64
Anal. Chem. 1903, 5 5 , 64-67
neous current within a single spark becomes so high that the signal to noise benefits gained through spatial dscrimination are defeated (34),we recommend synchronously time gating the detector integrator circuit during the valleys of the current wave form (35). The same microcomputer that is used to fire the spark and move the electrode can also do this, even though the situation today is economically such that it is quite sensible to design for several machines being active at once (36). The signal to noise can then be increased again, further compensating for the higher background that would come from using higher power on more difficulty sampled alloys. The net picture here then is one of substantial enhancements in overall analytical performance, all based on prior experimental research and all possible at expected modest cost due to the simpler electronic, computer-controlled spark sources presently possible. We look forward to reporting the first results of such combined techniques in the near future.
ACKNOWLEDGMENT The assistance of Robert J. Lang and Robert M. Schmelzer in instrument fabrication is appreciated, as is that of Patricia Brinkman in financial management. LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8)
(9) (10)
Sacks, R. D.; Walters, J. P. Anal. Chem. 1970, 42, 61. Walters, J. P.; Malmstadt, H. V. Anal. Chem. 1965, 3 7 , 1484. Walters, J. P. Anal. Chem. 1968, 4 0 , 1540. Walters. J. P. Science 1977, 198, 787. Scheellne, A.; Walters, J. P. I n "Contemporary Toplcs In Analytlcai and Clinical Chemistry"; Hercules, D. M., Hieftje, G. M., Snyder, L. R., Evenson, M. A., Eds.: Plenum Press, New York, Vol. 4, pp 295-372. Washburn, D. N.; Walters, J. P. Anal. Chem. 1981. 53, 1644. Eklmoff, D.; Walters, J. P. Anal. Chem. 1981, 53, 1644. Walters, J. P. Appl. Spectrosc. 1977, 31, 36. Walters, J. P.; Goldstein, S. A. ASTM Spec. Tech. Publ. 1973, STP 540, 45-71. Thackeray, D. P. C. Nature (London) 1957, 180, 913.
(11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36)
van der Piepen, H.; Schroeder, W. W. J . Phys. D 1972, 5 , 2190. Olesik, J. W. Ph.D. Thesis Unlversity of Wisconsin, 1982. Olesik, J.; Walters, J. P. Appl. Spectrosc., in press. Walters, J. P.; Goldstein, S. A,; Eaton, W. S. US. Patent 3815995, June 1, 1974. Kiueppel, R. J.; Coleman, D. M.; Eaton, W. S.; Goldstein, S. A,; Sacks, R. D.; Walters, J. P. Spectrochim. Acta, Part 8 1978, 338,1. Walters, J. P. Appl. Spectrosc. 1972, 26, 17. Barnhart, S. G.; Farnsworth, P. B.; Walters, J. P. Anal. Chem. 1981, 53,1432. Walters, J. P. Anal. Chem. 1968, 40, 1672. Takahashi, T. Bunko Kenyu 1966, 15, 164. Hlrokawa, K.; Goto, H. Spectrochlm. Acta, Part 8 1970, 258, 419. Strashelm, A.; Blum, F. Spectrochlm. Acta, Part B 1971, 268, 685. Farnsworth, P. B.; Walters:,J. P. Anal. Chem. 1962, 5 4 , 885. Mika, J.; Torok, T. Analytical Emission SpectroscopyFundamentals"; Crane, Russak & Co.: New York, 1974; p 423. Kuznetsova, L. A.; Petrova, N. G.; Podmoshenskaya, S. F. Zh. Prlkl. Spektrusk. 1976, 2 4 , 576. Holler, P.; Thoma, C.; Brost, U. Spectrochlm. Acta, Part 8 1972, 2 7 8 , 365. Yamane, T.; Matsushita, S. Spectrochlm. Acta, Part 8 1972, 2 7 8 , 27. Mathews, S. M.; Walters, J. P. Appl. Spectrosc., in press. Fassel, V. A. Sclence 1978, 202, 183. Klueppel, A. J.; Walters, J. P. Specfruchim. Acta, Part 8 1980, 368, 431. Mathews, S. M. Ph.D. Thesis University Of Wisconsin, 1982. Eklmoff, D. Ph.D. Thesis Universlty of Wisconsin, 1981. Washburn, D. N. PhD. Thesis Unlversity of Wisconsin, 1981. Holdt, G.; Strashelm, A. Appl. Spectrosc. 1960, 14, 64. Goldsteln, S. A. Ph.D. Thesis University of Wlsconsin, 1973. Barnhart, S. 0.; Walters, J. P. U.S. Patent Appl., 1982. Walters, J. P. 2nd Chemical Congress, North America, ACS, Las Vegas, NV, Aug 1982.
RECEIVED for review July 16, 1982. Accepted October 1,1982. We acknowledge the continual support of the National Science Foundation during the time that this work was done and as its initial results were independently verified under Grants GP-13975, GP-35602X, CHE76-17557, CHE77-05294, and CHE79-15195.
Analysis of Pharmaceuticals by Fluorine-19 Nuclear Magnetic Resonance Spectrometry of Pentafluoropropionic Anhydride Derivatives Gary E. Zuber," Davld B. Staiger, and Richard
J. Warren
Smith Kline & French Laboratories, P.O. Box 7929, Philadelphia, Pennsylvania 19 10 1
A quantitative method for the fiuorlne-I9 NMR analysis of pentafiuoroproplonic anhydride derlvatized pharmaceuticals Is presented. The procedure le based upon chromatographic derlvatization methods. Reactions were carried out in deuterated chloroform using approximately 50 mg of sample. The samples analyzed were bulk pharmaceutical materials and drug dosage forms containing hydroxyl and amlno groups. I n some cases, the catalyst pyridine at a reaction temperature of 55 OC was used to shorten the reaction times and to assure complete derlvatlzatlon. This fluorine derlvatizatlon technique results In fluorine-19 NMR spectra of pharmaceuticals which are greatly simpllfled in comparison to their more complex proton spectra. The major advantage of the method is the speed wlth which the analysis can be carried out since most derivatirations are completed In 10 min. The broad application of this technique to pharmaceutical analysis Is reported along with accuracy and preclslon data.
The quantitative analysis of pharmaceuticals as commonly done by gas chromatography is often a time-consuming and
difficult procedure (I). The use of proton nuclear magnetic resonance (NMR) for drug determinations is sometimes limited due to the complex NMR spectrum of some molecules. Consequently, we decided to develop an analytical procedure using fluorine derivatization of drugs containing active hydrogens and subsequent analysis of their fluorine-19 NMR spectra. Fluorinated derivatives are extensively used in analyses by gas chromatography (GC) and mass spectrometry (MS) (243, but their application in quantitative fluorine-19 NMR spectrometry, especially for pharmaceutical analyses, has been somewhat limited. A great deal of work has been done with hexafluoroacetone (HFA) and fluorine NMR to quantitate and characterize active hydrogen compounds (6-9). Since this initial work, some recent applications in the areas of food and coal product analysis have been made (IO, 11). The disadvantage associated with these HFA methods is that they require the use and storage of a reagent gas which is a potential toxicity hazard. In addition, fluorine-19 NMR analysis of trifluoroacetyl derivatives has been used to determine organic compounds and also some biologically related materials (12-17'). Trifluoroacetyl derivatives can be formed by using
0003-2700/83/0355-0064$01.50/0@ 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983 65
a variety of different derivatization reagents, the most popular ones being trifluoroacetyl chloride and trifluoroacetic anhydride. Use of trifluorostcetylchloride still requires the possibly undesirable storage of this reagent gas, and the use of volatile trifluoroacetic anhydride also requires careful reagent storage. The method presented here was based upon information and techniques found within these previous NMR, GC, and MS studies. We adapted the fluorinated anhydride derivatization methods usedl in GC/MS for use in a suitable fluorine-19 NMR method. The derivatization reagent (chosenfor use was pentafluoropropionic anhydride. This reagent is readily available and lis less volatile than trifluoroacetic anhydride, thus allowing for easier storage. The formation of pentafluoropropionyl derivatives also has another (advantage in that in most cases each derivatization reaction results in a derivatized drug which yields a fluorine NMR where quantitation is possible in both the CF3 and CF2 regions of the spectra. Consequently, an internal check of integral BCcuracy is present. This is not the case when trifluoroacetyl derivatives are formed This technique was designed to avoid any preliminary preparation steps so that accurate and precise results could be quicQy obtained. In addition, it was felt that quantitative analyses of anhydride derivatives by fluorine-19 NMR spectrometry should be possible even for complex drug mixtures provided that the components of the derivatized mixture yield well-separated adduct signals.
EXPERIMENTAL SECTION Apparatus. The instrument used waB a Perkin-]Elmer R32 equipped with a fluorine-19 accessory. The instrument operates at a magnet field strength of 21.1 kG with resonance hequencies for fluorine-19 at 84.6 IBHz and proton at 90 MHz. All spectra were obtained at a 100-ppm sweep width initially and also a 10-ppm sweep width for quantitation. A 180-s sweep rate was used for the integrations. Samples were each run at various spin rates in order to eliminate any chance of interference from spinning side bands. Reagents. Pentaflu'oropropionic anhydride (PFAA) was obtained from Pierce Chemicals, Rockford, E. The free acid content of these anhydrides is reported as being 1% or less. The anhydride reagent was kept undeir nitrogen and in a refrigerator between use to prevent any decomposition. The solvent used for all analyses was deuteriochloroform (minimum isotopic ]purity 99.6 atom % D) which was obtained from Merck and Co., Rahway, NJ. The internal standard used was trifluoroacetanilide (purity = 99.9% by GC) which was obtained from BDH Chemicals, Poole, England. AU of the pharmaceuticals analyzed were of the highest purity. All other chemicals were high-grade commercial products and were used without further purification. Procedure. In cases not requiring catalysis, approximately 50 mg of the sample and 30 mg of the internal standard were weighed into a 5-mL vial. This mixture was then dissolved with 0.5 mL of deuteriochloroform and transferred to a 5-mm NMR tube. An excess amount of the derivatization reagent (PFAA) was then introduced, and the reaction was allowed to proceed for 10 min at room temperature. Equation 1 illustrates the basic reaction. After this reaction period, the resultant mixture was
Rl-OH or
drug
cF,cFzc
+
n
\
0
PFAA
RlOCCFzCF3
--
or
derivative
analyzed by fluorine-19NMR at sweep widths of 100 and 10 ppm. The results were then analyzed, and additional reaction time was allowed if necessary. In cases (e.g., phenols and some amines) where a simple catalyst was required, an excess amount of either 3..0 M triethylamine or
I
I
1
PPM From Trilluoroacetanilide
Figure 1. Fluorine19 NMR spectrum of derivatized cholesterol in CDCI, (sweep width = 100 ppm).
pyridine in deuteriochloroform was added immediately following the addition of PFAA. In cases which required both pyridine and heat, an excess amount of 1.0 M pyridine was added to the NMR tube containing the sample, reference, and PFAA. This tube was then placed in a water bath kept at about 55 "C for the proper reaction period (usually 10 min) and then analyzed by fluorine-19 NMR. After the initial fluorine-19 NMR analyses were made, each sample was washed with a saturated sodium bicarbonate solution to eliminate any excess PFAA and pentafluoropropionic acid present. Each sample was then reanalyzed under the same NMR conditions previously described.
RESULTS AND DISCUSSION An example of a typical reaction between the drug cholesterol and PFAA appears below. 0 II
k-
CFICFIC\
HO
Excess PFAA
Cholesterol
dF+ +
tiO-CCF2CF,
11
t
(CFsCF2C12-0
II
(2)
cF~CF2c-0 ,
I
I7 21 146 0)
Derivative
O
Unreacted PFAA
The numbers in parentheses represent the chemical shifts in parts per million upfield from the reference trifluoroacetanilide. Figure 1 shows a 100-ppm fluorine-19 NMR spectrum of derivatized cholesterol. This spectrum is first order with quantitation possible either in the CF3 region (-6.8 to -7.4 ppm) or in the CF2 region (-45.6 to -46.4 ppm). Figure 2 is a 10-ppm expansion of the reference and both the CF3 and CF2 signal regions of the cholesterol derivative. Signal a at -7.2 ppm represents the CF3group of the cholesterol derivative and signal b at -46.0 ppm the CF2. Each of these signals was integrated five times vs. the reference standard trifluoroacetanilide. The results were then analyzed by means of the following basic equation. integration (sample) wt (std) X
X
integration (std) wt (sample) equiv wt (sample) X 100% = % determined (3) equiv w t (std) In cases where both the CFSand CF2 signals of the derivative
66
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
-
Table I. Quantitative Results for Alcohols % re1 deter- std reaction pharmaceutical mined dev time cholesterol (-OH) 99.8 1.8 10 rnin norethindrone (2-OH) 94.3 1.4 1day mestranol (-OH) 96.5 2.3 1 day menthol (-OH) 97.2 2.5 10 rnin 99.8 1.7 10 rnin oxycodone (-OH) bulbocapnine (PhOH) 97.5 1.9 10 min
catalyst none none none none none pyridine
Table i l Chemical S h i h For P F A A Derivative of Pharmaceuticals (Alcohols) Drug Name -68
-72-74
- 4 5 6 -480
CFI -
PPM From Trltluoroacetanilide
Figure 2. A 10-ppm expanded fluorine19 NMR spectrum of derivatized cholesterol in CDCI, (integral sweep rate
Peak Position ( P P M I
Structure
-464
1.
Cholesterol
2. Mestranol
-Cholesterol
-46 0
-6.90
-45.7
-692
-45.6
-7.05
-45.6
-7 10
-45 8
"0
= 80 s).
Reference
CFZ -
-7.20
Derivallve-CF, 3. Norethindrone
Cholesterol Derivative-CF,
\I
Q;.
4 . Menthol
I
0
-10
I
-20
I
I
-30
-40
I
H C
5. Oxycodone
-6.37
-
6 . Bulbocapnine
-7.10
-
-50
PPM From Trillouroacelanlllde
Figure 3. Fluorine19 NMR spectrum of derivatized cholesterol In CDCI, after
a
sodium bicarbonate wash (sweep width
=
100 ppm).
are observed, both are individually quantitated vs. the reference and the results averaged. The relative standard deviation for each analysis was calculated by means of the following: std dev X 100% = re1 std dev av integration
(4)
Figure 2 also illustrates the CF3 and CF2 signals for the unreacted PFAA reagent present with chemical shifts of -6.8 ppm and -45.6 ppm, respectively. The signals a t -7.4 and -46.4 ppm represent the respective CF3 and CF2 groups of pentafluoropropionic acid which is a byproduct of the anhydride reaction. Signal assignments were made on the basis of reference spectra of pentafluoropropionic acid, pentafluoropropionic anhydride, and a derivatized cholesterol sample which had been washed with saturated sodium bicarbonate. Figure 3 shows a 100-ppm fluorine-19 NMR spectrum of derivatized cholesterol which has been subjected to a bicarbonate wash to eliminate the excess PFAA and pentafluoropropionic acid interfering signals. The sodium bicarbonate wash resulted in a more simplified spectrum which was much easier to analyze. In Figure 3, the two derivative signals which remain have chemical shifts of -7.2 ppm and -46.0 ppm and represent the respective CF, and CF2groups of the cholesterol derivative. Alcohols. Table I illustrates the quantitative results obtained for six pharmaceuticals containing OH groups. The steroid cholesterol, alkaloid oxycodone, and menthol all undergo complete derivatization within 10 min. The alkaloid bulbocapnine which possesses a phenolic OH function requires the presence of the catalyst pyridine for a rapid and complete derivatization reaction. The presence of a base such as pyridine enhances reactivity by serving as an acid acceptor in the derivatization reaction (18). The steroid mestranol
possesses a cyclic OH group a t the C-17 position which is sterically hindered by the close proximity of an alkyne group. Consequently, the reaction of this compound at this site takes 24 h to near completion. It was also found that 2.0 M pyridine and heat did not catalyze this reaction to any significant extent. The steroid norenthidrone possesses two reaction sites which undergo derivatization at different rates. As in mestranol, a slow reacting sterically hindered OH group which requires 24 h for derivatization is present at the '2-17 position. In addition, an active hydrogen site is present at the C-3 position in norenthidrone. Keto-enol tautomerization of this C-3 carbonyl as seen below results in the formation of a fast reacting OH group.
a- n
0
HO
1 Reaction Site
The resultant active hydrogen found here reacts almost immediately with the anhydride reagent. Table I1 illustrates that the chemical shifts for the CF3 groups of these alcohol derivatives all are within the range of -6.90 to -7.20 ppm with the exception of oxycodone (-6.37 ppm). The CF, group derivative signals were unobservable for mestranol, norenthindrone, oxycodone, and bulbocapnine prior to washing with sodium bicarbonate and were believed to be overlapped by either the excess PFAA signal or the pentafluoropropionic acid signal. After bicarbonate washing, the mestranol and norethindrone signals did become observable, but the fluorine-19 NMR spectra obtained for the derivatized alkaloids, oxycodone and bulbocapnine, indicated that these compounds decomposed in the presence of sodium bicarbonate. Sodium bicarbonate washing of cholesterol and
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
Table 111. Quantitative Results for Amines % % reaction deter- preci- time, pharmaceutical mined sion min propylhexedrine 98.3 2.1 10
Table V. I9F NMR Analysis of a Standard Derivitized Mixture of Propylhexedrine a n d Menthol catalyst pyridine
98.3
0.9
10
+ pyridine +
98.9
1.6
10
none
(-",)
A
phentermine (-",)
benzocaine
A
(-",)
Table IV. Chemical Shifts Tor PFAA Derivatives of Pharmaceuticals (Amines)
Peak Position (PPM)
Drug Name
Structure
1. Propylhexedrine
3. Benzocaine
CH,CH:,-O-C~NH,
U 0
milligrams of Propylmilligrams of hexedrine menthol --__ NMR NMR method method reaction ( % deter( % deterconditions actual mined) actual mined) 24 h with 1.0 M 51.0 50.3 53.0 53.1 pyridine present (98.6) (100.2) 10 min with 51.4 50.8 50.6 50.9 1.0 M pyridine (98.8) (100.5) present and heat
C - -
readily analyze drug mixtures.
-6.20
CONCLUSIONS The above results demonstrate that a variety of different pharmaceutical types can be rapidly quantitated by fluorine-19 NMR analysis of their pentafluoropropionic anhydride derivatives. This method was shown to be applicable to simple alcohols and amines as well as complex drugs such as steroids and alkaloids. Steroids and alkaloids which normally give highly complex proton NMR spectra can be derivatized quickly under the proper reaction conditions and their simplified fluorine-19 NMR spectra used for accurate quantitations. This method of analyzing pure drugs and drug mixtures offers many advantages over previous analytical techniques. The analysis offers the advantages of speed, specificity, and accuracy (a relative standard deviation always less than 2.5%). Registry No. PFAA, 356-42-3;cholesterol,57-88-5;mestranol, 72-33-3;norethindrone, 68-22-4; menthol, 1490-04-6;oxycodone, 76-42-6; bulbocapnine, 298-45-3; propylhexedrine, 101-40-6; phentermine, 122-09-8;benzocaine, 94-09-7.
CF3
Fz
-38.9
2. Phentermine
67
-5.82
-40.8
-6.85
-46.:2
menthol did eliminate the interfering signals without causing derivative decomposition. Amines. Table I11 illustrates the quantitative results obtained for three pharmaceuticals containing NH, groups. The aromatic amine benzocaine was found to react rapidly with no catalysis required. The aliphatic amine propylhexedrine was found to react to the 80% level after a reaction time of 24 h both without a catalyst and with the catalyst triethylamine present. With 1.0 M pyridine added to the reaction mixture, a near complete (95%) reaction was achieved after 24 h. Finally, it was found that the addition of both pyridine and heat (55 "C) drove the reaction to completion within 10 min. On the basis of this information, the aliphatic amine phentermine was Completely derivatized within 10 min by using the same establlished reaction conditions. Table IV illustratee that all of the chemical shifts for the CF, groups of these dlerivatized amines are within the broad range of -5.82 to -6.85 ppm. The CF2 signals which were all observable are within a range of -38.9 to -46.2 ppm. All of these derivatized amine compounds underwent apparent decomposition when washed with saturated sodium bicarbonate. Drug Mixture. Table V illustrates the results obtained for a derivatized drug mixture of propylhexedrine and menthol. Menthol was flound to undergo rapid and complete derivatization under all reaction conditions. Propylhexedrine, however, requires the presence of 1.0 M pyridine and a reaction temperature of 55 "C to be completely derivatized. Once the proper catalyzed reaction conditions are established, quantitation of both drugs is possible in both the CF, and CF, signal regions. Accurate and precise results are possible because of the large chemical shift differences between the derivative signals of psopylhexedrine and menthol. The CF, signals for propylhexedrine and menthol were at -6.20 and -7.10 ppm, respectively, and the CF, signals at -38.9 and -45.8 ppm, respectively. Whenever this type of derivative signal separation is encountered, this technique can be used to
LITERATURE CITED Internal communication; Gas Chromatography Section; Smith Kline & French Laboratories, 1981. Ehrsson, H.; Walle, T.; Brotell, H. Acta Pharm. Suec. 1971, 8 , 3 19-328. Walle, T.; Ehrsson, H. Acta Pharm. Suec. 1971, 8 , 27-38. Walle, T.; Ehrsson, H. Acta Pharm. Suec. 1970, 7 , 389-406. Ervik, M.; Walle, T.; Ehrsson, H. Acta Pharm. Suec. 1970, 7 , 625-634. Leader, G. R. Anal. Chem. 1970, 4 2 , 16-21. Leader, G. R. Anal. Chem. 1973, 45, 1700-1706. Ho, F. F.-L. Anal. Chem. 1974, 46, 496-499. Ho, F. F.-L.; Kohler, R. R. Anal. Chem. 1974, 4 6 , 1302-1304. Gaffield, W.; Lundln, R. E. IARC Sci. Publ. 1978, 19, 87-95. Bartle, K. D.; Matthews, R. S.; Stadelhofer, J. W. Appl. Spectrosc. 1980, 3 4 , 615-617. Ilda, T.; Tamura, T.; Matsumoto, T. Nihon Dalgaku Kogakubu Kiyo, Bunrui, A 1980, 2 1 , 223-226. Brown, W. E.; Seamon, K. 9. Anal. Blochem. 1978, 8 7 , 211-222. Sleevi, P.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1979, 57, 1931-1934. Manatt, S. L. J. Am. Chem. SOC. 1966, 8 8 , 1323-1324. Manatt, S L.; Lawson, D. D.; Ingham, J. D.; Rapp, J. D.; Hardy, J. D. Anal. Chem. 1986, 3 8 , 1063-1065. Voelter, W.; Brletmaier, W.; Jung, G.; Bayer, E. Org . Mag. Reson, 1970, 2 , 251. "Pierce Handbook and General Catalog"; Pierce Chemical Co.: Rockford, IL, 1979-1980; p 189.
RECEIVED for review August 10, 1981. Resubmitted August 24, 1982. Accepted September 27, 1982. Presented in part at the 1981 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy in Atlantic City, NJ, March 11, 1981.
