Liquid Chromatography-circular Dichroism Research Articles

Development of a certified reference material for D-phenylalanine with evaluation of enantiomeric purity.

D-Phenylalanine (D-Phe) is a small chiral organic molecule that is both an important pharmaceutical intermediate and used as a calibrator for quantifying amino acids in liquid chromatography-circular dichroism. We have developed a process for a national certified reference material (CRM) for D-Phe following ISO 17034:2016. The identity of D-Phe was confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR), infrared, and ultraviolet (UV) spectroscopy. The absolute optical conformation was also determined using circular dichroism (CD) spectroscopy and optical rotation measurements. Impurities were identified via liquid chromatography (LC) with a UV-Vis detector and a charged aerosol detector (CAD) and LC-MS. Both mass balance and quantitative NMR were employed for value assessment, and the associated uncertainty was evaluated. The certified purity was determined to be 0.995 ± 0.003g/g, a validation that was confirmed by CD using L-Phe CRM as a calibrator. Twenty milligrams of raw material was packed in sealed brown glass tubes for storage, and no inhomogeneity was observed. Stability tests revealed that the D-Phe CRM remained stable at -20°C for at least 26months, at 4°C for at least 14days, and at 25°C and 60°C for at least 7days. The D-Phe CRM can be used to ensure the accuracy and reliability of D-Phe quantitation in the pharmaceutical field and also as a calibrator to ensure traceability to the International System of Units (SI) for L-Phe quantitation and protein purity analysis using LC-CD methods. The approach outlined in this paper also has potential for use in the development of other chiral CRMs.

Development of a human insulin certified reference material with SI-traceable purity.

A human insulin (hINS) certified reference material (CRM) was developed by the National Institute of Metrology (NIM). Three milligrams of purified solid hINS was packed into a brown sealed tube. The candidate material was identified by de novo sequence using mass spectrometry and Edman degradation methods. The content of insulin-related impurities, aggregation, moisture, volatile organic compounds (VOCs), anions, and ignition residues was also determined. Both mass balance (MB) and amino acid analysis-based isotope dilution mass spectrometry (AAA-IDMS) were used for the certified value assessment, which was determined to be (0.857 ± 0.024) g/g. The certified value was validated by liquid chromatography-circular dichroism spectroscopy (LC-CD) and quantitative nuclear magnetic resonance (qNMR) methods, which were in good agreement. No inhomogeneity was observed during a homogeneity examination. A stability examination showed that the CRM was stable for at least 12months when stored at - 70°C, and for 7days when stored at 4, 25, or 40°C. The CRM is expected to be used as a primary calibrator for matrix insulin CRM development and for quality control in biopharmaceutical production and clinical diagnostics.

A novel potential primary method for quantification of enantiomers by high performance liquid chromatography-circular dichroism

Primary methods play an important role in metrology. They can be used for the value assignment of certified reference materials, enabling the accuracy and comparability of the measurement. A novel potential primary method for enantiomer quantitation based on high-performance liquid chromatography-circular dichroism is described using L-phenylalanine as an example. The optimal quantitation range of L-Phe was from 0.1 mg/g to 1.2 mg/g, where both the relative bias and method variance were lower than 1%. The LOD and LOQ were 4 μg/g and 30 μg/g, respectively. The proposed method was also applied to the determination of the mass fraction of pure porcine insulin in solid. The average mass fraction obtained was 0.922 g/g with a RSD of 1.5%, and the associated relative uncertainty is 3.8% (k = 2), which agreed well with that obtained from the traditional isotope dilution mass spectrometry method. The LOD and LOQ for insulin quantitation were found to be 0.12 mg/g and 0.44 mg/g, respectively. The proposed method can be entirely described and understood by equations and a complete uncertainty statement can be defined in SI units.Therefore, it may be a potential primary method useful for the quantification of chiral compounds and proteins, and a supplementary method to the traditional isotope dilution mass spectrometry approach.

Adulterated pharmaceutical chemicals in botanical dietary supplements: novel screening approaches

AbstractThe increased availability and use of botanical dietary supplements (BDS) has been accompanied by an increased frequency of adulteration of these products with pharmaceutical chemicals. These adulterated products are a worldwide problem, and their consumption poses health risks to consumers. The main focus of this paper is to highlight novel screening approaches utilized in the detection of adulterants in BDS marketed for different therapeutic purposes. We summarize spectroscopic methods involving near-infrared, infrared, Raman, and nuclear magnetic resonance spectroscopy as feasible and interesting in-field screening tools for the analysis of suspected products (adulterated or not) before being sent to a laboratory for deeper inspection. In addition, the new approaches based on chromatographic methods such as liquid chromatography-circular dichroism, liquid chromatography-mass spectrometry, thin layer chromatography-surface enhanced Raman spectroscopy, and thin layer chromatography-mass spectrometry are discussed and reviewed. Novel analysis strategies from targeted analysis to post-targeted and non-targeted analysis allowing simultaneous determination of the number of multiclass pharmaceuticals are discussed.

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography-circular dichroism (LC-CD) method was developed for the simple and rapid identification of N-octylnortadalafil [(6R, 12aR)-6-(1,3-benzodioxol-5-yl)-2-octyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione; RR-OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC-CD using Chiralcel OD-RH (4.6 × 150 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR-OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR-OTDF and (6R, 12aS)-6-(1,3-benzodioxol-5-yl)-2-octyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione were detected in a dietary supplement.

Determination of atropisomeric configurations of macrocyclic bisbibenzyls by HPLC-CD/UV and quantum chemical calculations

Isoriccardin C (1) and riccardin D (2), isolated from the liverwort Reboulia hemisphaerica, were first characterized to be a mixture of two enantiomeric atropisomers by online chiral high-performance liquid chromatography-circular dichroism (HPLC-CD) analysis. Exemplarily for bisbibenzyls of the diarylether-biphenyl type, the absolute atropisomeric configurations of compunds 1 and 2 were determined by the analysis of their CD data coupled with quantum chemical CD calculations.

Simultaneous determination of optical and chemical purities of a drug with two chiral centers by liquid chromatography-circular dichroism detection on a non-chiral stationary phase

The need for a rapid and cheap chromatographic technique for the simultaneous determination of diastereoisomeric and chemical purities of a drug has led to develop a non-enantioselective HPLC method on a porous graphitic carbon (PGC) sorbent and by using circular dichroism (CD) detection. Among a lot of non-chiral chromatographic supports tested, PGC was the only one allowing the separation of all benzoxathiepin diastereoisomers. After optimization of the chromatographic conditions, a mobile phase composed of chloroform, acetonitrile and methanol was suitable to elute the diastereoisomers with their related chemical impurities in less than 20 min by gradient mode. CD detection was found to be compatible with elution gradient and both the UV, CD and g anisotropy factor signals were recorded without disturbance. The determination of chemical purity was achieved with UV detection (254 nm) whereas the diastereoisomeric purity was determined using CD detection (258 nm) by plotting the anisotropy factors ( R, S– S, R and R, R– S, S) versus the enantiomeric excess of each couple of enantiomers. A mathematic model was developed in order to express the diastereoisomeric excess versus the enantiomeric excess of each couple of enantiomers. Hence, the HPLC-UV/CD method was convenient to give access simultaneously to the optical and chemical purities of a chiral drug.

Separation and structure determination of two diastereomeric pairs of enantiomers from Dracocephalum rupestre by high-performance liquid chromatography with circular dichroism detection

All of the four stereoisomers of an unprecedented type of flavanones, the dracorupesins, were separated from the aerial parts of Dracocephalum rupestre simultaneously. The dracorupesins were characterized as two diastereomeric pairs of enantiomers by on-line chiral high-performance liquid chromatography-circular dichroism (HPLC-CD) analysis, which was a reliable stereoanalytical tool for natural products. The planar structures of the compounds were elucidated by means of spectroscopic methods including IR, MS, one-dimensional NMR (1D-NMR) and two-dimensional NMR (2D-NMR) techniques.

Determination of Enantiomeric Excess Using the Ultraviolet—Circular Dichroism and the High-Performance Liquid Chromatography—Circular Dichroism Methods

Two useful numerical methods using ultraviolet (UV) and circular dichroism (CD) spectroscopies are proposed to determine enantiomeric excess (e.e). An algorithm is also proposed to generate self-consistent pure R and S enantiomer reference spectra. After all pure reference spectra are generated, a simulated annealing algorithm is applied to minimize the mismatch between the experimental spectra and the spectra after a least-squares fit. Optimal factors for R and S enantiomers are then used to determine e.e. The ultraviolet-circular dichroism (UV-CD) method uses the combined UV and CD spectra in a composite form, while the high-performance liquid chromatography (HPLC)-CD method only employs the CD spectra with the total concentrations of R plus S enantiomers provided by HPLC using a non-chiral stationary phase. Both methods were successfully tested on mixtures with known composition and then applied to real experimental data (unknown compositions). Compared with the UV-CD method, the results show that excellent results are more readily obtained using the HPLC-CD method. With the systems studied, the latter usually provided outstanding estimations of e.e with low error percentages.

Roles of the enantioselective glutathione S-transferases in cleavage of beta-aryl ether.

Cleavage of the beta-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from low-molecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the beta-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of beta-aryl ether, alpha-(2-methoxyphenoxy)-beta-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and alpha-glutathionyl-beta-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the beta position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, beta-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.