Simultaneous determination of caffeine,paracetamol, and ibuprofen in pharmaceutical formulations by high‐performance liquid chromatography with UV detection and by capillary electrophoresis with conductivity detection

Paracetamol, caffeine and ibuprofen are found in over‐the‐counter pharmaceutical formulations. In this work, we propose two new methods for simultaneous determination of paracetamol, caffeine and ibuprofen in pharmaceutical formulations. One method is based on high‐performance liquid chromatography with diode‐array detection and the other on capillary electrophoresis with capacitively coupled contactless conductivity detection. The separation by high‐performance liquid chromatography with diode‐array detection was achieved on a C₁₈ column (250×4.6 mm², 5 μm) with a gradient mobile phase comprising 20–100% acetonitrile in 40 mmol L^?1 phosphate buffer pH 7.0. The separation by capillary electrophoresis with capacitively coupled contactless conductivity detection was achieved on a fused‐silica capillary (40 cm length, 50 μm i.d.) using 10 mmol L^?1 3,4‐dimethoxycinnamate and 10 mmol L^?1 β‐alanine with pH adjustment to 10.4 with lithium hydroxide as background electrolyte. The determination of all three pharmaceuticals was carried out in 9.6 min by liquid chromatography and in 2.2 min by capillary electrophoresis. Detection limits for caffeine, paracetamol and ibuprofen were 4.4, 0.7, and 3.4 μmol L^?1 by liquid chromatography and 39, 32, and 49 μmol L^?1 by capillary electrophoresis, respectively. Recovery values for spiked samples were between 92–107% for both proposed methods.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.     

Creatinine determination in serum by capillary electrophoresis

Creatinine in human serum was separated in a fused-silica capillary with H3PO4 (75 mmol/L, pH 2.5) as BGE, followed by UV detection at 200 nm. Serum with methylimidazole added as internal standard was deproteinized with acetonitrile and the supernatant, after dilution with water was injected at pressure mode. Creatinine and methylimidazole were baseline-resolved in 6.5 min. Linearity in the 0-880 micromol/L range gave an r2 > or = 0.998, recovery was 102 +/- 2.8% (n = 6). Enzymatic breakdown with creatininase confirmed that serum does not interfere. The within-day and between-days coefficient of variation (CV) were < or = 2.16 and 2.7%, respectively. The accuracy, determined for lyophilized samples by isotope dilution gas chromatography-mass spectrometry was < or = +/- 2.0%. The results were compared with HPLC for 32 lyophilized samples and on 27 serum pools. Capillary electrophoresis, rapid and inexpensive, seems a promising alternative to high-performance liquid chromatography (HPLC) for creatinine determination in human serum.     

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.     

Chiral separation of quinolones by liquid chromatography and capillary electrophoresis

The quinolones are derivatives of oxoquinolines and mostly known for their antibacterial and antiviral activities. Many quinolones are chiral compounds having asymmetric centers and important due to their enantioselective biological activities. In order to study the biological activities of quinolone enantiomers, to control the manufacturing of homochiral drugs and to prepare necessary quantities of pure enantiomers for preclinical or clinical trials, respective chiral separation methods are urgently needed. In this context, the present review discusses chromatographic and electrophoretic methods for the enantioseparation of chiral quinolones and provides some useful information on their physical and pharmaceutical properties. The drawbacks of currently used techniques are revealed and ways to overcome them are outlined. Moreover, recommendations for an optimal choice of a separation protocol are given.     

Determination of diflubenzuron and chlorbenzuron in fruits by combining acetonitrile‐based extraction with dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography

In this study, a simple and low‐organic‐solvent‐consuming method combining an acetonitrile‐partitioning extraction procedure followed by “quick, easy, cheap, effective, rugged and safe” cleanup with ionic‐liquid‐based dispersive liquid–liquid microextraction and high‐performance liquid chromatography with diode array detection was developed for the determination of diflubenzuron and chlorbenzuron in grapes and pears. Ionic‐liquid‐based dispersive liquid–liquid microextraction was performed using the ionic liquid 1‐hexyl‐3‐methylimidazolium hexafluorophosphate as the extractive solvent and acetonitrile extract as the dispersive solvent. The main factors influencing the efficiency of the dispersive liquid–liquid microextraction were evaluated, including the extractive solvent type and volume and the dispersive solvent volume. The validation parameters indicated the suitability of the method for routine analyses of benzoylurea insecticides in a large number of samples. The relative recoveries at three spiked levels ranged between 98.6 and 109.3% with relative standard deviations of less than 5.2%. The limit of detection was 0.005 mg/kg for the two insecticides. The proposed method was successfully used for the rapid determination of diflubenzuron and chlorbenzuron residues in real fruit samples.     

Pesticides analysis by liquid chromatography and capillary electrophoresis

Nowadays, a wide range of pesticides are used in agricultural production, and their monitoring in samples of environmental and alimentary interest is of extreme importance to ensure, among others, the safety of consumption of foods. The aim of this work is to provide updated information about the major developments in CE and HPLC in pesticide analysis, covering relevant publications between 2004 and early 2006. The use of different sample pretreatment steps to provide a suitable extraction of these compounds from the different matrices as well as to increase the sensitivity of the determination is also discussed.     

Capillary electrophoresis,high‐performance liquid chromatography,and thin‐layer chromatography analyses of phenolic compounds from rapeseed plants and evaluation of their antioxidant activity

Rapeseed plants, known for oil production, are also known to contain phenolic compounds such as phenolic acids and flavonoids, with potential antioxidant and anticancer activities. The separation and identification of 11 phenolic acids in rapeseed extracts (including leaves, flowers, Chinese seeds, Belgian seeds, and cake) by capillary electrophoresis were investigated. The results were compared with those obtained with high‐performance liquid chromatography and thin‐layer chromatography and showed that the capillary electrophoresis technique offers several advantages for the identification of phenolic compounds in various rapeseed extracts. The antioxidant activity of rapeseed extracts and reference compounds was evaluated using four different approaches, namely, 2,2′‐azinobis‐ (3‐ethylbenzohiazoline‐6‐sulfonic acid assay, free radical 2,2‐diphenyl‐1‐picrylhydrazyl assay, electron paramagnetic resonance spectroscopy and the measurement of the total polyphenol content. The contents of total polyphenols in the tested extracts were ranging between 5.4 and 21.1% m/m and ranked as follows: Chinese seeds ? Belgian seeds ? Flowers ? Cake ? Leaves.     

Determination of Doxepin in human plasma using cloud‐point extraction with high‐performance liquid chromatography and ultraviolet detection

A new straightforward method based on cloud‐point extraction has been developed, optimized, and validated for the determination of doxepin in human plasma by high‐performance liquid chromatography separation and UV detection. The nonionic surfactant Triton X‐114 was chosen as the extraction solvent. Chromatography separation was performed on a μBondapak^R C₁₈ column (4.6 mm id × 300 mm, 3 μm particle size), which was used for isocratic elution at a detection wavelength of 289 nm. Under the optimum conditions, the linear range of doxepin in human plasma was 0.1–0.9 μg/mL. Also, the detection limit, preconcentration factor, and enrichment factor were 0.08 μg/mL, 50, and 49.0, respectively.     

Method development for separating organic carbonates by ion‐moderated high‐performance liquid chromatography

An ion‐moderated partition high‐performance liquid chromatography method was developed for the separation and identification of common organic carbonates. The separation of organic carbonates was achieved on an ion exclusion column with an exchangeable hydrogen ion. An isocratic, aqueous mobile phase was used for elution and detection was performed with a refractive index detector. The developed method was validated for specificity, linearity, limits of detection and quantification, precision and accuracy. All calibration curves showed excellent linear regression (R² > 0.9990) within the testing range. The limits of detection were 3.8–30.8 ppm for the analyzed carbonates. Improvements in the peak resolution of the chromatograms were achieved by decreasing the column temperature. Addition of the organic modifier, acetonitrile, to the eluent was found to have insignificant effects on the peak resolution. The developed method was demonstrated for analyzing organic carbonate components in the electrolyte system of a commercial lithium ion battery.     

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Capillary zone electrophoresis (CZE) and reverse phase high‐performance liquid chromatography (RP‐HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety ‐PO₂^–‐CH₂‐ derived from the structure N‐Ac‐Val‐AlaB(‐CH₂)Leu‐His‐NH₂ synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris‐phosphate background electrolytes in the pH range 1.1–3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP‐HPLC method (C₁₈‐silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP‐HPLC it was possible to check the purity of these RP‐HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP‐HPLC. The results obtained by CZE and RP‐HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.     

Extraction and preconcentration of formaldehyde in water by polypyrrole‐coated magnetic nanoparticles and determination by high‐performance liquid chromatography

In this study, a simple and rapid extraction method based on the application of polypyrrole‐coated Fe₃O₄ nanoparticles as a magnetic solid‐phase extraction sorbent was successfully developed for the extraction and preconcentration of trace amounts of formaldehyde after derivatization with 2,4‐dinitrophenylhydrazine. The analyses were performed by high‐performance liquid chromatography followed by UV detection. Several variables affecting the extraction efficiency of the formaldehyde, i.e., sample pH, amount of sorbent, salt concentration, extraction time and desorption conditions were investigated and optimized. The best working conditions were as follows: sample pH, 5; amount of sorbent, 40 mg; NaCl concentration, 20% w/v; sample volume, 20 mL; extraction time, 12 min; and 100 μL of methanol for desorption of the formaldehyde within 3 min. Under the optimal conditions, the performance of the proposed method was studied in terms of linear dynamic range (10–500 μg/L), correlation coefficient (R² ≥ 0.998), precision (RSD% ≤ 5.5) and limit of detection (4 μg/L). Finally, the developed method was successfully applied for extraction and determination of formaldehyde in tap, rain and tomato water samples, and satisfactory results were obtained.     

Determination of multiple phytohormones in fruits by high‐performance liquid chromatography with fluorescence detection using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling

Plant hormone determination in food matrices has attracted more and more attention because of their potential risks to human health. However, analytical methods for the analysis of multiple plant hormones remain poorly investigated. In the present study, a convenient, selective, and ultrasensitive high‐performance liquid chromatography method for the simultaneous determination of multiple classes of plant hormones has been developed successfully using dispersive liquid–liquid microextraction followed by precolumn fluorescent labeling. Eight plant hormones in fruits including jasmonic acid, 12‐oxo‐phytodienoic acid, indole‐3‐acetic acid, 3‐indolybutyric acid, 3‐indolepropionic acid, gibberellin A₃, 1‐naphthylacetic acid, and 2‐naphthaleneacetic acid were analyzed by this method. The conditions employed for dispersive liquid–liquid microextraction were optimized systematically. The linearity for all plant hormones was found to be >0.9993 (R² values). This method offered low detection limits of 0.19–0.44 ng/mL (at a signal‐to‐noise ratio of 3), and method accuracies were in the range of 92.32–103.10%. The proposed method was applied to determine plant hormones in five kinds of food samples, and this method can achieve a short analysis time, low threshold levels of detection, and a high specificity for the analysis of targeted plant hormones present at trace level concentrations in complex matrices.     

Optimized determination of polybrominated diphenyl ethers by ultrasound‐assisted liquid–liquid extraction and high‐performance liquid chromatography

A method based on ultrasound‐assisted liquid–liquid extraction and high‐performance liquid chromatography has been optimized for the determination of six polybrominated diphenyl ether congeners. The optimal condition relevant to the extraction was first investigated, more than 98.7 ± 0.7% recovery was achieved with dichloromethane as extractant, 5 min extraction time, and three cycles of ultrasound‐assisted liquid–liquid extraction. Then multiple function was employed to optimize polybrominated diphenyl ether detection conditions with overall resolution and chromatography signal area as the responses. The condition chosen in this experiment was methanol/water 93:7 v/v, flow rate 0.80 mL/min, column temperature 30.0°C. The optimized technique revealed good linearity (R² > 0.9962 over a concentration range of 1–100 μg/L) and repeatability (relative standard deviation < 6.3%). Furthermore, the detection limit (S/N = 3) of the method were ranged from 0.02 to 0.13 μg/L and the quantification limit (S/N = 10) ranged from 0.07 to 0.35 μg/L. Finally, the proposed method was applied to spiked samples and satisfactory results were achieved. These results indicate that ultrasound‐assisted liquid–liquid extraction coupled with high‐performance liquid chromatography was effective to identify and quantify the complex polybrominated diphenyl ethers in effluent samples.     

Ultrapreconcentration and determination of organophosphorus pesticides in water by solid‐phase extraction combined with dispersive liquid–liquid microextraction and high‐performance liquid chromatography

Solid‐phase extraction coupled with dispersive liquid–liquid microextraction was developed as an ultra‐preconcentration method for the determination of four organophosphorus pesticides (isocarbophos, parathion‐methyl, triazophos and fenitrothion) in water samples. The analytes considered in this study were rapidly extracted and concentrated from large volumes of aqueous solutions (100 mL) by solid‐phase extraction coupled with dispersive liquid–liquid microextraction and then analyzed using high performance liquid chromatography. Experimental variables including type and volume of elution solvent, volume and flow rate of sample solution, salt concentration, type and volume of extraction solvent and sample solution pH were investigated for the solid‐phase extraction coupled with dispersive liquid–liquid microextraction with these analytes, and the best results were obtained using methanol as eluent and ethylene chloride as extraction solvent. Under the optimal conditions, an exhaustive extraction for four analytes (recoveries >86.9%) and high enrichment factors were attained. The limits of detection were between 0.021 and 0.15 μg/L. The relative standard deviations for 0.5 μg/L of the pesticides in water were in the range of 1.9–6.8% (n = 5). The proposed strategy offered the advantages of simple operation, high enrichment factor and sensitivity and was successfully applied to the determination of four organophosphorus pesticides in water samples.     

Online solid‐phase extraction with high‐performance liquid chromatography and mass spectrometry for the determination of five tannins in traditional Chinese medicine injections

A rapid analytical method based on online solid‐phase extraction with high‐performance liquid chromatography and mass spectrometry has been established and applied to the determination of tannin compounds that may cause adverse effects in traditional Chinese medicine injections. Different solid‐phase extraction sorbents have been compared and the elution buffer was optimized. The performance of the method was verified by evaluation of recovery (≥40%), repeatability (RSD ≤ 6%), linearity (r² ≥ 0.993), and limit of quantification (≤0.35 μg/mL). Five tannin compounds, gallic acid, cianidanol, gallocatechin gallate, ellagic acid, and penta‐O‐galloylglucose, were identified with concentrations ranging from 3.1–37.4 μg/mL in the analyzed traditional Chinese medicine injections.     

Enantioseparation of aldols by high‐performance liquid chromatography on polysaccharide‐based chiral stationary phases that bear chlorinated substituents

Two families of aldols, obtained from the condensation of aromatic aldehydes with cyclohexanone or acetone (ten examples in each group), were analyzed by high‐performance liquid chromatography in normal phase elution mode on three polysaccharide‐based chiral stationary phases of the Lux series, namely, Lux Cellulose‐2, Lux Cellulose‐4 and Lux Amylose‐2, which share the common feature of chlorinated substituents in the chiral selectors. Following simple optimization steps, the enantioseparation of all aldols derived from cyclohexanone was achieved and the highest values of separation factor (α, 1.32 < α < 2.20) and resolution (R_s, 4.5 < R_s <17.2) were observed on Lux Cellulose‐2, with the only exception of the 4‐nitro‐substituted derivative that was better resolved on Lux Cellulose‐4. On the contrary, Lux Amylose‐2 was the best choice for aldols derived from acetone and only specific analytes in this group were resolved on the cellulose‐based supports. A variable‐temperature study of selected compounds allowed us to determine thermodynamic parameters of the enantioseparation process, which was enthalpy‐controlled in all the cases except one.     

Identification,characterization, and high‐performance liquid chromatography quantification of process‐related impurities in vonoprazan fumarate

High‐performance liquid chromatography analysis of vonoprazan fumarate, a novel proton pump inhibitor drug revealed six impurities. These were identified by liquid chromatography with mass spectrometry. Further, the structures of the impurities were confirmed by synthesis followed by characterization by mass spectrometry, NMR spectroscopy, and infrared spectroscopy. On the basis of these data and knowledge of the synthetic scheme of vonoprazan fumarate, the previously unknown impurity was identified as 1‐[5‐(2‐fluorophenyl)‐1‐(pyridin‐3‐ylsulfonyl)‐1H‐pyrrol‐3‐yl]‐N‐methyldimethylamine, which is a new compound. The possible mechanisms by which these impurities were formed were also discussed. A high‐performance liquid chromatography method was optimized in order to separate, selectively detect, and quantify all process‐related impurities of vonoprazan fumarate. The presented method has been validated in terms of linearity, limits of detection, and quantification, and response factors and, therefore, is highly suitable for routine analysis of vonoprazan fumarate related substances as well as stability studies.     

Quantitative analysis of hydroxylated polymethoxyflavones by high‐performance liquid chromatography

Since the hydroxylated polymethoxyflavones were isolated from orange and other citrus peels, they have been found to exhibit potent anti‐cancer and anti‐inflammatory activities. Hydroxylated polymethoxyflavones, especially 5‐demethylnobiletin and 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone, exert more potent activities than their counterpart polymethoxyflavones (PMFs). These findings have led to a new era of exploration of hydroxylated polymethoxyflavones and PMFs from the citrus genus for their potential application in nutraceutical, pharmaceuticals and functional foods. A practical and efficient analytical method for quantitatively characterizing the composition of PMFs has only recently been developed, and has applications both in academic research laboratories and quality controls. However, chemical analyses of the hydroxylated polymethoxyflavones have not been previously described. This paper reports the quantitative analysis of six 5‐demethylated PMFs in various commercial orange peel extracts, using high‐performance liquid chromatography with UV detection. Copyright © 2009 John Wiley & Sons, Ltd.