ACKNOWLEDGMENT
''
The authors thank Barbara G' Urban' and Stephen P. Levine for their professional assistance a t various stages of this project. LITERATURE CITED E. C. Horning and M. G. Horning. Clin. Chem., 17, 802 (1971). E. C. Horning and M. G. Horning. J. Chromatogr. Sci,, 9, 129 (1971). J. E. Mrochek. W. C. Butts, W. T. Rainey, Jr., and C. A. Burtis, Clin. Chem., 17, 72 (1971). R . Teranishi, T. R. Mon, A. B. Robinson, P. Cary, and L. Pauling, Anal. Chem., 44, 18 (1972). M. G. Horning, A. Hung, R . M. Hill, and E. C. Horning. Clln. Chim. Acta, 34, 261 (1971). C. H. L. Shackelton, J.-A. Gustafsson, and J. Sjovall, Steroids, 17, 265 (1971). R. Reinmendal and J. B. Sjovall, Anal. Chem., 45, 1083 (1973). H. Haga. T. imanari. A. Tamura, and A. Momose. Chem. Pharm. Bull., 20, 1805 (1972). S. P. Markey, W. G. Urban, A. J. Keyser. and S. I. Goodman, Adv. Mass Spectrom., 6, 187 (1973). 0. A. Mamer. J. C. Crawhill. and S. S. Tjoa, Clin. Chim. Acta, 32, 171184 (1971). E. Jellum. 0. Stokke. and L. Eldjarn, Clln. Chem., 18, 800 (1972). "Handbook of Biochemistry", H. A. Sober Ed., Chemical Rubber Co., Cleveland, OH, 1968, Section B. P. B. Hamilton and B. Nacy, Space Life Sci., 3, 432 (1972). S. W. Fox, K. Harada, and P. E. Hare, Space Life Sci., 3, 425 (1972). C. W. Gehrke, R . W. Zumwalt, K. Kuo, J. J. Rash, W. A. Aue, D.L. Staliing, K. A. Kvervolden, and C. Ponnamperuma, Space Life Sci.. 3, 439 (1972). C. E. Costello. H. S. Hertz, T. Sakai, and K. Biemann, Clin. Chem., 20, 255 (1974). K. B. Hammond and S. I. Goodman, Clin. Chem., 16,212 (1970). T. A. Witten, S. P. Levine. J. 0. King, and S. P. Markey, Clin. Chem., 19, 586 (1973). T. A. Witten. S. P. Levine. M. T. Killian, P. J. R . Boyle, and S. P. Markey, Clin. Chem., 19, 963 (1973). R. A. Chalmers and R. W. E. Watts, Analyst (London), 97, 951 (1972). R . A. Chaimers and R. W. E. Watts, Analyst (London), 97, 224 (1972).
(22) R . A. Chalmers and R. W. E. Watts, Analyst(London), 97, 958 (1972). (23) A. M. Lawson. F. L. Mitchell, R . A. Chalmers, P. Purkiss. and R. W. E. Watts, Adv. Mass Spectrom., 6, 235 (1973). (24) S. P. Levine, J. L. Naylor, and J. P. Pearce, Anal. Chem., 45, 1560 [ 1973). (25) A. White, P. Handler, and E. Smith, "Principles of Biochemistry", McGraw-Hill Book Co., New York, NY, 1973, p 942. (26) D.O'Brien, F. A. Ibbott, and D. 0. Rodgerson. "Laboratory Manual of Pediatric Microbiochemical Techniques", Harper and Row, New York. NY. 1968, pp 114-116. (27) J. A. Thompson and J. L. Holtzman. J. Pharmacol. Exp. Ther., 186, 640 (1973). (28) M. G. Horning in "Biomedical Applications of Gas Chromatography". H. A. Szymanski Ed.. Vol. 2, Plenum Press, New York. NY, 1968, p 56. (29) G. Lancaster. P. Lamm, C. R . Scriver, S. S. Tjoa, and 0. A. Mamer, Clin. Chim. Acta, 48, 279 (1973). (30) J. R. Planner and S. P. Markey, Org. Mass Spectrom., 5, 463 (1971). (31) S. P. Markey. Anal. Chem., 42, 306 (1970). (32) "Handbook of Silylation". Pierce Chemical Co. Handbook GPA-30, Rockford, IL, 1972. (33) Bio-Rad Laboratories Tech. Bull. 1005, Richmond, CA, 1973. (34) S. Ohashi, N. Yoza, and Y. Veno. J. Chromatogr., 24, 300 (1966). (35) D. W. Baker, J. Assoc. Off. Anal. Chem., 56, 1257 (1973). (36) "Solubilities of Inorganic and Metal Organic Compounds", W. F. Linke, Ed., Vols. I and 11, American Chemical Society, Washington, DC, 1958. (37) B. Wengle. Acta Chem. Scand.. 18, 65 (1964). (38) "Mass Spectra of Compounds of Biological interest", S. P. Markey, W. G. Urban, and S. P. Levine Ed., National Technical information Service, U S . Department of Commerce, Springfield, VA. 1974. (39) G. Peterson, Tetrahedron. 3413 (1970). (40) G. Peterson, Org. Mass Spectrom., 6, 565 (1972). (41) C. M. Williams and C. C. Sweeley in "Biomedical Applications of G a s Chromatography". H. A. Szymanski Ed., Plenum Press, New York, NY, 1964. (42) A. L. German, C. D. Pfaffenberger, J-P Thenot, M. G. Horning, and E. C. Horning, Anal. Chem., 45, 930 (1973).
RECEIVEDfor review November 25, 1974. Accepted March 6, 1975. This work was supported by NIH Grants HD04870 and HD-04024 in addition to MCH Project Grant 252.
Gas Chromatographic Determination of Apomorphine in Urine and Feces Robert V. Smith Drug Dynamics Institute, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712
Andrew W. Stocklinski Division of Medicinal Chemistry and Natural Products, College of Pharmacy, The University of lowa, lowa City, IA 52242
A method for the determination of apomorphine in rat urine has been devised based on liquid-liquid extraction with ethyl acetate, derivatization with N,O-bis(trimethylsily1) acetamide and gas chromatography on an OV-17 column. The developed method can be incorporated into a scheme that permits selective determination of apomorphine and its isomeric 0-methyl metabolites. Attempts to devise similar procedures for the determination of these compounds in rat feces were only partially successful. Urinary elimination of apomorphine in Sprague-Dawley rats, following intraperitoneal injection of this drug, was determined.
T h e mammalian metabolism of aporphine alkaloids is being systematically investigated in these laboratories ( I 3 ) . As part of these studies, a procedure for measuring apomorphine (1) in rat urine and feces in the presence of its potential metabolites, apocodeine (2), isoapocodeine (3),
and norapomorphine (4) was desired. We have previously demonstrated that 2 and 3 can be separated from 1 by extraction with 1%isomyl alcohol in n-heptane (1%INH) a t p H 8.6. 2 and 3 were subsequently quantiated as such by gas chromatography (GC) ( 4 ) . A method for analyzing 1 and 4 in urine, based on thin-layer chromatographic fluorescence quenching ( 5 ) has also been developed though it is somewhat tedious to perform since it requires over twentyfour hours to complete. An earlier method used to estimate I in biological fluids requires mercuric chloride oxidation to the ortho-quinone, 5 , and subsequent colorimetric measurement (6). Although this method provides sufficient sensitivity to detect 1 a t sub-microgram levels, it is not entirely suitable since i t cannot distinguish 1 from a number of its potential metabolites. T h a t is, Cava et al. ( 7 ) have recently shown t h a t compounds such as 2 , 3 , and 4 can be converted to 5 (or its homolog) upon treatment with mercuric chloride. Other colANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1321
orimetric (8-11) and fluorometric (12) methods for I are either insufficiently sensitive and/or selective for determinations in urine and feces. I t was proposed that a selective and sensitive GC procedure might be devised for 1. Parker et al. (13) reported that 1 could be satisfactorily developed on a 5% SE-30 column. In our hands, however, GC experiments with 1 (even on glass columns) clearly indicated that decomposition was occurring a t temperatures necessary for elution of this material. Alternatively, it was thought that suitable GC development of 1 might be affected following conversion to its 0,Obis(trimethylsily1) (TMS) ether derivative (7). A method utilizing the latter, is the subject of this report.
EXPERIMENTAL Apparatus. A Hewlett-Packard 5750B gas chromatograph equipped with dual flame ionization detectors was used throughout. 3% OV-17 on 100/120 mesh Gas Chrom Q glass columns (6-ft X ?$-in. i.d.1 were obtained from Supelco, Inc., and were flow conditioned a t 280' for 1 2 hr prior to use. Operating conditions were: column and injection port temperature, 260'; detector temperature, 320'; carrier gas (He) flow, 70 ml/min; hydrogen flow, 40 ml/ min; air flow, 440 ml/min; range 10'; attenuation, 16. A model 1015 S/L Finnigan quadrupole mass spectrometer interfaced to a Varian 1400 gas chromatograph was used for structure determinations. The GC was operated as follows: a glass column (6-ft X Ih-in. i.d.1 packed with 3% OV-17 on Gas-Chrom Q was maintained a t 220'; injection port temperature, 270'; carrier gas (Nz) flow rate, 20 ml/min. The mass spectrometer conditions were: ionization energy, 70 eV; ion source temperature, 160'; emission current 200 mA; accelerating voltage, 3 kV; sensitivity, scan rate, 1 sec. Reagents. The preparation and purification of compounds 2, 3, 4, and 6 have been described elsewhere (2,4, 14). Apomorphine hydrochloride hemihydrate (1-HC1) was obtained from S. B. Penick and Co. and used without further purification after establishment of its homogeneity by GC (see below). The silylating reagent, N,Obis(trimethylsily1) acetamide (BSA), was purchased from Pierce Chemical Co. /3-Glucuronidase containing aryl sulfatase (type H-1) was purchased from Sigma Chemical Co. Enzyme activity of this preparation was determined according t o the supplier's recommended assay procedure. A solution of 1.5% trimethylchlorosilane in benzene was used to silylate all glassware used in the extraction procedures. Procedures. Stock solutions of 1-HC1 were prepared in 3-, 5-, or 10-ml volumetric flasks by dissolving 1.0 to 20.0 mg of the salt in double glass-distilled water. For studies extending beyond 1 hr, solutions were made to contain l ml of 5% HC1 per 10 ml to minimize oxidation of 1. Standard GC solutions were prepared either by directly adding BSA to weighed quantities of 1-HCl contained in graduated Chromoflex tubes (Kontes Glass Co.), or by reducing 1-ml aliquots of stock solutions to dryness in vacuo and treating the residues with BSA. Final volumes of the BSA treated standards were adjusted to 1 ml using BSA and the resulting reaction mixtures were gently shaken for 20 min. T o study the kinetics of the BSA-mediated trimethylsilyation of 1, 1 ml aliquots of BSA were added to 0.10 to 5.00 mg of 1-HC1 contained in 1-ml Microflex tubes (Kontes Glass Co.) fitted with 1322
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
Teflon-lined caps. The reactions were allowed to proceed a t room temperature, with and without shaking. Two-pl aliquots of the reaction mixtures were injected into the GC immediately after addition of BSA and continued until constant peak height was obtained. Injections were made with Hamilton 701N 10-pl syringes. Standard curves were prepared by GC of 1-pl aliquots of BSA-1 solutions (0.07 to 3.40 mg/ml 1-HCl). Curves of peak height vs. equivalent amount of 1 chromatographed were constructed. To measure recovery of 1 as a function of pH, 1-ml aliquots of aqueous stock solutions (0.97 to 1.10 mg/ml 1-HCl) were added to 10 ml of 0.4M acetate (final pH 5.52); 0.2M citrate (final pH's 6.00 and 6.71); 0.2M imidazole (final pH's 6.99, 7.08, and 7.18); 0.2M TES (final pH's 7.05, 7.25, and 7.43); and 0.1M Tris (final p H 8.55) buffers. The mixtures were then extracted with four 5-ml portions of ethyl acetate using 30-ml separatory funnels equipped with Teflon stopcocks. The organic layers were reduced to dryness in vacuo and the residues reacted with three 0.3-ml portions of BSA, and final volumes were adjusted to 1 ml in Chromoflex tubes for GC analysis. Recovery of 1 as a function of concentration was performed by adding 1-ml aliquots of 1-HC1 stock solutions (0.10, 0.50, 0.75, 1.00, and 2.00 mg/ml 1-HCl) to 10 ml of 0.2M T E S or 0.2M imidazole buffers (final pH, 6.99 to 7.10) and extracting with ethyl acetate as described above. Recovery of Apomorphine from Urine. Twenty-four-hr rat urines (mean volumes, 10 ml) were collected in 18- X 150-mm test tubes containing 1 ml 5% HC1 solution. The urine was frozen on collection using cold plates (Thermoelectronics). After equilibrating to room temperature, the urine samples were spiked with 1-ml aliquots of 1-HCl solution as described above. The spiked samples were adjusted to p H 7.00 (f0.05) with 10N NaOH and 5% HC1 (final volumes -13 ml) and immediately extracted with ethyl acetate. Emulsions were broken by centrifugation for 5 min. a t -2000 grams. Two to 5-pl aliquots of the resulting BSA-treated solutions were gas chromatographed. Recovery of 1, 2, and 3 from Hydrolyzed Urine. Urine samples (-10 ml) were spiked with 0.1- to 2.0-mg quantities 1-HCl, 2-HCl and 3-HC1, and divided into two equal portions; the samples were hydrolyzed with P-glucuronidase, or varying concentrations of HC1 according to the method of Kaul et al. (15). Acid hydrolysates were neutralized in an ice bath with 10N NaOH. Enzymatic hydrolyses were performed by first adjusting the urine samples to p H 5.40 (f0.20) with 5% HC1, adding 3 ml of 0.4M acetate buffer (pH 5.40) and P-glucuronidase (3000 Fishman U. per 10-ml sample) and shaking in a Dubnoff shaker for 20 hr a t 37'. The hydrolyzed samples were adjusted to pH 7.00 (10.05) for 1, and 8.60 (f0.05) for 2 and 3 using 2.5N NaOH, and extracted with ethyl acetate and 1%INH respectively. Determination of 1 in Urine. Urine specimens are obtained for 24 to 72 hr in 18- X 150-mm test tubes (containing 1 ml of 5% HC1 solution per tube) from rats injected with 8 to 30 mg of 1-HCl/kg, and frozen upon collection with cold plates. Following thawing, 24-hr specimens (mean volume, -10 ml) are adjusted to pH 7.00 (fO.05) with 10N NaOH and 5% HC1 (final volume, -13 ml) and immediately extracted with four 5-ml portions of ethyl acetate using 30-ml separatory funnels equipped with Teflon stopcocks. The combined organic extracts are reduced to dryness in vacuo and residues treated with three successive 0.3-ml portions of BSA; final volumes are adjusted to 1 ml in Chromoflex tubes. TWOto 5-pl aliquots of the resulting BSA-treated solutions are gas chromatographed. Peak heights resulting from 7 are compared to a standard curve prepared by GC of 1- to 5-pg quantities of 7. During analyses of 1, a standard solution (in BSA) of 7 (-3 wg) is gas chromatographed after every fifth sample to revalidate the working standard curve for a given day. Determination of 1 in Hydrolyzed Urine. Five-ml aliquots of 24-hr rat urines (mean volume, -10 ml; collected as indicated in the preceding paragraph) are treated with 4-ml portions of 9 N HCl, covered with parafilm, and heated on a water bath set a t 100' for 30 min. The hydrolysates are adjusted to pH 7.00 (fO.05) with ION NaOH and 5% HCl (final volume, -13 ml) and immediately extracted with four 5-ml portions of ethyl acetate using 30-ml separatory funnels equipped with Teflon stopcocks. The combined organic extracts are reduced to dryness in vacuo and the residues treated with BSA and analyzed as indicated in the preceding paragraph. Determination of 2 and 3 in Hydrolyzed Urine. Five-ml aliquots of 24-hr rat urines (mean volume, -10 ml) are collected as indicated under the paragraph on the determination of 1 in urine. The urine specimen is adjusted to p H 5.40 (40.20) with 5% HC1; 3 ml of 0.4M acetate buffer (pH 5.40) and 3000 Fishman U. of P-glu-
Y 3
13
20 2
Time [ m i r l
5
Figure 1. Derivatization of apomorphine with BSA
A r n ' t of
~(JJQ)
Figure 3. Standard curve for determination of apomorphine as TMS derivative
PH
Figure 4. Recovery of apomorphine by ethyl acetate extraction as a function of DH
T i me (m i n)
T I me (m i n)
Figure 2. GC of 5-pl aliquots of BSA derivatized extracts of blank urine ( A ) and blank hydrolyzed urine (5). Position of authentic TMS derivative of apomorphine (7) indicated by peak at observed retention time
curonidase (Sigma) is added and the resulting mixture is incubated with shaking in a Dubnoff shaker at 37' for 20 hr. The hydrolyzed sample is adjusted to pH 8.60 (h0.0L5)with 2.5N NaOH, extracted, and analyzed by GC as described previously ( 4 ) . Determination of 1, 2, and 3 in Feces. Analyses were performed with 24-hr rat feces (mean wet weight, 6.8 g) collected from individual animals. Fecal samples were frozen in a Dry Ice-acetone bath for 10 min and lyophilized. The dried feces samples (mean dry weight 3.9 g) were ground to a fine powder using a glass mortar and pestle. Two- g aliquots were spiked with 1-mg quantities of 1HC1,2-HCl, or 3-HC1, then hydrolzyed for 30 min at 90-100" using 5 ml of 4.8N HC1. Samples were adjusted to pH 7.00 (f0.05) with 10N NaOH and analyzed for 1 as indicated above. 2 and 3 were determined by GC of l%INH extracts of hydrolysates adjusted to pH 8.60 (h0.05) with 10N NaOH. Biological Studies. All experiments were performed with male Sprague-Dawley rats (obtained from Sprague-Dawley Co., Madison, WI) weighing 200-250 g. The animals were individually housed in stainless steel metabolism cages (Acme Steel Co.) and maintained on a diet of Purina Rat Chow. Food and water was given ad libitum except during the first 24 hours following injection of drug. During this time, food was completely excluded while water was withheld for 2 hr post injection. Animals were maintained on a schedule of 1 2 hr artificial light and 1 2 hr darkness. Rats were injected intraperitoneally with 0.5 ml solutions of 1-HCl in sterile water (2.1 to 5.8 mg/injection/animal). Urine samples were pooled, divided in half, hydrolyzed with HC1 (for I ) , or enzyme (for 2 and 31, and analyzed according to the methods described above. Feces samples were hydrolyzed with 4N HC1 in all cases then analyzed as previously indicated.
RESULTS AND DISCUSSION A time-course analysis of the reaction of BSA with 1-HC1 is given in Figure 1. T h e reaction was performed at room temperature and mixtures were shaken after each injection. Curves of similar shape were obtained for concentrations of 1-HC1 ranging from 0.10 to 5.00 mg/ml. T h e chemical identity of t h e peak obtained in t h e GC analysis of BSA-treated
samples of 1-HCl was established by means of GC/MS. Only one sample derived component was detected when total ion current was monitored over time, therefore confirming t h e homogeneity of the derivatized sample (see Figure 2). Mass spectra recorded from various portions of the single peak of the total-ion current chromatogram showed t h e compound t o have a parent ion a t m / e 411, in accord with the molecular weight of the 0,O- bis(trimethylsilyl) ether of 1. No peaks or responses were observed for a mono-silyl ether of 1, which would have a parent ion at mle 339. When 1-HC1 or its free base are reacted with BSA, the same results are observed. This derivatization procedure, therefore, has particular utility since standard curves can be obtained with solutions prepared with t h e hydrochloride salt (actually t h e hydrochloride hemihydrate) of 1, thereby obviating a n extraction step in this portion of the determination. Since apomorphine free base is quite unstable in solution, this fact is especially fortuitous. Persumably, the hydrochloric acid and water components of the standard reference 1 are consumed by the BSA reagent forming lower molecular weight materials (e.g., trimethyl silanol) that co-chromatograph with BSA. Standard Curves. Standard curves were prepared by plotting GC response as a function of amount of 1 chromatographed (Figure 3). T h e range over which linearity was observed coincides with levels of 1 that are likely to arise in extracts obtained during in vivo metabolism studies. At the range and attenuation used in preparing these plots (see Experimental), concentrations as low as 60 ng of 1 could be detected. Correlation coefficients were consistently found t o be equal t o or greater than 0.997. After repeated injections of BSA solutions, however, the slopes of these curves decreased, presumably because of t h e build u p of silicon dioxide deposits in the detectors since disassembly and cleaning completely restored original responses. Recovery of 1 as a Function of pH. Earlier experiments indicated t h a t 1 is essentially quantitatively recovered from buffer at p H 7.0 by extraction with ethyl acetate (5, 8, 15). During the current studies, it seemed useful t o determine the width of the p H range over which quantitative recovery is affected. As indicated in Figure 4, maxim u m recovery of 1 (97.0-99.4%) occurred only over the p H range of 6.99 t o 7.18. Both TES and imidazole buffers provided identical results over this range. Furthermore, recovANALYTICALCHEMISTRY,
VOL. 47, NO. 8, JULY 1975
1323
Table 1. Accuracy and Precision of GC Determination of Apomorphine i n Urine Percent recoveryb
Concn of apomorphine, m g l m l a
0.01 0.05-0.2
Mean
Std dev
76.9'
2.5 3.8
Range
73.6-82.7 80.6100.0
88.5d
In spiked one-day rat urines (mean volume, -10 mlj. In unhydrolyzed urine; values consistently decreased 10% by acid hydrolysis (see Experimental). 8 determinations. 34 determinations ( N = 7 to 10 at levels of 0.05, 0.08, 0.1, and 0.2 mg/ml).
Figure 5. GC of compound 6 and the TMS derivatives of 1, 2, 3, and 4
-
Urine
Table 11. Determination of Apomorphine 0-Methyl Metabolites i n Urine Following Enzymatic Hydrolysisa
Sample
1
divtde
two
H' hydro1
Adjust to pH 7 0
Adjust
Extr w EtOAc
Ag
discard
GC
I
BsA
?
Compound
mg/mlb
2 3
0.06 0.05
$-glucuronidase to p H 8 . 6
I'.INH
Extr w
Range
82.5-88.3 69.5-79.9
Mean
Std dev
85.1' 75.5d
2.4 3.8
Urine samples assayed after 20-hr incubation with d-glucuronidase-arylsulfatase preparation. In one-day rat urines (mean volume, -10 mlj. 4 determinations. 5 determinations. (1
Org Extr
I
Percent recovery
Concn, in
Aq - d i s c a r d
Org E x t r
(
I GC,
2
and
S
Figure 6. Selective determination of 1, 2, and 3 in urine
Table 111. Determinations of Apomorphine and its 0 - M e t h y l Metabolites in Feces Percent recovery
eries were essentially the same over the concentration range, 0.01 to 0.2 mg l/ml of buffer (the range of concentrations expected to occur in urine of rats during metabolism studies). I t should be noted t h a t recoveries of 1 were considerably poorer when phosphate buffers were employed. This fact may be a source of apparent losses of 1 in recovery experiments with biological media (see below). T h e narrow pH range required for maximum recovery of 1 reflects the amphoteric character (involving ionization of the tertiary amine and phenolic groups, respectively, in acidic and basic media) of this compound. In addition, the recovery of 1 from aqueous solutions above p H 7.0 is complicated by its facile decomposition a t alkaline pH's (16). Indeed, solutions of 1 exceeding pH 7.2 rapidly acquire a blue-green coloration indicative of aporphine oxidation (one product of which is compound 5 ) (16). Determination of 1 i n Urine. Results of determinations of 1 spiked a t several levels in one-day r a t urines appear in Table I. As should be noted, consistently higher recovery va!ues were obtained a t levels of 0.05 to 0.2 mg/ml compared to the 0.01 mg/ml concentration. Precision a t all levels, however, was satisfactory. Determinations of 1 following acid hydrolysis (as might be performed to free apomorphine from its 0-glucuronide conjugates) according to the procedure of Kaul e t al. (15)consistently decreased recoveries by ten percent. Hydrolysis of urine specimens with a p-glucuronidase preparation (see Experimental) produced a substance that caused an interference in the GC determination of 1. Determination of 1 in the P r e s e n c e of Its 0 - M e t h y l Metabolites. As indicated in Figure 5 , the T M S ether derivatives of 2 and 3 co-chromatograph but the composite peak is well separated from 7. Thus, while analyzing 1 in urine, one can a t the same time screen specimens for its 0methyl metabolites, 2 and 3. If specific determination of 2 and 3 is desired, then a modified procedure must be utilized. As indicated in Figure 6, this operation can be affected by initially splitting a urine specimen in two, analyzing one portion for 1 as indicated above and analyzing the second for 2 and 3 as described previously ( 4 ) . Since hy1324
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
Cornpa
1
2 3
hlethod
A, B, C, D, E, E,
liq-liq, EtOAcb continuous liq-liq, EtOAcd continuous liq-liq, CHC1,I continuous liq-solid, EtOAc' liq-liq, 1%INHi liq-liq, 1% INHi
hlean
Std dev
14.8'
...
27.4'
4.05 (6.60)
33.1g 35.6' 39.0' 63.1'
... ... .. .
1-mg quantities of 1-HC1, 2-HCI and/or3-HCl spiked into 12-hr rat feces (mean dry weight, -2.0 gj. Acid hydrolysate adjusted to pH 7.0 and extracted with five 5-ml portions of ethyl acetate. 2 determinations. Continuous ethyl acetate extraction of neutralized acid hydrolysate for 24 hr. e 8 determinations. f Continuous chloroform extraction of neutralized acid hydrolysate for 24 hr. g 3 determinations. Continuous ethyl acetate extraction (liquidsolid; soxhlet) of lyophilized residue of acid hydrolysate for 24 hr. z Acid hydrolysate adjusted to p H 8.6 and extracted with five 5-ml portions of 170I K H .
drolysis conditions for glucuronide conjugates of 2 and 3 were not formerly described ( 4 ) they were investigated in this study. In contrast to the results found with 1, hydrolysis with P-glucuronidase provided the least background interference in the GC determination of 2 and 3 after extraction a t p H 8.6 with 1%INH. Recovery and precision data for 2 and 3 using the procedure outlined in Figure 6 are given in Table I1 and compare favorably with those published previously ( 4 ) for analyses in unhydrolyzed urine samples. Determination of I , 2, a n d 3 in Feces. Gas chromatographic development of feces extracts obtained in a similar fashion to those from urine (see Experimental and Figure 6) revealed no interference a t a retention time coincident with that of 7. However, as indicated in Table 111, poor recoveries were obtained. The development of a suitable assay for 1 in feces might be facilitated by use of an appropriate internal standard. Experiments aimed at the synthesis and evaluation of potentially suitable compounds are currently being pursued in these laboratories. A marginally acceptable assay for 2 and 3 was possible by GC ( 4 ) of 1%INH extracts of feces samples previously hy-
Table IV. Excretion of Apomorphine in Urine of Sprague-Dawley Rats Administered 1-HC1 Amt of Np. of lrHCl ( m ) animals lnlected? Exp. No. injected animal
1 2= 3 4 5 6e
ve
2 2 3 3 3 3 3
2.1 3 .O 4.1 5.2 5.8 4.2 5.1
Amount of 1" (mg) excreted in w i n e 0-24 k
0.37 0.86 0.92
0.82 1.62 1.92 2.13
24-28 k
... ...
48-72 hr
...b
...
d
0.22 0.22 0.29
...
b
...b
... ... 0.11 ... d
...
During the development of the GC method for 1, compounds 4 (as its T M S derivative) and 6 were found to chromatograph with retention times different than those obtained for the T M S derivatives of 1,2, and 3 (see Figure 5 ) . Attempts were made to detect compounds 4 and 6 in hydrolyzed urine samples derived from rats injected with 1HC1. However, neither 4 nor 6 was observed in 72-hour samples.
d
CONCLUSION
b
b
a No more than trace levels of free 1 were detected in any experiment; values obtained from urine hydrolyzed to free 1 from its conjugates (probably glucuronides, see Ref. 18). Samples not examined. < In this experiment only, a 5.6% excretion of 1 as apocodeine was determined (qualitatively by TLC (Ref. 2 ) and GC (Ref. 4 ) ; quantitatively by GC ( 4 ) ) . No apomorphine detected. e During these experiments, animals drank 1%ammonium chloride solutions ad libitum in lieu of water.
drolyzed in 4N hydrochloric acid and subsequently adjusted to p H 8.6. Though recoveries are low (as indicated in Table 111), approximately 5 to 10% conversions of 1 to 2 or 3 could be detected in feces, assuming principal elimination via this route and dose levels of 16 t o 20 mg/kg of 1 in rats. Metabolism Studies. The developed GC methods were employed in determining amounts of I (combined free and conjugate) excreted in the urine of Sprague-Dawley rats injected with 1-HCl. Results of these investigations appear in Table IV. Over all, the values obtained are about one-half those reported by Kaul e t al. (15, 17) for a different inbred strain (Long Evens) of rat. Interestingly, significant increases in elimination of apomorphine in urine were observed in animals whose urinary p H was decreased by oral administration of ammonium chloride. This effect has also been observed in rabbits (15). The amount of I excreted over 72 hours by Sprague-Dawley rats seemed to be independent of dosage over the range studied (see Table IV). In the above experiments, the identity of 1 excreted in urine was confirmed by GC/MS of its T M S derivative. In one experiment, 2 was detected as a urinary metabolite of 1 (see Table IV). These results along with those of related experiments will be the subject of a future report. Pooled feces (collected for 72 hours following injection of 1-HC1) were examined for compounds 1, 2, and 3. Compound 1 was detected while its 0-methyl metabolites, 2 and 3, were not observed, within the limits of detections for these compounds.
GC methods have been developed for determining apomorphine and some of its potential metabolites in urine. Further work is required t o devise reliable assays for these compounds in feces. T h e methods described should permit further investigation of the metabolic fate of apomorphine in mammals.
ACKNOWLEDGMENT We are grateful t o Linda M. Lizak for excellent technical assistance during certain stages of this work. We also wish to express our thanks to Joseph G. Cannon of the University of Iowa for a gift of norapomorphine.
LITERATURE CITED (1) R. V. Smith and S. P. Sood. J. Pharm. Sci., 60, 1654 (1971). (2) J. G. Cannon, R. V. Smith, A. Modiri, S. P. Sood, R. J. Borgman. M. A. Aleem, and J. P. Long, J. Med. Chem., 15, 273 (1972). (3) R. V. Smith and M. R. Cook, J. Pharm. Sci., 63, 161 (1974). (4) R. V. Smith and A. W. Stocklinski, J. Chromatogr., 77, 419 (1973). (5) R. V. Smith, M. R. Cook, and A. W. Stocklinski, J. Chromatogr., 87, 295 (1973). (6)P. N. Kaul, E. Brochmann-Hanssen, and E. L. Way, J. Am. Pharm. Assoc., Sci Ed.. 48, 638 (1959). (7) M. P. Cava, A. Venkateswarbu, M. Srinivasan, and D. L. Edie, Tetrahedron, 28, 4299 (1972). (8) R. V. Smith and S. P. Sood, Anal. Lett.,5, 273 (1972). (9) K. Rehse and G. Dreke, Fresenius'Z. Anal. Chem., 248, 179 (1969) (10) K. Rehse, Arch. Pharm. (Weinheim),302, 487 (1969). (11) K. Rehse. Arch. Pharm. (Weinheim),305, 625 (1972). (12) W. K. VanTyleand A. M. Burkman, J. Pharm. Sci., 60, 1736 (1971). (13) K. Parker. C. R. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (14) M. V. Koch, J. G. Cannon, and A. M. Burkman, J. Med. Chem., 11, 977 (1968). (15) P. N. Kaul, E. Brochmann-Hanssen. and E. L. Way, J. Pharm. Sci.. 50, 244 (1961). (16) H. H. A. Linde and M. S. Rageb, Helv. Chim. Acta, 51, 683 (1968). (17) P. N. Kaui, E. Brochmann-Hannssen, and E. L. Way, J. Pharm. Sci, 50, 248 (1961). (18) P. N. Kaul, E. Brockmann-Hanssen, and E. L. Way, J. Pharm. Sci., 5 0 , 840 (1961).
RECEIVEDfor review December 23, 1974. Accepted March 12, 1975. This work was supported in ?art by grants NS04349 and NS-12259, National Institute of Neurological Diseases and Stroke.
Chromatographic Determination of Phenols in Water Colin D. Chriswell, Richard C. Chang, and James S. Fritz Ames Laboratory-USAEC and Department of Chemistry, lowa State University, Ames, /A 500 10
Phenols in natural waters and treated drinking water are determined by sorption on macroporous anion-exchange resin, elution with acetone, and measurement by gas chromatography. Techniques are given for preventing phenol losses caused by chlorination, oxidation, and other reactions during their determination. Common inorganic ions and many organic substances cause no interference; neutral or-
ganics that are retained by the resin can be removed by a methanol wash. The method gives accurate results for phenol, alkyl-, and chloro-substituted phenols in the ppb to ppm concentration range.
As part of an extensive effort to develop improved, practical methods f o r determining trace concentrations of orANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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