Quantitative Gas Chromatographic Analysis of Leucine Enantiomers. A Comparative Study William A. Bonner, Mark A. Van Dort, and Jose J. Flores Department of Chemistry, Stanford University, Stanford, Calif. 94305, and Ames Research Center, NASA, Moffeff Field, Calif. 94035
Three general methods for the gas chromatograhic resolution of amino acid enantiomers are described in the literature: l)conversion to N-TFA-L-prolyl dipeptide methyl ester diastereomers, or to 2) N-TFA-( +)-2-butyl ester diastereomers, followed by separation on conventional stationary phases, and 3) conversion into N-TFA-symmetrical alkyl ( e.g., 2-propyl) ester enantiomers, followed by separation on optically active stationary phases. Our need to develop a precise and reliable method for the quantitative gas chromatographic estimation of the full range of compositions of enantiomeric mixtures of amino acids has prompted us to examine these three methods in detail experimentally, using mixtures of leucine enantiomers as prototypes. Each method shows comparable accuracy (0.03-0.7 % absolute error) and precision (0.03-0.6 % standard devlation) when applied to leucine mixtures of known enantiomeric composition. Evidence is presented indicating the absence of racemization during the preparation of the N-TFA esters involved in methods 2 and 3.
The resolution of amino acid enantiomers by gas chromatography was first suggested less than a decade ago on the basis of two general techniques, each involving prior conversion of the enantiomers to a mixture of volatile diastereomers. In 1965 Pollock, Oyama, and Johnson ( 1 ) converted several racemic amino acids into their N- trifluoroacetyl-(f)-2-butyl ester diastereomers (I) and showed that these could be separated with reasonable completeness on both capillary and packed columns loaded with several conventional stationary phases. Concurrently and indepenFCCOXH
CH,
I
RC*HCOOC*HR' I
I. R'=CHICH? I1 R = C H (CH,),
-
R
C?.TACOZH&HCOOCHI
I
F,CCO
111
dently Gil-Av and coworkers ( 2 ) extended this method to embrace N - TFA-2-octyl esters (11) as well, demonstrating the diastereomer separations for I and I1 again with totally racemic systems. In the same year, Halpern and Westley ( 3 ) introduced the use of diastereomeric N- TFA-L-prolyl methyl ester derivatives of amino acids (111) for the actual partial resolution of racemic proline, leucine, valine, and alanine, using columns packed with 5% SE-30 phase. The early applications of these techniques have recently been reviewed ( 4 ) ,and more recently the N-TFA-2-butyl ester diastereomer procedure has been used to examine the amino acid content of hydrolysates from certain geological (5-7) and marine (8)sediments as well as to various mete(1) G. E. Pollock, V. I. Oyama, and R. D. Johnson, J, Gas Chromatogr., 3, 174 (1965). (2) E. Gil-Av, R. Charles-Sigler, and G. Fischer, J. Chromatogr., 17, 408 (1965). (3) B. Halpern and J. W. Westley. Biochem. Biophys. Res. Commun., 19, 361 (1965); 20, 710 (1965). (4) W. A. Bonner, J. Chromatogr. Sci., 10, 159 (1972).
2104
orite specimens (9-14), to study the occurrence and racemization of amino acids in bone fossils ( 1 5 ) and soil samples (16, 1 7 ) and to determine the configuration of alloisoleucine in the serum of a patient suffering from branched chain ketouria (181, a metabolic disease. This procedure has also been suggested ( 1 7 ) as part of a wet chemical probe for the detection of extraterrestrial life in future space exploration. Shortly after the development of the diastereomer technique, Gil-Av and coworkers (19) introduced still another method for the gas chromatographic resolution of amino acids, namely, the direct separation of volatile enantiomeric derivatives on high molecular weight optically active stationary phases (such as N - TFA-L-isoleucine lauryl ester (19) and the dipeptide derivative, N- TFA-L-valyl-L-valine cyclohexyl ester ( 2 0 ) ) .Subsequently, additional N - TFAL-L-dipeptide ester derivatives have been synthesized and evaluated (21) as optically active stationary phases both from practical and theoretical viewpoints, and very recently N- lauroyl-L-valyl-tert- butylamide has been recommended (22,23)as a superior stationary phase for the resolution of racemic N- TFA-amino acid esters. In connection with other areas of our current research ( 2 4 ) ,we have been confronted with the general problem of determining the exact enantiomeric composition of certain individual amino acids in the presence of unspecified quantities of extraneous byproducts, either optically active or not. The customary determination of enantiomeric compo-
(5) K. A. Kvenvolden, E. Peterson, and G. E. Pollock, Nature (London), 221, 141 (1969). (6) K. A. Kvenvolden, E. Peterson, and F. S. Brown, Science, 169, 1079 (1970). (7) K. A. Kvenvolden. E. Peterson, and G. E. Pollock, Advan. Org. Geochem., 387 (1972). (8) K. A. Kvenvolden, E. Peterson, J. Wehmiller, and P. E. Hare, Geochim. Cosmochim. Acta, 37, 2215 (1973). (9) J. G. Lawless, K. A. Kvenvolden, E. Peterson, C. Ponnamperuma, and E. Jarosewich, Nature (London), 236, 66 (1972). (10) J. G. Lawless, K. A. Kvenvolden, E. Peterson, C. Ponnamperuma, and C. Moore, Science, 173, 626 (1971). (11) K. A. Kvenvolden, J. Lawless, K. Pering, E. Peterson, J. Flores, C. Ponnamperuma, I. R. Kaplan, and C. Moore, Nature, (London), 228, 923 (1970). (12) K. A. Kvenvolden, J. G. Lawless, and C. Ponnamperuma, Proc. Nat. Acad. Sci. U.S., 68, 486 (1971). (13) G. E . Pollock, Anal. Chem., 44, 2368 (1972). (14) J. G. Lawless, Geochim. Cosmochim. Acta, 37, 2207 (1973). (15) J. L. Bada, K. A. Kvenvolden, and E. Peterson, Nature (London), 245, 308 (1973). (16) G. E. Pollock and L. H. Frommhagen, Anal. Biochem., 24, 18 (1968). (17) G. E. Pollock, A. K. Miyamoto, and V. I. Oyama, Life Sci. Space Res., VIII, 99 (1970). (18) B. Halpern and G. E. Pollock, Biochem. Med., 4, 352 (1970). (19) E. Gil-Av, B. Feibush, and R. Charles-Sigler, Tetrahedron Lett., 1009 (1966). (20) S. Nakaparksin, P. Birrel, E. Gil-Av, and J. Oro, J. Chromatogr. Sci., 6, 177 (1970). (21) See W. Parr and P. Y. Howard, Anal. Chem., 45, 71 1 (1973) for earlier references. (22) B. Feibush, Chem. Commun., 544 (1971). (23) E. Gil-Av. Addresses to the "4th International Congress on the Origin of Life," Barcelona, Spain (June 1973) and to the "International Symposium on Generation and Amplification of Asymmetry in Chemical Systems," Julich, Fed. Rep. Germany (September 1973). (24) W. A . Bonner, J. Chromatogr. Sci., 11, 101 (1973).
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 14, DECEMBER 1974
sition based on optical rotation (or rotary disperson) was here inapplicable, because of the small quantities of product often available, the highly impure nature of many of the samples investigated, and the likelihood of the presence of optically active contaminants. There are fortunately two analytical methods which are potentially capable of circumventing all of these difficulties-namely, quantitative gas chromatography utilizing one of the above resolution techniques, and quantitative resolution on an amino acid analyzer (25). Each method copes adequately with minute samples, and impurities are generally excluded automatically either in the derivatization procedure or during the course of the subsequent analysis itself. Between the two methods, we chose to investigate gas chromatography as being simpler, faster, less expensive, and potentially more accurate. A precise quantitative evaluation of the above gas chromatographic resolutions of amino acids has not hitherto been undertaken. T o be sure, many of the above applications of the N-TFA-(+)-2-butyl ester diastereomer technique to geological or meteoritic samples have included an approximate estimation of enantiomeric ratios based on the simple criterion of relative peak heights, but the validity of these estimates has not been tested. In 1963 Weygand and coworkers (26),investigating racemization during peptide synthesis, found with known mixtures of N - TFA-Lvalyl-D-valine and N - TFA-L-valyl-L-valine methyl esters an exact correspondence between the ratios of gas chromatographic peak areas and the weight ratios of the diastereomeric components, and in 1966 Gil-Av and coworkers (27) analyzed an N-TFA-(-)-Z-octyl amino acid ester mixture containing 40.3% L-valine and 12.3% D-valine (along with D,L-leucine and D,L-isoleucine) gas chromatographically, finding an enantiomeric composition of 37.1% L- and 13.9% D- for the valine components (3.2% and 1.6% total error, respectively). Gil-Av (27) also analyzed diastereomeric mixtures of some 17 lactic acid ester derivatives and compared the percent compositions observed gas chromatographically with those measured polarimetrically. Discrepancies ranged from 0.0 to 5.5%, with an average of 1.9%. Finally, Vitt et al. (28) analyzed known enantiomeric mixtures of N - TFA-(-)-methyl esters of valine, alanine, and leucine on a 4-m 5% PEGA column, finding in 12 analyses errors ranging from 0.0 to 2.0% and averaging 1.1%. For our studies, we felt that we required an analytical precision of &0.5% or better in determinations of the enantiomeric compositions of amino acid mixtures. We have accordingly undertaken a quantitative evaluation of the three most widely used techniques for the gas chromatographic resolution of amino acids (ie., using the diastereomeric derivatives I and I11 as well as the most promising optically active stationary phase (22, 23)) to see if such precision might be feasible. T o this end we have already reported ( 4 ) on the experimental conditions required to prepare NTFA-L-prolyl-D-(or L)-leucine methyl ester derivatives (111: R = (Ch3)2CHCHz) quantitatively and without racemization and on the quantitative gas chromatographic analyses of such diastereomer mixtures, where an analytical precision of 0.6% or better proved attainable (see Table I). We now extend this study to include the N-TFA-(+)-2butyl ester diastereomers of leucine (I, R = (CH:j)&HCH2), a n d to the analytical resolution of NJ. M. Manning and S. Moore, J. Bo/.Chem., 243, 5591 (1968). F . Weygand, A . Prox, 1.. Schmidhammer.and W. Koenig, Agnew. Chem. Int. Ed. €ng/., 2, 183 ( 1963).
E . Gil-Av. R . Charles-Sigler, G. Fischer, and D. Nurok, J. Gas Chromatogr., 4, 51 (1966). S . V. Vitt. M . €3. Saporowskaya,I. P . hedron Lett, 2575 (1965).
Gudkova, and V. M. Belikov, Tetra-
Table I. Gas Chromatographic Analyses of D- and LLeucine Mixtures Using Volatile Diastereomeric or Enantiomeric Derivatives Absolute % D-Leu ( h 0 - 1 ) ~
% D-Leu (found)a
A . “-TFA-L-Prol,l-Leucine
10.77
20.76 30.72 40.65 50.55 60.42 70.26 80.07 89.85
Uo. of a n d ‘
error,
(t),
Methyl Ester Diastereomers
10.42 20.51 30.91 40.70 51.14 60.49 70.78 80.73 90.04
0.11
0.35 0.25 0.19 0.05 0.59
0.10 0.04
0.08 0.06 0.57
2 2 2 2 2 3 2 2 2
0.07 0. 52
0.05 0.09 0.03
0.66 0.09 A V 0.31
T
0 23h
B. + - T F A - L e u c m e - ( t )-Z-Butyl Ester Diastereomers
20.48 35.55 50. OOd 63.98 78.85 C.
20.35 35.06 49.97 63. 58 78.63
0.20
0.13
0.08
0.49 0.03
0.03 0.21 0.11
5 5
4
0.40
5
0.22 Av 0.25
5 0.19
V-TFA-Leucine-2-Propyl Ester Enantiomers
11.44 33.83 50. OOd 66.28 89.66
10.98 33.13 50.12 66.93 89.88
0.29 0.36 0.17 0.26 0.29
0.46
7 7 3
0.70 0. 12 0.65 0. 2 2 A v 0.43
7 6 i
0.26
70L-Leu = 100.00 - % o-Leu. Standard deviation Number of separate GC analyses on the same sample d Prepared from D,L-Leu
TFA-leucine isopropyl ester enantiomers on optically active N - lauroyl-L- valyl-tert -butylamide phase.
EXPERIMENTAL N-TFA-L-Prolyl-D(or L)-Leucine Methyl Esters (111, R = isobutyl). These derivatives were prepared as previously described ( 4 ) , then were dissolved in methanol to make solutions of accurately known concentrations. Carefully measured aliquots of each solution were mixed to give a series of solutions having the range of known enantiomeric compositions seen in column 1 of Table I(A). These known mixtures then were analyzed as described earlier(4 ), such that the two diasteriomeric derivatives were separated with baseline resolution, and with a peak maxima separation of CQ. 3 min. Peak areas for quantitative estimation of diastereomer composition (Table I(A), column 2) were measured using a Hewlett-Packard Model 3370A electronic digital integrator. N-TFA-D(or ~)-Leucine-(+)-2-ButylEsters (I, R = isobutyl). Samples of D- and L-leucine were carefully weighed and combined to give mixtures having the compositions shown in column 1 of Table I(B). Approximately 100 mg of each mixture was treated with 1 ml of a saturated solution of hydrogen chloride in ( + ) - 2 butanol (Norse Laboratories, Inc., Santa Barbara. Calif.; [cY]D*” + 13.42’, corresponding to 98.27% (+) and 1.73% (-) enantiomer) (mole ratio BuOH/Leu ca. 15:1), and the mixture was heated under reflux (CaC12 tube) for three hours. The solvent was removed under vacuum with a rotary evaporator (50’) and the residue was “chased” by adding 5 ml of dichloromethane and vacuum evaporating twice, then was dissolved in dichloromethane ( 2 ml), and was treated with trifluoracetic anhydride ( 3 ml). The solution was heated under reflux for 15-30 minutes, then stripped of solvent under vacuum. The residue was “chased” twice as before, weighed (98-100% crude yield), and dissolved in nitromethane to give a CQ. 0.15M solution. Such solutions were diluted 1:10 with nitromethane for analysis. The above procedure is a modification of that of Pollock e t a / . ( I ) .
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50.00% 50.00%
49.81%
ANALYSIS:
In
I;
umn packed with 7% N- lauroyl-L-valyl-tert-butylamide phase (Research Division, Miles Laboratories, Inc., Kankakee, Ill.) on 60-80 mesh Chromosorb-W, installed in the above Hewlett-Packard 5700A gas chromatograph. Baseline separation of the enantiomers was achieved (Figure l ( C ) ) operating isothermally at 120' with a helium flow rate of ca. 9 ml/min. The two enantiomer peaks (D, then L) were integrated with the Autolab 6300 electronic integrator, and the resulting analytical data are shown in column 2 of Table I(C). Analogous results have more recently been obtained by us using 150-ft X 0.02-in. capillary columns coated with this same phase (Figure 1(D)).
50.10%
16.8 21.4
25.7 21.0
ELUTION TIME, min
COLUMN
A
B
C
D
Figure 1. Resolution of D, L-Leucine derivatives on several gas chromatographic phases. ( A ) KTFA-(+)-P-butyl ester on 1504 X 0.02-in. column coated with Carbowax 20 M phase; 95 to 120' at 2'1min program: He, 5 ml/min: chart 0.5 inhin. (6)KTFA-(+)-P-butyl ester on 1504 X 0.02-in. column coated with Ucon 75H 90M phase; 110' isothermal: He, 3 ml/min; chart 0.25 in./min. (C) KTFA-isopropyl ester on 7-R X %&. column packed with 7% Klauroyl-Lvalyl-tert-butylamide on Chromosorb-W; 120' isothermal: He, 9 ml/min: chart 0.1 in./min. (D)KTFA-isopropyl ester on 1504 X 0.02-in. column coated with Klauroyl-L-valyl-tert-butylamide phase: 109' isothermal; NP, 5 ml/min; chart 0.25 in./min.
Gas chromatographic analyses on these and similar samples have been conducted using 150-ft X 0.02-in. capillary columns coated either with Carbowax 20M phase (Applied Science Laboratories, Inc., State College, Pa.) or with Ucon 75H 90M phase (Perkin-Elmer Corp., Norwalk, Conn.). The former column, providing the data in Table I(B), was used isothermally at 122' or temperature programmed from 95' to 120' at 2'/min with a helium flow of 3-4 ml/min, giving base-line diastereomer peak separations (D(+), then L ( + ) ) as shown in Figure 1(A). The Ucon column was used isothermally a t 110' with a helium flow of 3 ml/min to achieve similar peak separations (again D ( t ) , then L ( + ) ) , as seen in Figure 1(B). The columns were installed in a Hewlett-Packard 7500A gas chromatograph, and peak area integration was accomplished with an Autolab 6300 digital electronic integrator, while monitoring with the aid of a Varian A25 20-speed recorder. The analytical data in Table I(B) have been corrected for the 1.73% (-)-2-butanol present in the derivatizing alcohol. Non-Racemization d u r i n g P r e p a r a t i o n of N-TFA-L-Leucine-(+)-2-Butyl Ester. L-Leucine (98.6 mg) in (+)-2-butanol saturated with hydrogen chloride (1 ml) was heated under reflux as above. After one hour, a 0.15-ml aliquot was removed and trifluoracetylated by processing as above, affording 30.5 mg (95.4%) of crude product. Similar aliquots were removed after three and seven hours and treated identically, yielding 30.6 mg (95.7%) and 31.2 mg (97.6%) of crude product, respectively, In a separate experiment, 100.5 mg of L-leucine and 1 ml of HC1-saturated (+)-2-butanol were refluxed for 23 hours, then processed similarly to yield 219.7 mg (101.2%) of crude N-TFA-ester. These products, dissolved as before in nitromethane, were analyzed as above using the Carbowax 20M column. The percentage of L-leucine derivative (corrected for 1.73% (-)-2-butanol in the starting 2-butanol) found in each sample was: 1 hour, 99.74 f 0.02%; 3 hours, 99.94 f 0.06%; 7 hours, 99.81 & 0.08%;23 hours, 99.31 f 0.17%. N-TFA-D(or L)-Leucine 2-Propyl Esters. Mixtures of carefully weighed quantities of D- and L-leucine (totalling ca. 120 mg; column 1, Table I(C)) were treated with 6.9-ml portions of a saturated solution of hydrogen chloride in 2-propanol (mole ratio 2PrOH/Leu ca. 71:l) and the solutions were heated under reflux for three hours. The solvents were stripped and the residues were trifluoracetylated as described above, ultimately affording 75-80% yields of the desired enantiomeric ester mixtures, which were dissolved in nitromethane to make 0.1M solutions. Gas chromatographic analyses of these were conducted using a 7-ft. X 'h-in. col2106
RESULTS AND DISCUSSION Table I indicates that the three most important methods in the literature for the resolution of amino acid enantiomers gas chromatographically can, with electronic peak area integration, be adapted to the quantitative estimation of the enantiomeric composition of D- and L-leucine mixtures with both comparable accuracy (ca. 0.03-0.7% absolute error) and comparable reproducibility (ca. 0.03-0.6% standard deviation in replicate analyses). Our choice of leucine as the amino acid to investigate in this study was dictated by our use of leucine as a substrate in a number of other investigations ( 2 4 ) and the consequent need to evaluate our analytical techniques in terms of this particular amino acid. While the use of other synthetic conditions and/or gas chromatographic parameters would undoubtedly be necessary in applying these techniques to other amino acids, there is every reason to believe that similar enantiomeric mixtures of other amino acids could be analyzed with comparable precision (26, 2 9 ) . As to a choice between the three resolution methods depicted in Table I, there is clearly little reason for a preference in terms of analytical precision, a t least as regards leucine. Our earlier study ( 4 ) of the N-TFA-L-prolyl methyl ester procedure, however, has emphasized the ease of racemization of the N - TFA-L-prolyl chloride reagent, both during its preparation and in its subsequent reaction with leucine methyl ester hydrochloride in the presence of triethylamine, and has indicated the resulting analytical errors which such racemization could engender. For this reason, as well as the laborious procedures needed to circumvent this difficulty ( 4 ) , we feel that the N - TFA-L-prolyl dipeptide technique suffers in comparison with the other analytical methods shown in Table I. Our decision ( 4 ) to investigate the N - TFA-L-prolyl dipeptide procedure initially, instead of the (+)-2-butyl ester method, was dictated by the supposition that the latter procedure might engender some racemization of the amino acid due to the action of the hydrogen chloride catalyst in the refluxing (+)-2-butanol (bp 100') reagent. This possibility, which appeared to have some precedent, a t least, in the known (30) racemization of amino acids with hot aqueous hydrochloric acid, has never been tested experimentally. This same possibility of racemization applies, of course, to the preparation of enantiomeric N - TFA-2-propyl esters for analysis on optically active stationary gas chromatographic phases, and here also could lead to unexpected analytical errors. For these reasons, we have investigated this possibility of racemization by subjecting L-leucine to the refluxing (+)-8-butanollHC1 esterification step for increasing lengths of time, reasoning that if racemization occurred, the amount of D-leucine ester found in the final product should increase regularly with time. The amounts of D-leucine ester present (as determined gas chromatographically) after 1, 3, 7 , and 23 hours were 0.26, 0.06, 0.19, and 0.69%, respectively, indicating that no detectable racemization at(29) W. A. Boner and J. Flores, Currents Mod. Biol., 5, 103 (1973). (30) S. Nakaparksin. E. Gil-Av, and J. Oro, Anal. Biochem., 33, 374 (1970).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 14, DECEMBER 1974
tended the conversion of L-leucine into its N-TFA-(+)-2butyl ester during the customary 3-hour reflux period, and that only negligible (less than 1%) racemization occurred even after 23 hours.
RECEIVEDfor review May 31, 1974. Accepted August 14, 1974. We are indebted to the Nationai Aeronautics and Space Administration for a Research Grant (No. NGL-05020-582) which supported a portion of this work.
Determination of Morphine in Biologic Fluids by Electron Capture Gas-Liquid Chromatography Jack
E. Wallace and Horace E. Hamilton
Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Kenneth Blum Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Clayton Petty Division of Anesthesiology, University of Arizona College of Medicine, Tucson, Ariz. 8572 1
A method that permits the quantitative determination of morphine at therapeutic levels in 1-2 mi of serum or plasma is described. Morphine levels at less than twenty-five nanograms per mi can be effectively assayed providing an internal standard of nalorphine is employed. Both the opiates are measured by electron capture detection (63Ni) as their respective trifluoroacetyl derivatives. Sensitivity of the technique is sufficient to permit the forensic scientist to establish the cause of death in “opiate sensitivity reactions” as well as those intoxications that involve high blood levels of morphine. The procedure has sufficient reliability for utilization in pharmacokinetic studies of morphine.
Eddy e t al. (1) described a quantitative gas-liquid chromatographic determination of morphine in 1961, one year following the demonstration by Lloyd et al. (2) that morphine analysis by gas-liquid chromatography was feasible. A plethora of gas chromatographic procedures for the detection of morphine has subsequently been introduced to the scientific literature. The earlier methods were directed to chromatographic separation of the free base (3-10) and explored the use of various column packings and treatment, but wereulimited by the nonlinear adsorption of the compound on the column. Investigators have derivatized morphine with a variety of reagents, achieving both superior chromatographic characteristics and enhanced sensitivity. Several authors utilized on-column derivatization techniques, forming morphine (1) N. B. Eddy, H. M. Fales, E. Haahti, P. F. Highet, E. C. Horning, E. L. May, and W. C. Wildman, United Nations Secretariat, ST/SOA/SER.K/114/ Corr. 1, Oct. 1961. (2) H. A. Lloyd, H. M. Fales, P. F. Highet, W. J. A. Vanden Heuvel, and W. C. Wildman. J. Amer. Cbem. Soc., 82, 3791 (1960). (3) L. Kazyak and E. C. Knoblock, Anal. Chem., 35, 1448 (1963). (4) K. D. Parker, C. R. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (5) E. Brochmann-Hanssen and T. Furuya, J. Pharm. Sci., 53, 1549 (1964). (6)J. L. Massingill, Jr., and J. E. Hodgkins, Anal. Cbem., 37, 952 (1965). (7) E. Brochmann-Hanssen and C. R. Fontan, J. Cbromatogr., 19, 296 (1965). (8) E. Brochmann-Hanssen and C. R. Fontan, J. Cbromatogr., 20, 394 (1965). (9) C. McMartin and H. V. Street, J. Cbromafogr., 22, 274 (1966). ( I O ) H. V. Street, J. Chromatogr., 29, 68 (1967).
acetate or propionate by injecting the appropriate anhydride immediately after the morphine injection (11-13); Anders and Mannering (11) in 1962 extended this technique to include trifluoroacetyl morphine and the trimethylsilyl ether. Most recent work, however, has centered on applications of the pre-chromatographic derivatization of morphine. The application of acetic anhydride to convert the free base to diacetylmorphine (heroin) was introduced in 1966 ( 1 4 ) and subsequently utilized by the authors (1.5). Trimethylsilyl ethers which utilize reagents such as hexamethyldisilazane (HMDS) (16-18), bis(trimethylsily1) acetamide (BSA) (19-20), and the trifluoro analog of BSA (21,22) have proved to be applicable to morphine analysis. Nalorphine, a structural analog of morphine, was observed to be an excellent internal standard for gas chromatographic determination of opiates. Street (10) first used nalorphine as an internal standard in the chromatography of free morphine base. Ikekawa e t al. (20) later demonstrated that nalorphine was excellent for derivatization applications in that the synthetic opiate served as a control for the derivatizing reactions as well as for the chromatographic techniques. With the exception of a few applications of argon ionization detectors (3, 1 6 ) , most gas chromatographic determinations of morphine have utilized flame ionization detectors (FID). Consequently, with morphine derivatives, the sensitivity of published methods is of such low magnitude that many procedures require 10 to 50 milliliters of urine or (11) M. W. Anders and G. J. Mannering, Anal. Chem., 34, 730 (1962). (12) S. J. Mule, Anal. Chem., 36, 1907 (1964). (13) H. W. Elliot, N. Nomof. K. Parker, M. L. Dewey, and E. L. Way, Clin. Pharmacol. Ther., 5 , 405 (1964). (14) E. Schmerzler, W. Yu. M. I. Hewitt, and I. J. Greenblatt, J. Pbarm. Sci., 55, 155 (1966). (15) J. E . Wallace, J. D. Biggs. and K. Blum. Clin. Cbim. Acta, 36, 85 (1972). (16) E. Brochmann-Hanssen and A. 6. Svendsen, J. Pharm. Sci., 51, 1095 (1962). (17) E. Brochmann-Hanssen and A. B. Svendsen, J. Pharm. Sc;., 52, 1134 (1963). (18) G. E. Martin and J. S. Swinehart. Anal. Cbem., 38, 1789 (1966). (19) F. Fish and W. D.C. Wilson, J. Cbromatogr., 40, 164 (1969). (20) N. Ikekawa. K. Takayama, E. Hosoya, and T. Oka, Anal. Biochem.. 28, 156 (1969). (21) G. R. Wilkinson and E. L. Way, Biochem. Pharmacol., 18, 1435 (1969). (22) H. E. Sine, N. P. Kubasik. and J. Waytash. Clin. Chem., 19, 340 (1973).
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