Table I. Retention Volumes for Selected Sulfur Compounds Compound Retention volume, m W b
cos
385
HzS
507 1761
sc12 CH3SH
2253
so2
2477 3429 3780 7425 9347
CH3CHzSH
cs2
CHzSCHz CC13SCI Thiophene
19560
Carrier (He): 60 ml/min, column temperature 22 “C. Retention volumes reported are relative to air which appears at 300 ml after injection. The precision of thesevalues is & l o % .
mixture a t liquid nitrogen temperature. Recovery of the sulfur-containing components was excellent. With the detection system employed, however, it was not possible to study the recovery of these gases a t levels below 100 ppm in the air. Recent reports showing a method for analysis in the range below 1 ppm have appeared and indicate that a nonmetallic system is important at these low concentrations (15, 16). Presumably a system of this type can be constructed utilizing the switching technique described here. TIME ( m i n t
Figure 2. Chromatogram of sulfur compound mixture analyzed on switching system with helium carrier flow, 60 ml/min; temperature, 22 “C. Switching occurred after 15 min (arrow) (1) Air, (2) carbonyl sulfide, (3) hydrogen sulfide, (4) methyl mercaptan,
(5)sulfur dioxide, (6) ethyl mercaptan, (7) ethylene sulfide
Received for review February 7, 1973. Accepted April 23, 1973. The authors acknowledge support of this research by NATO under Research Grant No. 592 and by the United States AEC under Contract No. AT (11-1)-2190. LK would also like to acknowledge the Phillips Petroleum Company for graduate fellowship support. LDS is a Camille and Henry Dreyfus Teacher-Scholar (1971-76).
Determination of Dicyclomine in Plasma by Gas Chromatography Peter J. Meffin, George Moore, and Jack Thomas Department of Pharmacy, University of Sydney, Sydney, N. S. W. 2006, Australia A method was required to determine plasma concentrations of dicyclomine (P-diethylaminoethyl-l-cyclohexylcyclohexanecarboxylate hydrochloride) following its administration at normal therapeutic dose levels to human subjects in order to carry out bioavailability studies on different dose forms of dicyclomine. The oral dosage contemplated was either 20 mg in a “normal” tablet formulation or 40 mg in a sustained release tablet formulation. A previous report ( I ) indicated that maximum plasma concentrations of dicyclomine following oral administration of 30 mg to adult volunteers was in the order of 30 X g/ml. This value was obtained using 14C-labeled dicyclomine and was an estimate based on counts/min/ml of plasma. A method of analysis of dicyclomine was required, therefore, which was sensitive and quantitative to 1-2 x 10-9 g/ml of plasma. Since it was important to be able to discriminate between dicyclomine and its metabolites, a technique based on total radioactive counting ( 1 ) I . E. Danhof, E. C. Schreiber, D. S. Wiggans, and H . M. Leyland, Toxicoi. Appi. Pharrnacoi., 13, 16 (1968).
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methods was not satisfactory. It was also considered to be undesirable to administer radioactive drug to subjects for a routine analytical procedure. Further, multiple blood sampling would be required during the course of bioavailability experiments; hence, the volume of blood required for each determination had to be kept to a minimum, preferably 10 ml or less. A method is described which satisfies the above criteria. A gas chromatographic procedure is described which uses a nitrogen sensitive flame detector and a novel method of concentrating the extracted sample of dicyclomine prior to injection into the gas chromatograph. EXPERIMENTAL Equipment. A Hewlett-Packard 5750 series gas chromatograph fitted with a nitrogen sensitive flame detector (Hewlett-Packard, model 15161A)was used. Gas Chromatograph Conditions. The column was glass Ys-in. 0.d. and 5 feet long, packed with 3% O.V. 225 on Gas Chrom Q (100-120mesh). Column efficiency was 600 plates per foot. It was important to ensure that the column was well conditioned since
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
the liquid phase was cyanopropylsilicone which would produce a large response by the nitrogen sensitive detector. The temperature of the oven was 180 "C, the detector 380 "C, the inlet port 250 "C; the carrier gas was helium a t a flow rate of 60-70 ml/minute, and the air flow rate to the detector was 130-150 ml/minute. The internal standard was cyclizine hydrochloride solution (100 X 10-9 g p . 1 ml). Retention times under these conditions were dicyclomine 2.8 minutes and cyclizine 3.4 minutes. These retention times were maintained by making slight adjustments t o the oven temperature as required, to compensate for the changes in carrier gas flow rate which were periodically made to optimize the detector response. Detector. Adjustments of the various parameters involved with the nitrogen sensitive flame detector were extremely critical, and the only method to obtain the sensitivity and selectivity required was practical experience with the particular instrument. The four variables which determine the sensitivity and selectivity of the detector are the height of the collector electrode above the flame, the hydrogen, air, and carrier gas flow rates. The following recommendations for optimizing the performance of the detector are based on experience gained in the quantitative estimation of dicyclomine. They are quite different from the manufacturers recommendations. The position of the collector electrode above the flame is controlled by an ungraduated adjustment screw. The distance between the electrode and the flame was critical and had a profound influence on the response of the detector. The optimum position of the collector electrode for the application described in this paper was arrived a t by the following procedure. The electrode was set a t its highest point. The hydrogen flow rate was adjusted to give a low standing current and the back off controls were set to practically zero. This overcame the problems of baseline drift at low attenuation and negative peaks immediately following the solvent peak which occurred when the standing current was high. A solution of dicyclomine in ether (10 ng/lO pl) was injected into the gas chromatograph (10 pl) and the detector response noted. The collector electrode was moved along its complete traverse in small increments by means of the adjusting screw and a t each position the above procedure was repeated. At high collector electrode settings, there was low sensitivity and selectivity, whereas a t low collector electrode settings, the noise to signal ratio was unacceptably high. Selectivity was assessed by the relative heights of the solvent and dicyclomine peaks. The optimum setting of the collector electrode and hydrogen flow rate was t h a t a t which the noise to signal ratio was acceptable and the sensitivity was such that more than 0.75 deflection of the recorder was produced by 10 ng of dicyclomine. The sensitivity was such that the solvent peak did not intrude onto the dicyclomine peak. Further minor adjustments to carrier gas and air flow rates had minor effects but were made to produce optimum sensitivity and selectivity. The hydrogen flow rate was most critical and it was necessary to control it by means of a high quality differential flow valve. Deterioration of Detector Response. Decreases in the sensitivity of the detector of the order of 30% were observed after continuous operation for several weeks. There was no significant difference in calibration curves established a t the original or decreased sensitivity of the detector. Minor adjustments to the hydrogen and air flow rates were made which returned the detector to its original sensitivity. The response of the detector was monitored regularly by injecting a standard solution of dicyclomine, cyclizine (internal standard for the assay), and a hydrocarbon (CWJ dissolved in cyclohexane, into the gas chromatograph. Peaks heights gave information about the sensitivity and selectivity of the detector. Glassware. All glassware, with the exception of the heparinized blood collection tubes, was cleaned with chromic acid mixture, silylated with Siliclad (Clay Adams, Parsippany N.J.) and washed with distilled water. Extraction of Dicyclomine from Plasma. Blood samples were collected in 10-ml tubes containing heparin (100 units) and were centrifuged at 3000 rpm for 5 minutes as soon as possible after collection. Plasma (1 to 5 ml) was transferred to a glass stoppered centrifuge tube (15 ml). Sodium hydroxide solution (0.5 ml, lOM), cyclizine hydrochloride solution (100 X 10-9 g/O.1 ml) and ether (5 ml, anaesthetic grade recently distilled) were added to the plasma; the tube was agitated for 1 minute on a Vortex mixer. The tube was placed in a dry ice/acetone bath to freeze the plasma, and the ether was transferred to a stoppered centrifuge tube ( 5 ml) which contained hydrochloric acid (0.2 ml, 1M).
n
j/ Figure 1. Hypodermic syringe with spacer fitted as used for the
final extraction of dicyclomine from aqueous to ethereal solution
The contents of the tube were mixed, centrifuged, frozen, and separated as described above and the ether phase was discarded. The last traces of ether were removed by placing the tube in a water bath maintained a t 40 "C. The aqueous solution which remained was drawn up into a 1-ml, all-glass hypodermic syringe, the syringe was inverted, the needle removed, and a rubber spacer fitted (Figure 1) to the plunger. Sodium hydroxide solution (0.05 ml, 10M) and ether (0.05 ml) were added by means of all-glass hypodermic syringes which were fitted with needles which could pass into the capillary a t the top end of the inverted extraction syringe. The plunger was withdrawn to the 1-ml mark and held in that position while the syringe was agitated on a Vortex mixer for 1 minute. The plunger was then advanced as much as the rubber spacer would allow and the syringe centrifuged a t 1000 rpm for 2 minutes to separate the two phases. The rubber spacer prevented the plunger from being pushed home by centrifugal force during centrifugation. The rubber spacer was removed and the syringe was placed in a holder so designed that the syringe was held vertically and inverted and the plunger could be advanced by a fine screw. The micrometer section of a n Agla syringe unit (Burroughs Wellcome & Co.) held in a laboratory stand, was found suitable. The plunger was advanced until the ether layer was pushed into the capillary at the end of the syringe barrel. Ether (10 pl) was then withdrawn into a suitable syringe and injected into the gas chromatograph.
RESULTS A calibration curve was established by plotting peak height ratio of dicyclomine hydrochloride to cyclizine hydrochloride as internal standard against the concentration of dicyclomine hydrochloride added to drug-free plasma. Five-milliliter samples of plasma were used containing 10 to 180 x g of dicyclomine hydrochloride and 13 concentrations of dicyclomine were examined. The regression equation for the curve was y = 0.0094~+ 0.0025 with r = 0.9365. A typical curve of plasma concentration of dicyclomine against time after administration of an oral dose is given in Figure 2.
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TIME(hr)
Plasma concentration time curves obtained in a human subject following the oral administration of dicyclomine Figure 2.
(0)40 mg dicyclomine in a sustained release tablet. ).( mine in 2 X 10 mg normal tablets
20 mg dicyclo-
DISCUSSION The problem of developing an analytical procedure capable of estimating dicyclomine in plasma in nanogram quantities fell into two parts. The first part was the extraction of dicyclomine from plasma and subsequent concentration of the extract prior to injection into the gas chromatograph. The second part was to separate dicyclomine from possible metabolites in the gas chromatograph and then provide a detector system with adequate sensitivity and linearity of response over the concentration range anticipated. Dicyclomine is a base and is readily separated from plasma by appropriate adjustment of pH and extraction with ether. However extraction of nanogram quantities of dicyclomine from plasma and concentration by slow evaporation of the ethereal solution on a water bath (40 "C) prior to injection into the gas chromatograph did not yield reproducible results. This was presumably due to losses during the evaporation process either by volatilization or adsorption of dicyclomine onto the glass surfaces involved. Silylation of the glassware improved this but not sufficiently for quantitative work. Attempts were made to modify the concentration procedure by extracting the dicyclomine into a small volume of an organic solvent ( e . g . , carbon disulfide, methylene dichloride, etc.) which was heavier than water and from which samples could easily be obtained by centrifugation of the mixture to collect the organic phase a t the bottom of a tapered tube. This approach was unsatisfactory because an appropriate solvent could not be found. The characteristics of the solvent required were that dicyclomine should partition into it readily and that it should produce a low response in the nitrogen sensitive detector. Solvents containing hetero
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atoms such as sulfur and the halogens produced too large a response in the detector (2) for a satisfactory assay procedure. Extraction of a large volume of aqueous solution with a small volume of ether in a conventional centrifuge tube was unsatisfactory because of surface tension effects with a relatively large area of contact between water and ether. Extraction of aqueous solutions with ether in tubes of small diameter did not provide adequate mixing of the two phases. These problems of extraction and concentration were overcome by use of the equipment as described in the Experimental section. Three different experimental techniques were examined in an attempt to provide a system which gave adequate detector response to dicyclomine which was required. A flame ionization detector was limited by the fact that the solvent peak was so large on the low attenuation required to provide adequate sensitivity that the peak heights of dicyclomine and internal standard could not be accurately measured. This problem could be overcome to some extent by exchanging the ether for carbon disulfide prior to injection into the gas chromatograph. This was accomplished by adding carbon disulfide to the ethereal solution and evaporating the ether off at 40 "C. The method was not satisfactory because of losses of dicyclomine during the evaporation step and the difficulty in removing all traces of ether from the carbon disulfide. A second approach was to attempt to hydrolyze dicyclomine to its constituent acid, l-cyclohexylcyclohexanecarboxylic acid, and alcohol, 2-diethylaminoethanol, and then form a derivative of either of these which could be used with an electron capture detector. The ester group is quite stable and attempts a t quantitative hydrolysis on submicrogram quantities were unsuccessful. The third approach which proved to be the one of choice was to use an ethereal solution of dicyclomine for gas chromatography in combination with a nitrogen selective flame detector. By suitable adjustments of the parameters which control the selectivity and sensitivity of this detector, it was possible to obtain the sensitivity required. A combination of the use of the nitrogen selective flame detector together with a concentrating technique of dicyclomine which did not involve a n evaporation procedure provided adequate sensitivity and linearity of response necessary for quantitative estimation of dicyclomine in plasma at concentrations of 1-2 x 10-9 g/ml of plasma. Received for review October 25, 1972. Accepted February 20, 1973. (2) W . A. Aue, K. 0. Gerhardt. and S. Lakota. J. Chromatogr.. 63, 237 (1971).
S E P T E M B E R 1973
