Practical Quality Control: Comparision of Methods on the Quantification of Stationary Phases in Paper and Thin-Layer Chromatographic Systems

Introduction: Paper and thin layer chromatography methods are frequently used in Classic Nuclear Medicine for the determination of radiochemical purity (RCP) on radiopharmaceutical preparations. An aliquot of the radiopharmaceutical to be tested is spotted at the origin of a chromatographic strip (stationary phase), which in turn is placed in a chromatographic chamber in order to separate and quantify radiochemical species present in the radiopharmaceutical preparation. There are several methods for the RCP measurement, based on the use of equipment as dose calibrators, well scintillation counters, radiochromatografic scanners and gamma cameras. The purpose of this study was to compare these quantification methods for the determination of RCP. Material and Methods: 99mTc-Tetrofosmin and 99mTc-HDP are the radiopharmaceuticals chosen to serve as the basis for this study. For the determination of RCP of 99mTc-Tetrofosmin we used ITLC-SG (2.5 x 10 cm) and 2-butanone (99mTc-tetrofosmin Rf = 0.55, 99mTcO4- Rf = 1.0, other labeled impurities 99mTc-RH RF = 0.0). For the determination of RCP of 99mTc-HDP, Whatman 31ET and acetone was used (99mTc-HDP Rf = 0.0, 99mTcO4- Rf = 1.0, other labeled impurities RF = 0.0). After the development of the solvent front, the strips were allowed to dry and then imaged on the gamma camera (256x256 matrix; zoom 2; LEHR parallel-hole collimator; 5-minute image) and on the radiochromatogram scanner. Then, strips were cut in Rf 0.8 in the case of 99mTc-tetrofosmin and Rf 0.5 in the case of 99mTc-HDP. The resultant pieces were smashed in an assay tube (to minimize the effect of counting geometry) and counted in the dose calibrator and in the well scintillation counter (during 1 minute). The RCP was calculated using the formula: % 99mTc-Complex = [(99mTc-Complex) / (Total amount of 99mTc-labeled species)] x 100. Statistical analysis was done using the test of hypotheses for the difference between means in independent samples. Results:The gamma camera based method demonstrated higher operator-dependency (especially concerning the drawing of the ROIs) and the measures obtained using the dose calibrator are very sensitive to the amount of activity spotted in the chromatographic strip, so the use of a minimum of 3.7 MBq activity is essential to minimize quantification errors. Radiochromatographic scanner and well scintillation counter showed concordant results and demonstrated the higher level of precision. Conclusions: Radiochromatographic scanners and well scintillation counters based methods demonstrate to be the most accurate and less operator-dependant methods.

Development of novel brush-type chiral stationary phases based on terpenoid selectors: HPLC evaluation and theoretical investigation of enantioselective binding interactions

The terpenoid chiral selectors dehydroabietic acid, 12,14-dinitrodehydroabietic acid and friedelin have been covalently linked to silica gel yielding three chiral stationary phases CSP 1, CSP 2 and CSP 3, respectively. The enantiodiscriminating capability of each one of these phases was evaluated by HPLC with four families of chiral aromatic compounds composed of alcohols, amines, phenylalanine and tryptophan amino acid derivatives and beta-lactams. The CSP 3 phase, containing a selector with a large friedelane backbone is particularly suitable for resolving free alcohols and their derivatives bearing fluorine substituents, while CSP 2 with a dehydroabietic architecture is the only phase that efficiently discriminates 1, 1'-binaphthol atropisomers. CSP 3 also gives efficient resolution of the free amines. All three phases resolve well the racemates of N-trifluoracetyl and N-3,5-dinitrobenzoyl phenylalanine amino acid ester derivatives. Good enantioseparation of beta-lactams and N-benzoyl tryptophan amino acid derivatives was achieved on CSP 1. In order to understand the structural factors that govern the chiral molecular recognition ability of these phases, molecular dynamics simulations were carried out in the gas phase with binary diastereomeric complexes formed by the selectors of CSP 1 and CSP 2 and several amino acid derivatives. Decomposition of molecular mechanics energies shows that van der Waals interactions dominate the formation of the diastereomeric transient complexes while the electrostatic binding interactions are primarily responsible for the enantioselective binding of the (R)- and (S)-analytes. Analysis of the hydrogen bonds shows that electrostatic interactions are mainly associated with the formation of N-(HO)-O-...=C enantio selective hydrogen bonds between the amide binding sites from the selectors and the carbonyl groups of the analytes. The role of mobile phase polarity, a mixture of n-hexane and propan-2-ol in different ratios, was also evaluated through molecular dynamics simulations in explicit solvent. (c) 2006 Elsevier Ltd. All rights reserved.

Optimization of photo-polymerized sol gel monolithic stationary phases prepared in polyacrylate-coated fused-silica capillaries for capillary electrochromatography

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Characterization of cation exchanger stationary phases applied for the separations of therapeutic monoclonal antibodies.

Cation exchange chromatography (CEX) is a well established strategy for the characterization of monoclonal antibodies (mAbs). The optimization of mobile phase conditions is well described in the literature, but there is a lack of information about CEX stationary phases for the analysis of therapeutic proteins. The aim of this study was to compare five state-of-the-art CEX stationary phases based on the retention, selectivity and resolving power achieved in pH- and salt-gradient modes, with various therapeutic mAbs and their variants. The Sepax Antibodix WCX-NP3, Thermo MAbPac SCX-10 RS, YMC BioPro SP-F, Waters Protein-Pak Hi Res SP and Agilent Bio mAb NP1.7 SS were considered in this study. In terms of retention, the YMC Bio Pro SP-F material was the less retentive one, while the Agilent Bio mAb NP1.7 SS provides the highest retention. Regarding the selectivity achieved between the main mAbs isoforms and their variants, the Thermo MabPac SCX column generally gave the highest selectivity. Finally, it was hard to rank columns in term of kinetic performance since their performance is strongly solute (mAb) and elution mode (pH or salt gradient) dependent. However, the highest resolution--in most cases--was observed on the strong cation exchanger YMC Bio Pro SP-F material.

PROLINE-BASED CHIRAL STATIONARY PHASES: INSIGHTS INTO THE MECHANISM OF CHIRAL SELECTIVITY

Proline (Pro) is a unique amino acid that has been examined previously as a potential chiral selector for high-performance liquid chromatography. In recent years, a new class of promising Pro based enantioselective stationary phases has been studied and the longer peptides were found to be competitive with commercial chiral stationary phases (CSPs). Here, we aim to perform a comprehensive examination of a t-butoxycarbonyl- (t-Boc-) terminated monoproline selector. This selector was grafted through an amide linkage to an aminopropyl siloxane-terminated Si (111) wafer and to a silicon atomic force microscopy tip. To ensure a flat, homogeneous overlayer of selectors suitable for force spectrometric measurements, the prepared surfaces were characterized using XPS, AFM and contact angle measurements. Chemical force spectrometry (CFS) has been used to examine the chiral discrimination in our monoproline CSP by measuring the interaction forces between two D- or L-monoproline monolayers in water and in the presence of a series of amino acids in solution to explore the degree to which binding of amino acids impacts self-selectivity. Chemical force titration (CFT) has been used to observe the influence of variations in pH on the binding interaction of proline modified chiral surfaces. Here we aim to explore the connection between side-chain hydrophobicity and differences in the nature of the binding between different ionic forms of amino acids and the t-Boc-Pro interface, and thereby to gain insight into the mechanism of chiral selectivity. The CFS results show several trends for different proline selector/amino acid combinations and indicate that the binding characteristics of amino acid to the proline surface is strongly dependent on the amino acid side chain where hydrophilic side chain amino acids exhibit a selectivity opposite to that seen for those with hydrophobic side chains. The CFT studies also provide valuable insights into interactions between the proline selector and the amino acids under a wide range of pH conditions, indicating that protonated amine groups of alanine and serine are closely involved in the binding mechanism to proline surfaces. On the other hand, the presence of the second carboxylic group in aspartic acid plays an important role while interacting with proline.

CROMATOGRAFIA POR INTERAÇÕES HIDROFILICAS (HILIC): ESTADO DA ARTE E APLICAÇÕES

Hydrophilic interaction liquid chromatography (HILIC) has been gaining increased attention for its effective separation of highly polar compounds, which include carbohydrates, amino acids, pharmaceutical compounds, proteins, glycoproteins, nucleosides, etc. Polar compounds are usually poorly retained on reverse-phase liquid chromatography (RP-HPLC) columns or have poor solubility in the apolar mobile phase of normal-phase high performance liquid chromatography (NP-HPLC). Since HILIC uses organic solvents such as ACN or MeOH ( > 70%), also used in RP-HPLC and polar stationary phases similar to NP-HPLC (bare silica, diol, amino, amide, saccharide, zwitterionic stationary phases, etc.), it represents a hybrid of the two separation modes. The high organic content in the MP leads to good compatibility with mass spectrometry (MS), increasing the detectivity. This review describes the fundamentals of HILIC and highlights some interesting applications.

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel.

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

Estado da arte da cromatografia gasosa de alta resolução e alta temperatura

The developments in stationary phase synthesis and capillary column technology, have opened new perspectives in analysis of high molecular mass compounds (³600 daltons) and thermolabile organic compounds by High Temperature High Resolution Gas Chromatography (HT-HRGC). HT-HRGC is a new analytical borderline and its application to the analysis of high molecular mass compounds is still in its infancy. The apolar and medium polar gum phases can now be operated at temperatures up to 400-480ºC, being used for the analysis of n-alcanes up to C-100, lipids, oligosaccharides, industrial resins, polyglycerols, cyclodextrins, porphyrins, etc. This technique should play a leading role as a powerful tool, for many different analysis types, in multidisciplinary fields of Science.

Novas fases estacionárias à base de sílica para cromatografia líquida de alta eficiência

The present work reviews recent advances in the preparation of new reversed phase packing materials such as sterically protected, bidentate, hybrid organic-inorganic and monolithic phases and phases containing embedded polar groups. The bonding chemistry involved in the preparation of these phases as well as their advantages over conventional C8 and C18 reversed phases are discussed. Understanding the reasons behind the development of these newer column packings helps analysts select the best stationary phase for a given application.

Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa

This review first discusses the limitations of many of the supports and stationary phases used in reversed phase high performance liquid chromatography and then describes those, developed more recently, that present better stabilities and more versatile selectivities. Emphases will be given to stationary phases that use higher purity silicas, hybrid silicas, monolithic silicas, metallic oxides and mixed oxides as supports and those that have embedded polar groups or contain phenyl or fluoro groups as the stationary phase as well as the phases used for mixed mode or hydrophilic interaction separations. These modern stationary phases facilitate the analysis of complex mixtures.

Fases estacionárias monolíticas para separações cromatográficas

Monolithic stationary phases represent a new generation of chromatographic separation media. These phases consist of a continuous separation bed prepared by in situ polymerization or consolidation inside the column tubing. In recent years, their simple preparation procedure, unique properties and excellent performance have attracted quite remarkable attention in liquid chromatography and capillary electrochromatography. This review summarizes the preparation, characterization and applications of monolithic stationary phases. The analytical potential of these columns is demonstrated with separations involving various families of compounds in different separation modes.

Cromatografia líquida capilar: estado da arte e aplicações

This review describes the advantages and disadvantages of using capillary liquid chromatography (CLC), which is considered the newest member in the analytical separation science arsenal. Although CLC has tremendous potential for being the next major innovation in separatory analysis, it has not yet obtained great popularity compared to conventional high performance (and ultra-high performance) liquid chromatography. Comparisons are made between these techniques and some of the reasons that CLC has not yet reached its potential will be advanced.

O desafio de analisar solutos básicos por cromatografia líquida em modo reverso: algumas alternativas para melhorar as separações

This review considers some of the difficulties encountered with the analysis of basic solutes using reversed-phase chromatography, such as detrimental interaction with stationary phase silanol groups. Methods of overcoming these problems in reversed-phase separations, by judicious selection of the stationary phase and mobile phase conditions, are discussed. Developments to improve the chemical and thermal stability of stationary phases are also reviewed. It is shown that substantial progress has been made in the manufacturing of stationary phases, enabling their use over a wide variety of experimental conditions. In addition, general measures to significantly extend their lifespan are discussed.