the mutarotation equilibrium but-to the extent that it controls the length of the long cross section of a conformer-could narrow the distribution of the conformer population of the labeled solute in a manner that would enhance the dialysis rate. Such control could account for the isotope effect during dialysis being greater than that observed during countercurrent distribution ( I , 2, 7 ) . An alternate explanation could be advanced if the control extended to the hydration of the conformer or hydration and length of the long axis of it. Such control would relate isotope effects during dialysis to the rate enhancements in reactions (9) that result from structural modifications caused by improvement in the angular relation of nucleophilic and electrophilic centers and exclusion of solvent between reaction partners as well as their desolvation. It would then seem that the inclusion of considerably larger rate changes in theories for kinetic isotope effects than those assumed for molecules that do not change their conformation is mandatory. The difference in the dialysis rates of the a and p anomers can have implications in long term dialysis techniques to correct chemical changes ( I O ) through hemodialysis, because any anomeric imbalance could influence activity of enzymes that are specific for one or the other anomer. Enzymes that catalyze reactions involving carbon 1 of phosphate esters of glucose are known to have such specificity ( 1 1 ) .
4 1.90-
1.80-
1.
m E
.-c E W
a W
u L
W
m 0
A
1.0
Received for review February 16, 1973. Accepted April 13, 1973. This research was supported in part by General Research Support Grant 5Sol-RR05361-10 of the United States Public Health Service and a grant from E. I. Du Pont de Nemours and Company.
1 15
45
75
105
135
Time in min First-order plots of the second diffusate sample of [6-14C]glucose
Figure 5.
D-
The conditions for the dialysis were the same as those for Figure 3 except t h a t t h e 30-min d i a l y s a t e was d i v i d e d i n t o parts, One).-.( was dialyzed at once the other ( B - B ) after anomeric equilibrium. Escape rates, expressed as in Figure 3, for the sample at once and after equilibrium, respectively, are (a) before 60 min, 8.1 X and 6.2 X ( b ) after 60 min, 7.1 X and 4.5 X
(9) Such enhancement is called stereopopulation control by Cohen: S. Milstien and L. A . Cohen, J. Amer. Chern. SOC., 94, 9158 (1972); R . T . Borchardt and L. A . Cohen, ibid., 94, 9166 (1972);ibid., 94, 9175 - - (19771 (lo) P. E, Teschan, Amer. J. Med.. 48,671 (1970). (11 ) M . Salas, E. Vinuela, and A . Sols, J. Bioi. Chern., 240,561 (1965). \
- I .
High Speed, High Pressure Liquid Chromatography of Similar Tropane Alkaloids Martin H. Stutz and Samuel Sass Analytical Chemistry Branch, Chemical Research Division. Chemical Laboratory, Edge wood Arsenal, Aberdeen Proving Ground, Md
21010
Liquid chromatography as a separate analytical technique has seen a rebirth during the past few years. A good summary Of Some Of the recent effort can be found in the compilation by Kirkland (11, and through a Perusal o f t h e recent literature (2-17). This paper deals with the application of high speed liquid chromatography to the separation and quantitative determination of some physically active compounds of similar structure. ~t demonstrates the applicability and usefulness of liquid chromatographS for differentiating between compounds which might have similar chemical structure but differing physiological effects. 2134
EXPERIMENTAL Apparatus. All experiments were performed on a ?;ester-Faust Model 1200 Liquid Chromatograph (Perkin-Elmer, Norwalk, Conn.). The chromatograph was equipped with dual high pressure (2000 psi) pumps and a n automatic solvent transport system which controls selection of pumps and eliminates the need to make changes in system plumbing when changing modes of ~ e r ation (i e , from step to gradiant elution). It also makes it possible to pre-program an elution profile and have the analysis carried out automaticallv. Sample application to the column was made ~ 1 syringe( ~ ~ company, ~ i whittier, l ~ ~ with a ~ 5 - Hamilton Calif.) through a septum injector directly above the column, A 4.6-mm (i.d.) X 1-meter stainless steel column was dry packed
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 1 2 , OCTOBER 1973
~
Table I. Quantitative Analysis of Atropine Sulfate
Compound
Scopolamine hydrobromide Eucatropine hydrochloride Homatropine sulfate Tropine Apoatropine hydrochloride Scopolamine hydrobromide Scopolamine hydrobromide 0 5
10
Figure 1. Separation of
15 MINUTES
20
25
tropane alkaloids
Conditions: Column: SiI-X, 1M X 4.5 mrn, stainless steel; mobile phase: ammonium hydroxide-tetrahydrofuran 1 : 100, v / v ; flow rate: 0.82 ml/ min; column iniet pressure: 500 psi; wavelength: 254 n m ; full scale: 0.05 absorbance unit; size 4 pi, A ) Scopolamine hydrobromide, 25.7 pg; 8 ) Eucatropine hydrochloride, 25.7 pg; C) Tropine, 27.5 pg; D ) Apoatropine hydrochloride. 25.1 pg, E ) Homatropine sulfate, 30.0 pg; F ) Atropine sulfate, 26.4 pg; G ) Hyoscyamine hydrochloride, 27.6 p g with sil-X adsorbent (Nester/Faust Corp.). Detection was accomplished through the use of a Differential Refractive Index Detector (Nester/Faust Model KFLC-100) having a range of 1.30-1.45 RI units a n d a high sensitivity UV Detector (Nester/Faust Model NFLC-250). A dual channel, dual pen recorder (Varian Model G4020, Varian Aerograph, Walnut Creek, Calif.) equipped with a disc integrator on one channel was used for monitoring the detector output and in t h e quantitative studies. R e a g e n t s . T h e alkaloids (chromatographically pure) used in this study were Apoatropine hydrochloride (K&K Laboratories, Inc., Plainview, N.Y.); Atropine sulfate (K&K); Eucatropine hydrochloride (K&K); Homatropine sulfate (K&K); Hyoscyamine hydrochloride ( M a n n Research Laboratory, New York. N.Y.); Scopolamine hydrobromide USP (Inland Alkaloid Inc., Tipton, Ind.); a n d Tropine (Nutritional Biochemicals Corp., Cleveland, Ohio). P r o c e d u r e . T h e column was dry-packed with the sil-X adsorbent and primed with eluant for 2 hours prior t o use (which was determined repeatedly t o be sufficient t o reach equilibrium). T h e eluant used was a mixture of ammonium hydroxide (28% NH3 by weight in water) a n d tetrahydrofuran in t h e ratio of 1:lOO v/v. T h e column effluent was monitored first through the UV detector ( a t 254 n m ) a n d then through t h e RI detector. Samples were dissolved in methanol and injected directly into the column through the on-column injector without interrupting the solvent flow.
RESULTS AND DISCUSSION The chromatogram shown in Figure 1 exemplifies the type of separation achieved utilizing this system with detection by the UV detector. Full scale absorbance of 0.05 was used for all the compounds with the exception of apoatropine which required a 10-fold attenuation. "Modern Practice of Liquid Chromatography," J . J. Kirkland. E d . , Interscience, New York, N . Y . . 1971 L. R. Snyder, Ana/. Chem.. 39,698, 705 (1967). T . W . Smuts, F. A. Van Niekerk, and V. Pretorius, J. Gas Chromatogr.. 5 , 190 (1967). D. C. Locke, Anal. Chem.. 39,921 (1967). J. C. Giddings, Anal. Chem.. 37,61 (1965). J . C. Giddings, Anal. Chem.. 35,2215 (1963). R. P. W . Scott, D. W. J . Blackburn, and T. Wilkins, J . Gas Chromatogr.. 5 , 183 (1967). C. Horvath and S. Lipsky, J. Chromatogr.. 7,109 (1969). L. R . Snyder, J. Chromatogr.. 7,595 (1969). R. E . Majors, J. Chromatogr.. 8, 338 (1970). C. G . Scott and P. Bommer, J . Chromatogr.. 8, 446 (1970). W . F. Beyer,Ana/,Chem.. 44, 1312 (1972) C-Y W u and S . Siggia, Anal. Chem., 44, 1499 (1972). J . F. K . Huber. J . Chromatogr. Sci.. 7, 172 (1969). H . Felton, J. Chrornatogr. Sci.. 7, 13 (1969) T . B. Davenport, J . Chrornatogr.. 42, 219 (1969). R . E. Jentoft, and T. H . Gouw, Anal. Chem.. 40, 923 (1968).
AtroCornpine pound, added, Pg 4.1 23.9 1.3 27.9 6.8 26.1 17.8 26.8
20]
1 9 23 6
Atropine Refound, covery, Pg ?h 22.5 25.9 24.7 23.6
94 93 94.5 88
234
227
97
64
56
87
As the RI cell had a maximum pressure rating of 100 psig and the pressures generated during the experimentation a t times exceeded 1000 psig, it was decided to use this detector with the reference cell only in the static mode. The cell was therefore filled with eluting solvent and capped off. The pressure drop between the pump and the end of the column was sufficient to allow the RI detector to be used with no hazard to the cell. Since the UV cell was designed to withstand pressures upward of 2000 psig, it could therefore be used with a dynamic reference. In dual detector operation, the eluting solvent was pumped through the reference cell of the UV detector, then through the column, the sample cell of the UV detector, and the sample cell of the RI detector, in that order. Dual detection has obvious advantages over the use of a single detector especially when used in a combination of UV and RI as has been done here. The compounds or impurities that have little or no UV absorption would still be detected by the RI detector. This was the case with a common decomposition product of the tropane alkaloids, tropine, which showed no UV absorption but had a strong peak in the RI. During the course of the experimentation, a study was made on determining the limits of detection of the individual compounds. Under the operating conditions of solvent flow and detector output, shown in Figure 1, 50 pg of each component could easily be detected through the RI detector and 1pg through the UV detector. Quantitative analysis was performed under the same operating conditions described above. Before quantitation on a routine basis, the response of a given compound was checked for reproducibility. This was accomplished by making a number of injections containing the same concentration of each component and comparing the integration of the peak area. The average deviation in integrator counts, over a concentration range of 5-50 micrograms, was within *170 for all the alkaloids examined in this study. The quantitation itself can be accomplished by first chromatographing a set of standards containing known quantities of the alkaloids. The concentration of the standards are varied over the range within which the unknown samples are expected to fall. Calibration curves are then plotted depicting standard concentration us integrator count. It is a simple matter to then chromatograph the unknown and read its concentration directly off the appropriate standard curve. As a test of the quantitative procedure, mixtures of known amounts of atropine sulfate with some of the other tropane alkaloids were made. The quantitative recovery after comparison with the standard curve for atropine sulfate is summarized in Table I. Ta6le I1 shows the tropane alkaloids used in this study along with their retention time, elution volume, and sensitivity expressed as integrator counts per microgram.
A N A L Y T I C A L CHEMISTRY, VOL. 4 5 , NO. 1 2 , OCTOBER 1 9 7 3
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Table I I . Tropane Alkaloids
Name
Scopolamine hydrobromide Eucatropine hydrochloride Apoatropine hydrochloride Homatropine sulfate Atropine sulfate Hyoscyamine hydrochloride (opticallyactive form of atropine) Tropine
Reten- Retention tion time, volume. min mil 7.2 7.5 14.4
20 21
21.6 12
IJV sensitivity. integrator counts per p g
5.9 6.2 11.8 16.4 17.2
23.4 29.8 1320 18.7 45.5
17.7
16.5
9.9
CONCLUSION The reported results show the separation and quantitation of a number of pharmacologically important similar
alkaloids utilizing an industrially available high speed, high pressure liquid chromatograph. The work reported here shows the applicability and usefulness of high speed, high pressure liquid chromatography to the field of pharmaceutical analysis. It is especially valuable in the case where the sample would have to be converted to its free base before it could elute from a gas chromatography column, as would be true with the above alkaloid salts. Other advantages over gas chromatography would be the performance of the analysis a t room temperature and the ability to scale up the system, with relatively few modifications, in order to obtain preparative samples. With the utilization of high speed, high pressure liquid chromatography, the shortcomings of gas chromatography can be bypassed without loss of efficiency or time.
Received for review November 30, 1972. Accepted April 30, 1973.
Formation of Trimethylsilyl Derivatives of Tetracyclines for Separation and Quantitation by Gas-Liquid Chromatography Kiyoshi Tsuji and John H. Robertson Control Analytical Research and Development, The Upjohn Company, Kalamazoo, Mich. 49001 Following reports which implicated degradated tetracycline compounds, especially 4-epianhydrotetracycline, in renal dysfunction (1, 2 ) , a large number of chromatographic procedures concerning the separation and identification of tetracyclines has appeared. The analytical methods reported are based on thin layer chromatography (38), paper chromatography ( 9 ) , gel filtration ( I O ) , or partition column chromatography (11-15). Recently, an automated liquid chromatographic method has been reported (16). The method is an automation of the previously reported diatomaceous earth column method (12). In 1969 the British Pharmacopoeia (BP) (17) established a strict limit for 4-epitetracycline (ETC), anhydrotetracycline (ATC), and 4-epianhydrotetracycline (EATC)
J. M. Gross, Ann. Intern. Med., 58, 523 (1963) L. I. Ehrlich and H. S. Stein, Pediatrics, 31, 339 (1963). P. P. Ascione. J. B. Zagar, and G. P. Chrekian, J. Pharm. Sci., 56, 1393 (1967). I. C. Diykhuis and M. R. Brommet, J, Pharm. Sci., 59, 558 (1970). A. A. Fernandez. V. T. Noceda, and E. S. Carrera. J. Pharm. Sci., 58, 443 (1969). P. B. Lloyd and C. C. Cornford, J. Chromatogr., 53, 403 (1970). Y . Nishimoto, E. Tsuchida. and S. Toyoshima, J. Pharm. SOC.,Jap., 87, 516 (1967). D. L. Simmons, R. J. Ranz, H. S. L. Woo, and P. Picotte, J. Chromatogr., 43, 141 (1969). A. Sina, M . K. Youssef, A. A. Kassem. and I . A. Attia, J. Pharm. Sci., 60, 1544 (1971). B. W. Griffiths, R. Brunet, and L. Greenberg, Can. J. Pharm. Sci., 5 , 101 (1970). P. P. Ascione and G. P. Chrekian, J. Pharm. Sci., 59, 1480 (1970). P. P. Ascione. J. B. Zagar, and G. P. Chrekian J. Pharm. Sci., 56, 1396 (1967). W. W. Fike and N. W. Bake. J Pharm. S c : , 61. 615 ( 1 9 7 2 ) . R. G. Kelly, J. Pharm. Sci., 53, 1551 (1964). V . C. Walton, M. R. Howlett, and G. B. Selzer. J. Pharm. Sci., 59, 1160 (1970). P. P. Ascione, J. 8. Zagar. and G. P. Chrekian, J. Chromatogr., 65, 377 (1972). "British Pharmacopoeia 1968," Addendum, 1969, p 77.
2136
in tetracycline (TC) based principally on the procedure reported by Ascione et al. ( 3 ) . However, the detection limit of the method (5%) (3) and the procedure, according to Lloyd and Cornford ( 6 ) , is tedious, the results are variable, and, in particular, the limit of detection, according to them, appears to be optimistic. The detection limit of the diatomaceous earth column method is approximately 1% due primarily to the spectrophotometric read-out system in use (18). The proposed FDA limit for EATC is not more than 3% (19). Obviously, there is a need for a sensitive method which separates and precisely quantitates various tetracycline entities with speed, thereby making the method suitable for a laboratory where a large quantity of tetracycline preparations are analyzed routinely. Gas-liquid chromatography (GLC) has been applied as a sensitive method for the separation and quantitation of various antibiotics and their isomers and derivatives (2026). This report is, to the best of our knowledge, the first to describe trimethylsilylation of tetracyclines and their separation and quantitation by GLC.
EXPERIMENTAL Apparatus. An F & M M o d e l 400 gas c h r o m a t o g r a p h w i t h a f l a m e i o n i z a t i o n detector was used: gas flow rate, h y d r o g e n 40 ml/min, a i r 600 ml/min, a n d h e l i u m 55 ml/min; c h a r t speed, 0.25
(18) J. D. Hettinger, The Upjohn Company. Kalamazoo, Michigan, personal communication, 1968. (19) Fed. Regisf., 34, (141) 12286, (1969). (20) T. Endo and H. Yonehara, J. Antibiot., 23, 91 (1970). (21) C. Hihta, D . L. Mays. and M. Garofalo, Anal. Chem., 43, 1530 (1971). ( 2 2 ) R. L. Houtman, D . G. Kaiser, and A. J. Taraszka, J, Pharm. Sci., 57,693 (1968), (23) M. Margosis, J. Chromatogr., 47, 341 (1970). (24) K. Tsuji and J. H. Robertson, Anal. Chem., 41, 1332 (1969). (25) K. Tsuji and J. H. Robertson, Anal. Chem., 42, 1661 (1970). (26) K. Tsuji and J. H. Robertson, Anal. Chem., 43, 818 (1971).
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