Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.     

Quantitative determination of L-DOPA in tablets by high performance thin layer chromatography

A densitometric high performance thin-layer chromatographic (HPTLC) method was developed and validated for quantitative analysis of L-DOPA in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone-chloroform-n-butanol-acetic acid glacial-water (60:40:40:40:35 v/v/v/v/v) as mobile phase. Quantitative analysis was carried out at a wavelength of 497 nm. The method was linear between 100 and 500 ng/microL, with a correlation coefficient of 0.999. The intra-assay variation was between 0.26 and 0.65% and the interassay was between 0.52 and 2.04%. The detection limit was 1.12 ng/microL, and the quantification limit was 3.29 ng/microL. The accuracy ranged from 100.40 to 101.09%, with a CV not higher than 1.40%. The method was successfully applied to quantify L-DOPA in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision, and accurate for the quantitative determination of L-DOPA in tablets.     

Metabolic fingerprinting of high-fat plasma samples processed by centrifugation- and filtration-based protein precipitation delineates significant differences in metabolite information coverage

Metabolomics and metabolic fingerprinting are being extensively employed for improved understanding of biological changes induced by endogenous or exogenous factors. Blood serum or plasma samples are often employed for metabolomics studies. Plasma protein precipitation (PPP) is currently performed in most laboratories before LC–MS analysis. However, the impact of fat content in plasma samples on metabolite coverage has not previously been investigated. Here, we have studied whether PPP procedures influence coverage of plasma metabolites from high-fat plasma samples. An optimized UPLC-QTOF/MS metabolic fingerprinting approach and multivariate modeling (PCA and OPLS-DA) were utilized for finding characteristic metabolite changes induced by two PPP procedures; centrifugation and filtration. We used 12-h fasting samples and postprandial samples collected at 2 h after a standardized high-fat protein-rich meal in obese non-diabetic subjects recruited in a dietary intervention. The two PPP procedures as well as external and internal standards (ISs) were used to track errors in response normalization and quantification. Remarkably and sometimes uniquely, the fPPP, but not the cPPP approach, recovered not only high molecular weight (HMW) lipophilic metabolites, but also small molecular weight (SMW) relatively polar metabolites. Characteristic SMW markers of postprandial samples were aromatic and branched-chain amino acids that were elevated (p < 0.001) as a consequence of the protein challenge. In contrast, some HMW lipophilic species, e.g. acylcarnitines, were moderately lower (p < 0.001) in postprandial samples. LysoPCs were largely unaffected. In conclusion, the fPPP procedure is recommended for processing high-fat plasma samples in metabolomics studies. While method improvements presented here were clear, use of several ISs revealed substantial challenges to untargeted metabolomics due to large and variable matrix effects.     

电泳和质谱技术研究4种胰脏铁蛋白的亚基类型和等电点特性

采用电泳和质谱技术对所制备的鸡、鸭、牛和猪胰脏铁蛋白的亚基类型和等电点特性进行了研究。采用天然聚丙烯酰胺凝胶电泳(PAGE)技术研究的结果表明,上述4种铁蛋白呈现不同的迁移率,据此可知鸡胰铁蛋白的相对分子质量(Mr)>鸭胰铁蛋白的Mr>黄牛胰铁蛋白的Mr>猪胰铁蛋白的Mr,而且均大于马脾铁蛋白(HSF)的Mr。采用十二烷基硫酸钠(SDS)-PAGE技术研究的结果表明,上述4种铁蛋白均由H(heavy chain)和L(light chain)类型的亚基组成,但H和L亚基的相对数量（即H/L亚基数量的比值）有差别。采用肽指纹图谱技术分别鉴定各铁蛋白的H和L亚基。选用变性等电聚焦方法研究发现,上述4种铁蛋白分别由3～6种不同等电点的亚基聚合体组成,说明铁蛋白的H和L亚基之间呈现复杂的相互作用和不同的聚合体。不同陆生动物胰脏铁蛋白亚基之间相互作用的强度和聚合态存在着差异,这一差异特性可能与调控铁蛋白释放铁的速率有关,也与动物对铁的需求和铁解毒速率有关。     

Linear optical realization of unambiguous quantum state comparison

In this paper, we propose an experimental scheme for unambiguous quantum state comparison assisted by linear optical manipulations, twin-photons produced from parametric down-conversion, and postselection from the coincidence measurement. In this scheme the preparation of the general two mixed qubit states with arbitrary prior probabilities and the realization of the optimal POVMs for unambiguous quantum state comparison are presented. This proposal is feasible by current experimental technology, and may be used in single-qubit quantum fingerprinting.     

Quantification of astaxanthin in salmons by chemiluminescence and absorption after TLC separation

Astaxanthin is a keto-carotenoid, belongs to the chemical class of terpenes and is a yellow lipid soluble compound. The compound is present in marine animals like salmons and crustacean. Its colour is due to conjugated double bonds and these double bonds are responsible for its antioxidant effect. Its antioxidant activity is ten times stronger than other carotenoids and nearly 500 fold stronger than vitamin-E. We present a new thin layer chromatography (TLC) method to measure astaxanthin on TLC-plates (Merck, 1.05554) in the visible absorption range as well as by using chemiluminescence. For separation a solvent mixture of cyclohexane and acetone (10?+?2.4, v/v) was used. The RF-value of astaxanthin is 0.14.The limit of detection in vis-absorption is 64?ng / band and the limit of quantification is 92?ng/band. In chemiluminescence the values are 90?ng / band and 115?ng/band. The method offers two independently working measurement modes on a single plate which increase the accuracy of the quantification.     

Quality consistency evaluation of commercial transfer factor injections by chromatographic fingerprint combined with multivariate statistical analysis

The current quality control methods relying mainly on chromogenic reaction can hardly ensure the quality and safety of the biochemical drug with complex chemical composition. Therefore, a chromatographic fingerprint method was developed for the quality evaluation of a multicomponent biochemical drug, transfer factor injection. High‐performance liquid chromatography fingerprint was measured by using a C₁₈ column (250 × 4.6 mm, 5 µm) with a mobile phase composed of 0.1% trifluoroacetic acid–water and 0.085% trifluoroacetic acid–acetonitrile under gradient elution. The developed method was validated and was subsequently applied to 57 batches of commercial products which were sampled by National Drug Assessment Program. High‐resolution mass spectrometry analysis was performed on characteristic peaks of fingerprints, and a series of amino acids, nucleosides, and deoxynucleosides were identified. In the fingerprint assessments, principal component analysis and Hotelling T² analysis yielded the best results. The results generally indicated that there was a significant difference among products of batch‐to‐batch or from different manufacturers. Abnormal samples and its discriminatory components were also explored. In summary, the established fingerprinting method with multivariate statistical analysis could offer an efficient, reliable, and practical approach for quality consistency evaluation of transfer factor injection, providing a reference for the quality control of other multicomponent biochemical drugs.     

Determination of metabolites relating to the tryptophan-NAD pathway in rat urine by cellulose high performance thin-layer chromatography

High performance thin-layer chromatography (HPTLC) was used to determine 12 metabolites of the tryptophan-NAD pathway in rat urine. The method of separation described in the previous report was improved. The metabolites were separated on the cellulose plate by using five multiple developments with four solvent systems. The plate was scanned by the TLC scanner at 254 nm and the amounts of the metabolites were calculated by comparing peak areas of the scanning curves obtained for the urine sample and for the authentic standards. This procedure can be used as a semiquantitative determination method for the metabolites in biological fluids.     

Anticircular high performance thin-layer chromatography

The principal analytical details for the third of three possible modes in high performance thin-layer chromatography are given, namely the anticircular mode. Separation is achieved by allowing the mobile phase to enter the plate layer on a precise outer circle line, from where it flows towards the centre with nearly constant speed. This technique is theoretically and practically the fastest of all three possible in HPTLC. It permits maximum sample capacity with a minimum of time, layer and mobile phase consumption. It is therefore the most economical HPTLC technique. A new carrier-free mobile phase transfer principle is used. The conditions for qualitative and quantitative analysis are good: repeatability, reproducibility and accuracy of routine TLC analyses are superior to those achieved by the classical trough technique. The specially narrow spot-path in anticircular HPTLC facilitates automated quantitation. Compared with the linear and circular modes, the anticircular mode shows better separation and significantly increased sensitivity at higher Rf-values. The drawback, however, is that the separation power (expressed by the separation number) is lower compared with the other two modes.     

Systematic and statistical errors in quantitative evaluation in TLC and HPTLC. 3. The determination of the primary statistical errors

The paper describes the measurement and calculation of the primary statistical errors in the evaluation of TLC or HPTLC using in situ reflectance measurements. The primary errors are the errors of the sample volume spotted onto the plate, errors caused by the chromatographic process itself, the positioning error of the plate with respect to the centre of the light beam and the error in the light measurement. By scanning the same spot, the same track or the same plate under different conditions, the various errors can be determined.