detection limits of about 1pg per ml. Because of the inherent specificity and sensitivity of the technique, it was decided to pursue a gas chromatographic approach. Here too, though, published methods tended to be somewhat insensitive or tedious. Either sensitivity was low because of sample loss during lengthy “clean up” steps, or great caution had t o be taken to prepare especially inert columns which caused little or no tailing of barbiturates. To overcome the problems of adsorption or decomposition of barbiturates resulting from interaction with the column, one must either chemically modify the drugs to (4)
L
I Braddock and
N
Marec J Gas Chrornatogr
3, 274 (1965)
make them less polar ( 5 - 3 , heavily silanize the column with repeated injections of a silanizing agent, infuse a compound such as formic acid into the column ( 8 ) , or find a liquid phase which, by its nature, causes little or no adsorption of the drug. Since the first two of these approaches are time-consuming, and the third requires the use of specialized apparatus, it was decided to try to find a liquid phase which would consistently produce good columns with a minimum of effort. The first column tested with "clean" solutions was 5% methyl silicone polymer (OV-101) on SO/lOO mesh Gas Chrom Q, similar to that described by Brochmann-Hansson and Baerheim-Svendsen (9). With this and other silicones such as OV-17, OV-225, and OV-210, the thiopental peak tailed excessively. In all cases, a silanized support (Gas Chrom Q) was used as recommended by these authors to help eliminate peak tailing, but this was not sufficient. In similar fashion, the suggestion of Parker e t al. (20) to use an SE-BO/Carbowax-BOM mixed liquid phase, and of Cieplinski ( 1 2 ) to use a combination of methyl silicone and C54 tricarboxylic acid to reduce tailing were tried and found unsatisfactory. Although some acceptable columns could be made, they are difficult to reproduce. The first successful and consistently reproducible column found was similar to that described by Byars and Jordan (12). These authors recommended the use of 10% Carbowax-20M-Terephthalic acid on 70/80 mesh DMCS-AW Chromosorb W for the separation of C2 through CIS free fatty acids. Because of the low volatility of barbiturates, it was deemed desirable to use a column of 2% Carbowax-20M-Terephthalic acid on SO/lOO mesh Gas Chrom Q. Although good columns could consistently be prepared with this combination, the chief limitation was low thermal stability. At the high temperatures used, columns would begin to deteriorate after about a week of use, requiring frequent replacement. When new polyamides such as Poly A-103, which are intended for use as liquid phases ( I 3 ) , became available, it was found that they worked equally as well as the above Carbowax combination, and afforded a higher degree of heat stability. Columns produced with Poly A-103 on Gas Chrom Q consistently performed well with no special deactivating precautions necessary. They could be used for extended lengths of time at 250 "C without noticeable deterioration in performance. With a satisfactory liquid phase chosen. attention was focused on methods of removing thiopental from plasma and achieving a high level of sensitivity. Although a large number of procedures are available in the literature for the gas chromatographic determination of barbiturates in blood or urine, virtually all are concerned with toxicological screens or autopsies (5. 6, 1416), and the concomitant high concentrations of drug. Gudzinowicz and Clark (17) reported a potential route to (5) M . J . Barrett. Clin. Chem. Newslett.. 3, 1 (1971). ( 6 ) J MacGee, Clin. Chem.. 1 7 , 587 (1971). (7) G . Kananen, R. Osiewicz, and I . Sunshine. J. Chromatogr. S c i . . 1 0 , 283 (1972) (8) R. F . Adams, Clin Chem Newslett . 4, 15 (1972) (9) E Brochmann-Hanssen and A . Baerheim-Svendsen, J . Pharm. SCi.. 50, 804 (1961) (10) K . D. Parker, C . R Fontan. and P. L K i r k , Anal. Chem.. 35, 1418 ( 1963) (11) E . W Cieplinski, Anal Chem.. 35, 256 (1963) (12) B . Byars and G . Jordan. J Gas Chromatogr , 2, 304 (1964) (13) R . G Mathews. R D Schwartz. J . E. Stouffer,and B. C. Pettite, J Chromatogr. S o . . 8, 508 (1970) (14) D. Blackmore. Perkin-ElmerAnalytical News. 3, 1 (1971) (15) H. F. Proelssand H . J . Lohmann, Ciin. Chem.. 1 7 , 222 (1971) (16) L . Kazyak and E. C . Knoblock, Anal. Chem.. 35, 1448 (1963) (17) B J. Gudzinowicz and S. J . Clark, J . Gas Chromatogr.. 3, 147 (1965)
increased sensitivity with the use of an electron capture detector for barbiturate analysis, but did not achieve the sensitivity required for the proposed work. The first approach in this laboratory consisted of extracting the thiopental with chloroform or other organic solvents, concentrating the extract to 100 pl, and subjecting a 4-pl aliquot to gas chromatography with a hydrogen flame ionization detector (HFID). The resulting chromatograms were so complex that it was impossible to resolve the thiopental peak from those of extraneous materials. Several clean-up procedures were investigated, including washing the organic extract with 5% aqueous sodium bicarbonate to remove the more strongly acid contaminants (18). This helped somewhat, but was still not satisfactory . It was then decided to investigate the use of an alkali flame ionization detector (AFID) to learn whether its selectivity for nitrogenous compounds would reduce the apparent background. As hoped, the sensitivity of the AFID for thiopental was about the same as the HFID, but the extraneous background peaks diminished tremendously. The background was now so low that it was possible to extract 5 ml of plasma with an organic solvent, evaporate the organic phase to dryness, reconstitute to 100 p1 with methanol and inject 4-pl aliquots a t a sensitivity of 16 x A/mV with tolerably low backgrounds. Under these conditions, it was possible to detect thiopental a t levels as low as 50 ng per ml of plasma. The low backgrounds also eliminated the need to remove high boiling materials from the column by programming the oven to a high temperature and cooling back to operating temperature after each injection ( 1 9 ) .
EXPERIMENTAL The instrument used for this work was a Varian Aerograph Model 2100, equipped with a n alkali flame ionization detector. The column used was a borosilicate glass U-tube, 5 f t long and %-in. o.d., packed with 3% Poly A-103 on 80/100 mesh Gas Chrom Q. T h e temperatures in the injection port, column, and oven, were all 220 "C. The flow rates were a s follows: carrier gas, nitrogen, 45 ml per min; detector gas, hydrogen. 33 ml per min; and air, 195 ml per min. All injections were made with a micro syringe, using a n injection volume of approximately 4 pl. The instrument was operated a t a sensitivity of 16-256 X A/mV. To 5 ml of plasma in a 20-ml screw capped test tube were added 1 ml of 1M tartaric acid, 1 ml of distilled water or 10 pg per ml aqueous thiopental standard, and 10 ml of reagent grade benzene. The tube was then capped, gently shaken about 5 min, and centrifuged for 15 min at 1500 g. An 8-ml aliquot of the upper organic phase was transferred to a conical centrifuge tube and evaporated to dryness a t 50 "C under a stream of anhydrous nitrogen. The residue was reconstituted to 100 pl with absolute methanol, and a 4-pl aliquot injected into the chromatograph. Calculations were made on the basis of comparisons of sample peak heights with those of a 2 pg per ml plasma standard carried through the procedure.
RESULTS AND DISCUSSION The procedure outlined in the Experimental portion above, describes sample, reagent, and injection volumes that were found to give high sensitivity for thiopental. The optimum amount of drug to be injected was felt to be about 300 ng on column. Thus, if one were dealing with very small plasma samples, high concentrations of drug, etc., reagent volumes and/or size of sample injection could be readily changed. When diethyl ether was used to extract blank plasma Smith. L. W . Dittert. W 0. Griffen, and J . T . Dolucio, J. Pharmacokinetics Biopharrnaceutics. 1 , 5 (1973) R . C . Driscoll, F. S. Barr, B J Gragg. and G . W . Moore, J . Pharm S O . 60, 1492 (1971)
(18) R . E. (19)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, M A Y 1974
753
-
0
2
4
6
TIME (Minuter)
Figure 1. Elution pattern on a Poly A-103 column of sample prepared from 0.5 ml of dog plasma found to contain about 5 pg per ml of thipental. Electrometer attenuation = 256 X l o - ” AFS
samples to which thiopental had been added a t levels between 0.1 and 10 pg per ml, anomalous results were obtained. Recoveries of about 20-30% were realized, and in each chromatogram an unidentified peak appeared, the height of which was approximately proportional to the amount of thiopental present in the original plasma sample. This peak had the same retention time as pentobarbital. I t was suspected that peroxide present in the ether might be reacting with the thiopental to produce an artifact. To test this hypothesis, three identical plasma samples were prepared, which contained thiopental a t a concentration of about 2 pg per ml. Nothing more was added to the first two, but to the third was added a few drops of 30% hydrogen peroxide. These three samples were extracted with diethyl ether, benzene, and benzene, respectively. As expected, the first and third samples contained large amounts of the unknown material, with appropriate losses of thiopental. The chromatogram from the second sample indicated none of the unknown, and quantitative recovery of thiopental. The use of benzene as the extraction solvent obviated this problem, and no further work was done on the question. Figure 1 shows a chromatogram obtained using blood from a dog which received a 15 mg per kg intravenous dose of thiopental. The tracing represents a concentration of about 5 pg thiopental per ml of plasma and required less than 1 ml of plasma. Figure 2 shows chromatograms obtained from a 5-ml human plasma blank and a 5-ml human plasma sample to which thiopental had been added to a level of 100 ng per ml. Previous reports in the literature describe determinations at levels of 15 pg per ml ( 6 ) , 2.5 pg per ml (20), 8 pg per ml (21),etc. The proposed procedure allows one to analyze large plasma samples t o levels as low as 50 ng per ml. Such sensitivity allows one to analyze large plasma samples for low levels of drugs, or to use smaller samples. Twelve 5-ml plasma samples were prepared, four each a t concentrations of 0.4, 1.0, and 5.0 pg per ml. These were analyzed over a three-day period. Recoveries were 98.9 f 6.970, 100.2 f 4.270,and 98.9 4.0%, respectively. (20) G W Stevenson, Ana/ Chem , 33, 1375 (1961) (21) M W Anders, A n a l Chem 38, 1945 (1966)
754
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, M A Y 1974
TIME (Minutes)
Figure 2. Elution patterns on a Poly A-103 column of samples prepared from 5.0 ml of blank human plasma (left) and 5.0 ml
of human plasma to which 0.5 wg of thiopental had been added (right). Electrometer attenuation = 16 X l o - ’ * AFS Table I. Data for Determination of Linearity of Plasma Thiopental Determination Theoretical plasma thiopental concn, pg/ml
Thiopental peak height, arbitrary units
0 .o 0.1 0.2 0.4 1.o 2 .o 10 .o
21 40 82 188 374 1848
0
Recalculated plasma thiopental concn, pg/ml
Per cent of theory
-0.02
0.09 0.20 0.42 1.oo 2.01 10 .oo
90:0% 100 .O% 105 .O% 100 .O% 100.5% 100 .O%
The linearity of the procedure was determined by preparing a series of 8 plasma standards, ranging in concentration from 0 to 10 pg thiopental per ml of plasma. These were carried through the analytical procedure, and concentrations determined by comparing the peak heights to those of methanolic thiopental standards. A least squares best fit was then calculated for a plot of each peak height us. the corresponding plasma thiopental concentration. The peak height was plotted on the Y axis, and the plasma drug concentration on the X axis. The equation of the 3.67. Each of the peak resulting line was Y = 184.5X heights used to calculate the equation of the line was inserted back into the equation, and concentrations were calculated. This gave an additional indication of precision, and a numerical idea of the linearity of the line. Results are summarized in Table I. The use of an internal standard was briefly investigated. Amobarbital proved an excellent choice, being eluted from the Poly A-103 column before thiopental, and being completely resolved from it. It was felt though that for our work the use of an external standard would afford adequate precision and accuracy, while simplifying the procedure. Another drawback to the use of an internal standard was the additional record-keeping necessitated by the use of a second controlled drug substance. This approach would obviously be available though to an investigator who desired additional precision and accuracy. It is interesting to note that the sensitivities of the AFID to thiopental, secobarbital, amobarbital, and pento-
+
barbital were virtually identical to the sensitivities of the HFID to the same compounds. Nevertheless, both detectors appeared to be about half as sensitive to thiopental as to the other three barbiturates on h weight basis. In summary, the proposed procedure was more reliable,
faster, and more precise than others previously reported in the literature, and presents possibilities of investigation with pediatric patients. Received for review August 30, 1973. Accepted January 11,1974.
Modified Electrolytic Conductivity Detector Cell for Gas Chromatography James F. Lawrence and Alan H. Moore Food Research Laboratories, Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario K 1 A OL2
The adaption of electrolytic conductivity to the detection and quantitation of nitrogen-, chlorine- or sulfur-containing compounds in the effluents from gas chromatographs was first accomplished by Coulson ( I , 2). Patchett ( 3 ) incorporated a number of refinements into the system which increased the sensitivity of detection to 0.1 ng of organic nitrogen. Cochrane and Wilson (4, Cochrane, Wilson, and Greenhalgh ( 5 ) , Cochrane and Greenhalgh (6), and Laski and Watts ( 7 ) have examined the response of the Coulson conductivity detector (CCD) (Tracor Inc.) to a wide variety of compounds. The comparison of the CCD to E.C. (8, 9), alkali flame ( I O , I I ) , and flame photometric (sulfur mode) (6) detectors has been carried out. Although the CCD was less sensitive, its extreme selectivity allowed a much greater freedom from interferences which made it very suitable for pesticide residue analysis. The response characteristics of the CCD in the pyrolytic and oxidative modes have recently been investigated (5, 6). The detector response increased with pyrolysis furnace temperature. Oxygen flow caused an initial increase in sensitivity followed by a gradual decrease at higher flow rates. The effects of furnace temperature and hydrogen flow rates in the reductive (nitrogen) mode has recently been examined (12). They were found to be similar to that obtained in the pyrolytic and oxidative modes. The flow rate of water through the CCD system also influenced detector response ( 2 2 ) . By having the gas-water contact area flow rate equivalent to the detector cell flow rate, sensitivity was increased 2 - to %fold for the CCD (12). Equal flow rates were used by Jones and Nickless (23) who described an electrolytic conductivity detector system in which dilute HC1 was used for conductivity measurements instead of deionized water. The overall sensitivity of their system was less for nitrogen-containing compounds than that obtained with the CCD. (1) (2) (3) (4) (5)
D. M . Coulson, J. D. M Coulson. J. G . G . Patchett, J.
GasChromatogr., 3, 134 (1965). Gas Chromafogr., 5 , 285 (1966). Chromafogr. Sci., 8, 155 (1970) W . P. Cochrane and 8. P. Wiison, J. Chromatogr., 63, 364 (1971). W. P. Cochrane, B. P. Wilson, and R. Greenhalgh. J . Chromatogr.,
75, 207 (1973).
(6) W. P. Cochrane and R . Greenhalgh, Intern. J . Environ. Anal.
Chem., in press, 1973. (7) R. R . Laski and R. R. Watts, J. Ass. Otfic. Ana/. Chem., 56, 328 (1973). (8) D. M . Coulson, J. E. DeVries. and B. J. Walther, J. Agr. Food Chem., 8, 399 (1960). (9) R. Purkayastha and W . P. Cochrane, J. Agr. Food Chem., 21, 93 (1973). (10) R. Greenhalgh and W. P. Cochrane, J. Chromatogr.. 70, 37 (1972). (11) J. F. Palframan, J. McNab. and N . T. Crosby, J. Chromatogr., 76, 307 (1973). (12) J. F. Lawrence, J. Chromatogr., 86, 333 (1973) (13) P. Jonesand G . Nickless, J. Chromatogr., 73, 19 (1972).
The present report describes the effort to further increase sensitivity of electrolytic conductivity detection for use with gas chromatography. For this purpose, a new cell was constructed, evaluated and compared to the Coulson conductivity cell (Both cells are covered by U.S. Patent No. 3,309,845, March 21, 1967).
EXPERIMENTAL Apparatus. An Aerograph HI-FY Model 600-C gas chromatograph fitted with a Coulson conductivity detector system (Tracor Inc., Austin, Texas) was used. The 6-ft X 6-mm 0.d. glass column was packed with 4% SE30 on 80jlOO mesh Chromosorb W/HP. Operating conditions were: column temperature, 185 "C; transfer unit temperature, 210 "C; pyrolysis furnace temperature, 780 "C; helium carrier, 60 ml/min; helium sweep, 60 ml/min; hydrogen, 50 ml/min; dc bridge potential, 30 V. A 1-cm plug of strontium hydroxide coated glass wool was placed in the end of the quartz pyrolysis tube. A 2-cm flattened nickel wire coil was inserted 2 inches from the end of the pyrolysis tube. A 1.0-mV strip-chart recorder operating a t 0.25 in./min was employed. Peak height was used to measure detector response. The herbicide, atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) a t a concentration of 1 pg/ml in hexane was used as the test compound. Temperature control was obtained with a constant temperature bath and circulator (Forma Scientific, Marietta, Ohio). The modified cell is depicted in Figure 1. The overall length was 5% inches. The water-jacket was constructed from 13h-inch diameter glass tubing. The dimensions of the glass capillary tubing were 0.5-mm diameter for the water entrance to the gas-water contact area and 1.0-mm diameter for the gas-water contact area and the cell arm. The electrodes consisted of platinum wire approximately 1.0 cm apart. A ball and socket joint was used to connect the effluent gas entrance to the pyrolysis tube. The pyrolysis tube, furnace, bridge circuitry, pumping system. ion exchange resin, and water were the same for both the modified cell and the Coulson cell. Tygon was used for all connective tubing. Water flow through the gas-water contact area of the modified cell was controlled by means of an adjustable screw-clamp (see Figure 1). The cell flow rate was controlled by siphon action. Both flows were equivalent so that no water escaped through the vent tube. The Coulson cell was run as outlined in the Tracor instrument manual but with the reservoir water level maintained 5'2 inch above the pump entrance. Temperature of the water was varied by inserting a 14-turn glass cooling coil, constructed from Y4-inch glass tubing, into the reservoir and connecting to the temperature bath and circulator. Sample Extraction. The extraction of atrazine spiked in potatoes (0.1 ppm) was carried out using 50 grams of sample and extracting with 250 ml benzene:acetone:lN sulfuric acid (190:10:4). The cleanup technique employed consisted of the cold bath precipitation method described by McLeod and Wales (14). The cleanedup extracts were dissolved in hexane for gas chromatography.
(14) H. A .
McLeod and P. Wales, J. Agr. FoodChem., 20, 624
(1972)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
755
