Analysis Of Basic Compounds Research Articles

Synthesis of a stationary phase based on silica modified with branched octadecyl groups by Michael addition and photoinduced thiol-yne click chemistry for the separation of basic compounds.

A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.

Development of Amine Capillary Column Applied to the Analysis of Basic Compounds by Electrochromatography

An amine capillary column was developed in a fused-silica capillary as stationary phase and applied to separation of basic compounds by capillary electrochromatography (CEC). The functionalized capillary was prepared by inmobilization of 3-Aminopropyl)triethoxysilane (APTES). The CEC conditions including APTES and buffer concentration, pH and applied voltage were investigated to obtain the optimal CEC system for the separation of three anesthetics as basic test molecules. The capillary column provided an efficiency of up to 20,000 plates/m. Lidocaine, ketamine and xilacine were baseline separated under the running conditions with 10 mM Na2HPO4 pH 9.0 as BGE. The applied voltage was 5 kV temperature was set at 25oC and UV detection was performed. The resolutions were 4.97 and 1.53 for ketamine, lidocaine and xilacine, respectively. The column used in CEC mode showed better separation of the anesthetics compared with those used in the capillary zone mode. The comparison with reversed stationary phases used in HPLC in terms of resolution (Rs), sensitivity (LOD), efficiency (N), precision (RSD), asymmetry (T) was also performed. This study provided an alternative way for the CEC separation of basic compounds and demonstrated the improvement in the chromatographic parameters.

Analysis of basic compounds by supercritical fluid chromatography: Attempts to improve peak shape and maintain mass spectrometry compatibility

While neutral and acidic compounds are well separated by supercritical fluid chromatography (SFC), basic analytes are more challenging to separate and often problems occur with their peak shapes. Two different methods were explored in the present paper to reduce these problems and maintain compatibility with mass spectrometry (MS). Five different, commercially available 2-ethylpyridine (2-EP) stationary phases were tested without a mobile phase additive using 92 pharmaceutical compounds with basic properties. The kinetic performances of the 5 columns were nearly identical, but the peak shapes of the basic drugs were strongly affected by the stationary phase. The PrincetonSFC 2-EP and Zymor Pegasus 2-EP phases clearly outperformed the other stationary phases, with 77% and 69% of the compounds having Gaussian peaks (and asymmetries between 0.8 and 1.4), respectively. Comparatively, the Waters Viridis Silica 2-EP, Waters Viridis BEH 2-EP and ES industries GreenSep 2-EP phases provided only 52%, 44% and 22% of the compounds with Gaussian peaks, respectively. These differences were attributed to the significant dissimilarities in their silica matrix properties. An alternative strategy was also performed with a hybrid silica stationary phase, Viridis BEH, using 20mM ammonium hydroxide in the mobile phase, which was a mixture of CO2 and MeOH. With these conditions, 81% of the peaks observed for the basic analytes were Gaussian; however, this value dropped to 17% and 10% in the absence of additive and in the presence of 20mM formic acid, respectively. Finally, the use of a hybrid bare silica stationary phase in the presence of 20mM ammonium hydroxide is quite an interesting solution as this system is compatible with both ultra high performance SFC (UHPSFC) columns packed with sub-2μm particles and with MS detection. The overall applicability of this system was demonstrated with various mixtures of basic drugs.

The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds

The hydrophilic interaction chromatography (HILIC) like properties of an ACE cyano (CN) HPLC column was studied for the separation of some basic compounds. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. When Trishydroxymethyl aminomethane (Tris) (p K a 8.0), which is a weaker amine than ammonia (p K a 9.3), was used as an additive in the mobile phase retention of the basic compounds was increased. The ACE CN column gave excellent peak shapes for all the basic compounds. The utility of the column for impurity profiling of two basic drugs was tested and some impurities in oxprenolol were characterised by interfacing with Fourier transform mass spectrometry. It was also observed that ACE butyl and ACE phenyl columns retained basic compounds when the columns were eluted with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate.

Analysis of basic compounds by reversed-phase liquid chromatography–electrospray mass spectrometry in high-pH mobile phases

The separation of basic compounds in high-pH mobile phases results in extended retention, excellent peak shapes and good chromatographic efficiency. A severe decrease in sensitivity with electrospray mass spectrometric detection in positive ion mode (ESI +-MS) is expected under conditions that suppress analyte ionization in solution. We compared the responses of a large number of various basic drugs covering a wide range of hydrophobic (log P 0.09 to 7.6) and basic character (p K a 6.8–10) in LC–ESI +-MS/MS in 0.1% formic acid in water and acetonitrile, to responses in 10 mM ammonium hydrogencarbonate buffers of different pH (7.8–11), and acetonitrile. Contrary to common expectations, high-pH mobile phases do not affect negatively the responses of basic compounds in ESI +. Analyte responses and limits of detection are comparable, or most often better in high pH compared to acidic mobile phases.

Analysis of basic compounds by reversed-phase high-performance liquid chromatography using hybrid inorganic/organic phases at high pH

The retention and mass overload of five bases and a quaternary ammonium compound were studied on a bridged ethyl hybrid inorganic/organic phase (XBridge C18) over a pH range 2.7–12.0 using acetonitrile–phosphate and carbonate buffers. Some comparisons were drawn with results on a methyl hybrid (XTerra) phase. At low pH, rapid mass overload was observed with severe decreases in efficiency as sample mass was increased over the range 0.04–5 μg of solute. At intermediate pH ( pH 7 w s .0 ), generally good peak shapes for small sample mass were still obtained on the ethyl hybrid, but with somewhat increased tailing of bases compared with pH w s 2.7 . At higher pH ( pH 10 w s ), good peak shape and improved loadability were obtained for moderately strong bases, due to their occurrence mostly as neutral species. However, stronger bases gave poor efficiency, attributed to interaction of the charged basic solute with increasingly ionised column silanol groups. Results were broadly similar on the methyl hybrid at pH 10 w s . At pH 11 w s , unusual profiles of increasing followed by decreasing efficiency were obtained on the ethyl hybrid for some bases as sample mass was increased; improved results were obtained at pH 12 w s . While the column loadability increased substantially at the highest pH studied, tailing for small sample mass was still more severe than at low pH, even though all compounds were <1% ionised in the mobile phase.

An Effective Tool for Column Evaluation in the Analysis of Basic Compounds

A chromatographic test is described for the evaluation of base-deactivated stationary phases. Seven test basic compounds, selected on their physico-chemical properties, were injected with two different mobile phases (one at pH 7.0 and the other at pH 3.0), on 45 chromatographic supports. Thanks to the measured chromatographic parameters (k and As), it was possible to evaluate both silanol activity and hydrophobic character of the base-deactivated columns. In addition, the validity of this chromatographic test was assessed by measuring the fundamental properties of the same supports with a general test protocol, issued from the literature.

Simplification of a chromatographic test methodology for evaluation of base deactivated supports

Today reversed-phase liquid chromatography (RP-LC) is the most employed chromatographic technique for analysis of basic compounds. Unfortunately, the strong ionic interactions of basic compounds with residual silanols result in asymmetric peaks and non-reproducible retention. This problem and the continous increase in the use of RP-LC techniques have furthered the need for a new generation of “base deactivated” stationary phases.

The analysis of basic pharmaceutical compounds by capillary electrochromatography using continuous bed stationary phase

Capillary electrochromatography (CEC) is classed as a hybrid technique between CE and HPLC and it combines the advantages of both these techniques. However, in some cases the disadvantages are also brought to light and some of these are difficult to resolve. For example the analysis of basic compounds using CEC. The problems of tailing peaks during HPLC analysis of basic compounds was resolved by end capping the residual silanol groups, but in CEC these are the groups that generate the electroosmotic flow. The analysis of basic compounds is crucial within the pharmaceutical industry where a high percentage of the drug actives are basic. Specially designed Continuous Beds stationary phases (CB) can mean that each application can have a specific stationary phase. In order to overcome the problem associated with the analysis of basic compounds using electrochromatography, we have designed a CB stationary phase with a positive charge, which could be operated using negative voltage. The resulting chromatography showed almost gaussian peaks for bases like nortriptyline which tail significantly using stationary phase typically used in CEC.

Silica and other materials as supports in liquid chromatography. Chromatographic tests and their importance for evaluating these supports. Part II

Reversed-phase chromatography is the method of choice for the analysis of basic compounds. However, because of the problems encountered with these compounds, “base deactivated” stationary phases have been developed. This review focuses on a major problem encountered by the chromatographer which is to effectively choose, for a particular separation, the appropriate reversed-phase packing. Often, columns of the same type but provided by various manufacturers vary in their chromatographic behaviour. It is known that differences in the purity of column packings, in the properties of silica gel supports and the density of silanols on the surface can be responsible for chromatographic differences. Unfortunately, in the case of “base deactivated” columns there is no universal test and chromatographic effects are compound dependent. The aim of this review is to describe the various chromatographic tests proposed in the literature for the differentiation of the many stationary phases on the market.

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.

Comparison of aqueous and non-aqueous capillary electrochromatography for the separation of basic solutes

The analysis of basic compounds by capillary electrochromatography (CEC) on silica-based materials using conventional HPLC stationary phases has failed to address the problem of severe peak tailing and non-reproducible chromatography. Several new generation stationary phases were evaluated using aqueous and non-aqueous mobile phases. The best results were obtained in the aqueous mode using Waters Symmetry Shield RP-8, a material in which the residual silanol groups were shielded by an octylcarbamate function. For comparison, experiments were carried out using unmodified silica.

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile–phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6×10 4 plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1×10 5 plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8×10 5 plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1×10 5–3×10 5 plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.

The selection of buffer salts for the high-performance liquid chromatography of basic compounds

The use of silica with reversed-phase type eluents at alkaline pH offers a useful approach to the analysis of basic compounds by highperformance liquid chromatography (HPLC). The original buffer salt was ammonium nitrate [l], which has been used widely, and for which considerable retention data are available [2, 31. Whilst being considered a volatile salt and a good buffer at alkaline pH, the use of ammonium nitrate gives one major draw-back. The high ultraviolet (UV) absorbance of the nitrate anion at low wavelengths limits the use of such a system to detection at >250 nm, which can be a serious limitation in drug analysis. The obvious replacement anions; sulphate and chloride (both of which are strong acids) give non-volatile ammonium salts volatility being a useful feature in preparative HPLC and combined HPLC mass spectrometry (LCMS) and are not fully UV transparent. Chloride also has the disadvantage of dissolving stainless steel. The other common HPLC buffer anion, phosphate does not give soluble ammonium salts in aqueous methanol solutions. Turning to the organic acids; citric, acetic and trifluoroacetic which are commonly used as HPLC eluent constituents, only trifluoroacetic and acetic acid give volatile ammonium salts. The present work evaluates and compares the use of ammonium acetate, and trifluoroacetate (both of which are widely used in HPLC) with nitrate in terms of their physical, UV, buffering and electrochemical properties as well as their effect on chromatographic performance.

Analysis of basic compounds on a silica column with an aqueous methanol eluent : The use of quantitative structure-retention relationships in metabolite identification

High-performance liquid chromatography data for 69 mono-functional aryl-alkyl amines was generated using a silica column and an aqueous methanol eluent