(9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
J. W. Russell, Environ. Sci. Technol., 9, 1175 (1975). J. S. Parsons and S. Mitzner, Environ. Sci. Technol., 9, 1053 (1975). D. Ellgehausen, Anal. Lett., 8, 11 (1975). M. Novotny, M. L. Lee, and K. D. Bartle, Chromatographia, 7, 333 (1974). A. Zlatkis, H. A. Lichtenstein, and A. Tishbee. Chromatographia, 6 , 67 (1973). D. Schuetzle, A. L. Crittenden, and R . J. Charlson, J. Air follut. Control Assoc., 23, 704 (1973). P. W. Jones, "Analysis of Nonparticulate Organic Compounds in Ambient Atmospheres", 67th Air Pollution Control Association Meeting, Denver, Colo.. Paper No. 74-265, June 1973. K. Grob and G. Grob, J. Chromatogr., 62, 1 (1971). W. Bertsch, R. C. Chang, and A. Zlatkis, J. Chromatogr. Sci., 12, 175 (1974). A. Raymond and G. Guiochon, Environ. Sci. Techno/., 8, 143 (1974). E. D. Pellizzari, "Development of Analytical Techniques for Measuring Ambient Atmospheric Carcinogenic Vapors", Publication No. EPA60012-75-076, Contract No. 68-02-1228, November 1975, p 187. E. D. Pellizzari, "Development of Method for Carcinogenic Vapor Anal-
(21) (22) (23) (24) (25) (26) (27) (28)
ysis in Ambient Atmospheres", Publication No. EPA-650/2-74-12 1, Contract No. 68-02-1228, July 1974, p 148. Eight Peak Index of Mass Spectra, Vol. I (Tables 1 and 2) and II (Table 3), Mass Spectrometry Data Centre, AWRE, Aldermaston, Reading, RG74PR, UK, 1970. J. L. Cresch, Jr., and M. N. Johnson, J. Occup. Med., 16, 150 (1974). I. R . Tabershaw and W. R. Garrey, J. Occup. Med., 16, 509 (1974). R . R . Monson. J. M. Peterson, and M. N. Johnson, Lancet, 397 (1974). H. J. Martsteller, W. K. Lelbach. R . Muller, and P. Gedigke, presented at the Working Group, Toxicity of Vinyl Chloride-Polyvinyl Chloride, New York Academy of Sciences, New York, May 10-11, 1974. Chem. Eng. News. 53,41 (May 19, 1975). Chem. Eng. News, 53,6 (May 5 , 1975). R. West and E. Carberry. Science, 189, 179 (1975).
RECEIVEDfor review September 22, 1975. Accepted February 5, 1976. Research supported by EPA Contract No. 6802-1228 from the Environmental Protection Agency, Health, Education, and Welfare.
Determination of Debrisoquin and Its 4-Hydroxy Metabolite in Plasma by Gas Chromatography/Mass Spectrometry Susan L. Malcolm and Timothy R. Marten* Department of Biochemistry, Roche Products Ltd, Broadwater Road, Welwyn Garden City, Herts. England
An analysis of debrlsoquin and its 4-hydroxy metabolite In plasma has been developed. After derivatization with hexafluoroacetylacetone, samples, containing deuterated internal standard, were examined by gas chromatography/mass spectrometry. Linear responses for the drug and metabolite to 1 ng/ml arld 5 ng/ml plasma, respectively, were observed. The method has been applied to the measurement of plasma levels after therapeutic dosing.
Metabolic studies ( I ) on the anti-hypertensive drug, debrisoquin sulfate (Ro 05-3307) (I), have shown that the major metabolite is the 4-hydroxy compound (11) (Ro 037594). This compound has also been shown to exhibit some hypotensive activity in anaesthetized cats (I. L. Natoff and T. C. Hamilton, personal communication), and it is therefore desirable that an analytical method for debrisoquin in blood should also be capable of measuring the metabolite. Two methods for the determination of debrisoquin in blood have been described, but neither is suitable for the estimation of the metabolite. The method of Medina et al. (2) involves the direct extraction of the drug a t high pH and measurement of the fluorescence of the ninhydrin derivative. The sensitivity of the method which is only 50 ng/ml plasma, is not sufficient to measure the levels found in patients receiving therapeutic doses of the drug. It also suffers from the disadvantage that the 4-hydroxy metabolite cannot be extracted quantitatively into organic solvents at any pH ( 1 ) . The alternative method ( 3 ) relies on the hydrolysis of the amidino group with strong base, followed by gas chromatography with an electron capture detector or gas chromatography/mass spectrometry (GC/MS) of the derivatized tetrahydroisoquinoline. However, under the hydrolysis conditions employed, the 4-hydroxy compound is unstable. The method described here is based on the extraction procedure ( I ) used to isolate metabolites from biological samples. By condensing the amidino group with acetylace-
tone to form a pyrimidino compound, the polarity was reduced sufficiently for the drug and metabolites to be extracted into organic solvents. The use of hexafluoroacetylacetone in a two-phase reaction mixture as described by Erdtmansky and Goehl ( 4 ) made it possible to analyze low levels of debrisoquin and its 4-hydroxy metabolite by monitoring for single ions characteristic of the bis(triflu0romethy1)pyrimidines (111) and (IV) on a GC/MS system, using derivatized decadeuteriodebrisoquin (V) as an internal standard. In all cases, the monitored peaks corresponded to the base peaks in the mass spectra of the individual compounds (Figure 1). The method has been successfully applied to the measurement of drug and metabolite levels in plasma taken from a patient treated with a single dose of debrisoquin sulfate. R
&N-CZ"
R
"2
I
R = H
111
R = H
11
R
=
IV
R=OH
v
o,olll
OH
EXPERIMENTAL General. A F i n n i g a n 1015D gas chromatographiquadrupole mass spectrometer f i t t e d w i t h a glass c o l u m n (1.5 m, i.d. 2 m m ) packed w i t h 3% OV-17 o n Gas C h r o m Q was used. H e l i u m was used as carrier gas (flow rate 20 m l / m i n ) a n d t h e oven was p r o grammed f r o m 150-190 O C a t 4 " l m i n . A single stage glass j e t separ a t o r a t 210 "C was employed. T h e m a n i f o l d temperature was 60 O C a n d t h e electron voltage, 70 eV. I o n currents a t m / e 344, 347, a n d 356 (see Figure 1) were m o n i t o r e d w i t h a 4-channel automatic peak selector w i t h a channel w i d t h o f 0.5 a m u a n d a sampling t i m e o f 10 mdchannel. T h e integrated signals were o u t p u t o n t o a rnultichannel recorder. T h e derivatives were characterized o n a Varian CH7 mass spectrometer (Figure 1). ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, M A Y 1976
807
%
I
anlU
Figure l a . Mass spectrum of the hexafluoroacetylacetone derivative
t
of debrisoquin (Ill) % 100
'I
t
344
I,
2 100
200
400
300
amu
Figure lb. Mass spectrum of the hexafluoroacetylacetone derivative
4
6
8
1 0 1 2 hrs after dose-
24
Figure 2. Levels of debrisoquin ( 0 )and 4-hydroxydebrisoquin (0)in plasma after a single oral dose of the drug (20 mg)
of 4-hydroxy debrisoquin (IV)
All glassware was washed in boiling 50% nitric acid and thoroughly rinsed with distilled de-ionized water. All solvents were distilled prior to use. Hexafluoroacetylacetone was obtained from Fluorochem (Glossop, Derbyshire) and was stored under nitrogen. Preparation of Dlo-debrisoquin Sulfate for Use as Internal Standard. Isoquinoline (2 g) was suspended in deuterium oxide (24 ml) and heated in a sealed steel calorimeter at 200 "C in the presence of 5% platinized asbestos for 72 h ( 5 ) .The cooled solution was Ciltered and the asbestos thoroughly rinsed with ether. The aqueous filtrate was extracted three times with ether and the combined organic layers were dried and evaporated. The above procedure was then repeated using fresh deuterium oxide and catalyst. T h e isolated isoquinoline (600 mg) was dissolved in Dd-acetic acid (10 ml) and reduced with deuterium in the presence of Adam's catalyst (0.3 9). After 2.5 h, uptake of gas ceased (180 ml; theoretical 208 ml) and the mixture was filtered to give a clear solution. After adjusting to pH 10 with 10 N sodium hydroxide, the solution was extracted three times with ether. Evaporation of the dried solution gave a pale yellow oil (350 mg) (calcd for CgDloN: M+ 142; found, M+ 142, 14%). The oil was dissolved in water (2 ml) and S-methyl isothiourea (450 mg) was added (6). After stirring for 18 h a t room temperature, methanol was added and the precipitated solid collected by filtration. Crystallization from aqueous acetone afforded the desired salt (180 mg) (calcd for C ~ O D ~ O H ~N ~ :found, M+ 185 M+ 185; (26%), M' - 1 (31.5%) (base peak), M+ - 10 (0.2%)). Preparation of the Pyrimidino Derivatives (111), (IV), and (V). The pyrimidino compounds (III), (IV), and (V) were prepared essentially as described by Erdtmansky and Goehl ( 4 ) . Debrisoquin sulfate (I) (10 mg) in saturated sodium bicarbonate solution (0.5 ml) was heated with a solution of hexafluoroacetylacetone (0.2 ml) in toluene (5 ml) at 100 OC for 2 h. The organic layer was then removed, dried, and evaporated to give a solid (111) which was crystallized from hexane a t 4 "C. Mass spectra of these compounds showed the base peaks to be at mle 347 (III),344 (IV), and 356 (V) (Figure 1). Plasma Derivatization and Analysis. Centrifuge tubes (10 ml) fitted with air condensers were used for the derivatizations. A solution of the Dlo-debrisoquin sulfate internal standard (10 ng) in water (10 p1) and saturated sodium bicarbonate solution (50 p1) 808
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
was added to plasma (100 ~ 1containing ) the drug and metabolite (0.1-10.0 ng). Hexafluoroacetylacetone (50 pl) in toluene (500 pl) was then introduced and the samples were heated a t 100 "C for 2 h. The organic layer was removed with a Pasteur pipet and blown to dryness with a stream of argon. A solution of this material in toluene (7 pl) was then injected onto the GLC. Deuterated standard (10 ng) was added to plasma samples (100 pl), collected from a previously untreated hypertensive patient after receiving an oral dose of debrisoquin sulfate (20 mg). These were then analyzed for the drug and 4-hydroxy metabolite as described above (Figure 2).
RESULTS AND DISCUSSION The biphasic system produced very clean samples, whereas the single phase system which had been employed ( I ) to isolate metabolites gave many interfering peaks a t low levels of the drug. However, when acetylacetone was used in the biphasic system, there were still interfering peaks present, due to the self-condensation of this diketone in base. Thus, the combination of the two-phase reaction and derivatization with the fluorinated diketone was preferred as it afforded a clean, quantitative analysis. Figure 3 shows a typical trace obtained after derivatization of a plasma sample containing debrisoquin sulfate (2 ng), 4-hydroxydebrisoquin sulfate (2 ng), and internal standard (10 ng). A linear response was obtained with good reproducibility as shown in Figure 4 where the results of three separate series of experiments were plotted. The lower limit for quantitative estimation was found to be 1 ng/ml plasma for debrisoquin and 5 ng/ml plasma for the metabolite. Lower levels were detectable but the accuracy was reduced. A set of samples was analyzed using 5 plasma aliquots each containing 2 ng of the drug and the metabolite (i.e., 20 ng/ml of plasma). Good reproducibility was shown by both compounds, giving internal standard peak ratios of 0.39 &
Detector response
I
11
J , ,
1 2.5
I
Debrisoquin
/
f 20. ~ ~ t n t e r n Standard a l
a d
a
15.
70.
Debrisoquin Derivative
-
d Mass 356 1.0 v
I-Hydroxy debrisoquin
4.Hydroxy Debrisoquin Derivative ng ml plasma
I
Figure 4. Plot of the response obtained after derivatization of three
Mass 347 0.5 V
Mass 3 4 4
0.2
v
L 2
4
6
8
sets of plasma samples spiked with debrisoquin and 4-hydroxydebrisoquin sulfates
10 rnin
Figure 3. Mass fragmentographic GLC trace after derivatization of plasma containing debrisoquin sulfate (2 ng), 4-hydroxydebrisoquin sulfate (2ng) and Dlo-debrisoquin sulfate (10 ng)
0.02 (5%) for debrisoquin and 0.11 f. 0.01 (9%) for 4-hydroxydebrisoquin. The lower relative value obtained for the hydroxylated species can be explained by the base peak (mle 344) in the mass spectrum (Figure I b ) being a smaller proportion of the total ion current in this compound than the corresponding peak in the parent molecule. Significant enhancement of the base peak value in 4-hydroxydebrisoquin can be achieved by derivatization of the sample with pentafluoropropionic anhydride prior to injection onto GLC. However, incomplete derivatization was obtained and the lack of absolute reproducibility led us to use underivatized samples. Using this method of analysis, levels of debrisoquin and the 4-hydroxy metabolite were measured in the plasma of patients receiving therapeutic doses of the drug. Figure 2 shows the levels of the drug and metabolite which were
found in plasma after a single oral dose, each point being the average of two determinations. Although levels of 4hydroxydebrisoquin are higher than the parent drug, it is more rapidly eliminated and was not detectable 24 h after administration.
ACKNOWLEDGMENT We are indebted to G. T. Tucker of the Section of Therapeutics, The Royal Infirmary, Sheffield, for supplying the plasma samples and for helpful discussions.
LITERATURE CITED (1) J. G. Allen, P. B. East, R. J. Francis, and J. L. Haigh, Drug Metab. Dispos., 3, 332 (1975). (2) M. A. Medina, A. Giachetti, and P. A. Shore, Biochem. fharm., 18, 891 (1969). (3) J. H. Hengstrnann, F. C. Falkner. J. T. Watson, and J. Oates, Anal. Chem., 48, 34 (1974). (4) P. Erdtrnansky and T. J. Goehl, Anal. Chem., 47, 750 (1975). (5) G. Fischer and M. Puza, Synthesis, 4, 218 (1973). (6) British Patent No. 1,018,381 and 1,057,280.
RECEIVEDfor review September 29, 1975. Accepted December 16,1975.
N,N’- Bis[ p- but oxybenzy Iidene]- a ,a’-bi-p- t o I uidine: T hermaI ly Stable Liquid Crystal for Unique Gas-Liquid Chromatography Separations of Polycyclic Aromatic Hydrocarbons George M. Janini,” Gary M. Muschik, and Walter L. Zielinski, Jr. NCI Frederick Cancer Research Center, Frederick, Md. 2 170 1
This paper is the third in a series dealing with applications of high-temperature nematogenic liquid crystals as stationary phases for gas chromatography. The synthesis and successful utilization of N,N’-bis[pbutoxybenzylldene]-cr,a’bi-p-toluidine (BBBT) in its nematic range ( 188°-3030C) are illustrated for the resolution of geometric isomers of three-five-rlng polycycilc aromatic hydrocarbons (PAH). In comparison with its methoxy homologue, this new liquid crystal achieves comparable separations and exhibits diminished bleed levels at elevated column temperatures over prolonged operating periods. The theoretical plate count and retention time of chrysene have been reproducible to less than 3 % over a period of 150 h of continuous operation at 260 OC on column packings of 2.5% (w/w)
BBBT on 100-120 mesh HP Chromosorb W. Under the same conditions, over the same period of contlnuous operation, a 2.5% packing of the methoxy homologue lost about 16% of its liquid phase loading.
The use of liquid crystals in gas-liquid chromatography (GLC) has been recently reviewed by Schroeder ( 1 ) who cited numerous attempts a t separating benzene positional isomers using nematic, smectic, and cholesteric mesogens as GLC stationary phases. The first practical demonstrations of the usefulness of liquid crystals for GLC separations were presented by Kelker (2, 3) and Dewar and Schroeder ( 4 ) . In a review by Kelker and Von-Schivizhoffen ( 5 ) , a list of 19 GLC-tested liquid crystalline comANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
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