Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.     

Application of Computer-Aided Spectral Deconvolution Technique in Validation of High Performance Liquid Chromatographic Peaks

Abstract

Reliable analysis with high performance liquid chromatography (HPLC) requires purity of the eluting peak. The present work has combined the advantages of the availability of full spectral data from HPLC photodiode array UV detector and computer algorithms to perform chromatographic peak purity check. A deconvolution technique based on multicomponent analysis has been applied to the UV spectra of co-eluting components. This method employs residual error (Relative-fit-error, RFE) between predicted spectrum and analyte's spectrum to detect presence of other component or contaminant. Typical RFE values for uncontaminated chromatographic peaks of norethisterone and ethynyloestradiol range between 1 and 3, while contaminated peaks have RFE values as large as 145. A systematic increase in ‘relative-fit error’ from 1.10 to 145 was observed for peaks of norethisterone when contaminated to varied extent with ethynyloestradiol. Extent of peak overlap in chromatogram was also mapped out with this technique. The co-prescribed oral contraceptive, norethisterone and ethynyloestradiol were used as model in this work. An advantage of the method is its applicability when the contaminant's spectrum is unavailable. The method, unlike several earlier techniques, is also applicable to chromatograms with concidental elution time for the components.     

Natural lycopene from Blakeslea trispora: all-trans lycopene thermochemical and structural properties

Structural and thermal properties of all-trans lycopene are described in the present paper. Different crystalline lycopene samples obtained from fermentation process and recrystallized lycopene were analyzed. Structural properties (NMR, mass spectrometry and powder X-ray diffraction) of lycopene were clarified with recent techniques. High purity sample analysis by differential scanning calorimetry was used to study thermal behavior of pure lycopene and with traces of isomers. But also this was correlated with HPLC method for determine lycopene purity and isomers in low proportion.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

Separation techniques for biotechnology in the 1990s.

Scientists are constantly looking for better and cheaper separation techniques to replace or complement the current technology. Over the past few decades, and in particular the last 10 years, new separation techniques or modifications of existing techniques have become available for separating compounds from complex sample matrices. There are many areas, however, where the separation technology is not sufficient to achieve high purity and yield while remaining cost effective. In the area of biotechnology, separation techniques are urgently needed to meet demands for ultra-high purity and yield. Thus, a variety of techniques are being developed to address these needs. Generally, biological compounds for the pharmaceutical and biotechnology industries must be obtained at greater than 99.9% purity (sometimes greater than 99.99%) while maintaining high yield. In any area of chemistry this degree of purity would cause problems; in biotechnology it is even more difficult to achieve because of the complex sample matrices. In addition, the compounds of interest may be very similar to impurities or contaminants in the sample matrix, and the compounds could be denatured (or even destroyed) by certain solvents and/or high temperature. In particular, three areas of biotechnology have presented scientists with problems in separations: cell separations, DNA-RNA separations, and protein-peptide separations. The current technology available and possible future trends in these areas are discussed, and also problems to be solved in the future.     

2-DE and MALDI-TOF-MS analysis of therapeutic fusion protein abatacept

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.     

辉光放电质谱法在高纯材料分析中的应用

高纯材料是现代高新技术发展的基础,在电子、光学和光电子等尖端科学领域发挥着重要作用。采用固体样品直接分析的辉光放电质谱法(GDMS),在高纯金属、高纯半导体材料的痕量和超痕量杂质分析中有着非常广泛的应用。综述了GDMS法对高纯金属、高纯半导体材料进行的元素分析,并对分析过程中工作参数、溅射时间、干扰峰等因素的影响进行了阐述。同时,也详述了应用GDMS法对高纯金属钛、镉,高纯半导体硅,分别进行的痕量杂质元素分析,结果显示放电稳定性良好,典型元素含量的相对标准偏差均在较为理想范围内。GDMS应用前景广泛,未来,GDMS将在除固体样品之外的其他样品类型的分析领域中发挥重要作用。     

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods

The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg⁻¹ are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.     

Spectroscopic Investigation for Purity Evaluation of Detonation Nanodiamonds: Experimental Approach in Absorbance and Scattering

Nanodiamond powders have excellent mechanical, chemical, physical, and optical properties. In this research, two non-pure groups of nanodiamond particles labeled as ND1 and ND2 have been selected for purity evaluation by means of absorbance and scattering analysis. The nanodiamond powders have been used for Raman and dynamic light scattering (DLS), Fourier transform infrared (FTIR) and UV–Vis absorbance approaches. The Raman spectra show a weak diamond signal in ND2 as purity and 4 basic set of bands. FTIR absorbance spectroscopy was used in the spectral range of 400–3600 cm^?1. The results show that ND2 is almost non-absorbance with wavelength. It means that the diamond phase purity of ND2 is greater than ND1, and ND2 shows a better structure of diamond. UV–Vis absorption spectra of the sample have been recorded in the spectral range of 200–800 nm by means of nanodiamond suspended in methanol (NDM). The UV–Vis absorbance of NDM1 is stronger than NDM2 at the same concentration. Therefore the diamond phase purity of ND2 is greater than ND1. Particle size distribution and zeta potential of DNDs were investigated by DLS method. Finally the structure and phase of samples have been evaluated by X-ray diffraction (XRD) for confirmation.     

毛细管电泳分析中的富集技术及其应用

毛细管电泳（CE）具有分析速度快、分离效率高、样品消耗少、成本低廉等优点,已被应用于无机离子、有机小分子、蛋白质、核酸及细胞等的分析中。CE中最常用的检测方式是紫外检测（UV）,但由于常规进样样品体积小、检测光程短,CE-UV的灵敏度往往不能满足复杂样品中痕量物质直接分析的要求。CE中的在柱富集技术包括堆积、动态pH界面、吹扫和瞬间等速电泳等,可在很大程度上提高CE-UV的检测灵敏度;另外,固相和液相微萃取技术及其与在柱富集技术相结合应用在CE中也能净化样品基质,进一步提高富集倍数,改善分析灵敏度,从而拓宽了CE-UV在复杂样品分析中的应用范围。     

Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract

The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.     

Synthesis,structural investigations and biological studies on symmetrically substituted 4,4′,4″,4″′- tetra-methoxyphenylimino phthalocyanine complexes

The synthesis and characterization of novel metal(II) 4,4′,4″,4″′-tetra-methoxyphenylimino substituted phthalocyanines (MImPc) of copper(II), cobalt(II), nickel(II) and zinc(II) by condensing the 4,4′,4″,4″′-tetra amino phthalocyanines with anisaldehyde are described. The dark bluish green colored tetraimino substituted phthalocyanine derivatives are characterized by various physico-chemical techniques, elemental analysis, magnetic susceptibility, electronic, and IR spectra, powder X-ray diffraction and thermo gravimetric analysis (TGA) to check the structural integrity and purity. The variations of magnetic moment as a function of field strength indicated the presence of intermolecular co-operative interactions. The complexes are also evaluated for their antifungal and antibacterial activities.     

Broad range purity analysis by melting point depression using a singular feature common to all DSC purity scans

DSC purity assay by melting point between has had a limited range of less than several percent impurities. Step-mode heating techniques have increased the range to 6–8%. However, empirical corrections were used to linearize the purity slope of the van't Hoff plot, temperatures vs. reciprocal of fraction melted (1/F).A unique feature common to all dynamic purity scans has been discovered which matches a point on the non-linear van't Hoff plot to the heating rate. This point, or reciprocal of the melt fraction (1/F), occurs where the melting rate has attained maximum acceleration. The melting rate at this point is used as a tangential straight line projection from the curve to become a function of mole % impurity. The melting rate at maximum acceleration is independent of reference material, heating rate, sample size, thermal conductivity, or specific heat.Phenacetin, spiked with 50 mole % benzamide, has been measured to ±2 mole % at heating rate of 5 and 10°C min^?1; the restriction on upper detection limits is adequate resolution between preliminary eutectic events and the final melt. Impurities in indium, with a reported purity of 99.9999%, have been measured at 4 × 10^?5 mole % by this technique.     

Efficient recovery of electrophoretic profiles of nucleoside metabolites from urine samples by multivariate curve resolution

Chemometric techniques usually employed in purity assessment and resolution of multicomponent peaks have been applied to analytical data from complex biological samples obtained with CE‐DAD. In the assessment of the purity of the electrophoretic peaks, the orthogonal projection approach, the orthogonal projection approach with Durbin–Watson criterion, and the simple‐to‐use interactive self‐modeling mixture analysis method have been employed. Multivariate curve resolution with alternating least squares has been successfully implemented to resolve co‐migrating peaks of metabolites in CE‐DAD and to recover qualitative and quantitative information about co‐migrating components of urine extract. The main challenge consisted of developing high‐quality multivariate curve resolution with alternating least squares models of multicomponent peaks acquired during the CE analysis of nucleoside patterns in 18 urine samples. The recovered ultraviolet visible (UV–Vis) spectra have been employed to identify additional nucleosides, such as 1‐methylinosine, 2‐methylguanosine, and 1‐methylguanosine, whose presence in the metabolic profile produced by the applied CE‐DAD method has not yet been recognized. Concentration profiles of these compounds can be used in metabonomic studies.     

Open-access liquid chromatography/mass spectrometry in a drug discovery environment

The use of open-access mass spectrometry to monitor synthetic chemistry reactions, and also the integrity and purity of new chemical entities, has been a part of the medicinal chemist's tool-box for more than 5 years. Originally in our group at Wyeth Research there were two open-access methods available to the chemists, flow injection analysis (FIA) and liquid chromatography/mass spectrometry (LC/MS). The FIA method was approximately 3 min long, while the LC/MS method was approximately 20 min long (including an 8 min gradient). Within the first 2 years, the total number of open-access analyses increased by approximately 125%. It is interesting, however, that the number of LC/MS analyses increased by more than 285%. This is attributed to the fact that the chemists began using the LC/MS data to monitor reactions and also to check final product integrity and purity. In addition, the number of chemists performing parallel synthesis reactions has increased; thus, individual chemists can produce sample sets of up to 100 vials. This paper describes the implementation of new methodology, which accommodates the need for much faster run times and also the ability to acquire alternating positive and negative ion spectra within the same run. In addition, the instrument has been configured to e-mail the resulting processed data report to the submitting chemist. Several methods have been developed, including structure elucidation using in-source collision-induced dissociation (CID) and night-time analysis. The LC/MS methods for this system are described herein and are applicable to both industrial and academic synthetic chemistry optimization efforts.     

On-line coupling of ion chromatography and atomic spectrometry and use of direct graphite furnace atomic absorption spectrometry as new tools for ultra trace determinations in refractory metals

On-line coupling of inductively coupled plasma (ICP) techniques such as ICP-AES and ICP-MS with ion chromatography (IC) offers unique features for ultra-trace analysis. An on-line preconcentration procedure based on cation exchange enables sub-ng/g analysis in complex matrices like molybdenum and tungsten. The best dissolution reagent for these matrices is hydrogen peroxide, which can be cleaned to ultra high purity with the same metal free chromatography equipment used for the preconcentration. Preconcentration is possible for elements that show cationic reactions within acidic peroxide containing solutions. In this study 28 elements detrimental for microelectronics applications are observed. A comparison of the combinations IC-ICP-AES and IC-ICP-MS with glow discharge mass spectrometry (GDMS) for the analysis of today's purest tungsten samples shows the analytical power and accuracy of the coupled devices. Graphite furnace atomic absorption spectrometry (GFAAS) as an extremely sensitive analytical technique is applied with and without the same sample pretreatment as used for the on-line coupling. Direct GFAAS measurements of alkali metals are complementary to IC-ICP techniques. The data evaluated with these wet chemical techniques are compared to the usual manufacturers characterisation technique GDMS. With respect to the low concentrations present in these high purity materials (ng/g level in the solid) the discrepancies between all methods are acceptable. The sensitivity of IC-ICP-MS is in most cases far superior to IC-ICP-AES and for some elements also to GDMS. Furthermore the specific advantages of on-line coupling such as the elimination of isobaric interferences in ICP-MS or spectral interferences in ICP-AES are shown for ICP-AES and ICP-MS determinations.     

Applications of capillary electrophoresis in biotechnology

Capillary electrophoresis (CE)-related techniques are increasingly being used as a matter of routine practice in the biotechnology discipline. Since recombinant DNA-derived proteins and the antisense oligonucleotides constitute a large portion of the applications of these techniques, they have been emphasized in this review. Analyses by CE of Escherichia coli-derived proteins and glycosylated proteins derived from mammalian cell cultures are summarized, as well as those of the carbohydrate chains that have been enzymatically removed from the protein. Applications of CE in the analysis of the antisense oligonucleotides for the determination of purity and the analytical studies on the metabolism of these modified oligonucleotides, by CE are reviewed. The literature mainly covers the period from 1996.     

气相色谱法分析高纯异丁烯中的微量烃类杂质

采用HP Al2O3/S PLOT(50 m×0.32 mm i.d.×8 μ m) 毛细管色谱柱对高纯异丁烯中的微量烃类杂质进行了气相色谱方法研究。结果表明,该色谱柱能很好地分离异丁烯中的C1~C4烃类杂质;采用液相进样阀液态直接进样,保证样品不失真,所测定的结果有良好的准确度和精密度。     

Industrial applications of instrumental neutron activation analysis

Neutron activation analysis is shown as a useful diagnostic technique in semiconductor industry. A better acceptance of the method for applications in industry has been achieved through a specialized analytical service. Its main application is the characterization of high purity silicon in all stages of production. Irradiation of large sample volumes allowes a very sensitive detection of impurities in silicon with detection limits down to 10^–16 g/g. Other applications discussed are the analysis of silicon carbide, quartz, pure water and titanium. Special techniques described are autoradiography, depth profiling and surface analysis. In semiconductor process technology NAA was used to monitor contamination of silicon wafers.     

Simultaneous purification and spectrophotometric determination of nickel present in as-prepared single-walled carbon nanotubes (SWCNT)

The quality of single-walled nanotubes (SWCNT) is usually assessed by means of electron microscopic techniques or Raman spectroscopy. However, these sophisticated techniques are not widely available and do not reliably estimate the impurities in highly heterogeneous samples containing metal particles, fullerenes and other carbonaceous materials. We have developed a simple, inexpensive and convenient spectrophotometric method to assess the purity of arc-discharge grown as-prepared SWCNT. Purification process consists of initial gas phase oxidation and refluxing with nitric acid at the optimal conditions including short time period during acid refluxing. We have shown that this method could remove the metal particles effectively with a good yield of high quality SWCNTs, as shown by the spectrophotometric and scanning tunneling microscope studies described here. The extent of removal of the nickel present in as-prepared carbon nanotube sample is followed by spectrophotometeric analysis of the dissolved nickel analyte. The composition of nickel in the SWCNT sample is found to be 17.56%. The method is based on the chelating of Ni²⁺ with dimethylglyoxime in ammoniacal citrate medium to form nickel dimethylglyoxime complex. A second stage purification of SWCNT eliminates the residual metal particles. The purified SWCNT has been studied using scanning tunneling microscopy which shows clearly resolved individual carbon nanotubes.