Determination of diastereomeric composition of drugs
Since the chiral nature of the living systems has on the one hand implications on biological active compounds interacting with them, the stereoisomers of drugs can differ in both the pharmacodynamic and the pharmacokinetic action [1]. On the other hand from synthesis procedures drugs, having one or more centres of chirality, were often obtained as isomeric mixtures of both diastereomers and/or enantiomers. Owing to the fact, that diastereomers have different chemical and physical properties, the determination of the isomeric composition is possible in achiral medium. Herein, 1H and 13C NMR spectroscopy has been shown to be a powerful tool to elucidate the configuration and isomeric purity of drugs. Applying 1H NMR spectroscopy integrals of corresponding signals of the isomers can be directly used for the evaluation in the case the signals are well separated. When 13C NMR spectroscopy is used it has to be checked whether the "response" of the signals considered is comparable.
Response: The area of NMR signals is directly proportional to the molar amount of the isotope considered, e. g. 1H or 13C. The ratio between two different signals of one molecule should be 1: 1, with the number of represented atoms being taken into account. In practice, there are differences caused by different relaxation times. This is the time an excited atom needs to fall down to the ground state. In case of heteronuclear decoupling, the Nuclear Overhauser Effect (NOE) can also cause different response factors. These response problems are influenced by the measuring parameters, they disappear or minimise with the correct (problem oriented) choice. Within a family of atoms in similar chemical surroundings these effects may be neglected. The response factors change from ±10% in 1H NMR spectra up to ±50% in 13C NMR spectra [2, 3].
Examples
Quinine / quinidine
Cortex Cinnamoni consists of several structural related alkaloids, e.g. the quinine, an antimalaria antibiotic, and quinidine, an antiarrhythmic agents. Even though the substances are diastereomers due to the different configuration at hydroxylated C9 the separation from the plant extracts is difficult. Thus, the current edition of the European Pharmacopoeia limits the content of either other china alkaloids to 2.5 percent using an HPLC method. In order to check whether NMR spectra are principally appropriate to evaluate the isomeric purity both alkaloids were recorded separately for assignment [4, 5] and as a mixture for working out which signals can be used for evaluation. As can be seen from Fig. 1, the signals of the hydrogens at C11 of quinine and quinidine are well separated and can be, therefore, used for quantification by means of integration. In a mixture of the two diastereomers an amount 2% of quinine can be easily evaluated in presence of 98% of quinidine. Interestingly most of the signals of the other hydrogens of both diastereomers show almost the same chemical shift and are most well resolved.
Figure 1: 1H NMR spectrum of a mixture of quinine and quinidine in DMSO-d6,
containing 2. 198% quinine
containing 2. 198% quinine
Parameters quinine/ quinidine:
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Fluvoxamine
Fluvoxamine is an antidepressant drug consisting of an oxime ether functions which can exist in an E and Z configuration. The pharmacological effect is associated with the E-isomer; thus, the European Pharmacopoeia limits the Z-isomer to less than 0.2 percent. The 1H NMR spec-trum of a 1: 1 mixture (for spectrum and structural formula see Fig. 2) reveals the resonance signals of the hydrogen at C10 (δ = 4.2-4.5 ppm) and C2 (δ = 2.5-3.0 ppm) to be well separated and, therefore, appropriate for evaluation. In order to find out the optimal pulse repetition period the T1 relaxation time was measured first. A recovery of the z-magnetization of > 99.9% was achieved using seven times the longest T1 which results in a pulse repetition time of 8 sec. Using the optimized parameter (see below) and after checking the linearity, the limit of quantification of the Z-isomer could be determined to be 0.15 %, which meets the limit demanded by the European Pharmacopoeia. For details see ref. [6].
Figure 2: 1H NMR spectrum of a mixture of the isomers of fluvoxamine, containing 47.6% E-fluvoxamine
and 53.2% Z-fluvoxamine, in MeOH-d4
and 53.2% Z-fluvoxamine, in MeOH-d4
Parameters fluvoxamine:
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Captopril
The antihypertensive agent captopril inhibits the angiotensin-converting enzyme. Due to two centres of chirality four isomers can be expected. The pharmacological interest resides with the SS-isomeric form (see structural formula in Fig. 3) [7]. Thus the RS diastereomer, epicaptopril, is an impurity, which results from incomplete removal during the syntheses [7, 8]. Other impurities to be expected are methylmercaptopropionic acid (impurity of synthesis) and an disulfide analogue (oxidation product of two molecules of captopril) [9, 10]. In addition two amido conformers were observed in solute state [8].
1H NMR spectroscopy is able to distinguish between captopril (SS), the two amido conformers, epicaptopril (RS) and the disulfide analogue [8]. The signal for analytical determination is the hydrogen (H-2) representing the methine hydrogen attached to C-2 of the pyrrolidine ring. This hydrogen (H-2) shows in DMSO-d6 a clearly separated signal in the range between 4.1 and 4.7 ppm. Its chemical shift depends on configuration and conformation. The dublication of the H-2 resonance in a major and a minor part in solution is due to the existence of a major and a minor amount of the two amido conformers [8].
Figure 3: 400 MHz 1H NMR spectrum of captopril (14 mg/0.5 ml DMSO-d6); M = major, m= minor
Parameters captopril:
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The ratio between these two signals of captopril in DMSO-d6 was reported to be about 5.
Resonance dublication can also be seen for the H-5 multiplet and the doublet of the methyl group. Both signals are analytically critical because the minor part of H-5 arises in the region of the broad water resonance in DMSO-d6 and the signals of the major and minor methyl doublet overlap. In the case of well separated signals quantification should not be a problem even in routine analysis. A field strength of 9.4 Tesla (400 MHz) is sufficient for operating.
Casy and Dewar describe [8] the NMR characteristics of a mixture of captopril (16 mg) and its RS epimere, epicaptopril (4 mg), and of a secondary mixture of captopril (16 mg), epicap-topril (4 mg) and the disulfid analogue impurity (2 mg) in DMSO-d6 at 400 MHz. The binary mixture shows the major resonances of the amido conformers overlapped but the minor part signals are clearly separated (see Fig. 4). Also in the tertiary mixture the minor part signals of the components are resolved for detection.
The 1H NMR spectrum in Fig. 3 exhibits the two amido conformers of captopril in DMSO-d6. The signals of the minor conformer (m) at 4.46 ppm and the major (M) at 4.25 ppm are clearly separated; the ratio is about 5. Resonances of the RS diastereomer and of the disulfide analogue are not visible in the sample studied. The broad response of the -COOH group was measured at 12.40 ppm.
Figure 4: Binary mixture: 16 mg captopril (SS), 4 mg epicaptopril (RS), tertiary mixture:
16 mg cap-topril (c), 4 mg epicaptopril(a), 2mg disulfid analogue(b); solvent: DMSO-d6
16 mg cap-topril (c), 4 mg epicaptopril(a), 2mg disulfid analogue(b); solvent: DMSO-d6
Examples described in the literature
The antipsychotic effect of chlorprothixene (Fig. 5) resides mainly with the cis isomer whereas the trans isomer is less active and has, thus, to be considered as an impurity. In addition, UV light induces a cis-trans-isomerization. Rosler et al. [9] developed a NMR method for determining the isomeric content based on the ratio of integrals of the cis-N-methyl signals in comparison to the trans-N-methyl resonances. Using a 300 MHz spectrometer, the signals occurring at 2.24 ppm (cis-isomer) and 2.23 ppm (trans-isomer) are separated; applying the deconvolution routine the limit of detection is "easily" below 5 percent of the trans isomer. Tablets studied did not show any content of the trans-isomer.
In order to determine the isomeric composition of the stilbene derivatives clomiphene and tamoxifen (Fig. 5), having ovulation-stimulating and antiestrogenic properties, respectively, the British Pharmacopoeia 1993 and United States Pharmacopoeia XXIII utilise HPLC meth-ods. Lindgren and Martin [10] showed that in both cases NMR spectroscopy using a 300 MHz spectrometer can be a more rapid and accurate tool for this quantitative determinations. The base of clomiphene citrate was liberated from tablets and the isomeric ratio determined by integration of the triplets of the OCH2 group appearing at δ = 3.98 ppm (E-isomer) and δ = 4.11 ppm (Z-isomer). All samples, clomiphene substance (Z = 33.3%), Pergotime® tablets (Z = 40.5%) and Clomivid® tablets (Z = 33.3%), were found to be in the range required by the BP and USP: 30 ≤ Z ≤ 50%. In the case of the Z-isomer of tamoxifen carrying the biological activity the E-impurity could be determined in the same manner down to and under the 1% level required by the Pharmacopoeia: E-impurity amounted to 0.5% in tamoxifen substance, to 0.4% in Tamoxifen NM® tablets and to 0.4% in Novaldex® tablets. In comparison to the USP XXII, the USP XXIII has tightened the maximum level down to 0. 3%. Thus, the samples stud-ied by Lindgren and Martin did not meet the requirement.
Figure 5: Structural formula of clomiphene, tamoxifen, chlorprothixen and nifuroxazide,
the diastereomers iloprost and isoiloprost, and atracurium besylate
the diastereomers iloprost and isoiloprost, and atracurium besylate
Care must be taken that sidebands of either isomer do not interfere with the integral traces over the peaks considered. The change of sample spinning can help to avoid the interfering spinning sidebands. In addition, the13C sidebands have to be taken into account.
Iloprost, a mimic of the prostacycline PGI2 and an inhibitor of the blood-plated aggregation [11], is characterised by an exocyclic E-configuration of the trisubstituted 5,6-double bond (Fig. 5) whereas isoiloprost having lower biological activity is the unnatural Z-isomer. Since the side chain is attached to the bicyclo[3.3.1] octanone system by a non-stereoselective Wittig reaction iloprost always contains a certain amount of the Z-isomer. The configuration of the double bond was elucidated using the 2D 13C, 1H relayed coherence transfer experiment and the g-effect occurring at double bonds. The isomeric composition was determined by integration of the corresponding carbon atoms [12].
Lartigue et al. [13] observed the photoisomerization of the iminofurane moiety of the antibiotic nifuroxazide. Since resonances of the imino hydrogen CH= N of both isomers are well separated (δ = 7.74 versus 8.41 ppm) the increase of the signal of the upfield-shifted hydrogen can be easily observed upon UV irradiation. The configuration of nifuroxazide was found to be E and of the photoisomer Z.
Since the muscle relaxants atracurium besylate consists of two racemic 1,2,3,4-tetrahydropapaverine molecules connect by a pentamethylene diester it is composed of various proportions of ten isomers, present as four enantiomeric pairs and two meso forms. Since the 1H NMR spectrum of the mixture suffers from overcrowding and heavy overlapping of corresponding signals it cannot be used for assignment and quantification. Thus, the hyphenation of a 750 MHz NMR spectrometer with chiral HPLC using the stop-flow-technique was utilised for full analysis. The fractions obtained from a Chiracel OD-H column (250 x 4.6 mm, 10 µm) with a mobile phase of 0.5 M NaClO4 (pH 2.0) and acetonitrile (60: 40) were send through a very small cell (4 mm i.d. of 65 µl volume) for NMR measurements by indirect detection with a z-gradient flow probe. Due to the small flow cell for nine out of ten peaks pure spectra could be recorded. In combination with HPLC-CD experiments, the peaks could be assigned to the different isomers [14].
Using the continuous-flow HPLC-NMR technique Albert and coworkers were able to separate and assign the five cis and trans isomers of vitamin A [15]. By means of the hyphenation of SPE-MS-NMR the isomers and various isomers and methylation products of gentamicin could be also separated and assigned [16]. Further examples can be found in ref. [15, 17].
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