Ultraviolet Derivatization of Digitalis Glycosides as 4Nitrobenzsates for Liquid Chromatographic Trace Analysis Frledrich Nachtrnann and Hans Spitzy Department of General-, Micro- and Radio-Chemistry, Technical University of Graz, Graz, Austria
Roland W. Frei" Analytical Research and Development, Pharmaceutical Depatiment, Sandoz Ltd., 4002 Basel, Switzerland
The derivatiration of 11 digitalis glycosides and their aglycones with 4-nitrobenzoylchloride (4-NBCI) has been studied. Completely substltuted and stable derivatives can be formed. The derivatization and extraction steps can be carried out in less than 1 h. The structures of the derivatives have been investigated by UV and NMR spectroscopy.The major advantage of this esterification procedure is an enhancement of UV absorbance. The absorption maximum is in the favorable range of 255 to 260 nm which permits a simple detection with filter photometers at the Hg line of 254 nm. The extinction values range from e = 14 800 (digitoxigenin) to 118 000 (desacetyllanatoside C). The reproducibility of the technlque is limited by the derivatization procedure and varies between 2.5 and 3.5 % rel. std. dev. The derivatizationresults in molecules with lower polarity and better adsorption chromatographic properties. This permits rapid isocratic separations with reduced tailing of complex mixtures by liquid chromatographic techniques.
Chemical, biochemical, and radiochemical methods have been used for the analysis of digitalis glycosides. T h e sugar moiety can be derivatized for photometric detection without t h e steroid portion of t h e molecule reacting (1).T h e CY-,punsaturated lactone ring has a tendency t o form colored complexes which can also be used for analytical purpose. Picric acid is frequently adopted to this end (2).Some fluorimetric methods (3-5) are based on dehydration reactions of the steroid moiety; the mechanism of these reactions is not known. A summary of chemical analysis procedures has appeared i n 1974 (6). Recently, some very sensitive enzymatic methods have been developed (7,8) and radioimmunoassay techniques of even better sensitivity and specificity are now also available (9-11).
T h e disadvantage of these methods, with the exception of t h e radioimmunoassay approach, is the necessity t o carry out a chromatographic separation prior to applying the above mentioned techniques. Paper or thin-layer chromatography has t o be used. Gas chromatographic techniques require tedious derivatization procedures which lead to fragmentation of the glycosides (12,13).T h e low vapor pressure and relative instability of digitalis glycosides would make high pressure liquid chromatography an ideal separation technique (14,15). Unfortunately these compounds have only a relatively weak absorption maximum at about 220 n m (lactone ring) which is not sufficient for detection of breakdown or by-products i n t h e nanogram concentration region in pharmaceutical formulations or for trace analysis of digitalis glycosides and metabolites in a biological matrix. The low wavelength of the absorption maximum (220 nm) also puts serious restrictions on t h e choice of weakly absorbing chromatographic solvents (15). It is for this reason that t h e present work has been under1576
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taken. T h e idea was t o introduce a suitable chromophor to improve t h e detectability of these digitalis glycosides and possibly also the chromatographic properties by reducing their polarity. For this purpose, esterification reactions with 4nitrobenzoylchloride (4-NBC1) have been investigated. 4NBCl has been used for some time as a derivatization reagent for non-aromatic hydroxy groups (16). It has also been applied with good success for hydroxy steroids (17). T h e problem in our study was to derivatize the entire molecule (steroid and sugar segments) without destroying the molecule. The suitability of such derivatives to liquid chromatographic separation was briefly investigated.
EXPERIMENTAL Reagents. For a good functioning of the derivatization, it is essential that the reagents be very pure. The 4-NBC1 analytical grade (Fluka) was recrystallized once from petroleum ether (Riedel de Haen) bp 60-70 "C. The melting point should be between 71-73 "C. Analytical grade pyridine (Fluka) was refluxed for 3 h with NaOH (Merck anal. grade), distilled, and stored over NaOH (bp 115-116 "C). 4-Dimethylaminopyridine purum (Fluka) and acetonitrile uvasol qualrty (Merck) were used. All other reagents and solvents were analytical grade (Merck). The digitalis glycosides and aglycones (see Table I) were provided by Sandoz Ltd. For TLC, Merck SIL G60 F254 silica gel plates were used. Silica gel Merckosorb SI 60 with 5 p average particle size was employed for HPLC. Instruments. UV measurements were carried out on a Zeiss-PMQ 11-Spectrophotometer. NMR spectra were recorded with a Bruker HX-90 E instrument. The HPLC work was done on a HewlettPackard UFC-1000 Chromatograph equipped with a DuPont 842 UV detector for 254 nm. Derivatization. A fresh reagent solution has to be prepared every day. One hundred mg of 4-NBC1were dissolved per 1 ml of pyridine with gentle warming. The glycosides were also dissolved in pyridine. The reaction was carried out in stoppered 10-mlcentrifuge tubes. To 50 pl of a glycoside solution containing not more than 0.5 mg of the glycoside, 150 p1 of reagent solution were added, well shaken, and reacted for 10 min at room temperature. After this time, the reaction was quantitative as shown by TLC screening. Extraction of the Derivatives. Aglycones. The method described by Fitzpatrick ( 1 7 )was somewhat modified. In order to remove the excess reagent, the reaction mixture was hydrolized with 1 drop of HzO. After 1min, 2 ml of a 5% NaHC03 solution and 2 ml of chloroform were added and the vial was shaken for 1min. A good separation of the two layers was obtained by centrifugation at 3000 U/min for 30 s. The supernatant aqueous layer was removed and the extraction step repeated with the same chloroform solution by adding another 2 ml of 5% NaHC03 solution. This was followed by treating the chloroform solution twice with 3 ml of a 1N HC1 solution. Glycosides and Mixtures of Glycosides and Aglycones. The instability of the glycosides demanded the development of an alternate procedure. It was necessary to remove the pyridine prior to hydrolizing the excess reagent. The centrifuge tubes were imbedded in a beaker filled with sand at a temperature of 50 "C. This sand bath was placed in a desiccator and the pyridine removed by a water suction vacuum within 10 min. The centrifuge tubes were flushed with an air or nitrogen stream and 2 ml of a 5%NaC03 solution was added, which also
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Table I. Chemical Structures of the Digitalis Glycosides and Aglycones Investigated
12
Digitoxigenin Digitoxin Acetyldigitoxin Lanatoside A Gitoxigenin Lanatoside B Digoxigenin Digoxin Acetyldigoxin Lanatoside C Desacetyllanatoside C a D = Digitoxose; AcD
14
16
H OH H H OH H H OH H H OH H H OH OH H OH OH OH OH H OH OH H OH OH H OH OH H OH OH H = Acetyldigitoxose; G
Ra
H D-D-DAcD-D-DG-AcD-D-DH G-AcD-D-DH D-D-DAcD-D-DG-AcD-D-DG-D-D-D= Glycose.
contained 5 mg of 4-dimethylaminopyridine.The excess reagent was hydrolyzed after 5 min of shaking or treatment in an ultrasonic bath. A blank treated simultaneously should yield a clear solution. The derivatives are poorly soluble in water and are giving turbid solutions. The derivatives were then extracted with 2 ml of chloroform and treated with 2 ml of a 5%1NaHC03 solution and twice with 3 ml of a 0.05 N HCl solution con1,aining 5% NaCl. This leads to a quantitative isolation of the derivatives and complete exclusion of excess reagent and pyridine. UV Measurements. Two hundred or 500 pl of the chloroform extract were diluted with 3 ml of chloroform and measured vs. a blank treated identically. All the measurements were done in a 1-cm quartz cuvette at room temperature. A slit width of 0.08 mm was used. NMR Measurements. The derivatives were dried in a vacuum and dissolved in CDCls. The spectra were recorded at 90 MHz using the Fourier technique. TLC of the Derivatives. Qualitativeseparations can be done directly from the reaction mixture. They were useful to control the derivatizationprocess and the uniformity of the derivatives. Two solvent systems were used: 1)Ethyl acetateln-propanolltoluene 66:14:20 and 2) Toluene/methanol5:1. The solutions were spotted (5 pl/spot) on commercial Merck silica gel plates SIL G 60 F254, dried, and developed. For detection at low concentration levels, the layers were sprayed with 10% ethanolic sulfuric acid and dryed at 110 O C for 15 min. This resulted in fluorescent spots due to dehydration of the steroid portion of the derivatives (Aex 366 nm). HPLC of the Derivatives. Separations were carried out on a column of 15 cm in length and 3-mm i.d. The column was packed with Merckosorb SI 60 silica gel with 5 p average particle size using an equal density packing procedure (18). Isocratic separations were carried out at -20 O C (room temperature) without thermostating. A solvent system n-hexanelmethylene chloride/acetonitrile, 10:3:3, was used as mobile phase. The flow-ratewas 1.5 mllmin with a pressure drop of 116 atm. A chromatronix loop injector was used for the injection of 25 111of chloroform extract.
RESULTS AND DISCUSSION Kinetics. T h e dependence of t h e derivatization reaction on reagent concentration and time can be seen in Figure 1with gitoxigenin. T h e reaction reaches a n equilibrium state after a relatively short period. A quantitative reaction is only possible with a large excess of reagent; hence, one can conclude t h a t t h e equilibrium constant rather than the kinetics of t h e reaction is t h e limiting factor. From t h e data in Figure 1i t becomes clear, t h a t with a reagent excess of about a hundred-fold, a quantitative esterification with 4-NBCl cain be achieved. The other aglycones and glycosides give similar results. The reactions were also verified by TLC methods (see
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Flgure 1. Formation of gitoxigenin-4-nitrobenzoate as a function of reagent concentration 116.8 pg of gitoxigenin are reacted with (X) 2.5 rng 4-NBCI: (0) 5.0 mg 4-NBCI;
(a)10.0 rng 4-NBCI; (A)20.0 rng 4-NBCI (for details of reaction conditions,see Experimental section)
n 1
2
3
4
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U Flgure 2. Thin-layer chromatogram of digoxigenin-4-NB derivatives (1) No 4-NBCI added; (2) 10 rng 4-NBCI added; (3) 4 mg 4-NBCI added: (4) 0.9 rng 4-NBCI added (for chromatographicdetails, see Experimental section)
Figure 2) and confirm t h e results observed in Figure 1. One has to realize, however, t h a t the significance of t h e results is limited by the visual detection limit on TLC which is around 100 nghpot. The spot with the highest Rf value corresponds to the completely substituted aglycone digoxigenin. This is to be expected since this sample contained t h e largest excess of reagent (10 mg 4-NBCl; row 2, Figure 2). With only 4 mg of reagent, the reaction is not complete (row 3, Figure 2); the two poorly resolved spots would correspond t o the two possible monosubstituted digoxigenins. T h e spot with the lowest Rf value corresponds to the non-reacted aglycone (rows 1,3,and 4,Figure 2). From t h e observations with other TLC experiments, one concludes t h a t digitoxigenin is only monosubstituted and digoxigenin, gitoxigenin are doubly substituted; consequently, one can show that the tertiary OH group in the 14 position (Table I) is not available for substitution, because of steric hindrance. Temperature Dependence of the Reaction. For an investigation of the influence of elevated temperatures on the reaction, a reagent concentration was chosen which, a t room temperature, would not give a complete substitution. The other reaction conditions were kept t h e same as for room temperature. T h e results are shown in Figure 3. T h e esterification is an endothermic reaction; hence, t h e equilibrium is shifted in favor of the derivatized product. T h e average of derivatization at higher temperatures is, however, offset by the inconvenience and the danger of partial decomposition
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Flgure 5. High-pressure liquid chromatogram of a mixture of digitalis glycosides and aglycones Column: SI 60, 5 p : 15-cm length, 0.3 cm4.d. Solvents: n-hexanelCH~CI~l CHBCN, 10:3:3 (isocratic). Pressure drop: 116 atm, flow: 1.5 mllmin. Detector: Du Pont 842, X = 254 nm. Injection: Valco valve: loop 25 pl. (l)gitoxigenln, (2) digitoxigenin, (3) digoxigenin, (4) acetyidigitoxin, (5) digitoxin, (6) acetyidigoxin, (7) digoxin, (8) unknown, (9) lanatoside B, (IO) lanatoside A, (1 1) lanatoside C, (12) desacetyi-lanatoside C
0.4-
0.3-
0.2-
0.1-
Flgure 4. UV spectra of mol/l. of (1) digoxin in ethanol, (2) digoxin-4-NB in chloroform (5 NB groups substituted), (3) digoxigenin-4-NB in chloroform (2 NB groups substituted)
and, hence, poor reproducibility as evidenced by preliminary tests. NMR Spectra. The NMR spectrum for the 4-NB derivative for digoxigenin (C37H40N2011) has been recorded. The aromatic protons can be seen in a multiplet between 8.1 and 8.5 ppm. The integration yields 8 aromatic protons for digoxigenin. This corresponds to the degree of substitution of the fully derivatized glycoside and confirms the TLC data. In addition, all protons of the lactone ring can be recognized. The ring remains intact. Digoxigenin is substituted in the C3 and Clz position. The signal of the free tertiary OH group in the 14 position cannot be seen in the spectrum. Another spectrum taken for the digitoxin derivative under identical conditions revealed clearly 16 aromatic protons which corresponds to the theoretically expected fourfold substitution for digitoxin. U.V. Spectra. A quantitative study of UV absorption revealed again, that with the exception of the OH group in the 14 position all other groups are reacting quantitatively. The digitoxigenin derivative, for example, gives an extinction value t of 14 800 (one NB group) using a wavelength of 260 nm, digoxigenin and gitoxigenin give an e of 29 500 (2 NB groups). The glycosides show greater extinction values depending on the degree of substitution. Further studies about the exact correlation of extinction values of 4-nitrobenzoates and 1578
structure of the molecules including mono- and disaccharides are in progress. The UV spectra of digoxin and the 4-NB derivative of digoxin and digoxigenin are shown in Figure 4. The strict additivity observed as a function of the degree of substitution is an indication for the complete and quantitative substitution of the glycosides. It also shows the improvement in absorption properties than can be obtained through derivatization. The adsorption maximum is for all derivatives a t 260 nm in chloroform and ethanol and 255 nm in cyclohexane/chloroform 2O:l. This would mean that, for example, in HPLC detection, a simple single wavelength detector (mercury line a t 254 nm) could be utilized. The stability of the derivatives is excellent also toward irradiation. No change of E values was observed after irradiation of a sample of 4-NB-digoxin a t 260 nm with a slitwidth of 2 mm for over 1h. Calibration Curves. In continuation of the UV studies, calibration curves were established a t 260 nm for all the compounds investigated. The linearity is excellent over a large concentration range and the plots go through the point of origin. The slope is indicative of the degree of substitution. The largest slope is obtained for desacetyl lanatoside C which has an &fold substitution. Lanatosid; B and C are identical, both with a 7-fold substituted molecule, and lanatoside B has a 6-fold substitution and hence the smallest slope value. It is interesting to note that the acetyl group is not split off the lanatosides during the derivatization procedure. The reproducibility of this technique is limited by the derivatization step and varies between 2.5 and 3.5% relative standard deviation for concentrations >lo0 ng/ml depending on the glycoside. The detection limits (3:1,signal-to-noise ratio) vary between 5 and 20 ng/ml a t an injection volume of 100 pl (19). Separation by HPLC. The potential of this derivatization technique in conjunction with liquid chromatography is demonstrated in Figure 5 . The isocratic separation of this
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
mixture of digitalis glycosides can be achieved in 12 min. The separation is sufficiently good for quantitative work (19). Impurities originating from the chloroform used in the extraction and traces of reagent are eluted before the derivatives. The first peak corresponds to t o (non-sorbed component, dodecylbenzene). The unidentified peak is an impurity of the desacetyl lanatoside C standard. The lowering of the polarity of the aglycones and glycosides due to the derivatization step results in a better chromatographic behavior. The eluents used can be of lower polarity and viscosity; peak broadening due to strong adsorbentadsorbate interactions is reduced and the improved selectivity permits an isocratic separation of very complex mixtures.
CONCLUSIONS Esterification of the digitalis glycosides with 4-NBC1 can be carried out simply and quantitatively. With a sufficient excess of reagent, completely substituted derivatives are obtained. The liquid-chromatographic properties of the glycosides are considerably improved. The strong absorption and the favorable ,A, (260 nm) of the 4-nitrobenzoates permits a very sensitive and simple detection both in HPLC and TLC. For all these reasons, this technique is superior to previously existing methods for trace analysis of digitalis glycosides. The method should be widely applicable t o the derivatization of non-aromatic OH groups which are not sterically hindered. I t seems particularly promising for the wide field of carbohydrate analysis where a more rapid and sensitive method could mean a real breakthrough in analytical knowhow. Further work on the application of this technique to the
separation and quantitation of glycosides and sugars is in progress.
ACKNOWLEDGMENT The authors thank H. R. Loosli for taking the NMR spectra.
LITERATURE CITED (1) J. W. Myrick, J. Pharm. Sci., 58, 1018 (1969). (2) T. Higuchi and E. Brochmann-Hanssen, "Pharmaceutical Analysis", Interscience Publishers, New York, London, 1961, p 56. (3) D. Wells, B. Katzung, and F. H. Meyers, J. Pharm. Pharmacoi., 13, 389 (1961). (4) L. F. Cullen, D.L. Packman, and G. J. Papariello, J. Pharm. Sci., 59, 697 (1970). (5) A. 2 . Britten and E. Njau, Anal. Chim. Acta, 76, 409 (1975). (6) D.P. Page, FDA By Lines, 1, 1 (July 1974). (7) S. Mardh, Clin. Chim. Acta, 44, 165 (1973). (8) G. H. Burnett and R. L. Conklin, J. Lab. Clin. Med., 78, 779 (1971). (9) R. E. Chambers, Ciin. Chim. Acta, 57, 191 (1974). (10) G. S.Ahluwahia and Z . J. Kuczala, Clin. Chem.. 21, 270 (1975). (1 1) R. C. Boguslaski and C. L. Schwartz, Anal. Chem., 47, 1583 (1975). (12) E. Watson and S. K. Kalman, J. Chromatogr., 56, 209 (1971). (13) E. Watson, P. Tramell, and S. K. Kalman, J. Chromatogr., 69, 157 (1972). (14) F. J. Evans, J. Chromatogr., 88, 411 (1974). (15) W. Lindner and R. W. Frei, J. Chromatogr., 117, 81 (1976). (16) S.Siggia, "Instrumental Methods of Organic Functional Group Analysis", Wiley-lnterscience, New York, 1972, pp 1-74. (17) F. A. Fitzpatrick and S. Siggia, Anal. Chem., 45, 2310 (1973). (18) R. M. Cassidy, D. S. Legay, and R. W. Frei, Anal. Chem., 46, 340 (1974). (19) F. Nachtmann, H. Spitzy, and R. W. Frei, J. Chromatogr., 122, 293 (1976).
RECEIVEDfor review January 15, 1976. Accepted June 14, 1976. F. Nachtmann thanks Sandoz Ltd. for a grant in support of his thesis work.
Exclusion Chromatography with Multiple Detectors for Following Compositional Changes of Petroleum Residuals during Desulfurization E. W. Albaugh" and R. C. Query Gulf Research & Development Company, Pittsburgh, Pa. 15230
The changes in size, molecular weight, sulfur distribution, and aromaticity during catalytic desulfurization have been shown. Also, a method for continuously monitoring the sulfur content of chromatographic effluents has been demonstrated.
Recently, the feasibility of rapidly following the changes in molecular weight distribution and aromaticity as a function of molecular weight by utilizing exclusion chromatography (GPC) with multiple detectors was reported for petroleum residuals ( I ) . In that work, samples of identical weight (mass) were separated with a set of exclusion columns and the effluent was monitored with a differential refractometer (RID), an ultraviolet absorption detector (UAD), and a flame ionization detector (FID). By comparing the chromatograms from the RID and UAD of samples taken a t different stages of desulfurization, estimates of the changes in aromaticity could be made. A comparison of the changes in the chromatograms from the FID gave an estimate of the changes in mass distribution as a function of molecular weight.
One of the problems with the previous work was the inability of the FID to produce equal response for the various classes of petroleum hydrocarbons. For saturates, the response was comparatively high; while with resins and higher molecular weight species, the response was low. In the current work, this problem has been minimized by incorporating the improved Pye LCM2 FID for following changes in weight distribution. Also included in this current work are preliminary data on instrumentation for obtaining, on an analytical wale, the sulfur distribution as a function of molecular weight.
EXPERIMENTAL The liquid chromatograph was fabricated at GR&DC and was of conventional design. It consisted of a solvent reservoir, degasser, pumping system, sample injection valve, three Waters 4-ft X 3/s-in. lo4linear Styragel columns, an oven, and detectors. Benzene was used as solvent and pumped at a flow rate of 1.7 ml/min. The experimental details of the system have been reported previously ( I , 2). The Pye System I1 flame ionization detector used previously ( I ) was replaced by the Pye LCM2. This instrument converts the sample to carbon dioxide and then to methane which is determined with a
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