Table I. Determination of Carbon in Phosphoric Acid-Alcohol Mixtures No. of determinations
Material
Phosphoric acid (AR) Phosphoric acid + 4.17% n -hexanol Phosphoric acid + 7.01% n -hexanol Phosphoric acid + 3.65% n - heptanol
Carbon content Determined
.. .
5 4 ppm 5 294 ppm,
293 ppm
C V = 0.8% 5 483 ppm, cv = 0.9% 5 273 ppm, c v = 1.0%
494 ppm 264 ppm
A comparison of results using the gas chromatographic and gravimetric techniques is shown in Table 11. There is no bias, and similar precisions are obtained from both methods. Although the method was developed principally for application to fertilizers and phosphoric acid, other materials have been examined. These include sulfur (70-850 pprn C), sulfuric acid (8500 ppm), phosphate rocks (300-5000 ppm), potassium chloride (1000 ppm), sand (200 ppm), kaolinite (3000 pprn), carboniferous limestone (11.8%), vehicle exhaust deposits (30.0%),and an oil (88.2%).
LITERATURE CITED Table 11. Determination of Carbon using Gas Chromatographic and Gravimetric Methods KO. of Material
determinations
Fertilizer 1
5
Fertilizer 2
5
Unfiltered Phosphoric Acid
5
Gas chrom. method
Gravimetric method
163 ppm, cv = 1.0% 131 ppm, CV = 5.8%
158 PPm, CV = 5.6% 143 PPm, CV = 5.8%
473 ppm, CV = 3.8%
447 PPm, CV = 4.1%
that the carbon dioxide was transferred quantitatively to the collecting flask. The accuracy of the method was assessed by analysis of pure phosphoric acid to which known amounts of n- hexano1 or n- heptanol had been added. The results, shown in Table I, are reproducible and in agreement with the theoretical values. The method described takes approximately 15 minutes per determination compared with two hours for the gravimetric method.
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
A. A. Duswalt and W. W. Brandt, Anal. Chem., 32, 272 (1960). 0. E. Sundberg and C. Maresh, Anal. Chem., 32, 274 (1960). D. R. Beuerman and C. E. Meloan, Anal. Chem., 34, 1671 (1962). You Sun Kim, Youn So0 Son, and Q. Won Choi, Korean J . Chem., 8, 188 (1964). 0. A. Sukhorukov and N. T. Ivanova, Zavod. Lab., 31, 1070 (1965). L. G. Berezkina. N. A. Elefterova and M. V. Lesnaya, Zavod. Lab., 35, 157 (1969). L. G. Berezkina and N. A . Elefterova, Zh. Anal. Khim., 24, 269 (1969). C. F. Nightingale and J. M. Walker, Anal. Chem., 34, 1435 (1962). M. L. Parsons, S. N. Pennington, and J. M. Walker, Anal. Chem., 35, 842 (1963). W. K. Stuckey and J. M. Walker, Anal. Chem., 35, 2015 (1963). J. M. Walker a n d C . W. Kuo. Anal. Chem., 35, 2017 (1963). D. L. West, Anal. Chem., 36, 2194 (1964). S. Pennington and C. E. Meloan. Anal. Chem., 39, 119 (1967). R. A. Dobbs, R. H. Wise, and R. B. Dean, Anal. Chem., 39, 1255 (1967). F. R. Cropper, D. M. Heinekey, and A. Westwell, Analyst (London), 92, 436 (1967). D. D. Van Slyke and J. Folch, J. Biol. Chem., 136, 509 (1940). R. M. McCready and W. 2. Hassid, hd. Eng. Chem., Anal. Ed., 14, 525 ( 1942). A. A. Houghton, Analyst (London), 7 0 , 118 (1945). I. Dunstan and J. V. Griffiths, Anal. Chem., 33, 1598 (1961). I. Dunstan and J. V. Griffiths, Anal. Chem., 34, 1348 (1962).
RECEIVEDfor review June 3, 1974. Accepted November 12, 1974.
Gas-Liquid Chromatographic Electron Capture Determination of Some Monosubstituted Guanido-Containing Drugs Paul Erdtmansky and Thomas J. Goehl Sterling- Winthrop Research Institute, Rensselaer, NY 72 144
Until recently, no sensitive method for the determination of guanido-type antihypertensive agents existed ( 1 - 4 ) . With the publication of a GLC method utilizing an FID or MID detector ( 5 ) ,this need was partially fulfilled. Unfortunately, for the analysis of nanogram/milliliter samples, a GLC-MS unit is needed. Their method also requires much sample manipulation and considerable time. Another method involving a tedious fluorescent assay has been proposed, but complete details have not been published (6). In developing a method for the determination of the mono-substituted guanido metabolite, 3,4-dihydro-l-methyl-2(1H)-isoquinolinecarboxamidine(IV), of the potential antihypertensive agent, 3,4-dihydro-l-methyl-2(lH)-isoquinolinecarboxamidoxime (111) (7, 8 ) , a general method for the determination of other compounds' of this type evolved. In the present paper, this extremely simple meth750
ANALYTICAL CHEMISTRY, VOL. 47, NO. 4, APRIL 1975
od for the determination of monosubstituted guanido-containing drugs in biological fluids is described. This method combines the extraction and derivitization steps into a single procedure. The derivative is quantitated by electroncapture detection in the nanogram/milliliter range. EXPERIMENTAL Apparatus. A Hewlett-Packard Model 402 gas chromatograph equipped with a tritium electron-capture detector (200 mCi) was used. The instrument was fitted with a 1.8-m glass column (id. 2 mm) packed with 3% OV-17 on 100-120 mesh Gas Chrom Q (Applied Science Labs, State College, PA). The flow rate of carrier gas (7% methane in argon) was 75 mlimin. The temperatures of the injection port, column, and detector were 200, 160, and 200 "C, re-
spectively. The mass spectral characterization of the derivatives was made with a JEOL Model JMSOLSC mass spectrometer.
Materials. Debrisoquin (I) (Hoffmann-La Roche), guanethidine (11) (Ciba-Geigy), 3,4-dihydro-l-methyl-2(1H)-isoquinolinecarboxamidoxime (111), and 3,4-dihydro-l-methyl-2(lH)-isoquinolinecarboxamidine (IV) (Sterling Drug), were kindly supplied by the
I0c
respective drug companies. 6-
; ? L \
0 9c
NH
ii:
NH
$
I1
r N - C H 2 CHZN-C-NHZ
4-
1 -
0-10
0 2 03 0 4 05 06 0 7 08 R A r I O O F A O U E O U S TO ORGANIC VOLUME
01
I CH3 N H
FH3 N O H
I O ,
Figure 1. Influence of the ratio of the aqueous to organic volume on the hexafluoroacetylacetonederivatization ( 0 )1,
m
09
(0) 11, (A) IV
Ip
Hexafluoroacetylacetone was obtained from Pierce Chemical Co., Rockford, IL and purified by distillation. The hexafluorodiacetyl derivatives of the drugs were prepared for mass spectral characterization by refluxing 1 mg of drug in 5 ml of 10% hexafluoroacetylacetone in methanol for one hour. The volume was reduced, an aliquot spotted on a silica gel TLC plate and developed in ethyl acetate or isopropyl acetate. The area corresponding to the derivatized drug was scraped and eluted with methanol. Blood samples were obtained from hypertensive patients who were being treated with 111. The blood was collected in oxalated tubes and centrifueed. The plasma was separated and stored froZen until analysis.-Urine was collected without preservative and also stored frozen until analysis. Methods. T o a 130- X 15-mm test tube fashioned with a 12/18 ground glass joint was added 0.1 ml of plasma (urine) and 5 pl of 0.01N HC1 containing the internal standard. The plasma (urine) was buffered with 50 p1 1M NaHC03, followed by the addition of 0.5 ml benzene and 50 p1 hexafluoroacetylacetone. A 140- X 10-mm open-ended tube with a 12/18 ground glass joint was connected to the test tube. This acted as a condenser during the heating process. The test tube-condenser assembly was then heated in an aluminum heating block a t 100 "C for two hours. They were then taken from the heating block and the condensers removed. Five ml of 3N sodium hydroxide was added to hydrolyze the excess hexafluoroacetylacetone. The samples were vortexed, centrifuged, and 2 p1 of the benzene phase was then injected onto the GLC. Where it was necessary to use larger plasma samples, the plasma protein concentration was reduced by precipitation with concentrated hydrochloric acid. An aliquot of the supernatant was then taken and neutralized with sodium hydroxide before beginning the analysis.
RESULTS AND DISCUSSION Initially, our efforts in developing a method for the analysis of IV were along routine lines. The drug was first extracted, then purified further by back-extraction into dilute acid, and then extracted again into an organic solvent. The derivatization was then carried out after the evaporation of the solvent. However, we found that large losses were being incurred in this procedure due possibly to binding of drug to the glass and/or volatility of the drug. Subsequently, it was discovered that the reaction could be carried out in a heterogeneous system of water and benzene. The next step was an attempt to run the derivatization by substituting plasma or urine for the water and avoid any preliminary purification step. The first try gave encouraging results and the procedure was pursued. Various parameters were examined. Benzene was chosen because it gave a very clean extract. The p H had little effect on the derivatization or the extraction of the derivative in the range 3 to 13. An intermediate p H of 9 was chosen to avoid problems of hexafluoroacetylacetone hydrolysis which would occur with the analysis of samples in which larger aqueous to benzene ratios were needed.
0 I5
30
45
60
120
240
OF R E A C T I O N (MINUTES;
of heating time on the hexaRuoroacetylacetone
Figure 2m
derivatization
0
I0
20
30 T E M P E R A T U R E OF R E A C T I O N
('0
Figure 3. Influence of temperature of the reaction on the hexafluoroacetylacetone derivatization ( 0 )1, ( 0 )11,
(4IV
In our studies an excess of hexafluoroacetylacetone was used. The volume was kept low, however, to avoid carryover of the derivatizing reagent's hydrolysis products into the benzene. The derivative of IV proved quite soluble in benzene so that the aqueous/benzene ratio was not a problem in the extraction. But it was a problem in relation to the derivatization of the drug. It was necessary to keep the aqueous/benzene ratio below 0.4 t o have complete reaction (Figure 1).This would seem to be related to the hydrolysis of the reagent in the buffer. The effect of reaction time is shown in Figure 2. After one hour, the reaction was virtually complete. In Figure 3 is shown the effect of temperature on the reaction, with 100 "C giving the best results. In the analysis of larger volumes of plasma, it was necessary to precipitate some of the proteins with strong acid and use the supernatant after neutralization for the analysis. If this were not done, the "protein plug" that formed A N A L Y T I C A L C H E M I S T R Y . VOL. 47, NO. 4, A P R I L 1975
751
.
‘I
i
I
0
1
1
2
3
4
5
L--0 1
:
2
3
4
5
MINUTES
Figure 4. Gas chromatograms of human plasma assayed as described Left: chromatogram from normal human plasma plus 200 ng/ml of I as internal standard. Right: chromatogram from human plasma containing 90 ng/ml of IV plus 200 ng/ml of i as internal standard
during the heating period sometimes resulted in loss of sample by “bumping.” No other problems such as coprecipitation were found using this technique. Mass spectral data were obtained for all derivatives and the derivatization was confirmed. For example, the structure of the derivative of IV was shown to be:
throughout this range. If greater sensitivity were needed for other work, this could be achieved by: 1) increasing the sample volumes or decreasing the volume of benzene within limits set by the data in Figure 1; 2) injecting a larger aliquot of benzene onto the GLC column; 3) transferring the benzene to another tube and reducing the volume; and 4) extracting a larger volume of biological fluid with a more polar solvent (e.g., ether), back-extracting into a small volume of dilute acid, and running the derivatization in the buffered aqueous phase with benzene present. The method as described is simple, sensitive, and specific. It has been used in the determination of IV (internal standard was I) in plasma and urine from patients receiving the potential antihypertensive agent 111. This compound did not interfere in the assay since it could not be derivatized using the above technique. It was also found that no naturally occurring substances in plasma or urine, or other metabolites, interfered in the assay (Figure 4). The procedure has also been examined for the determination of I and I1 and the conditions determined (Figures 1-3). I t should also be applicable for the determination of other monosubstituted guanido-containing compounds. I t has the advantages of using plasma or urine directly without preliminary extraction of drug and requiring only a GLC-EC instrument for determination of nanogram/milliliter levels.
ACKNOWLEDGMENT We would like to thank Stephen Clemans for obtaining the mass spectra of the derivatives, Mike McGrath for the statistical analysis of the data, and Denis M. Bailey for some invaluable suggestions. LITERATURE CITED S. L. Tompsett, Acta Pharmacol. Toxicob, 19, 365 (1962). E. C. Bose and R. Vijayvargiya. J. Pharm. Pharmacol., 16, 561 (1964). C. McMartin, P. Simpson. and N. Thorpe, J . Chromatogr., 43, 72 (1969). Y. H. Chang and R. Pinson, Jr., Biochem. Pharmacol., 16, 201 (1967). J. H.Hengstmann, F. C. Falkner, J. T. Watson, and J. Oates, Anal. Chem., 46, 34 (1 974). (6) C. N. Corder, T. Klaniecki. and R. H. McDonald, Jr., Pharmacologist, 15, 194 (1973). (7) T. J. Goehl, Sterling-Winthrop Research Institute, Rensselaer, NY, unpublished data, 1972. (8)D. M. Bailey, C. G. DeGrazia, H. E. Lape, R. Frering, D. Fort, and T. SkuIan, J. Med. Chem., 16, 151 (1973). (1) (2) (3) (4) (5)
The smallest on-column amount of derivatized IV that could be detected was 5 picograms. The lower limit of sensitivity of the procedure described here was 25 ng/ml of biological fluid. A nonlinear curve was found in the range 25 to 400 ng/ml with a relative standard deviation of 5%
RECEIVEDfor review September 13, 1974. Accepted December 16, 1974.
Reproducibility of Standards Carried through a Gas Chromatographic Procedure for the Assay of Plasma Neutral Lipids Martin Gold and George Mathew Department of Laboratory Medicine, Nazareth Hospital, 260 1 Holme Avenue, Philadelphia, PA 19 752
Kuksis et al. ( I , 2 ) have developed a method for the direct gas-liquid chromatography of plasma neutral lipids and phospholipid. They tested reproducibility with 3 plasmas of abnormally high lipid concentration processed through the whole procedure in duplicate. 752
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 4, A P R I L 1975
The analysis of the major plasma neutral lipids was of interest to us and by using the procedure of Kuksis et al. (Z), this could be carried out fairly expeditiously. However, it was necessary to check the applicability a t low lipid concentrations as well as high. Therefore, we undertook a
