Determination of furosine in milk samples by ion-pair reversed phase liquid chromatography

Summary An ion-pair, reversed-phase, liquid chromatographic procedure using UV detection for quantitation of furosine is described. The standard plot was linear (r>0.999) over a 5 ng range. An authentic synthesised sample of furosine was used for calibration. Commerical milk samples were analyzed by the described procedure.     

Peptide mapping with liquid chromatography using a basic mobile phase

A new peptide mapping with liquid chromatography (LC) using an ammonia-containing basic mobile phase was reported. As compared with a method under a traditional acidic condition with a mobile phase containing trifluoroacetic acid (TFA) or formic acid (FA), the new method exhibited excellent overall performance: it was advantageous over the TFA method in terms of the ultraviolet (UV) and mass spectrometry (MS) sensitivities and the sequence coverage for a tryptic map; it was superior to the FA method in terms of the UV sensitivity, the sequence coverage and the separation capacity. Due to a significant difference in the chromatographic selectivity, several important peptide mapping applications that were sometimes difficult to be conducted previously could now be carried out using the new method. For example, the baseline separation of peptides from the corresponding deamidated products could be achieved with confidence using the new method, a critical pre-requisite for definitive identification and quantification of the deamidation products with LC/MS. No on-column deamidation was observed with the conditions used for the separation. Complementary and confirmative information about a protein could be obtained by running its proteolytic digest under both the basic and acidic conditions.     

高效液相色谱手性流动相添加剂法分离3种黄酮类化合物对映体

采用反相高效液相色谱法(RP-HPLC),以磺丁基醚-β-环糊精(SBE-β-CD)作为手性流动相添加剂,建立了二氢黄豆苷原(dihydrodaidzein)、雌马酚(equol)和山姜素(alpinetin)3种黄酮类化合物的手性拆分方法。考察了环糊精的种类和浓度、有机相的种类和比例、缓冲盐的种类和浓度以及pH对3种化合物手性拆分效果的影响。结果表明:采用Kromasil 100-5C₁₈(250 mm×4.6 mm, 5 μm)色谱柱,流动相为乙腈-10 mmol/L SBE-β-CD水溶液(含20 mmol/L KH₂PO₄, pH值到4.0)(体积比为20:80)的条件下,二氢黄豆苷原、雌马酚和山姜素的对映体都达到了基线分离,分离度分别为1.8, 1.9和1.4。该方法简便,分离效果好,对黄酮类化合物的拆分具有应用价值。     

Rapid determination of acetaminophen in physiological fluids by liquid chromatography using SDS mobile phase and ED detection

Acetaminophen is determined in serum and urine samples by a rapid, sensitive, and precise chromatographic method without any pretreatment step in a C18 column using a pure micellar mobile phase of 0.02M sodium dodecyl sulfate at pH 7. Acetaminophen is eluted in less than 5 min with no interference of the protein band. The use of electrochemical and UV detection is compared. Linearities (r > 0.999), as well as intra- and interday precision, are studied in the validation of the method. Limits of detection (LOD) are also calculated to be 0.56, 0.83, and 0.74 ng/mL in micellar solution, serum, and urine using electrochemical detection. The developed micellar liquid chromatographic method is useful for the quantitation of acetaminophen in serum and urine. Recoveries in the biological matrices are in the 98-107% range and results are compared with those obtained using a reference method. Drug excretion (in urine) and serum distribution are studied in several healthy volunteers, and no interference from metabolites is found. The developed procedure can be applied in routine analyses, toxicology, and therapeutic monitoring.     

Selectivity in normal-phase liquid chromatography with binary mobile phase

A simple theoretical approach to normal-phase liquid chromatography with binary mobile phase is presented. In the model used the driving force for the retention is competitive adsorption of solutes and both solvents. A new expression for the separation factor is proposed and discussed. An influence of different parameters on the selectivity is shown. The theoretical concepts are illustrated by the selected experimental data.     

Chiral separation using cyclodextrins as mobile phase additives in open-tubular liquid chromatography with a pseudophase coating

The chiral separation of various analytes (dichlorprop, mecoprop, ibuprofen, and ketoprofen) was demonstrated with different cyclodextrins as mobile phase additives in open-tubular liquid chromatography using a stationary pseudophase semipermanent coating. The stable coating was prepared by a successive multiple ionic layer approach using poly(diallyldimethylammonium chloride), polystyrene sulfonate, and didodecyldimethyl ammonium bromide. Increasing concentrations (0–0.2 mM) of various native and derivatized cyclodextrins in 25 mM sodium tetraborate (pH 9.2) were investigated. Chiral separation was achieved for the four test analytes using 0.05–0.1 mM β-cyclodextrin (resolution between 1.11 and 1.34), γ-cyclodextrin (resolution between 0.78 and 1.27), carboxymethyl-β-cyclodextrin (resolution between 1.64 and 2.59), and 2-hydroxypropyl-β-cyclodextrin (resolution between 0.71 and 1.76) with the highest resolutions obtained with 0.1 mM carboxymethyl-β-cyclodextrin. %RSD values were <10%. This is the first demonstration of chiral open-tubular liquid chromatography using achiral chromatographic coatings and cyclodextrins as mobile phase additives.     

Determination of D-alanine and D-glutamic acid in biological samples by coupled-column chromatography using beta-cyclodextrin as mobile phase additive.

A high-performance liquid chromatographic assay for monitoring traces of D-alanine and D-glutamic acid in peptide and protein hydrolysates is presented, which uses a two-column set-up with column switching. The main advantage of the proposed method is the higher reliability of the results, compared with the analysis by derivatization and single-column chromatography of the diastereomers. A non-chiral alkyl-silica reversed-phase column is combined with a second column, in which beta-cyclodextrin is used as a chiral mobile phase additive. The amino acids of the hydrolysate are dansylated, and the amino acid of interest is separated from the others on the first column and transferred to the second column where chiral resolution is performed. The transfer volume of ca. 200 microliters is small enough not to cause any peak distortion or dilation in the second column.     

Simultaneous determination of three alkaloids in Huangbo using an ionic liquid as a mobile phase additive in reversed‐phase liquid chromatography

A reversed‐phase high‐performance liquid chromatography method for the simultaneous determination of jatrorrhizine, palmatine, and berberine in Huangbo, the dried bark of Chinese Corktree, was established by using 1‐hexyl‐3‐methylimidazolium tertafluoroborate as a mobile phase additive. The chromatographic behavior of the three compounds on the C₁₈ column was studied with four different types of 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as the mobile phase additives. The effect of 1‐hexyl‐3‐methylimidazolium tertafluoroborate was the best in the four investigated ionic liquids. The concentration of 1‐hexyl‐3‐methylimidazolium tertafluoroborate and the pH of the mobile phase, which influenced the chromatographic behaviors of the three bioactive compounds, were investigated. The linearity, precision, accuracy, repeatability, limit of detection, and quantification of the proposed method were found to be satisfactory. To explain the role of ionic liquids as the mobile phase additives, the possible mechanism was also explored and discussed.     

Retention and mass transfer characteristics in reversed-phase liquid chromatography using a tetrahydrofuran-water solution as the mobile phase.

The characteristics of the retention and the mass transfer kinetics in reversed-phase liquid chromatography (RPLC) were measured on a system consisting of a C18-silica gel and a tetrahydrofuran-water (50:50, v/v) solution. These parameters were derived from the first and the second moments of the elution peaks, respectively. Further information on the thermodynamic properties of this system was derived from the temperature dependence of these moments. Some correlations previously established were confirmed for this system, namely, an enthalpy-entropy compensation for both retention and surface diffusion and a linear free-energy relationship. These results are compared with those observed in other similar systems using methanol-water (70:30, v/v) and acetonitnile-water (70:30, v/v) solutions. The contribution of surface diffusion to intraparticle diffusion in C18-silica gel particles was shown to be important. The analysis of the thermodynamic properties of surface diffusion suggests that, in these three RPLC systems, its activation energy is lower than the isosteric heat of adsorption. The nature and the extent of the influence of the mobile phase composition on the parameters describing the retention and the mass transfer kinetics are different but the chromatographic mechanisms involved in RPLC systems appear similar, irrespective of the nature of the organic modifier in the mobile phase.     

Molecular interactions in adsorption liquid chromatography with mixed mobile phase

Summary The equation of Jaroniec was used to describe the molecular interactions in liquid adsorption chromatography with mixed mobile phases. The main object of the investigation was to demonstrate that the type of association in the bulk phase is independent of the nature of the stationary phase, and to examine the influence of the nature and number of components in the mobile phase upon the specific interactions in the system. The relationship between the type of association and the structure of the chromatographed substance was also taken into account.     

Retention behavior of cardiac steroids using cyclodextrin in the mobile phase in high-performance liquid chromatography

The retention behaviour of twenty cardiac steroids and four fluorescent derivatives was examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The addition of a suitable cyclodextrin improved the separation of isomeric cardiac steroids. The steroid A/B ring junction is the most important factor in the choice of the optimum cyclodextrin to be added; the C/D ring junction is less important. The hydroxyl group at the 3- or 12-position of the steroid enhanced the changes in retention times of these compounds. The effect of an unsaturated lactone ring at the 17 beta-position on the retention in the presence of cyclodextrin was also determined with cardenolide (five-membered ring) and bufadienolide (six-membered ring) but little difference was observed. Isomeric cardiac steroids, whose separation has not been done by the conventional method, were clearly separated by this method. The fluorescence intensity of 3-(1-anthroyl)-cardiac steroid was enhanced by the addition of cyclodextrin to the mobile phase.     

Retention behaviour in liquid and supercritical fluid chromatography using methanol or diethyl ether as mobile phase

Summary Effects of column temperature and pressure on the retention behaviour of aromatic hydrocarbons were investigated in liquid and supercritical fluid chromatography by using a packed column and methanol or diethyl ether as the mobile phase. Oligomers of styrene, polysiloxane and a non-ionic detergent were separated by temperature- and pressure-gradient elution.     

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse‐phase liquid chromatography at different methanol‐water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol‐water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.     

Dimethylformamide as a mobile phase component in reversed-phase liquid column chromatography on octadecylsilica

Summary N,N-Dimethylformamide selectively accelerates the elution of weakly basic compounds and reduces their tailing in reversed-phase liquid chromatography with aqueous organic mobile phases. This effect is demonstrated with two types of octadecylsilica packings for representative solutes, covering aromatic amines and N-heterocyclic compounds such as pyridine, pyrimidine and purine derivatives.     

Mixed-mode liquid chromatography of aliphatic anionic surfactants with a naphthalenetrisulfonate mobile phase

Summary Separation of three classes of anionic surfactants (alkyl sulfates, alkanesulfonates, and alkyl phosphates) are achieved on a mixed-mode reversed phase (RP) phenyl/-anion exchange column using a naphthalenetrisulfonate (NTS)-acetonitrile (ACN) mobile phase via indirect photometric, indirect fluorometric, direct or indirect conductivity detection. Mixtures of C₅–C₁₈ sulfates, C₆–C₁₈ sulfonates, and C₁–C₄ phosphates (mono- and di-ester) can be separated in less than 20, 24, and 20 min respectively. Although hydrophobic effects are more pronounced in mixed-mode chromatography, equivalent-per-equivalent exchange of analysis and eluent ion is still required for sensitive indirect photometric, fluorometric, or conductivity detection. The detection limits of alkyl sulfates and alkanesulfonates are in the range of 3–15 pmoles which are at least an order of magnitude better than suppressed conductivity detection. The determination of surfactants in a variety of real samples is also presented.     

手性流动相添加法拆分酮康唑外消旋体

采用C18反相色谱柱,利用在流动相中加入手性选择剂的方法实现酮康唑对映体的拆分。研究了手性选择剂的种类及浓度、流动相pH值、甲醇比例和柱温等因素对酮康唑手性分离的影响,结果表明磺丁基-β-环糊精可以使酮康唑对映体完全分离,最后选择的流动相组成为甲醇-0.02 mol/L磷酸二氢钠(体积比为60∶40,含0.02%三乙胺和1.0 mmol/L磺丁基-β-环糊精,用稀磷酸调节pH值到3.00)。酮康唑对映体在6 min内得到基线分离,分离度为2.05。方法简便,分离效果好,对酮康唑对映体的拆分具有应用价值。