Development of an analytical methodology to quantify melamine in milk using micellar liquid chromatography and validation according to EU Regulation 2002/654/EC

Melamine is a toxic triazine, illegally used as an additive in milk to apparently increase the amount of protein. A chromatographic procedure using a C18 column and a micellar mobile phase of sodium dodecyl sulphate (0.05 M) and propanol (7.5%), buffered at pH 3, and a detection set by absorbance at 210 nm, was reported for the resolution and quantification of melamine in liquid and powdered milk samples. In this work, samples were diluted with a SDS solution and were directly injected, thus avoiding long extraction and experimental procedures. Melamine was eluted in nearly 9.3 min without overlapping the protein band or other endogeneous compounds. The optimal mobile phase composition was taken using a chemometrical approach that considers the retention factor, efficiency and peak shape. Validation was performed following the European Commission's indications (European Decision 2002/657/EC), and the main analytical parameters studied were: linearity (0.02-100 ppm; r² = 0.999), limit of detection (5 ppb), intra- and inter-day precision (R.S.D. <7.6% and <9.7%, respectively) and robustness (R.S.D. <7.4% for retention time and <5.0% for area). Sensitivity was adequate to detect melamine under the safety limits proposed by the US FDA. Finally, recoveries for several milk samples were found in the 85-109% range.     

Separation and determination of aminophenols and phenylenediamines by liquid chromatography and micellar electrokinetic capillary chromatography

The separation and determination of aminophenols and phenylenediamines were investigated by liquid chromatography (LC) and micellar electrokinetic chromatography (MEKC) in this study. Aminophenols and phenylenediamines are commonly used components in commercial hair colorants. The problem of tailing peaks in LC was improved by the technique of using mobile phase containing 15 mM triethylamine at pH 8.0. The analysis of o-aminophenol was not succeeded with LC even though the modifier of triethylamine was added. But it could be quantitative successfully by MEKC. The optimum separation condition of MEKC was achieved by employing 55 mM cetyltrimethyl ammonium chloride in 50 mM borate buffer (pH 9.2) with electric field strength of −145 V cm⁻¹. Finally, the commercial hair dyes were analyzed by developing methods of LC and MEKC. From both the results, there is no significant difference presence at 99.5% confidence level. These two methods could give the complementary results.     

Development of a high‐performance liquid chromatography method with fluorescence detection for the routine quantification of tamoxifen,endoxifen and 4‐hydroxytamoxifen in plasma from breast cancer patients

To date, several methods for the quantification of tamoxifen and its metabolites have been developed, most of which employ liquid chromatography tandem–mass spectrometry (LC–MS/MS). These methods are highly sensitive and reproducible, but are also time‐consuming and require expensive equipment; one of their main disadvantages is matrix ionization effects. A more viable option, particularly in developing countries, is high‐performance liquid chromatography coupled with UV or fluorescence detection. We developed and validated a method for simultaneous quantification of tamoxifen, endoxifen and 4‐hydroxytamoxifen based on high‐performance liquid chromatography with fluorescence detection in a reverse‐phase column. The method is rapid (16 min plus 5 min of column re‐equilibrium), accurate (80–100%) and precise (0.23–6.00%), and does not require any additional irradiation process. Sample pretreatment consists of protein precipitation with acetonitrile under alkaline conditions, employing only 200 μL plasma. The validated method's wide range allowed quantification of steady‐state levels in patients under standard tamoxifen treatment (20 mg/day). This assay is ready for application in clinical studies and routine quantification of tamoxifen, endoxifen and 4‐hydroxytamoxifen in healthcare institutions.     

Development and validation of a liquid chromatography with tandem mass spectrometry method for the quantification of vitisin B in rat plasma and urine

A new, rapid, and sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of vitisin B and validated in rat plasma and urine using carbamazepine as an internal standard. The plasma (0.05 mL) or urine (0.2 mL) samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Eclipse Plus C₁₈ column (100 × 4.6 mm, 3.5 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid water (60:40, v/v) at a flow rate of 0.7 mL/min. Detection and quantification were performed by mass spectrometry in selected reaction‐monitoring mode with positive electrospray ionization. The calibration curves were recovered over the concentration ranges of 10?5000 ng/mL (correlation coefficients, r≥0.9833) in plasma and 5?2500 ng/mL (r≥0.9977) in urine, respectively. All validation data, including the specificity, precision, accuracy, recovery, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of vitisin B following intravenous administration of 0.5 and 1 mg/kg and intraperitoneal injection of 5, 10, and 25 mg/kg to rats. This is the first report on the pharmacokinetic properties of vitisin B. The results provide a meaningful basis to evaluate preclinical or clinical applications of vitisin B.     

Tamoxifen monitoring studies in breast cancer patients by micellar liquid chromatography

A simple micellar liquid chromatographic procedure is described to determine tamoxifen in plasma. To perform the analysis, tamoxifen solutions were diluted in water and UV-irradiated for 20 min to form the photocycled derivative with a phenanthrene core which shows intense fluorescence. Samples were then directly injected, thus avoiding long extraction and experimental procedures. The resolution from the matrix was performed with a mobile phase containing 0.15 M SDS–7% n-butanol at pH 3 running at 1.5 mL/min through a C18 column at 40 °C. Detection was carried out by fluorescence, and the excitation and emission wavelengths were 260 and 380 nm, respectively. The chromatographic analysis time was less than 15 min. The analytical methodology was validated following the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines. The response of the drug in plasma was linear and in the 0.5–15 μg/mL range, with r ² > 0.999. Accuracy and precision were <9% in both cases. The limits of detection and quantification (in nanograms per millilitre) were 50 and 150 in plasma, respectively. The method developed herein shows no interferences by endogenous compounds. Finally, the analytical method was used to determine the amount of tamoxifen in the plasma of several breast cancer patients from a local hospital.     

Determination of prednisolone and prednisone in plasma by liquid chromatography with fluorescence detection

After a single-step extraction from plasma (250 μl) with dichloromethane, the drugs and dexamethasone (internal standard) are oxidized by copper(II) acetate to the corresponding glyoxal and converted into the fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene. The quinoxalines are separated within 55 min by reversed-phase liquid chromatography with isocratic elution. The detection limits for prednisolone and prednisone added to plasma are 3 ng ml^?1 in plasma (signal-to-noise ratio=3).     

Effect of molecular structure on the solute-micelle and solute-stationary phase binding constants in micellar liquid chromatography

The effects of molecular structure on the solute-micelle and solute-stationary phase binding constants in micellar liquid chromatography (MLC) have been investigated. The following points have been observed. (1) There is quite a good linear relationship between the solute-micelle and solute-stationary phase binding constants in MLC with the cationic (CTAB) and anionic surfactants as the additives, which means that the contribution of physico-chemical properties of solutes on the solute-micelle and solute-stationary phase binding constants acts in a parallel way. (2) Good quantitative relationships between the solute-micelle and solute-stationary phase binding constants and the solvatochromic parameters have been obtained, which indicates that the distribution mechanism of the neutral solutes in MLC is determined via their molecular interactions. Both the cavity process and the hydrogen bond interaction play a very important role in the retention of neutral solutes in MLC. The contribution of the hydrogen bond interaction, especially the hydrogen donor ability of the solutes on those binding constants in anionic and cationic surfactant MLC, is determined in a different way. (3) Linear regression analysis of the solute-micelle and solute-stationary phase binding constants between the cationic and anionic surfactant MLC has been carried out. The obtained results suggest that the transfer of the non-polar solutes from the aqueous phase to the anionic and cationic surfactant micelles acts in a parallel way, but that of the polar solutes in a different way. A model of micelles with three different sites of solubilization, i.e., (1) the core of the micelle, (2) the surface of the micelle and (3) the palisade layer of the micelle, has been used to successfully explain the observed results. Finally, the retention behavior of solutes in MLC is compared with that in reversed-phase liquid chromatography (RP-LC). It has been observed that there is no difference in separation selectivity for the non-polar solutes between MLC and RP-LC; however, for the polar solutes, MLC provides a different separation selectivity compared to that in RP-LC.     

Separation of nonionic surfactants according to functionality by hydrophilic interaction chromatography and comprehensive two-dimensional liquid chromatography

It is shown, that amphiphilic polymers--such as polysorbates and fatty esters of polyethylene glycol can be separated by comprehensive two-dimensional liquid chromatography using a reversed phase column (under critical conditions for the polyoxyethylene chain) and a HILIC column, which may arranged in different order. The mobile phases in both dimensions can be 93-97 wt% acetone water. As the retention of higher esters on the reversed phase column is very strong, this column should be used as the first dimension. On the HILIC column all fractions elute within a reasonably short time (at a flow rate of 2.5 ml/min within 2 min). With a flow rate of 0.1 ml/min in the first dimension, a full separation can be achieved in 90 min.     

Determination of flurbiprofen in pharmaceutical formulations by UV spectrophotometry and liquid chromatography

In this study, a new and rapid UV spectrophotometric (UV) method and a reversed phase high performance liquid chromatographic (LC) method were developed for quantitative estimation of flurbiprofen, a non-selective, non-steroidal, anti-inflammatory drug (NSAID), in pure form and in pharmaceutical dosage form. The solvent system, wavelength of detection, chromatographic conditions were optimized in order to maximize the sensitivity of both the proposed methods. The linear regression equations obtained by least square regression method were Abs=7.5906×10⁻² concentration (μg/ml) + (−) 4.6210×10⁻² for the UV method, and peak area=1.2652×10² concentration (ng/ml) + 1.4830×10³ for the LC method. The detection limit as per the error propagation theory was found to be 0.34 μg/ml for UV method and 15 ng/ml for LC method. The developed methods were successfully employed with high degree of precision and accuracy for the estimation of total drug content in two commercial ophthalmic drops of flurbiprofen. The results of analysis were treated statistically, as per USP 2000 and International Conference on Harmonization (ICH) guidelines for validation of analytical procedures, and by recovery studies. The results obtained from UV method were comparable with those obtained by using LC. It was concluded that both the developed methods are equally accurate, sensitive, precise, reproducible, robust and rugged and could be applied directly and easily to the pharmaceutical preparations of flurbiprofen. However, LC method is useful at very low level (ng/ml), whereas UV method is suitable at μg/ml level.     

Development and validation of a liquid chromatography with mass spectrometry method to determine resveratrol and piceid isomers in beeswax

This paper represents the first report of a liquid chromatography coupled to electrospray ionization mass spectrometry method for simultaneously analyzing resveratrol and piceid isomers (cis and trans) in beeswax. An efficient extraction procedure has been proposed (average analyte recoveries were between 89 and 95%); this involved a solid–liquid extraction using a mixture of ethanol and water (80:20, v/v) and a concentration step in a rotary evaporator. The separation of all the compounds was achieved using a C₁₈ column and a mobile phase composed of ammonium formate 0.03 M in water and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification ranged from 1.0 to 1.7 and 3.5 to 5.5 μg/kg, respectively. Finally, the proposed method was applied to analyze beeswax samples collected from experimental and organic apiaries.     

Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.     

Determination of procaine and tetracaine in plasma samples by micellar liquid chromatography and direct injection of sample

Summary A liquid chromatographic procedure is proposed for the determination of procaine and tetracaine in plasma samples with direct injection. The method uses a Spherisorb octadecylsilane ODS-2 C₁₈ analytical column and a micellar mobile phase containing 0.15 M sodium dodecyl sulphate, 0.5% triethylamine at pH 2.5 and 10% propanol. The UV detection was carried out at 300 nm. Plasma sample preparation required only adequate dilution with the mobile phase before injection into the chromatographic system. The proposed method allows the determination of procaine and tetracaine in plasma at therapeutic levels.     

Development and validation of a method to determine amoxicillin in physiological fluids using micellar liquid chromatography

A simple and robust method was developed for the routine identification and quantification of amoxicillin by micellar LC. Amoxicillin, a beta-lactamase inhibitor, is one of the most commonly prescribed drugs in the treatment of urine and skin structure infections. In this work, amoxicillin was determined in urine samples without any pretreatment step in a phenyl column using a micellar mobile phase of 0.10 M SDS and 4% butanol at pH 3. A UV detection set at 210 nm was used. Amoxicillin was eluted at 5.1 min with no interference by the protein band or endogenous compounds. Linearities (r >0.9998), intra- and interday precisions were determined (RSD (%) 0.4-2.7% and 0.3-5%, respectively, in micellar media, and 0.14-2.6% and 0.13-6%, respectively, in urine), and robustness was studied in the method validation. LOD and LOQ were 0.04 and 0.1 microg/mL in micellar media and 0.14 and 0.34 microg/mL in urine, respectively. Recoveries in the urine matrix were in the range of 95-110%. The validated method proved to be reliable and sensitive for the determination of amoxicillin in urine samples.     

Multiresidue analysis of sulfonamides in meat by supramolecular solvent microextraction,liquid chromatography and fluorescence detection and method validation according to the 2002/657/EC decision

A multiresidue method was described for determining eight sulfonamides, SAs (sulfadiazine, sulfamerazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) in animal muscle tissues (pork, chicken, turkey, lamb and beef) at concentrations below the maximum residue limit (100 μg kg⁻¹) set by the European Commission. The method was based on the microextraction of SAs in 300-mg muscle samples with 1 mL of a supramolecular solvent made up of reverse micelles of decanoic acid (DeA) and posterior determination of SAs in the extract by LC/fluorescence detection, after in situ derivatization with fluorescamine. Recoveries were quantitative (98–109%) and matrix-independent, no concentration of the extracts was required, the microextraction took about 30 min and several samples could be simultaneously treated. Formation of multiple hydrogen bonds between the carboxylic groups of the solvent and the target SAs (hydrogen donor and acceptor sum between 9 and 11) were considered as the major forces driving microextraction. The method was validated according to the European Union regulation 2002/657/EC. Analytical performance in terms of linearity, selectivity, trueness, precision, stability of SAs, decision limit and detection capability were determined. Quantitation limits for the different SAs ranged between 12 μg kg⁻¹ and 44 μg kg⁻¹, they being nearly independent of matrix composition. Repeatability and reproducibility, expressed as relative standard deviation, were in the ranges 1.8–3.6% and 3.3–6.1%. The results of the validation process proved that the method is suitable for determining sulfonamide residues in surveillance programs.     

New method for the determination of four antiepileptic drugs in human plasma by high performance liquid chromatography

Summary The concurrent administration of several antiepileptic drugs for the treatment of seizure disorders has become common practice. Lamotrigine is a new antiepileptic given in combination with other antiepileptic drugs, but which is not routinely measured in clinical laboratories. An isocratic high-performance liquid chromatographic method is described for the simultaneous measuring lamotrigine, carbamazepine, phenobarbital and phenytoin within 10 minutes. The chromatographic system used an Hichrom Spherisorb CN column (20 cm×4 mm, i.d., 5 m particle size), a Bondapak CN precolumn, and a mobile phase consisting of methanol : acetonitrile : 5 mM sodium acetate (5:20:75: by volume, pH adjusted to 6.3 with acetic acid). BWA 725C was used as internal standard. The drugs were extracted from 200 l of plasma with ethyl acetate, acetonitrile and 5 mM sodium acetate. After evaporation of the organic layer and reconstitution in mobile phase, 25 l of extract was eluted with mobile phase at a flow rate of 1.2 ml/min. The eluted drugs were detected by their absorption at 205 nm and quantified from their peak heights. The method was found to be rapid, relatively simple to perform and sufficiently sensitive to determine each drug over its entire therapeutic range. Lower limits of detection varied from 50–100 ng/ml, absolute recoveries from 93–98%, and mean intra- and inter-assay CVs were <3.0%.     

Development and validation of a liquid chromatography with tandem mass spectrometry method for the determination of isomangiferin in rat plasma with application to a preclinical pharmacokinetic study

A sensitive and specific liquid chromatography with tandem mass spectrometry method was firstly developed for the measurement of isomangiferin in rat plasma. Chloramphenicol was selected as the internal standard. Sample preparation was carried out through a simple one‐step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reaction monitoring mode with transitions of m/z 421.1/301.1 for isomangiferin, and 321.1/151.9 for chloramphenicol. The calibration curve was linear over the range of 0.1–600 ng/mL, with a lower limit of quantification at 0.1 ng/mL. The intra‐ and interday precisions (relative standard deviation) were no more than 8.2% and accuracies (relative error) were within the range of –8.4 to 2.2%. The recovery, matrix effect and stability under different conditions were all proved acceptable. The validated method has been successfully applied to a preclinical pharmacokinetic study of isomangiferin in rats for the first time.     

Advances in capillary electrophoresis: the challenges to liquid chromatography and conventional electrophoresis

In the 1980s, capillary electrophoresis (CE) developed rapidly into a first-class analytical separation technique. Its advances in instrumentation and method development will not only enhance or complement existing mature separation techniques such as liquid chromatography and conventional slab gel electrophoresis, but will also severely challenge these separation methods. A brief overview of the most striking achievements of CE in the 1980s is given. which illustrates the challenges to liquid chromatography and conventional slab gel electrophoresis, and some detailed discussions are presented to highlight the advantages of CE. New developments in CE that can be expected for the 1990s include especially column technology, separation chemistry and instrumentation, which will serve further to diversify and improve the applicability of this technique in areas which are poorly addressed by other separation methods. This paper considers and speculates on the technological advancements that can be expected to emerge for CE in the 1990s.     

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 μm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.     

Determination of some banned stimulants in sports by micellar liquid chromatography

Summary A procedure has been developed for the determination, in <12 min, of several stimulants (amphetamine, ephedrine, methoxyphenamine, phenylephrine and phenylpropanolamine) in spiked urine samples after direct injection, using a hybrid micellar mobile phase of 0.15 M sodium dodecyl sulfate and 3% pentanol at pH 7, on a C₁₈ column with UV detection. Recoveries were 94–102% and limits of detection 4.5 ng·mL⁻¹ for methoxyphenamine and 0.39 μg·mL⁻¹ for amphetamine, similar to those obtained for aqueous solutions. Linearity reached 0.99 and intermediate precision was <8.4 and 5.3, for the two different concentrations tested.