Quantitative Determination of Guanethidine and Other Guanido-Containing Drugs in Biological Fluids by Gas Chromatography with Flame Ionization Detection and Multiple Ion Detection Jurgen H. Hengstmann, Fred C. Falkner, J. Throck Watson, and John Oates School of Medicine, Vanderbilt University, Nashville, Tenn. 37232
A general method for the analysis of the guanido-containing drugs used in the treatment of hypertension is described. This method utilizes extraction for purification of the parent compounds and hydrolysis and trifluoroacetylation of the resulting amines to obtain derivatives with excellent gas chromatographic properties. Detection is achieved by flame ionization (FID) or by multiple ion detection ( M I D ) , and quantification is done by use of an internal standard. Application of the general scheme to guanethidine has shown that biological samples with levels of guanethidine greater than 100 ng/ml can be analyzed easily by GLC and FID. Urine and plasma samples containing 1 to 100 ng/ml are readily amenable to analysis by GLC and M I D .
The treatment of hypertension with guanethidine (1) and other guanido-containing drugs like guanoxan (2), debrisoquin ( 3 ) , and bethanidine (4) is in widespread use.
1
2
enough. Until now, measurement of blood pressure was the only objective parameter available for assessment of drug therapy. The additional monitoring of blood levels of the drug and its urinary excretion during the course of treatment could facilitate adjustment of the maintenance dose as well as elucidate those pharmacokinetic parameters which explain the wide interindividual variation. In the present paper, we describe the isolation and purification of these drugs from biological fluids. Final separation of guanethidine is achieved by gas-liquid chromatography with quantification by flame ionization detection for highlevel samples ( > l o 0 ng/ml) and by multiple ion detection for low-level samples ( LC-FID procedure were necessary because the extraction of guanethidine from plasma according to the scheme for urine presenled unexpected difficulties due to gel formation with result a n t poor recovery. T o five milliliters of plasma are added 500 ng of debrisoquin and 20 ml of diethyl ether. The tube is shaken for 10 minutes and centrifuged. The organic layer is discarded. Then 0.5 ml of 50% NaOH are added and the contents thoroughly mixed on a Vortex-type mixer until a homogeneous suspension results. The subsequent steps are the same as those described for urine except that 20 t o 40 pl of ethyl acetate are used for the final solution.
RESULTS AND DISCUSSION Extraction of biological material with organic solvents a t defined p H values is often used as the first step for isolation and concentration of drugs. Therefore, the partition coefficients into eight different organic solvents were measured (Table I). Chloroform proved to be the most suit-
Figure 2. Extraction of the guanido-containing antihypertensive drugs as a function of pH
Table I. Partition Coefficients of Guanethidine, Guanoxan, Debrisoquin. and Bethanidine in Various Organic Solventsa Chloroform Dichlorornethane Ethylene dichloride Ethyl methyl ketone Ethyl acetate Diethylether Toluene Cyclohexane
Guanethidine
Guanoxan
1.44
0.26
0.64
0.33 0.35
0.32 2.29 0.11 0.08 0.03 0.0
0.12 0.07 0.24 0.02
...
Debrisoquin
0.93 0.91
0.70 0.32 0.03
0.15 0.07 ...
Bethanidine
7.1
6.2 19.6 9.3
0.04
1.6 0.09 ...
uPaftition between 2.5N sodium hydroxide and an equal volume of organic phase.
able solvent for guanethidine and debrisoquin, while ethylene dichloride was best for bethanidine and guanoxan. The curves for the amount of drug extracted a t different p H levels (Figure 2) showed a pK of 11.9 for guanethidine and debrisoquin, 12.3 for guanoxan, and 10.6 for bethanidine with its substituted guanido group. Therefore, the p H had to be adjusted to a t least 13.5 to ensure an efficient extraction into an organic solvent for guanethidine, guanoxan, and debrisoquin, and to 12.2 for bethanidine. The loss of compound during preliminary extraction with toluene a t p H 10 was less than 5%. The back extraction of all the amines into aqueous acid was 95% efficient. The overall recovery (60%) of guanethidine was constant throughout the concentration range of 0.1 to 5.0 Fg/ml of urine. Development of a GLC assay utilizing FID or electron capture detection (ECD) for intact guanethidine as a bis( perfluoroacyl) derivative was unsuccessful. The trifluoroacetyl (TFA) and pentafluoropropionyl (PFP) derivatives were very unstable and decomposed upon removal of the anhydride after 10 min a t room temperature. Additionally, substances which interfered with ECD could not be removed either by adsorption to aluminum oxide or Amberlite XAD-2 or by preliminary extraction with a variety of organic solvents. Therefore, we investigated the optimal conditions for removal of the guanido group by hydrolysis. Hydrolysis in 8% aqueous KOH for 4 hr a t 110 "C gave the best results.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, J A N U A R Y 1974
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0 GUANETHIDINE-GUANOXAN
o
GUANETHIDINE-DEBRISOCIUIN
cnci, cnci,
0 GUANETHIDINE
A GUANOXAN 0 DEBRISOQUIN BETHANIDINE Y
a
Y
n
I
.
'
1
1
2
4
-
6
8
12
18
+
24
TIME ( l i R )
Figure 3. Hydrolysis of the guanido-containing antihypertensive drugs as a function of time
01
Ob
10
20 COIUCENTRATION Iug/mll
5'0
Figure 5. GLC-FI D calibration curve for guanethidine extracted from urine with guanoxan or debrisoquin as internal standard
I
Table I I . Partition Coefficients of N-(2-aminoethyl)octahydroazocinea
0.06p g h l
1.4 p g h l
7.5 7.5 9.5 10.8 14.3 25.2
Partition between 8% aqueous potassium hydroxide and an equal volume of organic phase.
\
;I
Figure 4. Gas-liquid chromatographic traces of N - ( 2 trifluoroacetamidoethy1)octahydroazocine (first peak, from guanethidine) and 2-trifluoroacetyl-l,2,3,4-tetrahydroisoquinoline
(second peak, from debrisoquin). Concentrations for guanethidine in urine are given with each trace The yield of N-(2-aminoethyl)octahydroazocine (OHA, la) from guanethidine was calculated to be 100% by comparing the area of OHA obtained in this manner with the area of an equimolar amount of authentic OHA. Hydrolysis in sodium hydroxide and ammonium hydroxide gave lower yields. The influence of the time of hydrolysis on the yield of amine can be seen in Figure 3. When guanoxan was employed as the internal standard, a period of 15 hr for hydrolysis was necessary, whereas use of debrisoquin as internal standard required only 4 hr. Hydrolysis a t 90 or 140 "C resulted in lower yields, probably because of incomplete hydrolysis a t the lower temperature or decomposition of the compounds a t the higher temperature. To prevent loss of the resulting amines due to decomposition during hydrolysis in the aqueous alkali, an organic solvent was added to the hydrolysis mixture to provide continuous extraction of the amine as it formed. 36
Hexane Heptane Toluene Cyclohexane Benzene Ethylene dichloride
i.5 pg/ml
The amines, in contrast to their guanido-containing parent compounds, were very well extracted into nonpolar organic solvents. Representative partition coefficients are shown for OHA into six different organic solvents (Table 11). Prior to the evaporation of the organic solvent, formic acid was added to prevent loss of these volatile amines. Acyl derivatives of these amines were then easily prepared with trifluoroacetic, perfluoropropionic, or heptafluorobutyric anhydride and ethyl acetate a t room temperature. These derivatives have excellent GLC properties and may be separated by a variety of stationary phases (Table 111). A gas chromatogram of guanethidine with debrisoquin as internal standard processed from urine as described above is shown in Figure 4. The octahydroazocine TFA derivative which elutes first and the tetsahydroisoquinoline TFA derivative which elutes second, maintain their excellent peak shapes even down to the level of 100200 picograms injected on-column. The lack of biological interferences is a tribute to the specificity of the purification scheme. The calibration curve for GLC-FID analysis is shown in Figure 5 . It is linear from 0.1 to 5.0 pg/ml of urine with a correlation coefficient of 0.99. The precision of the method was evaluated by repeated analysis of a patient's urine with a mean value of 0.63 pg of guanethidine per ml of urine. For 11 determinations, the mean of the ratio of guanethidine to internal standard was 0.22 with a relative standard deviation of 4.6%. The specificity of the method was confirmed by 0))taining the mass spectra of the derivatives eluting with appropriate retention times in the GLC analysis of a patient's urine sample. Mass spectra identical with standards were obtained. GLC-MID Analysis. In order to monitor serum levels of guanethidine and to follow urinary excretion kinetics for several weeks after discontinuation of therapy, a more
ANALYTICAL CHEMISTRY, VOL. 46, NO. 1 , J A N U A R Y 1974
Table I l l . Retention Times (min) for Amine Derivatives
N- (2-Arninoethyl)octahydroazocine
OV-101 ( 1 5 0 ° C )
OV-17 (130°C)
4.2 ... ...
3.8 3.2 3.2
4.2
5.1
6.3 1.3
11.8
TFA PFP
HFB TFA TFA TFA
1,2,3,4-Tetrahydroisoquinoline
2-(Arninornethyl)benzo-l,4-dioxane Benzylarnine
-
.
IW
4.6 3.5 3.2 6.7
3.0 (180°C)
1.4
2.3
c
110
P o l y l - 1 1 0 (130°C)
408%
N-CHz+CHZNHTFA lj6j
126
/*
U
183
i
'
U
IW
I
251
250
I50 m I.
I
>
f
Lj
I
'
U z
lb
140
50 2
loo]
150
100
200
210
-I.
Figure 6. Mass spectra of the trifluoroacetyl derivatives of N-(2-aminoethyl)octahydroazocine (top) and 2-aminomethyl-l,4-benzodioxane (bottom)
50
I I
4b
134 69
7V 107
3 D
150
100
200
6
m/.
Figure 7. Mass spectra of the trifluoroacetyl derivatives of benzylamine (top) and 1,2,3,4-tetrahydroisoquinoIine (bottom)
sensitive detection system than FID was needed. The most logical technique to examine first was GLC with ECD. However, the TFA, PFP, and HFB derivatives of the perhydroazocine showed poor ECD response with the detection limit being essentially the same as that for FID. Attention was therefore turned to the technique of multiple ion detection (MID) which is capable of detecting picogram amounts of volatile substances. To study the feasibility of applying MID to the guanethidine assay, it was necessary to examine the mass spectra of OHA derivatives as well as the mass spectra of derivatives which were candidates for internal standard.
The mass spectrum of N-(2-trifluoroacetamidoethyl)octahydroazocine (lb) is shown in Figure 6. The facile alpha cleavage a t the site of the tertiary nitrogen predominates over all fragmentations to yield the abundant ion at m l e 126. There were no other ions above mass 70 with a relative abundance greater than 5%. Other derivatives of OHA, namely, the perfluoropropionyl, heptafluorobutyryl, and 2,4-dinitrophenyl, gave mass spectra which also showed the overwhelming predominance of the ion at m l e 126 and, hence, had no analytical advantages over the TFA derivative. Because the tertiary amine strongly directed fragmentation to form the ion of mass 126, only
ANALYTICAL C H E M I S T R Y , VOL. 46,, NO. 1, J A N U A R Y 1974
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n
J2d
e4
136
1
126 /116 AREA RATIO 0 1261117 AREA RATIO
20
10
20
30
40
50
60
CONCENTRATION ("9 I mll
Figure 9. GLC-MID calibration curve for guanethidine extracted from plasma with debrisoquin as the internal standard 0
IS
1 114 26
117
,
.
.
. .
.
..
Figure 8. Ion current profiles of patient plasma samples analyzed by GLC-MID. The first peak in each trace is due to the ion at m/e 126 (from l b ) . In the upper trace, the second peak is due to m / e 135 (from 2b) and in the lower trace, the second peak corresponds to the ions at m/e 116 and 11 7 (from 3b) single ion detection for guanethidine was possible. Because of instrumental parameters, the range of m / e values for which ions from an internal standard could be monitored was approximately 10% of m / e 126. Hence, the candidates for internal standard (guanoxan, debrisoquin, and bethanidine) were hydrolyzed, and ions within the range 115-139 were sought in the mass spectra of the TFA derivatives of the resulting amines. 2-(Trifluoroacetamidomethyl)benzo-l,4dioxane (2b) fragmented by loss of the C-2 substituent to give the ion a t m / e 135 which was the base peak in the mass spectrum and thus an excellent ion to monitor in conjunction with m / e 126. The ions a t m / e 115, 116, and 117 in the mass spectrum of 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline (3b, Figure 7 ) were all possible candidates for simultaneous monitoring with m / e 126. Although the prominent ions of benzyl trifluoroacetamide (4b) were the molecular ion ( m / e 203), the M - 1 ion ( m / e 202), and the tropylium ion ( m / e 91), an ion a t m / e 134 due to loss of the trifluoromethyl radical was of sufficient abundance to serve as internal standard. Of the three possible choices for internal standard, debrisoquin was the most suitable. The TFA derivative of the hydrolysis product (3a) had an excellent peak shape on the 3% Poly 1-110 phase. Its retention time, as well as that of the OHA-TFA derivative was less than 3 min under the column conditions employed. These short retention times permitted analysis of 25 samples per day assuming 3 injections per sample. With guanoxan as internal standard, the more polar benzodioxane TFA derivative necessitated the use of OV-101 phase. The longer retention time for 2b meant a reduction of one half in the total number of samples which could be analyzed in one 38
day. Debrisoquin also offered another feature not possible with either guanoxan or bethanidine in that two of its fragment ions could be monitored. The ability to monitor two ions, in this case m / e 116 and 117, had two advantages. First, any co-chromatographing impurity with ions a t 116 or 117 could be detected by a change in the peak ratio of 0.70 (116/117). Second, two calibration curves could be prepared, one using the ratio 126/116 and the other 126/117. If for some reason interference occurred on one of the internal standard ion profiles, the other could be used to calculate a ratio and, hence, the concentration of guanethidine (Figure 9). The collection, reduction, and processing of the data was done by means of a display-oriented computer system described by Watson et al. (7). Typical data profiles are shown in Figure 8 which is a photograph of data display taken directly from the oscilloscope of the PDP-12 computer. These result from analysis of patient plasma samples. In the top trace, guanoxan was used as internal standard and, in the bottom trace, the internal standard was debrisoquin. Area calculations were performed with the aid of the computer. A calibration curve for guanethidine in plasma with debrisoquin as internal standard is shown in Figure 9. The curve was linear over the range assayed, and the correlation coefficient was 0.99. The results for urine were similar and showed a correlation coefficient of 0.98. The slopes for the plasma and urine curves were comparable (0.044 us. 0.052), indicating the consistency of extraction from either medium. An absolute recovery of 48% was obtained for plasma in the range of 2-60 ng/ml. The relative standard deviation of the ratios of the areas of the ion profiles was determined to be 4.2% for 10 consecutive GLC-MID analyses of a patient's plasma sample. The precision of the entire GLC-MID method was obtained by analyzing nine plasma samples, each with a concentration of 20 ng/ml. A mean value of 21.8 ng/ml was obtained with a relative standard deviation of 8.6%. Guanethidine and related guanido-containing drugs are extensively used in the treatment of hypertension. Until now, investigations of its pharmacokinetics in hypertensive patients have been severely limited due to a lack of adequate analytical methodology. The availability, specificity, and sensitivity of the method described herein should be valuable in evaluating the physiological disposition of guanethidine. This should aid greatly in the establishment of a rational approach to the drug regimen. (7) J. Throck Watson, D. R. Pelster. B. J. Sweetman, J. C. Frolich, and John A . Oates. Anal. Chem., 45, 2071 (1973).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, JANUARY 1974
ACKNOWLEDGMENT
Received for review May 24, 1973. Accepted July 24, 1973. This work was supported by the Research Center for Clinical Pharmacology and Drug Toxicology (GM-15431). J.H.H. was supported by the Deutsche ForschungsgeWe wish to thank Mrs. Betty Fox for assistance in obmeinschaft. J.T.W. was Recipient of Public Health Service taining the mass spectral data and T. Thompson of CibaGeigy for the sample of N-(2-aminoethyl)octahydroazo- Research Career Development Award for 1973-78 (Nationcine. al Institute of General Medical Sciences)
Application of the Extractive Alkylation Technique to the Gas Chromatographic Determination of Chlorthalidone in Plasma in Nanogram Quantities Magnar Ervik and Klas Gustavii The Research Laboratories of A B Hassle, S-431 20 Molndal, Sweden
The polar molecule chlorthalidone is converted to its tetramethyl derivative by a process called extractive alkylation. This process takes place when the substrate in anionic form is extracted as an ion pair with a quaternary ammonium ion into an organic solvent such as dichloromethane in the presence of methyl iodide. The reaction is very rapid and goes to completion within 20 min when appropriate reaction conditions are used. The derivative formed is very sensitive to electron capture detection and is thus determined by gas chromatography. The method allows determination down to 2 ng/ml of plasma with a relative standard deviation below 6 % .
ammonium ion into an organic solvent-e.g., methylene chloride-containing. the alkylating reagent, an alkylhalogenide. The alkylation reaction takes place very rapidly. Procedures of this kind have been called extractive alkylation. Ehrsson (8) has used this method to convert some aliphatic carboxylic acids to pentafluorobenzyl derivatives, and obtained a quantitative and fast reaction in the millimole range. In the present paper, a gas chromatographic method, based on the extractive procedure, has been developed for the determination of chlorthalidone in the nanogram range.
EXPERIMENTAL There are very few methods published so far on the determination of chlorthalidone in biological materials. Pulver et al. ( 1 ) have used a spectrophotometric method for the determination in urine when studying the elimination of the drug in dogs. The method is not sensitive enough for human blood level determinations. A very sensitive method based on radioactivity measurements has been published by Beizenherz et a / . ( 2 ) . However, this method gives the total content of chlorthalidone and metabolites and the use of radioactive labeled drugs for extended studies in human subjects is very unsuitable. The chlorthalidone molecule, given in Figure 1, contains three polar active groups which make it impossible to determine the drug by direct gas chromatography. The active groups, however, contain four hydrogen atoms attached to hetero atoms. The molecule could thus be supposed to be subject to alkylation giving a derivative with properties suitable for gas chromatography. At our research laboratories, Brandstrom and Junggren (3-7) have evaluated very convenient methods for alkylation of different substrates in the preparative scale. The substrate is extracted as an ion pair with a quaternary ( 1 ) R. Pulver, H. Wirz, and E. G . Stenger, Schweiz. Med. Wochenschr.. 89, 1130 ( 1 9 5 9 ) . (2) G. Beisenherz, F. W. Koss, L. Klatt, and B. Binder, Arch. Intern. Pharmacodyn.. 161, 76 (1966) ( 3 ) A . Brandstrorn and U. Junggren. Acta Chem. Scand.. 23, 2203 (1969). ( 4 ) /bid.. p 2204. (5) / b i d . . p 2536. ( 6 ) /bid.. p 3585. ( 7 ) A . Brandstrorn and U. Junggren, Tetrahedron Lett. 6, 473 (1972).
Apparatus. A Varian 1740 gas chromatograph equipped with a 8.5-mC 63Ni electron capture detector at a dc electrode voltage of 90 V , connected to a 1-mV Philips recorder, Model 8000 was used. The operating parameters were: Column, 170-cm long glass column with internal diameter 2-mm packed with 3% J X R on Gas Chrom Q 100-120 mesh; column temp., 240 "C; injector temp., 240 "C; detector temp., 250 "C; carrier gas, purified nitrogen; and flow rate, 40 ml/min. Reagents. The column packing, 3% J X R on Gas Chrom Q 100-120 mesh, was obtained from Applied Science Lab. Methyl isobutyl ketone, Esso Chemical Co., was purified by distillation. Methyl iodide, Fluka AG, purum quality, was used without purification. Tetrahexylammonium hydrogen sulfate, commercially available from AB Hassle, S-431 20 Molndal, Sweden, was synthesized in our chemical research laboratories. It was used as solutions neutralized with an equivalent amount of sodium hydroxide. Dichloromethane p.a., E. Merck AG, was purified by distillation. Chlorthalidone was supplied by the courtesy of CIBA-
GEIGY AG. Method of Determination. The deep-frozen sample (plasma, serum, or urine) is allowed to thaw at room temperature. After shaking, 2 ml (maximum) of the sample is weighed into a 15-ml centrifuge tube and 0.2-0.3 g of sodium hydrogen carbonate and 5.00 ml of methyl isobutyl ketone are added. The tube is shaken for 10 minutes. After centrifugation, 4.00 ml of the organic layer is transferred to a 1 5 m l centrifuge tube, 2.50 ml of 0.1M sodium hydroxide is added, and the tube is shaken for 10 minutes. After centrifugation, 2.00 ml of the aqueous layer is transferred to a 15-ml centrifuge tube with a screw cap (with Teflon-faced rubber liner). Next 50 pl of 0.1M tetrahexylammonium solution in water and 5.00 ml of 0.5.W methyl iodide in dichloromethane are added. The tube is shaken at 50 "C for 20 minutes. After centrifugation for 5 (8) H . Ehrsson,Acfa Pharm. Suecica. 8, 113 ( 1 9 7 1 ) .
ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, JANUARY 1 9 7 4
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