Characterization of peak purity by fast multiscan circular dichroism detection

Summary A method has been developed to detect inhomogeneity of apparently homogeneous peaks of very similar analytes. The method utilizes the rapid scan feature of state-of-the-art spectrometers/detectors that allow the recording of up to 30 spectra in a single chromatographic peak. Sensitivity and selectivity are enhanced by chiroptical/optical detection. Thus, identification of “front” and “rear” components of the peak can be carried out. The method is exemplified by mixtures of codeine, hydrocodone and oxycodone as analytes. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Determination of the enantiomeric purity of epinephrine by HPLC with circular dichroism detection

Several hundred drug substances approved by the U.S. Food and Drug Administration are chiral molecules. For the enantiomeric purity assessment, current practice is to develop separation techniques using chiral columns or mobile phase modifiers to separate enantiomers before detection. An alternative approach is to use currently accepted HPLC assay methods and use chiral-specific detectors to confirm whether the correct enantiomer is present. In this paper, adding a circular dichroism (CD) detector to an achiral HPLC method from the US Pharmacopeia (USP) is shown to be amenable for the determination of the enantiomeric purity of epinephrine, a substance used to treat anaphylaxis. This HPLC-UV-CD approach was able to detect the inactive D-(+) enantiomer at 1% of the total epinephrine composition. The linearity, accuracy, and precision of HPLC-UV-CD were evaluated and compared to analyses using a chiral HPLC method. Additionally, an epinephrine drug product was analyzed for assay (concentration) and enantiomeric purity. The results from achiral and chiral methods were identical within the experimental error. Overall, achiral chromatography performed using a USP method with CD detection may serve as a general means of determining chiral drug enantiomer purity and avoids the need for the development of additional chiral-specific methods for each individual drug.     

Quantitative determination of formic acid in apple juices by H NMR spectrometry

A quantitative determination method of formic acid in apple juices is proposed by means of the proton nuclear magnetic resonance (¹H NMR) technique. Formic acid gives a singlet signal at the 8.2-8.4 ppm interval of the spectrum, and its area is used to determine the concentration of the acid. 1,3,5-Benzenetricarboxylic acid is added to the juice as an internal standard. Since the chemical shift of both species varies with the pH, ascorbic acid is also added to adjust it at 2.74 and to avoid the overlapping of the signals. Recoveries between 95 and 109% are obtained when the standard addition method is applied to the juices of five different cider apple varieties. The coefficient of variation obtained is 3.9% for intra-day repeatability (n = 5), and 4.6% for inter-day repeatability (n = 10). The limit of detection is 1.49 mg/l, calculated from “3S_y/x + intercept”. The described method is direct and no previous derivatization is needed.     

Ligand-exchange ion chromatographic determination of malic acid enantiomers in apple juice with photometric detection.

An ion chromatographic separation with photometric detection using a chiral copper(II) complex as the eluent has been developed for the resolution of enantiomers of malic acid in commercially available apple juices. The results obtained by this method were in good agreement with those by an enzymatic method with separation by high-performance liquid chromatography.     

Enantiomeric purity determination by NMR: proving the purity of a single enantiomer

The NMR spectra of separate samples of an analyte complexed with each enantiomer of a chiral solvating agent (CSA) give an accurate estimate of the chemical shifts of racemic analytes in the presence of a single enantiomer of the CSA. This effect allows a CSA-based chiral NMR method to be developed when only a single enantiomer of analyte is available. The ability to develop a method capable of discriminating between enantiomers in these circumstances is useful, for example, to resolve the question of whether racemization has occurred during the synthesis of a chiral molecule.     

Photoacoustic detection of circular dichroism

An analysis is presented for an experimental technique involving the measurement of circular dichroism using polarization-modulated photoacoustic spectroscopy. The technique is referred to as photoacoustic circular dichroism (PACD). In the PACD experiment, a photoacoustic signal is induced by using polarization-modulated excitation light which is alternately left-and right-circularly polarized. Expression appropriate for analyzing the PACD experimental observables (signal strength and phase) in terms of sample circular dichroism are developed within the general framework of the Rosencwaig and Gersho model for the photoacoustic effect in solids and liquids. Calculations based on these expressions are reported and the applicability of PACD for measuring the circular dichronic properties of optically opaque samples is discussed.     

Determination of enantiomeric purity by simultaneous dual circular dichroism and ultraviolet spectroscopy

A method is described for the determination of enantiomeric composition. The ellipticity and absorbance of the sample are measured simultaneously by CD and UV spectroscopies, and the resulting G value is determined. G is an intensive physico-chemical parameter, a close derivative of anisotropy factor. Its magnitude is identical with opposite sign for enantiomers. The experimental G value is concentration-independent, as long as both absorption and ellipticity are linear functions of concentration. The analytical procedure introduced here is simple, rapid, and inexpensive, even though it includes calibration with standards of established enantiomeric composition. Provided the sample contains some UV-active contaminant(s), the method can be used after achiral chromatographic purification. By virtue, the method lacks several sources of error, arising normally from concentration inaccuracies. Applicability of the principles is tested by the example of phenylglycine and mandelic acid. Advantages of the method allowed the determination of phenylglycine enantiomer purity with an accuracy of 0.1%.     

Enantiomeric separation of racemic neolignans on chiralcel OD and determination of their absolute configuration with online circular dichroism

Effective enantiomeric separations of erythro- and threo-8.O.4'-neolignans with different aromatic substitution pattern (1a-i, 2a-i) are achieved on the commercially available chiral stationary phase cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). It is shown that the chiral recognition of the stationary phase is significantly dependent on the substitution pattern of the racemic compounds. Online liquid chromatography (LC)-circular dichroism (CD) analysis allows for the establishment of a correlation between the absolute configuration of the separated erythro-8.O.4'-neolignans and their characteristic CD transitions, which could be used to determine or revise the configuration of previously isolated erythro-8.O.4'-neolignans. Although the absolute configurations of threo-isomers is not determined unambiguously from the LC-CD analysis, it is proven that both their elution order and chiroptical properties are significantly influenced by the substitution pattern of the aromatic rings.     

Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism

The four-stranded G-quadruplex motif is a conformation frequently adopted by guanine-rich nucleic acids that plays an important role in biology, medicine, and nanotechnology. Although vibrational spectroscopy has been widely used to investigate nucleic acid structure, association of particular spectral features with the quadruplex structure has to date been ambiguous. In this work, experimental IR absorption and vibrational circular dichroism (VCD) spectra of the model quadruplex systems d(G)(8) and deoxyguanosine-5'-monophosphate (5'-dGMP) were analyzed using molecular dynamics (MD) and quantum-chemical modeling. The experimental spectra were unambiguously assigned to the quadruplex DNA arrangement, and several IR and VCD bands related to this structural motif were determined. Involvement of MD in the modeling was essential for realistic simulation of the spectra. The VCD signal was found to be more sensitive to dynamical structural variations than the IR signal. The combination of the spectroscopic techniques with multiscale simulations provides extended information about nucleic acid conformations and their dynamics.     

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm x 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm x 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min(-1). The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.     

Stereochemical studies of sialic acid derivatives by vibrational circular dichroism

Systematic VCD studies of N-acetylneuraminic acid (Neu5Ac), a recognition-related unique carbohydrate, were performed for the first time. Two pairs of anomeric isomers regarding a quaternary C2 asymmetric carbon of Neu5Ac derivatives were synthesized. VCD spectral patterns around the ester carbonyl region, as well as other Mid-IR regions, would be practical markers to distinguish the C2 stereochemistry.     

Enantiomeric purity determination of ketoprofen by capillary electrophoresis: development and validation of the method

A simple, fast capillary electrophoresis method for determining the total ketoprofen content in an oral pharmaceutical formulation is proposed. The addition of 75 mM of heptakis(tri- O-methyl)-beta-cyclodextrin to the background electrolyte allows the quantitation of the enantiomeric impurity of ( R)-(-)-ketoprofen contained in the formulation. A relative limit of detection is proposed as a measure of the lowest detectable enantiomeric impurity and the results show that the method can detect the minor enantiomer at levels as low as 0.04% in ( S)-(+)-ketoprofen. The chiral method was validated following ICH recommendations and the quality parameters obtained show the suitability of the proposed method. The analysis of samples examined during the course of a stability study under chiral and achiral conditions revealed that the total ketoprofen content did not change significantly with time and the enantiomeric impurity range was 0.1-0.4%.     

Enantiomeric purity determination of propranolol by capillary electrophoresis using dual cyclodextrins and a polyacrylamide-coated capillary.

The use of a chirally selective capillary electrophoresis method is reported for the enantioselective purity determination of propranolol drug substance. The method employed a combination of both charged and neutral cyclodextrin. An internally coated capillary was used to suppress electroosmotic flow and potential peak tailing. The method was capable of monitoring below 0.1% m/m of the undesired impurity. Acceptable validation data was also obtained for recovery, linearity, and for both short and long-term injection precision.     

High-performance liquid chromatographic determination of furfural and hydroxymethylfurfural in apple juices and concentrates

Summary A rapid and sensitive method for determining 2-furaldehyde (FUR) and 5-hydroxymethyl-2-furaldehyde (HMF) in apple juices and juice concentrates has been developed. The method for FUR and HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to reversed-phase separation with detection at 280 nm. The mobile phase was acetonitrile-water (8/92, v/v) at a flow rate of 1.0 ml/min. Recoveries from apple juices and juice concentrates spiked at different levels ranged from 94.1 to 104.0 (FUR) and 94.5 to 100.5 (HMF). The quantification limit for both, FUR and HMF, was 5 ppb.     

High-performance liquid chromatographic determination of major organic acids in apple juices and ciders

Summary A reversed-phase high-pressure liquid chromatographic method is presented for the simultaneous separation and determination of malic, citric, lactic, succinic and ascorbic acids in apple juices and ciders. After filtration and/or degasification, the organic acids in the sample are separated on a LiChrosorb/C₁₈ column and quantified by using a rapid diode array detector. The method is considered to be a suitable choice for the accurate and precise determination (C.V. 5%) of these compounds.     

Spectrofluorimetric determination of malic acid with β-naphthol

A detailed study of the spectrofluorimetric determination of malic acid by reaction with /gb-naphthol is reported. As little as 0.1 /smg of malic acid can be determined. The optimum concentration of sulfuric acid is 80% (v/v). The interferences of citric, succinic and tartaric acids are discussed.     

Quantitative determination of (-)-epicatechin in cider apple juices by (1)H NMR

This paper reports a quantitative determination method of (−)-epicatechin in apple juices by measuring of its signal at 7.05 ppm in the ¹H NMR spectrum. It is a direct method that does not need any previous derivatization. 1,3,5-Benzenetricarboxylic acid was added to the juice as internal standard for the determination of the absolute concentration of (−)-epicatechin. Ascorbic acid was also added to avoid enzymatic oxidation of the phenolics and to adjust the pH at 2.74, since the chemical shift of some compounds varies with the pH. Standard addition method accomplished with the juices of two different varieties of apples gave recoveries between 95 and 109%. The precision of the method was tested for repeatability (n=5) and reproducibility (n=13) obtaining a coefficient of variation of 5.8 and 8.6%, respectively. Limit of detection, calculated from “3S_y/x+intercept”, is 24 mg l⁻¹.     

Optical circular dichroism of the fullerene amino acid derivatives

The dependence of the circular dichroism spectra of the amino acid derivatives of fullerene C₆₀ on the structure of the initial α-amino acid was studied.     

Dipeptide structure determination by vibrational circular dichroism combined with quantum chemistry calculations.

The solution structure and the local solvation environments of alanine dipeptide (AD, 1 a) and its isotopomer (AD*, 1 b, 13C on the acetyl end C==O) are studied by using infrared (IR) spectroscopy and vibrational circular dichroism (VCD). From the amide I IR spectra of AD* in various protic solvents, it is found that each of the two carbonyl groups is fully H-bonded to two water molecules. However, the number of alcohol molecules H-bonded to each C==O varies from one to two, and the local solvation environments are asymmetric around the two peptides of AD* in alcohol solutions. The amide I VCD spectra of AD and AD* in D2O are also measured, and a series of density functional theory (DFT, B3LYP/6-311++G**) calculations are performed to obtain the amide I normal-mode rotational strengths of AD and the intrinsic rotational strengths of its two peptide fragments. By combining the VCD-measurement and DFT-calculation results and employing a coupled oscillator theory, we show that the aqueous-solution structure of the dipeptide can be determined. We believe that the present method will be of use in building up a library of dipeptide solution structures in water.