Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparations

The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C(18) analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples.     

A review on development of analytical methods to determine monitorable drugs in serum and urine by micellar liquid chromatography using direct injection

Therapeutic drug monitoring is a common practice in clinical studies. It requires the quantification of drugs in biological fluids. Micellar liquid chromatography (MLC), a well-established branch of Reverse Phase-High Performance Liquid Chromatography (RP-HPLC), has been proven by many researchers as a useful tool for the analysis of these matrices. This review presents several analytical methods, taken from the literature, devoted to the determination of several monitorable drugs in serum and urine by micellar liquid chromatography. The studied groups are: anticonvulsants, antiarrhythmics, tricyclic antidepressants, selective serotonin reuptake inhibitors, analgesics and bronchodilators. We detail the optimization strategy of the sample preparation and the main chromatographic conditions, such as the type of column, mobile phase composition (surfactant, organic solvent and pH), and detection. The finally selected experimental parameters, the validation, and some applications have also been described. In addition, their performances and advantages have been discussed. The main ones were the possibility of direct injection, and the efficient chromatographic elution, in spite of the complexity of the biological fluids. For each substance, the measured concentrations were accurate and precise at their respective therapeutic range. It was found that the MLC-procedures are fast, simple, inexpensive, ecofriendly, safe, selective, enough sensitive and reliable. Therefore, they represent an excellent alternative for the determination of drugs in serum and urine for monitoring purposes.     

Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography-tandem mass spectrometry

This paper describes a fast method for the sensitive and selective determination of melamine in a wide range of food matrices, including several milk-based products. The method involves an extraction with aqueous 1% trichloroacetic acid before the injection of the 10-fold diluted extract into the liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) system, using labelled melamine as the internal standard. As melamine is present in aqueous media in the cationic form, the chromatographic separation in reversed-phase LC requires the use of anionic ion-pair reagents, such as tridecafluoroheptanoic acid (THFA). This allows a satisfactory chromatographic retention and peak shape in all the types of food samples investigated. The method has been validated in six food matrices (biscuit, dry pasta and four milk-based products) by means of recovery experiments in samples spiked at 1 and 5 mg kg⁻¹. Average recoveries (n = 5) ranged from 77% to 100%, with excellent precision (RSDs lower than 5%) and limits of detection between 0.01 and 0.1 mg kg⁻¹. In addition, accuracy and robustness of the method was proven in different soya-based matrices by means of quality control (QC) sample analysis. QC recoveries, at 1 and 2.5 mg kg⁻¹, were satisfactory, ranging from 79% to 110%. The method developed in this work has been applied to the determination of melamine in different types of food samples. All detections were confirmed by acquiring two MS/MS transitions (127 > 85 for quantification; 127 > 68 for confirmation) and comparing their ion intensity ratio with that of reference standards. Accuracy of the method was also assessed by applying it to a milk-based product and a baking mix material as part of an EU proficiency test, in which highly satisfactory results were obtained.     

Development and validation of micellar liquid chromatographic methods for the determination of antibiotics in different matrixes

Antibiotics are the most important bioactive and chemotherapeutic compounds to be produced by microbiological synthesis, and they have proved their worth in a variety of fields, such as medicinal chemistry, agriculture, and the food industry. Interest in antibiotics has grown in parallel with an increasingly high degree of productivity in the field of analytical applications. Therefore, it is necessary to develop chromatographic procedures capable of determining various drugs simultaneously in the shortest possible time. Micellar liquid chromatography (MLC) is an RP-HPLC technique that offers advantages over conventional HPLC as far as sample preparation, selectivity, and versatility are concerned. Its main advantage is that samples can be injected directly into the chromatographic system with no previous preparation step. This paper mainly focuses on the results of the authors' own recent research and reports the chromatographic conditions for determination of various antibiotics (penicillins, quinolones, and sulfonamides) in different matrixes (pharmaceuticals, biological fluids, and food). The work of other authors on MLC-based antibiotic determination has been included.     

Capillary electrophoresis with diode array detection as an alternative analytical method for paralytic and amnesic shellfish toxins.

In recent years the marine environment has been seriously damaged by the presence of several toxic phytoplanktonic species, such as dinoflagellates and other toxic algae, which contaminate shellfish and other marine products. Amnesic and paralytic shellfish toxins are examples of these contaminants. The search for sensitive methodologies for the analysis of such compounds is one of the aims of researchers working in the marine environment. High-performance liquid chromatographic methods have been used for this purpose, allowing the detection of very low levels of these toxins. Recently, capillary electrophoresis (CE) has been used as an alternative for the separation and analysis of these compounds. In this paper, we report the optimization of CE procedures for their analysis. Due to the complexity of the matrix, clean-up procedures are required for removing interferences which affect the electrophoretic resolution. The influences of electrophoretic parameters such as voltage, buffer concentrations and organic modifiers, were studied in order to optimize the electrophoretic system to achieve high resolution as well as an accurate quantitation. Extraction and other steps such as clean-up of samples prior to the electrophoretic analysis have been also studied. Different buffers and organic modifiers were used in order to improve the separation of the toxic components, and consequently to obtain accurate quantitative information about the amount of toxins present in the contaminated samples.     

Recent theoretical and practical applications of micellar liquid chromatography (MLC) in pharmaceutical and biomedical analysis

Micellar liquid chromatography (MLC) is an analytical technique belonging to the wide range of reversed-phase liquid chromatographic (RP-LC) separation techniques. MLC with the use of surfactant solutions above its critical micellar concentration (CMC) and the addition of organic modifiers is currently an important analytical tool with still growing theoretical considerations and practical applications in pharmaceutical analysis of drugs and other biologically active compounds. The use of MLC as an alternative, relatively much faster in comparison to conventional chromatographic separation techniques has several advantages, especially as being suitable for screening pharmaceutical analysis. The analytical data received from MLC analysis are considered a useful source of information to predict passive drug absorption, drug transport and other pharmacokinetics and physicochemical measures of pharmaceutical substances.     

Non-chromatographic atomic spectrometric methods in speciation analysis: A review

In recent years, knowledge of the different chemical forms of the elements has gained increasing importance. There has been significant progress in methods that hyphenate chromatographic separations with atomic spectrometry. These hyphenated methods can provide the most complete information on the species distribution and even structure. However, they can be lengthy, relatively costly and difficult to bring to the routine. On the other hand, it is important to remember that chromatographic techniques represent only a minor part of the separation procedures available and, in certain cases, the application of basic chemistry to sample treatments can give quantitative information about specific chemical forms. In this sense, non-chromatographic procedures can provide methods that offer sufficient information on the elemental speciation for a series of situations. Moreover, these non-chromatographic strategies can be less time consuming, more cost effective and available, and present competitive limits of detection. Thus, non-chromatographic speciation analysis continues to be a promising research area and has been applied to the development of several methodologies that facilitate this type of analytical approach. In view of their importance, the present work overviews and discusses different non-chromatographic methods as alternatives for the speciation analysis of clinical, environmental and food samples using atomic spectrometry for detection.     

Comparison of C18 silica bonded phases selectivity in micellar liquid chromatography

The paper describes a new test designed in micellar LC (MLC) to compare the commercial C18 stationary phase properties. This test provides the total hydrophobicity, hydrophilicity, steric selectivity, hydrogen bonding, and ion‐exchange capacity properties calculation of the ODS stationary phases. Both the test compounds and chromatographic separation conditions choice for column characterization in MLC are detailed. The chromatographic performance of several stationary phases that are used in MLC was evaluated with specific chromatographic test comprising nine test compounds, possessing different physico‐chemical properties, which were injected on different supports with two micellar mobile phases: one at pH 7.0 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol), and other at pH 2.7 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol adjusted to pH by TFA). Fundamental column chromatographic properties were obtained under these conditions and were treated by hierarchical cluster analysis. From the results of cluster analysis, two closely related groups of columns are distinguished, and it was shown that the chosen column characteristic parameters allow characterizing both sorbent and micellar chromatographic system properties. Eleven columns were analyzed by this test, which allows a comparison of columns with the aim of the selection of suitable and analogous column for the analysis with MLC.     

Melamine contamination

In the summer of 2008, serious illnesses and deaths of babies in China were linked to melamine-tainted powdered infant formula. Melamine contains several metabolites, such as ammeline, ammelide, and cyanuric acid, and has been used for the adulteration of foods or milk to increase their apparent protein content. It is assumed that melamine and its metabolites are absorbed in the gastrointestinal tract, and precipitate in the kidney to form crystals. A new tolerable daily intake of 0.2 mg kg^?1 body weight was adapted by the World Health Organization in 2008. This paper reviews the variety of analytical methods that have been used for the analysis of melamine in food. The limit of detection of these various methods is 0.05–100 ppm. The maximum acceptable concentration in food has been set at 50 ppb by the US FDA. A fast and ultrasensitive procedure for screening, detection, and characterization of melamine and its derivative compounds needs to be established. Currently, mass-spectrometry technologies provide an alternative to derivatization for regulatory analysis of food.     

Membrane sampling with microdialysis coupled to HPLC/UV for on-line simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer

An on-line microdialysis/high-performance liquid chromatography method was developed for the simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer. To collect these analytes from aqueous samples, the microdialysis system featured a microdialysis probe incorporating a polyarylethersulfone membrane and employed 0.05 M HCl in 0.1% (v/v) MeOH as the perfusate, with optimal efficiency obtained at a flow rate of 1 μL min(-1). The chromatographic conditions were optimized when using a reverse-phase phenyl column and a mobile phase of phosphate buffer solution in 10% (v/v) MeOH, buffered at pH 3.0. Good linearity relationship (r(2) > 0.9987), intra- and inter-day precisions (RSDs < 6.6%), recoveries (96.9 - 105.0%), and limits of detection (melamine, 3 ppb; cyanuric acid, 150 ppb) were observed for the two analytes. This method has been successfully applied to simultaneous determination of melamine and cyanuric acid in commercial creamers with the recoveries in the range of 97.5 to 102.6%.     

应用亲水作用色谱检测鸡蛋和肉中三聚氰胺及三聚氰酸二酰胺

应用亲水作用色谱(HILIC)对从市场上购买的鸡蛋和肉中的三聚氰胺及三聚氰胺降解产物三聚氰酸二酰胺进行了检测。采用的色谱柱为ZIC-HILIC柱,流动相为3 mmol/L磷酸二氢铵溶液(pH 6.9)-乙腈(20:80, v/v),流速为0.8 mL/min,检测波长为220 nm。在该体系下,三聚氰胺和三聚氰酸二酰胺的保留时间适中,与样品中的内源性物质有良好的分离。样品经0.1%磷酸提取,偏磷酸及乙腈沉淀蛋白质和糖类物质,以及P-SCX固相萃取柱净化。三聚氰胺和三聚氰酸二酰胺在0.4～40 mg/L范围内与峰面积呈良好的线性关系,样品定量限(按信噪比(S/N)不小于10计)为2 mg/kg,在2～10 mg/kg添加水平下的平均回收率为80%～105%,相对标准偏差小于10%。该方法具有良好的分离选择性,可用于鸡蛋和肉中三聚氰胺和三聚氰酸二酰胺的同时检测。     

The application of pseudo template molecularly imprinted polymer to the solid-phase extraction of cyromazine and its metabolic melamine from egg and milk

The combination of molecularly imprinted polymer with high performance liquid chromatography has been developed to determine cyromazine and its metabolic melamine in some samples. However, the potential risk of template leakage used in molecularly imprinted polymer is a major disadvantage. To solve this problem, 2-(4,6-diamino-1,3,5-triazin-2-ylamino) ethanethiol disulfide, a molecule that shares the similar imprinting sites with cyromazine and melamine, was selected as pseudo template to prepare molecularly imprinted polymer. Methacrylic acid, ethylene glycol dimethyl acrylate and toluene were selected as functional monomer, crosslinker and porogen, respectively. The molecular recognition property and binding capability of cyromazine and melamine were evaluated by adsorption test and Scatchard analysis. The results showed that the molecularly imprinted polymer based on pseudo template had more excellent affinity and selectivity for cyromazine and melamine. The resulting molecularly imprinted polymer was used as a solid-phase extraction material to enrich cyromazine and melamine in egg and milk samples for high performance liquid chromatography analysis. The solid-phase extraction process was carefully optimized. It was found that when different concentration of cyromazine and melamine standards were spiked into samples, satisfactory recovery rate of cyromazine and melamine were obtained as 85.6-98.8% with relative standard deviation <5.5%.     

Novel analytical procedures for screening of drug residues in water, waste water, sediment and sludge

Traces of pharmaceuticals are continuously introduced into the aquatic environment mainly by sewage treatment plant effluents. Final data about their impact on the ecosystem are still partly missing. Progress in instrumental analytical chemistry has resulted in the availability of methods that allow a monitoring of these pollutants at ng L⁻¹ levels. In this review the state-of-the-art of residue analysis of pharmaceuticals by chromatographic and electrophoretic techniques is summarized. Improvements in detection limits over the past years have mainly been due to sophisticated mass spectrometric detection techniques. Furthermore, robust sample preparation and preconcentration protocols based on solid-phase extraction and related procedures have contributed significantly to the achievements observed so far. This review also covers several immunochemical approaches which may serve as an inexpensive alternative for quick screening of samples.     

The determination of uranium in environmental samples using extraction chromatography

Many traditional methods for the determination of actinides in environmental samples have involved several solvent extraction and/or ion exchange steps that separate the radionuclide of interest from the bulk sample matrix. These methods are generally labor-intensive employ hazardous substances and generate considerable volume of toxic wastes. Extraction chromatographic resins have been developed that combine the power and selectivity of solvent extraction with the ease of use of ion exchange chromatographic resin. Some of these extraction chromatographic resins have been developed that will selectively sorb a particular radionuclide or groups of radionuclides from solution. A considerable amount of interest has recently been shown in these resins as they are claimed to be cost-effective in terms of increasing sample throughput, eliminating the need for expensive solvent extractants and being generally safer to handle. One such commercially available resin, U/TEVA.Spec^® will selectively absorb uranium and tetravalent actinides such as thorium and plutonium from an acidic nitrate solution. This paper describes the application of U/TEVA.Spec^® to the determination of uranium in a range of environmental samples.     

Development of melamine certified reference material in milk using two different isotope dilution mass spectrometry techniques

A new certified reference material (CRM) of melamine in milk GLHK-11-02 was developed aiming to address the great demand from the testing community after the melamine crises. The material was prepared by adding an appropriate quantity of melamine into the skimmed milk samples and the final product was in the form of fine lyophilized powder. Characterization of the material relied on two newly developed gravimetric isotope dilution mass spectrometry (IDMS) methods, one using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and another gas chromatography-mass spectrometry (GC-MS). Experimental parameters with crucial effects on the performance of the two IDMS methods were thoroughly investigated. These included purity of standard used, equilibration time of isotopes, efficiency of extraction methods as well as possible interferences from the matrix and melamine analogues. Precision was found to be excellent with a coefficient of variation of 2.5% for the LC-IDMS/MS (n=46) and 1.9% for the GC-IDMS (n=30) respectively. Using one-tail Student's t-test at 95% confidence interval, analytical data sets generated from the two methods were found to exhibit no significant difference. Measurement accuracy of the methods was further verified through an Asia Pacific Metrology Program (APMP) pilot study. Analytical results of the present LC-IDMS/MS for the two milk test samples at the concentration level of about 0.45 and 3.5 mg kg(-1) were proven to be very good. There were excellent overlaps between our results and the assigned reference values, and the absolute deviation was less than 3.2%. Both the LC-IDMS/MS and GC-IDMS methods were shown to be sufficiently reliable and accurate for certification of the melamine CRM. Certified value of melamine in dry mass fraction in GLHK-11-02 was 1.14 mg kg(-1). Expanded uncertainty due to sample inhomogeneity, long term and short term stability and variability in the characterization procedure was at 7.1% or 0.08 mg kg(-1). The CRM is primarily used to provide a complete method validation for and to improve the technical competence of melamine analysis to food and chemical testing laboratories.     

Determination of melamine in aquaculture feed samples based on molecularly imprinted solid‐phase extraction

This research highlights the application of highly efficient molecularly imprinted solid‐phase extraction for the preconcentration and analysis of melamine in aquaculture feed samples. Melamine‐imprinted polymers were synthesized employing methacrylic acid and ethylene glycol dimethacrylate as functional monomer and cross‐linker, respectively. The characteristics of obtained polymers were evaluated by scanning electron microscopy, Fourier transform infrared spectroscopy and binding experiments. The imprinted polymers showed an excellent adsorption ability for melamine and were applied as special solid‐phase extraction sorbents for the selective cleanup of melamine. An off‐line molecularly imprinted solid‐phase extraction procedure was developed for the separation and enrichment of melamine from aquaculture feed samples prior to high‐performance liquid chromatography analysis. Optimum molecularly imprinted solid‐phase extraction conditions led to recoveries of the target in spiked feed samples in the range 84.6–96.6% and the relative standard deviation less than 3.38% (n = 3). The aquaculture feed sample was determined, and there was no melamine found. The results showed that the molecularly imprinted solid‐phase extraction protocols permitted the sensitive, uncomplicated and inexpensive separation and pre‐treatment of melamine in aquaculture feed samples.     

Chromatographic methods for tetracycline analysis in foods.

The tetracyclines have served for decades as an important class of antibiotics in food animal health and production. As such, they have also been a source of concern for residue monitoring authorities around the world. In response to this concern a number of microbial inhibition, immunoassay and bacterial receptor methods have evolved for the detection of this class of compounds in various foods of animal origin. However, these methods often lack specificity and are subject to false positive and false negative results. For these reasons a number of chromatographic methods for the separation and determination of the tetracyclines isolated from foods have been developed that are capable of identifying and quantifying individual tetracycline drugs. We present here an overview of tetracycline analytical methods, including microbial inhibition, immunoassay and receptor technologies for detection, techniques for isolation from food matrices, and thin-layer chromatographic, high-performance liquid chromatographic, gas chromatographic and mass spectrometric procedures for determination of this class of compounds. A discussion of the variables involved in such methodology and a review of method criteria are offered.     

Determination of melamine and related triazine by-products ammeline, ammelide, and cyanuric acid by micellar electrokinetic chromatography

In this study, micellar electrokinetic chromatographic (MEKC) methods were developed for the detection of traces of melamine and its related by-products (ammeline, ammelide, and cyanuric acid). Two on-line sample concentration steps namely reversed electrode polarity stacking mode (REPSM) and cation-selective injection (CSI) were used for improving the detection sensitivity. For REPSM, a borate-NaOH buffer (pH 10, 35 mM) composed of 60 mM SDS and 10% (v/v) methanol, was used as carrier electrolyte, and samples were prepared in an aqueous solution of 10 mM NaOH. In CSI, a phosphate buffer (pH 2, 50 mM) containing 41 mM SDS was used as the carrier electrolyte, and samples were prepared with an aqueous solution of 10 mM NaOH and a phosphate buffer (pH 2.0, 25 mM) in a volume ratio of 1:9. The results indicated that REPSM enhanced all analyte signals except for melamine, which could be concentrated only by the CSI. The detection limit was reduced from 1.7 mg L⁻¹ to 2.8 μg L⁻¹ for melamine by the optimal CSI step, and from 0.23-1.2 mg L⁻¹ to 2.4-5.0 μg L⁻¹ for the other three analytes by the optimal REPSM step. Tableware made of melamine and samples of flour were used as test samples, and the results indicated that the proposed MEKC methods can successfully determine contaminations from melamine. The study also indicated that when the plastic made of melamine was exposed only once to an acidic solution (acetic or phosphoric acid) at 80 °C for 30 min, melamine continuously leached out from the test sample even without any further treatment with an acidic solution.     

Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review

Fluoroquinolones are one of the most promising and intensively studied drugs of contemporary anti-infective chemotherapy. New fluoroquinolone antibacterials with improved pharmacokinetic properties and a broad spectrum of activity have been developed, opening new windows of opportunity for clinical use. To our knowledge, no comprehensive and critical review of the analytical methods for the determination of these agents, which correspond to the third- and fourth-generation quinolones, has yet been published. This work summarizes for the first time most of the liquid chromatographic methods reported in the literature for the separation and quantification of the new fluoroquinolones in biological matrices and pharmaceutical formulations. A systematic and detailed survey of physicochemical properties, sample preparation procedures, and chromatographic and detection conditions is presented herein. In the course of this review several liquid chromatographic methods are discussed: reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IEX-HPLC), hydrophilic interaction liquid chromatography (HILIC), high-performance thin-layer chromatography (HPTLC) and other chiral chromatographic methods. Their advantages, applicability and limitations are also examined.     

Synthesis of magnetic polyphosphazene-Ag composite particles as surface enhanced Raman spectroscopy substrates for the detection of melamine

Magnetic polyphosphazene(MPZS) particles coated by Ag nanoparticles(MPZS-Ag) have been developed as surface enhanced Raman spectroscopy(SERS) substrates for sensitive detection of melamine in aqueous solutions and milk samples.5,5'-Dithiobis-(2-nitrobenzoic acid)(DTNB) was used as model analyte to test the SERS activity of the MPZS-Ag particles.The prepared MPZS-Ag particles possess both magnetic responsiveness and excellent SERS properties.SERS detection of different concentrations of melamine aqueous solutions and spiked milk samples were performed by the MPZS-Ag particles.The limit of detection(LOD) of the melamine in aqueous solutions was 10-7 mol/L(0.0126 mg/L) and 0.6 mg/L in real milk samples using the MPZS-Ag particles as SERS substrates.The LOD of the melamine are much lower than the safety values of Food and Drug Administration and Codex Alimentarius Commission.These results indicate that the MPZS-Ag particles have promising application prospect for SERS analysis in food safety fields.