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(3) M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum, and J. M. T. Raycheba, Anal. Chem.. 46, 374 (1974). (4) M. J. Milano and H. L. Pardue, Anal. Chem., 47, 25 (1975). (5) H. L. Pardue, A. E. McDowell, D. M. Fost, and M.J. Milano, Clin. Chem. (Winston-Salem, N.C.), 21, 1192 (1975). (6) G S. Wilson and L. Ramaley. Anal. Chem., 42, 611 (1970). (7) T. E. Hewitt, M. J. Milano, and H. L. Pardue, Clin. Chem. ( Winston-Salem, N . C . ) , 20, 1028 (1974). (8) M. J. Milano and H. L. Pardue, Anal. Chem., 47, 25 (1975).
(9) M. J. Milano and H. 1.. Pardue, Clin. Chem. ( Winston-Salem. N.C.).21, 2 1 1 (1975).
RECEIYED for review April 3, 1978. Accepted June 5, 1978. This work was supported by Grant No. CHE 75-1550 AD1 from the National Science Foundation.
Determination of Chenodeoxycholic Acid and Ursodeoxycholic Acid by Nuclear Magnetic Resonance Spectrometry Pranab K. Bhattacharyya“ and Yakub G. Bankawala Quality Control Department, Hoffmann-La Roche Inc., Nutley, New Jersey 077 70
NMR spectrometry provides an efficient method for rapid identification and quantitation of the free bile acid epimers, chenodeoxycholic acid and ursodeoxycholic acid. The NMR spectra of these bile acids are characterized, and the NMR technique of assaying these compounds is described. The identification and assay procedures outlined in thls report are aimed at providing an efficient alternative to nonspecific conventional techniques of separation and titration.
Table I. Chemical Shifts of the Proton Resonances of Chenodeoxycholic and Ursodeoxycholic Acids 6
protons -CH, (18) -CH, ( 1 9 ) -CH, (21) -CH,-X l o
T h e proton NMR spectra of the free bile acids are characterized, as in all steroids, by two ranges of linewidths, some of which are much greater than others. The sharp and intense lines in the spectra are attributed to methyl groups on quarternary carbon atoms. In practice, the NMR technique is an excellent method for the determination of the number of angular methyl groups which. in general, give rise to stereochemically diagnostic features in the NMR spectra. The broad lines in the range of 1-2.5 ppm, underlying the sharper peaks due to the methyl protons, are attributed to the strong spin-spin couplings and small chemical shifts associated with the numerous indistinguishable protons on the steroid skeleton. Although not subject to detailed interpretation, this background absorption serves as a characteristic “fingerprint” for the bile acids under investigation. The epimeric configuration change from ursodeoxycholic acid (UDCA) to chenodeoxycholic acid (CDCA) strikingly alters the single broad signal due to the C3 and Ci methine protons in UDCA to two distinct and separate signals due to the same protons in CDCA. In this report. the quantitation of the free bile acids is based on the utilization of the resonances due to the C3 and C;methine protons. Besides spectral characterization, the purpose of this work is to obtain a method for routine identification and quantitation, thereby providing an alternative to the nonspecific titration and separation procedures, the latter of which involve preparation of TMS-ether or TFA derivatives of the free bile acids. EXPERIMENTAL The samples of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) were dissolved in a mixture of CDC1, and DMSO-d6 (8:l v/v). Maleic acid was used as the standard for the purpose of quantitation. The NMR spectra were obtained with a Varian XL-lOO/Nicolet TT-100 NMR spectrometer. 0003-2700/78/0350-1462$0 1 OOiO
+CH X 6 >CH(3) >CH ( 7 ) -COOH -OH
CDCA 0.64 0.88 0.94
1 1
/ppm UDCA 0.66 0.95 0.92
-0.94-2.5
-0.95-2.4
=3.38 3.78
=3.45 “3.45
-4.0-7.0
-4.0-7.0
resonance character singlet singlet doublet [overlapping with -CH, (1911 multiplet broad relatively sham (CDCAL broad (UDCA) broad
R E S U L T S AND D I S C U S S I O N Characterization. The free bile acids, chenodeoxycholic and ursodeoxycholic acids, are epimers which differ only in the configuration of the chiral center at C7 (see Figure 1). The conformation of these acids is A/B cis or 5p. Both compounds exist in a normal chair conformation. The NMR spectra of CDCA and UDCA are presented in Figure 2. The spectral assignments are listed in Table I. The intense and narrow lines in the range of -0.5-1.0 ppm are attributed to the tertiary CISand C19 methyl groups which possess a rotational degree of freedom. Because of the equivalence of the three protons in the group, they give rise to resonances of rather large intensities. The chemical shifts of the angular methyl groups (CI9, in this case) are sensitive, in general, to subtle structural changes, such as the epimeric configurational change a t C7 from UDCA to CDCA. These shifts depend upon the sum of diamagnetic and paramagnetic shifts, the former being mainly due to the electron density around the methyl protons and the latter arising because of the departure from spherical symmetry of the electronic charge distributions around the same protons. Useful correlations of structures with spectra have been derived ( I , 2 ) in the literature from the spectra of several steroids. The identification of the signals due to the CI8 and C19methyl groups, respectively, is unambiguous. Comparison of the spectra of UDCA and CDCA show that the chemical shifts of the CIBangular methyl peaks are practically the same, 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11. SEPTEMBER 1978 I8
23
Figure 1. Stereochemical configurations of chenodeoxycholic and ursodeoxycholic acids. The @-bondsare indicated by the dotted lines going behind the plane of the paper, and the p-bonds by the solid lines
coming out of the plane of paper. The small letters "a" and "e" refer to "axial" and "equatorial" bonds, respectively
CDCA
UDCA
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between protons of similar chemical shifts, which manifests itself in a large number of unresolved multiplets. This region of the spectrum (the so-called "fingerprint" region) is highly characteristic of a given steroid in a given configuration and can be altered markedly by disturbing even a single proton. The epimeric configurational difference between UDCA and CDCA a t C7 has altered strikingly t h e appearance of the spectra in the fingerprint region. The structurally useful observation is that the splittings caused by axial-equatorial proton-proton couplings in UDCA and CDCA are dependent on the configuration of the hydroxyl substituent. In addition to the methyl groups, the C; (p) skeletal proton in CDCA gives rise to a rather sharp signal a t 3.78 ppm which can be explained on the basis of a rather small spin-spin coupling for this proton by the application of the Karplus relation for the dihedral angle dependence of t h e vicinal coupling constant. In UDCA, the methine proton a t C 7 ( a )is coupled strongly to the axial protons on Cs and C8 by virtue of having the trans-diaxial relationship. Three axial protons attached to alternate carbon atoms are spaced rather closely together when they are on the same side of t h e steroids. However, as it happens, they are about the same favorable distance apart (2.3 .&) as the other protons. Now, if a proton is replaced by a hydroxyl group, crowding occurs. The most severe crowding takes place among atoms held by t h e three axial bonds on t h e same side of the steroids-the resulting interaction is called the 1,3-diaxial interaction. A hydroxyl group has more room in an equatorial position than in a n axial position. I n UDCA, the C, ( p ) and C7 ( a ) methine protons are both in axial positions and, sterically speaking, almost equally shielded so that the resonance positions of these protons coincide at approximately 3.45 ppm. In CDCA, the C7 (8) and C3 (p) methine protons are in equatorial and axial positions. respectively. The C3 methine proton resonance is substantially a t the same position (6 3.38 ppm) while the C7 methine proton is shifted downfield (6 3.78 ppm) because of steric congestion between the hydroxyl group a t C7 and the proton a t C4, as shown below. CH3
PPM
I81
Figure 2. Proton NMR spectra of chenodeoxycholic acid (CDCA)and ursodeoxycholic acid (UDCA) in the mixture of solvents CDCI, and DMSO-d6 (8.1 v/v). The solvent peaks are marked with asterisks
but t h e shifts of the Clg methyl peaks are slightly different (3= 0.1 ppm) because of the epimeric change in the configuration of t h e hydroxyl group a t C;. In UDCA, t h e peak caused by the CI9 methyl group and the doublet caused by t h e Czl methyl group overlap. In CDCA, this doublet is discernibly resolved from the peak caused by the CI9methyl group. An important effect to notice, particularly in UDCA, is that, for the doublet arising from the CzLmethyl group, the peak appearing a t the lower field constitutes the higher peak of the pair (see Figure 2). The simple, first-order treatment of spin-spin coupling predicts two lines of equal intensity. In practice, second-order effects are operative and the resulting doublet is perturbed. T h e perturbation occurs so that the bigger peak of the doublet is the lower field signal when the proton t o which the group is coupled is a t lower field. This is usually the case for t h e secondary methyl resonances. Conversely, the higher field peak of the doublet will be bigger when the proton causing the splitting is at higher field than the center of the doublet, The portion of the spectra roughly between 1.0 and 2.5 ppm consists of a "hump"-like structure arising from the methylene and methine protons due t o extensive spin- spin coupling
The paramagnetic shift associated with the steric interaction of two groups arises because the effective shielding of the proton is decreased on asymmetrical distortion of the electron cloud because of van der Waals repulsion so that the electron density about the methine proton a t C7 (8) is reduced. Assay Technique. While steroids, in general, d o not readily lend themselves to quantitative analysis by PMR spectrometry, the presence of distinct and isolated signals caused by the C3 and C7methine protons in CDCA and UDCA is found to be very useful in the quantitation of CDCA and UDCA. By using approximately 1:8(v/v) mixture of t h e solvents DMSO-d, and CDCl, (CDCA and UDCA are not very soluble in chloroform), the broad envelope caused by the carboxyl proton resonance arising in t h e DMSO-d, solvent, wherein CDCA and UDCA are very soluble, can be narrowed strikingly and its peak position shifted away from interference with the spectra of CDCA and UDCA and, in particular, the C3 and C7 methine proton peaks which are used for quantitation. Maleic acid is found to be suitable 9s an internal standard. The resonance caused by its methine protons (6 = 6.34 ppm) does not interfere with the spectra of IJDCA or CDCA (Figure 3). Besides, in its presence, the resonances due to the
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
l I
7
CDCA
I 6
I
1
5 PPM
4
I
3
(SI
Flgure 3. Typical display of t h e spectral region used for quantitation by FTNMR spectrometry. The C3 and C, methine proton peaks of CDCA and the methine proton peak of maleic acid are labeled CDCA and MA,
respectively Table 11. Optimized Experimental Conditionsa for Quantitation of UDCA and CDCA observe offset 46240 Hzb 5.0 ,us (tip angle, 303)c pulse width 1 kHz sweep width filter (computer) 1 kHz acquisition time 8.192 s acquisition delay 0.1 s interferogram 16K 32 number of transients FID apodization (LB) 1.0 Hz The field modulator card in the spectrometer module is disconnected. Locked on DMSO-d,. 10-dB attenuator used in the transmitter channel. carboxylic protons of the sample and standard exchange and occur as a broad singlet at 8.19 ppm for CDCA and 8.52 ppm for UDCA. The purity of maleic acid, which is stated by the manufacturer to be 98% (minimum) by the titration method, is actually determined to be 98.1% by using benzoic acid as the standard (Fisher Scientific Co.; purity, 100.0%). This result was calculated from the known weights and the ratio of the peak areas due t o the methine protons of maleic acid and the aromatic protons of benzoic acid in the solvent DMSO-d, with D 2 0 added to eliminate spectral interferences due to the carboxylic proton peaks. I t should be noted t h a t benzoic acid is not suitable for use as a standard in the quantitation of UDCA and CDCA because of interference from its broad carboxylic proton peak. The optimized experimental conditions for quantitation of UDCA and CDCA are listed in Table 11. In the quantitation of compounds by FTNMR spectrometry, the following aspects of data generation and acquisition play a critically important role. Repetition rate (or the interval between successive pulses) should be long enough so that saturation (or T I )effects are negligible, acquisition time long enough so that errors due t o relaxation by spin dephasing (or T z )are insignificant, and t h e interferogram size (or FID data points) large enough to define accurately the line shapes of signals. In the present case for purpose of quantitation, a 16K interferogram (8K real display or 8 data points/Hz) is found to be necessary for defining t h e methine peak of the standard and the comparatively broad C3 and Ci methine peaks in UDCA and CDCA. Due to the rather small values of TI (less than 2 s) and T2(less than 0.5 s), the values of the repetition rate (-8.2
Table 111. Quantitative Analyses of CDCA and UDCA by FTNMR Spectrometry weight ratio of weight of of peak purity of standard,a sample, areas, samgle, sample WR/mg Ws/mg A s / i l R % CDCA 20.83 55.86 0.805 99.6 25.53 59.88 0.702 99.3 24.86 60.39 0.727 99.3 21.04 54.12 0.773 99.7 21.69 56.79 0.786 99.6 53.17 0.794 99.5 20.08 UDCA 19.54 52.71 0.801 98.5 22.68 56.75 0.745 98.8 24.45 60.82 0.737 98.3 22.14 58.44 0.786 98.8 23.36 65.73 0.709 98.6 21.20 54.19 0.758 98.4 a Purity = 98.1%. The standard deviation of a single measurement is estimated to be 0.3%.
__
Table IV. Comparison of the Purity Determinations of CDCA and UDCA Samples sample ________ CDCA UDCA purity test (lot =32121) (lot 7510 906) assay ( N M R ) 99.5 * 0.3% 98 6 2 0 3% not less than 98% purity (TLC)" assay (GLC)a 100.0% 98.8% total acidsa 100.14% a Obtained from the Certificates of Analysis supplied by the manufacturers.
s) and acquisition time are adequate. The quantitative information required is the purity of CDCA and UDCA samples. This is obtained from the following equation:
As x Abfsx WR x P A , X MR X Ws
% sample = ____----_
(1)
where W , and W,, respectively, are the weights of the sample and standard. M s ( = 392.56) and MK ( = 116.07) are the molecular weights of the sample and standard, respectively. As is the total area of the C3- and C7-methine proton peaks of CDCA or UDCA. AR is the area of the peak due to the methine protons of the standard. P is the purity of the standard (in percent). Approximately 20 mg of maleic acid and 60 mg of CDCA or UDCA are dissolved in 0.5 mL of 8:l (v/v) of CDC13 and DMSO-d,, and T M S (-,3 pL) is added. The NMR spectrum is obtained using the experimental conditions described above. Using the "zoom" routine in the computer, the C,, and Cy methine proton lines of the sample and the methine proton peak of the standard are individually selected for display, corrected for baseline tilt, if any. adjusted for slope and curvature of the integral display, and then integrated to obtain a printout of the peak areas on the Silent 700 terminal. The assay values obtained from Equation 1 are presented in Table I11 and it is shown in Table IV that the net NMR assays for the samples of CDCA and UDCA compare favorably with the assays obtained, for example, by the GLC technique.
CONCLUSION The NMR spectrometric technique provides a superb method for rapid identification and quantitat,ion of the C7epimers: chenodeoxycholic acid and ursodeoxycholic acids. Instant identification of the free bile acids is obtained from the number of the C3 and Cy methine proton peaks in the
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
NMR spectra since two distinct peaks (6 -3.38, 3.78 ppm) caused by these protons are observed in CDCA vs. only one peak (6 -3.45 ppm) caused by the same protons in UDCA. T h e fingerprint region of the spectrum (6 1-2.5 ppm) and the relative position of t h e CISmethyl peak with respect to t h a t of the doublet caused by the Cpl methyl group provide additional confirmation of t h e identity of the particular epimer. Accurate quantitative results are obtained from careful integration of t h e C3 and C7 methine proton peaks. The specific method of identification and quantitation described in this report provides an efficient alternative to the rather nonspecific identification and assay procedures by conventional techniques, some of which involve making derivatives of the bile acid epimers. This paper, which is the first publication of its kind dealing with the quantitation of
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steroids by NMR spectrometry, demonstrates t h a t NMR spectrometry can be a n excellent tool for assaying steroids.
ACKNOWLEDGMENT The authors thank S.Moros for his comments and support, A. Mlodozeniec and J. Sheridan for their support, and L. Rubia for helpful discussion, and Mrs. J. Comment for typing the manuscript.
LITERATURE CITED (1) J. N. Shoolery and M. T. Rogers, J. Am. Chem. Soc 80, 5121 (1958). (2) N. S. Bhacca and D. H. Williams, “Applications of NMR Spectroscopy in Organic Chemistry, Illustrations from the Steroid Field”, Holden-Day, San Francisco, Calif., 1964.
RECEIVED for review February 21, 1978. Accepted J u n e 19, 1978.
Isotope-Ratio-Monitoring Gas Chromatography-Mass Spectrometry D. E. Matthews’ and J. M. Hayes* Departments of Chemistry and Geology, Indiana University, Bloomington, Indiana 4740 1
A 750 OC cupric-oxide-packed combustion furnace is inserted between the gas chromatographic column outlet and a GC/MS interface attached to a computer-controlled beam-switching isotope ratio mass spectrometer. Monitoring of N2 and COP ion currents due to the combustion products allows continuous measurement of 15N/14Nor I3C/”Cratios, providing directly comparable isotopic analyses for all eluting compounds regardless of composition and mass spectrometric behavior of the parent compounds. Carbon and nitrogen isotope ratios can be measured with a precision of 0.5% or better with 20 nmol of C02 or 100 nmol of N,. Enrichments of I3C and I5N as low as 0.004 at. YO excess can be detected.
Isotope ratios of carbon and nitrogen can be determined with very good precision (better than 0.170relative standard deviation) by differential isotopic analysis of combustion products using the dual inlet, dual collector mass spectrometric technique developed by C. R. McKinney et al. ( I ) , and with poor to good precision (10.0-0.576, depending upon the conditions) by selected ion monitoring gas chromatography-mass spectrometry (SIM-GCMS) (2). The techniques are somewhat complementary, t h e former providing high precision but requiring relatively large samples, the latter handling smaller samples a t the cost of reduced precision. Differences between the approaches are fundamental, however, because the GCMS technique directly incorporates a process of sample purification and is capable of resolving mixtures of compounds, while conventional dual inlet, dual collector techniques require that even sample combustion, let alone any steps aimed a t t h e resolution of mixtures, be performed off-line. In practical terms, the integral separation process gives the GCMS technique enormous advantages in the rate of sample throughput and in the confidence which Present address, Department of Medicine, Washington University School of Medicine, St. Louis, Mo. 63110. 0003-2700/78/0350-1465$01 O O / O
can be placed in results which are intended to relate to individual, pure compounds. A second fundamental contrast involves the nature of the ion currents measured: the GCMS technique deals with large, multielement fragment ions, while the dual inlet, dual collector technique accepts only molecular ions of small species containing at most two elements. The advantage here lies decisively with the latter technique. Consider, for example, the problem of measuring nitrogen isotopic abundances in organic compounds by SIM-GCMS: an ionic species useable for nitrogen isotopic measurements must be identified in the spectrum of each compound of interest, and t h e resulting data must be corrected for (i) contributions due to other heavy isotopes (2H, 13C,1 7 0 ) , and (ii) possible contributions to the apparent I5N abundances by other ionic species (e.g., fragment + H). The uncertainties associated with these corrections can seriously degrade the nitrogen isotopic measurements. On the one hand, natural isotopic abundance variations in I3C, for example, could introduce significant errors. On the other hand, exclusion of errors due to the second correction could require the selection and careful study of as many as 20 different pairs of masses in a chromatogram involving 20 different compounds of interest. Here we report on t h e capabilities of a new technique (3) aimed a t combining the mixture-handling capacity and speed of SIM-GCMS with the isotopic clarity of the dual inlet, dual collector technique. T h e technique, which we term “isotope-ratio-monitoring gas chromatography-mass spectrometry” (IRM-GCMS), can measure nitrogen and carbon isotope ratios down to natural abundance levels for any organic component (or componenw) that can be resolved gas chromatographically. The IRM-GCMS system shown in Figure 1 is very similar to that described by Sano et al. ( 3 ) : a gas chromatograph, a combustion oven, and a mass spectrometer, connected in series. The effluent from the gas chromatograph is quantitatively combusted to COz, Nz, HzO, etc; a selective trap removes combustion products other than those of interest; and the effluent enters the mass spectrometer. Carbon isotope ratios are determined by mea1978 American Chemical Society
