Hydrophobicity parameters determined by reversed-phase liquid chromatography. XVI: A new hydrogen-accepting parameter for monosubstituted thiophenes and furans for correlating retention factors and octanol-water partition coefficients

We recently proposed a new hydrogen-accepting parameter, S(HA), for monosubstituted (di)azines on the basis of the heat of formation calculated by the conductor-like screening model (COSMO) method. In this work, S(HA) values for monosubstituted thiophenes and furans were calculated and the results were applied to the analysis of relationships between log P (P: 1-octanol/water partition coefficient) and log k (k: retention factor obtained by reversed phase HPLC). The S(HA) parameter was found to work effectively as a hydrogen-bonding parameter in a range of heteroaromatic compounds.     

除草剂双苯唑快的反相高效液相色谱法分析

采用反相高效液相色谱法分析除草剂——双苯唑快,色谱柱为Ｓｅｌｅｃｔｏｓｉｌ５Ｃ１８不锈钢柱,检测波长为２５４ｎｍ,用Ｖ（甲醇）Ｖ（水）＝６０４０（ｐＨ３）为流动相。方法的变异系数为０．４１％,平均回收率为９９．３４％,线性相关系数为０．９９９９。方法快速、准确。     

Rapid assay for tryptophanase using reversed-phase high-performance liquid chromatography.

A rapid and sensitive high-performance liquid-chromatographic assay for tryptophanase based upon the fluorometric measurement of the enzymatically liberated indole was developed. The total incubation time is 20 min, and the reversed-phase separation is fast (elution time of indole in 8 min) and reproducible. The sensitivity of the method is in the nanomole range. This method was tested in the assay of tryptophanase activity in E. coli, giving an average activity of 6589.6 U/g of cells. Because of its speed, high sensitivity and minimal sample preparation, this method circumvents several problems commonly encountered in standard spectrophotometric methods of analysis.     

Determination of pheophytinatonickel(II) by reversed-phase highperformance liquid chromatography

Summary HPLC determination of pheophytinatonicke(II) (Pheo-Ni) prepared by the replacement of magnesium(II) in chlorophyll with nicke(II) is described. The good separation of PheoNi was obtained by using chemically bonded C₁₈ as the stationary phase and acetone-methanol (50∶50, vol/vol) as the mobile phase. Conventional spectrophotometric method was also used for the determination of PheoNi. For the synthetic samples prepared by mixing (pheophytinato a) nicke(II) [(Pheo-a) Ni] and (pheophytinato b) nicke(II) [(Pheo-b) Ni], analytical values obtained by the spectrophotometric method were very high compared to those obtained by HPLC. In the proposed HPLC method, (Pheo-a) Ni and (Pheo-b). Ni could be determined in the concentration range of 0.028–30μg/ml and 0.038–30μg/ml with relative standard deviations (n=10) of 3.1% and 0.8%, respectively.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Evaluation of peptide-peptide interactions using reversed-phase high-performance liquid chromatography.

The separation of peptides during RP-HPLC depends mainly upon differential hydrophobic interactions of the individual peptides being separated with the C18 group of the stationary phase. We have examined the behavior of dimeric disulfide-linked model peptides during RP-HPLC in order to study self-induced conformational effects. A set of 18 analogues of the amphipathic alpha-helical sequence Ac-LKLLKKLLKKLKKLLKKL-NH2 was used for this study. These analogues differed only by the successive replacement of each position with a cysteine. Strong peptide-peptide interactions, occurring through interchain hydrophobic forces, resulted in a presenting face to the C18 group, consisting primarily of lysine residues and, in turn, in early retention times. Three homo-dimers were also found to be strongly alpha-helical in water as determined by circular dichroism spectroscopy.     

Hydrophobicity parameters determined by reversed-phase liquid chromatography. Xiv. Application of a new hydrogen-accepting scale of monosubstituted pyrazines to analysis of the relationship between octanol-water partition coefficients and retention factors measured in different mobile phases

We recently proposed a new H-accepting scale, SHA, for monosubstituted pyrazines, and demonstrated that this parameter works effectively in expressing the relationship between logP (P: 1-octanol/water partition coefficient) and logk' (k': retention factor derived from reversed phase liquid chromatography) with aqueous methanol solutions as the mobile phase, according to the equation: logk' = alogP+rhosigmaI+sSHA+const., where sigmaI represents the electronic substituent constant. In this work, we have extended the same treatment to analysis of logk' measured in mobile phases containing different organic modifiers such as 1-propanol, acetonitrile, and dioxane, and found that the above equation is still useful. By comparing the correlations obtained, it was confirmed that the parameter SHA could be universally utilized for representing the difference in H-bonding effects involved in different partitioning systems.     

Variability of column selectivity for reversed-phase high-performance liquid chromatography compensation by adjustment of separation conditions

Reversed-phase columns are widely used in assays based on high-performance liquid chromatography (HPLC). When such assays are repeated over time, it is often necessary to replace the column. In such cases, the selectivity of columns from different production batches may prove sufficiently variable to result in a failed separation. It is possible to compensate for differences in column selectivity by making small changes (adjustments) in separation conditions. The present paper describes an efficient procedure for choosing adjusted conditions and discusses its general applicability.     

Hydrophobicity of beta-lactam antibiotics. Explanation and prediction of their behaviour in various partitioning solvent systems and reversed-phase chromatography.

beta-Lactam antibiotics tend to undergo self-association in hydrophilic organic solvents, which leads to a strong dependence of their experimentally observable log P values on the partitioning conditions. As a result, most of the earlier obtained log P values for beta-lactam antibiotics cannot be applied as a common hydrophobicity measure, but they proved to be linearly related to each other and to a large body of reversed-phase chromatographic data. The retention of cephalosporins on reversed-phase liquid chromatographic columns is complicated by silanophilic interactions. However, under elution conditions that eliminate these silanophilic interactions, good correlations with log P data are observed, and a unified hydrophobicity scale for 90 penicillin and cephalosporin compounds could be evaluated. The Hansch and Leo additive scheme was shown to be valid for the calculation of hydrophobicities for penicillin and cephalosporin C-6(7) substituents, but it failed when applied to the prediction of cephalosporin C-3-substituent hydrophobicities. The hydrophobic increments for the sixteen most common cephalosporin C-3-substituents were empirically evaluated from literature data, and a simple equation was derived for an overall beta-lactam antibiotic hydrophobicity calculation. The proposed scale is valid for predicting the partitioning of most beta-lactam antibiotics in both hydrophilic and lipophilic organic-water systems, although it should be used with caution when applied to antibiotics containing additionally charged side-chains.     

The separation of nonapeptides by reversed-phase high-performance liquid chromatography.

The separation properties of five nonapeptides on commercial reversed-phase materials have been investigated and the effects of pH, salt concentration and solvent composition have been studied. With appropriate variation of the pH and salt concentration in the mobile phase, it is possible to resolve all of the peptides investigated and their by-products. Mixtures of water and organic solvents (acetonitrile, dioxan, methanol and n-propanol) have been used. The choice of the organic solvent does not strongly influence the separation pattern. The simplicity, speed and quality of the separations and the favourable detection limits (ca. 30 ng) at 220 nm render this technique suitable to routine quantitative analysis.     

Lipophilicity measurements of protonated basic compounds by reversed-phase high-performance liquid chromatography : II. Procedure for the determination of a lipophilic index measured by reversed-phase high-performance liquid chromatography

The retention behaviour of protonated basic compounds in reversed-phase high-performance liquid chromatography, using methanol-water mixtures as the eluent, is reported. A minimum is found in the relationship between the logarithm of the capacity factor (log k) and the percentage of methanol (x) in the eluent. The deviation from linearity is postulated to be caused by a dual retention mechanism, namely polar interactions between the solute and eluent molecules in water-poor eluents, and hydrophobic expulsion in water-rich ones. The influence of the pH, pK_a and lipophilicity on retention behaviour is also investigated.     

The application of gradient reversed-phase high-performance liquid chromatography to the pK(a) and log k(w) determination of polyprotic analytes

The purpose of this work was to propose a theoretical model of the combined pH/organic modifier gradient in reversed-phase high-performance liquid chromatography (RP HPLC) with special emphasis on its applicability to polyprotic analytes. The model was developed and approximated to be useful for a data set comprising organic modifier gradients obtained at different pH changes and different gradient durations. It was evaluated regarding its ability to describe experimental data. The chromatographic pK(a) and lipophilicity parameter, log k(w), were obtained by fitting to the proposed model and comparing to the literature values.     

Multivariate optimisation of the experimental conditions for determination of three methylxanthines by reversed-phase high-performance liquid chromatography

Caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine) and theophylline (1,3-dimethylxanthine) are the most important naturally occurring methylxanthines. Caffeine is a constituent of coffee and other beverage and included in many medicines. Theobromine and theophylline are formed as metabolites of caffeine in humans, and are also present in tea, cocoa and chocolate products.

In order to improve the chromatographic resolution (R_s) with a good analysis time, experimental designs were applied for multivariate optimisation of the experimental conditions of an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method used for the simultaneous determination of caffeine, theobromine and theophylline. The optimisation process was carried out in two steps using full three-level factorial designs. The factors optimised were: flow rate and mobile phase composition. Optimal conditions for the separation of the three methylxanthines were obtained using a mixture of water/ethanol/acetic acid (75:24:1%, v/v/v) as mobile phase and a flow rate of 1.0 mL min⁻¹. The RP-HPLC/UV method was validated in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, recovery and the precision, calculated as relative standard deviation (R.S.D.). In these conditions, the LOD was 0.10 μg L⁻¹ for caffeine, 0.07 μg L⁻¹ for theobromine and 0.06 μg L⁻¹ for theophylline. The proposed method is fast, requires no extraction step or derivatization and was suitable for quantification of these methylxanthines in coffee, tea and human urine samples.     

Quantitative analysis of forskolin in Coleus forskohlii (Lamiaceae) by reversed-phase liquid chromatography

A rapid method was developed for the evaluation of forskolin in Coleus forskohlii Briq. (Lamiaceae). Forskolin was quantitated in the root and stem of dried C. forskohlii and in 17 market products by reversed-phase liquid chromatography (LC) with a photodiode array detector at 210 nm. The temperature was held constant at 30 degrees C, and the retention time of forskolin was approximately 6.8 min. The samples were extracted with acetonitrile by sonication. The precision of the method was confirmed by a standard deviation < 5.0% (n = 3), and forskolin recovery was 99.1%. Limit of detection was 1.5 microg/mL, and the response was linear through zero from 6.3 to 630 microg/mL with a correlation coefficient (R2) of 0.9998. Identity of the marker compound was confirmed by an LC/mass spectrometry experiment. The method was successful in the qualitative and quantitative evaluation of the marker compound in C. forskohlii plant material and in market products claiming to contain C. forskohlii.     

Separtation of metal tetrakis(p-tolyl)porphine complexes by reversed-phase high-performance liquid chromatography

Summary The feasibility of reversed-phase high-performance liquid chromatography for the separation of several metal complexes ofmeso-tetrakis(p-tolyl)porphine (TTP) is described. A combination of an octadecyl-bonded stationary phase with a non-aqueous polar mobile phase, such as an acetone-acetonitrile mixture, has proved effective for the separation. Thus, the TTP complexes of Mg, VO, Ni, Cu, Zn, and Pd and also TTP free acid were successfully separated in about 10min on a Li-Chrosorb RP-18 column (7m, 250×4mm i.d.) with a 7030 (vol/vol) mixture of acetone and acetonitrile at a flow-rate of 1 mlmin^–1.     

Determination of phosphonoformate (foscarnet) in biological fluids by ion-pair reversed-phase liquid chromatography

Bioanalytical liquid chromatographic methods for the determination of phosphonoformate (foscarnet) have been developed. Biological fluids, after simple pre-treatment (ultrafiltration and/or treatment with charcoal), were injected into a reversed-phase liquid chromatographic system with electrochemical detection. Foscarnet was retained as an ion pair with tetrahexylammonium; addition of pyrophosphate was necessary in order to obtain an acceptable peak. This additive could also be used for the fine regulation of the retention to achieve the necessary selectivity.     

Separation and determination of synthetic impurities of sildenafil (Viagra) by reversed-phase high-performance liquid chromatography.

A simple and rapid high-performance liquid chromatographic method for the separation and determination of process-related impurities of sildenafil was developed. The separation was achieved on a reversed-phase C18 column using acetonitrile-0.05 M potassium dihydrogen orthophosphate (70:30 v/v) as a mobile solvent at a flow rate of 1.0 ml/min and UV detection at 230 nm. The method was used not only for quality assurance, but also for monitoring the chemical reactions during the synthesis of sildenafil. It was found to be specific, precise and reliable for the determination of all process-related impurities of sildenafil in bulk drugs and formulations.