material, the packing pressure, the column-to-vessel connection, and on the injection device used for testing. CONCLUSIONS T h e dynamic slurry packing technique offers several advantages over other methods when packing small particles ( < l o pm). It can be adapted to chemically bonded (reversed phase) and regular silica gel materials ( 4 ) . With the discussed design, up to 6 columns can be packed simultaneously which makes it useful for routine packing of large series. The choice of solvents and solvent mixtures is not very critical and nontoxic and inert solvents can be used. The solvents can be identical or a t least compatible with the mobile phase used for testing, which cuts down equilibration times before actual use. With the precautions mentioned, the technique can be absolutely safe. T h e columns within one packing batch are as a rule very similar in performance with occasionally some fluctuations in permeability due to differences in metal frit porosity at the outlet of the column. This means in practice that only one of three columns is tested which results in considerable time saving. One limitation of this packing technique is that it should only be used for relatively short columns 1 2 5 cm in order to assure a reasonably good quality (see also Ref. 4); but this can be justified since in industry the question is usually not how can one get the largest number of theoretical plates (N) but.
what is the smallest N necessary to solve a problem and what is the time needed for a routine separation. From this point of view, i t is understandable that more than 70% of our columns used are between 10 and 1.5 cm long. Short columns have the additional advantage of lower pressure drop which means a simpler pumping and injection system can be used; they are also practical and time saving for separation optimization both with and without gradient. ACKNO WLEDGMENT Mrs. Defanti and Mr. Hoyer are thanked for technical assistance in this project. LITERATURE CITED J. J. Kirkland, J. Chromatog. Sci., 9, 206 (1971). R. E. Majors, Anal. Chem., 44, 1722 (1972). R. M. Cassidy, D. S.Le Gay, and R. W. Frei, Anal. Chem., 46, 340 (1974). H. R. Linder. H. P. Keller, and R. W. Frei, J. Chromatog. Scl., 14. 234 (1976). (5) P. A. Bristow, P. N. Brittain, C. M. Riley, and B. F. Williamson, J . Chromatogr., 131, 57 (1977). (6) L. Berry and B. L. Karger, Anal. Chem., 45, 819A (1973). (7) R. G. Srownlee and D. Gere, Paper No. IO, presented at the Pittsburgh Conference on Analytical Chemisby and Applied Spciroscopy, Cleveland, Ohio. March 1976. (1) (2) (3) (4)
RECIVED for review April 18, 1977. Accepted July 6, 1977. Presented a t the Symposium on Liquid Chromatographic Techniques, Baden-Baden, GFR, September 1976.
Determination of 4,4’-Alkyl- and Alkoxy-Disubstituted Azoxybenzenes by lsocratic Reverse Phase High Performance Liquid Chromatography Satoru Shiono,
Titose Miyakura, Junzo Enomoto, and Takashi Imamura
Central Research Laboratory, Mitsubishi Electric Corporation, Amagasaki, Hyogo, Japan
An lsocratic reverse phase high performance liquid chromatographic method has been developed for the separation and determlnatlon of 4,4’-alkyl- and alkoxy-dlsubstltuted trans-azoxybenzenes. Chromatography was performed at 48 ‘ C with an octadecyl bonded phase column and methanolwater mobile phases, using a UV detector (254 nm). The preclslon Is less than 0.5% relatlve standard deviation and the detection limits are between 0.2 and 1.6 ng with a methanol mobile phase containing 10% (w/v) of water.
Several 4,4’-alkyl- and alkoxy-disubstituted trans-azoxybenzenes are nematic liquid crystals and various mixtures of them are increasingly used as the major constituent of the liquid crystal. The qualitative and quantitative determination of 4,4‘-disubstituted azoxybenzenes is of utmost importance for in-process and final product quality control. Methods previously reported for the problem of azoxybenzene analysis require thin-layer chromatography (1-3). Gas chromatography ( 4 ) is not suitable for precise analysis because of the thermal instability of azoxybenzenes. I n this report we describe a reliable method for the separation and determination of 4,4’-alkyl- and alkoxy-disubstituted azoxybenzenes by isocratic reverse phase high per-
formance liquid chromatography (HPLC). EXPERIMENTAL Apparatus. A Shimadzu (Kyoto, Japan)-Du Pont model 830 high pressure liquid chromatograph equipped with an ultraviolet detector (254nm) was used at 48 “C. The column was a Du Pont 25 cm X 2.1 mm Zorbax ODS column (octadecyl bonded silica). Methanol mobile phases containing a certain amount of water (from 10% (w/v) to 35% (w/v)) were used at a flow rate of 0.5 mL/min. A Shimadzu six-port valve with a 12-pLexternal sample loop was used to inject samples dissolved in methanol onto the analytical column. Chromatograms were integrated with a Shimadzu model 1.4 digital integrator. Mass spectra were taken on a Japan Electron Optics Laboratory (Tokyo, Japan) model 01SG-2mass spectrometer and infrared spectra on a Shimadzu model 430 infrared spectrometer. Reagents. The following nine disubstituted azoxybenzenes were studied; azoxybenzene (I),4,4’-dimethoxyazoxybenzene(II), 4,4’-methoxyethylazoxybenzene (111) (an isomeric mixture of 4-methoxy-4’-ethylazoxybenzene(IIIa) and 4-ethyl-4’-methoxyazoxybenzene (IIIb)),4,4’-methoxy-n-butylazoxybenzene (IV) (anisomeric mixture of 4-methoxy-4’-n-butylazoxybenzene (IVa) and 4-n-butyl-4’-methoxyazoxybenzene (IVb)), 4,4’-di-n-butylazoxybenzene (V), 4,4’-di-n-pentyloxyazoxybenzene (VI), 4,4’di-n-pentylazoxybenzene (VII),4,4’-di-n-hexylazoxybenzene (VIII), 4,4’-di-n-heptyloxyazoxybenzene(IX). Nuclear magnetic resonance analysis (5)showed that I11 consisted of IIIa (ca. 25%) and IIIb (ca. 75%) and that IV consisted of IVa (ca. 30%) and IVb ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1963
Table I. Slope of Calibration Curve, Relative Standard Deviation of Slope, Precision and Detection Limit for 4,4'-Disubstituted Azoxybenzenesn 4,4'-Disubstituted azoxybenzene (AB)
Slope of calibration curve, AU. sec/minb
Dimethoxy AB Methoxyethyl AB Methoxy-n-butylAB Di-n-butylAB Di-n-pentyloxyAB Di-n-pentyl AB Di-n-hexylAB Di-n-heptyloxyAB
0.862 1.056 0.783 0.624 0.607 0.590 0.513 0.435
Re1 std dev of slope, %
Precision, re1 std dev, %'
Detection limit, n$
0.29 0.54 0.51 0.49 0.54 0.46
0.2 0.3 0.4
0.2 0.2 0.3 0.5 0.6
0.4
0.5 0.4 0.4
0.94 0.99
0.7
1.2 1.6
0.4
a Mobile phase, 10% (w/v) of water in methanol; flow rate, 0.5 mL/min; temp., 48 " C ; amount of azoxybenzenes injected, 0.5-10 p g . A.U.: absorbance unit. Amount of azoxybenzenes injected, 3 pug. Signal-to-noise ratio, 3 : l .
0
20
40
0
50
20
Figure 1. HPLC chromatograms of 4,4'disubstituted azoxybenzenes. Mobile phase, (a) 10% (w/v) of water, (b) 15%, (c) 20%; flow rate, 0.5 mL/min; temp., 48 O C . Peak identity: (I) azoxybenzene(AB),(11) 4,4'dimethoxyAB, (111) 4,4'-methoxyethylAB (an isomeric mixture of 4-methoxy-4'-ethylAB (IIIa) and 4-ethyC4'-methoxyAB (IIIb)), (IV) 4,4'-methoxy-n-butylAB (an isomeric mixture of 4-methoxy-4'-n-butylAB (IVa) and 4-n-butyl-4'-methoxyAB (IVb)), (V) 4,4'-di-n-butylAB, (VI) 4,4'dl-n-pentyloxyAB,(VII) 4,4'd~n-pentylAB,(VIII) 4,4'd~n-hexylAB, (IX) 4,4'-di-n-heptyloxyAB
50
40 time,min
t i m e . min
Figure 2. HPLC chromatograms of 4,4'dlsubstituted azoxybenzenes. Mobile phase, (d) 25% ( w h ) of water, (e) 30%, (f) 35%; flow rate, 0.5 mL/min; temp., 48 O C (For peak identity, see Flgure 1)
(ca. 70%). These azoxybenzenes were purchased from E. Merck (Darmstadt, Germany), Wako Pure Chemical Industries (Osaka, Japan), Fuji Color (Osaka, Japan) and Eastman Organic Chemicals (Rochester, N.Y.). They were recrystallized from methanol or ethanol except I11 and IV, which were used without further purification. Spectroquality methanol (Dotite, Kumamoto, Japan) and distilled water were used as solvents.
RESULTS AND DISCUSSION Figure 1 and Figure 2 show the HPLC chromatograms of the 4,4'-disubstituted azoxybenzenes obtained with several methanol mobile phases containing from 10% (w/v) to 35% (w/v) of water. T h e capacity factors of the azoxybenzenes are increased with an increase in the carbon numbers of the 4-and 4'-substituents. The effect of an alkyl substituent with n carbon atoms on the capacity factor is approximately the same as that of an alkoxy substituent with n + 1carbon atoms. T h e unsymmetrically substituted azoxybenzenes (111and IV) yield two adjacent peaks with methanol mobile phases having higher water content, as shown in Figure 2. These two peaks are attributable to geometrical isomers. In order to prove this, infrared and mass spectrometric analysis of the two eluted peaks was carried out. Here we describe only the results of the analysis of the two peaks yielded by IV, because similar conclusions can be drawn for those yielded by 111. The two eluted peaks for IV were fraction-collected with a methanol mobile phase containing 35% (w/v) of water (a differential refractometer detector was used because the photoreaction of azoxybenzene derivatives might be induced 1964
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
40
80
120
160 mle
200
240
280
Mass spectra of the two eluted peaks for 4,4'-methoxyn-butylazoxybenzene. (a) 4,4'-methoxy-n-butylazoxybenzene (IV) (an isomeric mixture of 4-methoxy-4'-n-butylazoxybenzene and 4-n-butyl-4'-methoxyazoxybenzene (ca.70:30)),(b) the first peak ylelded by IV, (c) the second peak yielded by IV (See Figure 2f) Figure 3.
by UV irradiation (6)). The same infrared spectra were obtained for the first peak and the second one. The infrared spectra suggested that these compounds were azoxybenzene derivatives in which the two benzene rings were para-disubstituted and that one of their substituenb was a methoxy group. The mass spectra obtained for the two peak eluates
yielded by IV are shown in Figure 3 in comparison with that of IV. First, both these compounds are found to have the same molecular weight (Mf = 284). In view of the principle fragmentation of the two geometrical isomers of IV (7), shown below,
r, 133
135J" e107
161~" the mass spectra indicate t h a t the first peak eluate and the second one are regarded as IVa and IVb, respectively. From the results of the infrared and mass spectrometric analysis described above, it is concluded that the two peaks for IV are attributable to the geometrical isomers of IV. With respect to the separation of the 4,4'-disubstituted azoxybenzenes studied, there is no methanol-water mobile phase which permits all of the azoxybenzenes to be sufficiently separated for quantitative determination (as I is not liquid crystal, the separation and determination is discussed to the exclusion of I). The resolution of V and VI are in competition with t h a t of VI11 and IX. However, V and VI can be ade-
quately separated with a methanol mobile phase containing 20% (w/v) of water and on the other hand VI11 and IX can be separated with a methanol mobile phase containing 10% (w/v) of water. Consequently it is concluded t h a t for any mixtures of the azoxybenzenes, qualitative and quantitative determination can be made with two methanol mobile phases containing an appropriate amount of water. The capacity factors of the azoxybenzenes studied were between one and twenty with a methanol mobile phase containing 10% (w/v) of water, while the resolution of V and VI was poor (Figure l a ) . Hence in order to study the reproducibility, calibration curves were established with a methanol mobile phase containing 10% (w/v) of water between peak areas and amounts of the azoxybenzenes injected over the range of 0.5 to 10 pg. The linearity was good and the plots passed through the point of origin. The slope of the calibration curve, the relative standard deviation of the slope, the precision and the detection limit obtained for each azoxybenzene are shown in Table I. In Table I, the two unsymmetrically disubstituted azoxybenzenes, I11 and IV, are treated as a mixture of their isomers.
LITERATURE CITED (1) W. R. Edwards, Jr., 0.S. Pascual. and C. W. Tate, Anal. Chem., 28, 1045 (1956). (2) C. C. Jahiatt, An. Fac. Ouim. Farm., Univ. Chile, 18, 282 (1966). (3) F. H. Alvarado. J . Chromatogr., 42, 144 (1969). (4) T. Kawahara, S.Goto, and T. Kashiwa, BunsekiKagaku, 18, 1344 (1969). (5) R. E. Rondeau, U.S.P. 3, 730, 687. (6) G. G. Spence, E. C. T a r n , and 0.&char&, Chem. Rev., 70, 231 (1970). (7) J. H. Bowie, R. G. Cooks, and G. E. Lewis, Aust. J . Chem., 20, 1601 (1967).
RECEIVED for review July 7,1977. Accepted August 22,1977.
Quantitative Determination of Benzoylecgonine and Cocaine in Human Biofluids by Gas-Liquid Chromatography M. J. Kogan,* K. G. Verebey, A. C. DePace, R. B. Resnlck, and S. J. Mu16 New York State Office of Drug Abuse Services, Testing and Research Laboratory, Brooklyn, New York 11217, and New York Medical College, Department of Psychiatry, New York, New York 10029
Cocalne (COC) and Its prlnclpal metabollte In man, benroylecgonlne (BE) were determined by quantitative gas-liquid chromatographlc methods. Nitrogen detectlon was used for COC and electron capture detection for BE following extraction from human plasma. Using flame lonlzatlon detectlon, COC and BE were slmultaneousiy determlned from human urlne. The limits of detection for COC underivatlred and BE as the pentafluorobenzyl bromide derlvative In plasma were 10 and 5 ng/mL, respectlvely. I n urlne the sensltlvlty limits of the sllyl derivatives of COC and BE were 0.5 and 1.0 pg/mL, respectively. The coefflcient of varlation ranged between 0.9-2.2% and the coefficient of determination was 0.99 for these methods. Data on plasma and urine concentration of COC and BE collected over a tlme period of 24 h from three human subjects who recelved 1.0 to 1.9 mg/kg cocalne*HCI i.v. are presented.
The euphoric and stimulant effects of cocaine induce a high level of psychological dependence ( 1 ) and subsequent abuse
(2). Current detailed information has recently been published concerning the historical, chemical, physiological, sociological, and treatment aspects of cocaine use and abuse (3, 4 ) . Cocaine, a lipophilic drug, is extensively biotransformed in man to water soluble metabolites (5-7). The various analytic methods for the detection of cocaine and its metabolites have been critically reviewed (8, 9). A major biotransformation product of cocaine in man is benzoylecgonine (BE). Moderately sensitive gas chromatographic techniques have been reported recently for the detection of cocaine and benzoylecgonine in urine (10-15) and the quantitative determination of cocaine, but not benzoylecgonine, in human plasma (16, 17). Until now methods were not available for the quantitation of BE in plasma primarily because underivatized BE did not chromatograph well. A variety of electron capture derivatizing agents have been used t o improve the detection and quantitation of trace amounts of carboxylic acids (18-24). Benzoylecgonine, a carboxylic acid, will form an ester with the halogenating reagent pentafluorobenzyl bromide (PFB), thus providing a reliable and sensitive benzoylecgonine derivative for gas chromatography. ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1085
