Stability-Indicating HPTLC Method for Simultaneous Determination of Ezetimibe and Simvastatin

Simvastatin and ezetimibe are used to treat hyperlipidemia. A simple, selective and stability-indicating HPTLC method has been established for analysis of simvastatin and ezetimibe. The method has been validated so that both drugs can routinely be analyzed simultaneously. The method uses aluminum-backed silica gel 60F₂₅₄ TLC plates as stationary phase with n-hexane–acetone 6:4 (v/v) as mobile phase. Densitometric analysis of both drugs was carried out in absorbance mode at 234 nm. This system was found to give compact bands for simvastatin and ezetimibe (R _F 0.39 ± 0.05 and 0.50 ± 0.05, respectively). Linear relationships were obtained between response and amount of drug in the range 200–1,600 ng per band with high correlation coefficients (r ² = 0.9917 ± 0.0018 for simvastatin and r ² = 0.9927 ± 0.0021 for ezetimibe). The method was validated for precision, robustness, and recovery. The limits of detection and quantitation were 25 and 150 ng per band, respectively. Simvastatin and ezetimibe were subjected degradation by acid, pH 6.8 phosphate buffer, oxidation, dry heat, and wet heat. The degradation products were well resolved from the pure drug with significantly different R _F values. Because the method could effectively separate the drug from its degradation products, it can be used for stability-indicating analysis.     

A reliable high-performance liquid chromatography with ultraviolet detection for the determination of sulfonamides in honey

The sulfonamides are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value is a potential hazard to human health. Brazilian authorities have included in the National regulatory monitoring program, the control of the three most widely used sulfonamides in honey production, i.e., sulfathiazole, sulfamethazine and sulfadimethoxine. A method for the determination of residual sulfonamides in honey, using sulfapyridine as an internal standard has been developed, optimized and validated. Some changes were implemented on current available methodologies for the analysis of sulfonamides in honey in order to adopt such procedures to Brazilian honey samples. Sulfonamides were extracted from honey with dichloromethane after dissolution with 30% sodium chloride, and cleaned up with solid phase extraction on Florisil columns. The eluate was analyzed by high-performance liquid chromatography with ultraviolet detection. The limit of detection was determined at 3 μg kg⁻¹, 4 μg kg⁻¹ and 5 μg kg⁻¹ for sulfathiazole, sulfamethazine and sulfadimethoxine, respectively with average recoveries of 61.0% for sulfathiazole; 94.5% for sulfamethazine and 86.0% for sulfadimethoxine at the 100 μg kg⁻¹ level. As the final step of validation procedure, the analysts were submitted to a blind spiked sample prepared by the quality assurance officer which results were successfully obtained regarding recovery and deviations.     

Improved quantitative thin-layer chromatographic method for the separation of cobalamin derivatives

Summary A new quantitative thin-layer chromatographic method was developed for the separation and determination of cobalamin derivatives. A mixture of acetone, acetonitrile, isopropanol, diethylamine and aqueous ammonium hydroxide as the eluent and precoated Kieselgel 60 chromatoplate as the TLC plate was used. The evaluation of the separated spots was carried out by densitometry. The method can be used for the determination of cyanocobalamin in fermentatic mixture without any cleaning or concentration procedure as well as for the purity test of cyanocobalamin and hydroxocobalamin.     

A normal phase HPTLC method for the quantitative determination of fumonisin B1 in rice

Summary A normal phase HPTLC method has been validated by spiking, in quadruplet, uncontaminated extract of rice with fumonisin B₁ over the range 0 to 16g/g. The method utilises solid phase extraction using strong anionic exchange (SAX) cartridges, uni-directional normal phase high performance thin layer chromatography (HPTLC), novel visualisation by dipping into a 0.16% acidic solution ofp-anisaldehyde and quantification by scanning fluoro-densitometry. Response was linear only over the range 0 to 5 g/g (0 to 125 ng/spot) where recoveries averaged 81% for rice. Weighted linear regression yielded a limit of detection of 0.25 g/g for rice. Coefficients of variance were 15.4, 5.3, 2.8, 3.5, and 0.9% at fumonisin B₁ levels of 0.20, 0.5, 1.0, 2.0 and 5 g/g respectively, demonstrating good precision. This method claims to be the first fully quantitative HPTLC method for determining fumonisin B₁ in rice.     

A normal phase HPTLC method for the quantitative determination of fumonisin B1 in rice

Summary A normal phase HPTLC method has been validated by spiking, in quadruplet, uncontaminated extract of rice with fumonisin B₁ over the range 0 to 16 μg/g. The method utilises solid phase extraction using strong anionic exchange (SAX) cartridges, uni-directional normal phase high performance thin layer chromatography (HPTLC), novel visualisation by dipping into a 0.16 % acidic solution ofp-anisaldehyde and quantification by scanning fluoro-densitometry. Response was linear only over the range 0 to 5 μg/g (0 to 125 ng/spot) where recoveries averaged 81 % for rice. Weighted linear regression yielded a limit of detection of 0.25 μg/g for rice. Coefficients of variance were 15.4, 5.3, 2.8, 3.5, and 0.9 % at fumonisin B₁ levels of 0.20, 0.5, 1.0, 2.0 and 5 μg/g respectively, demonstrating good precision. This method claims to be the first fully quantitative HPTLC method for determining fumonisin B₁ in rice.     

Simple and rapid determination of sulfonamides in milk using Ether-type column liquid chromatography

A simplified and rapid determining/identifying method for residual sulfonamides (SAs) in milk by using Ether-type stationary phase, which made in our lab, was presented. The target analytes were extracted by mixing with ethanol-acetic acid (97:3, v/v) followed by centrifugation. The procedure used a Ether-type C8 column, isocratic elution with acetonitrile-water (5:95, v/v), and a photo-diode array detector. The linear range of determination was 50-10,000 μg/L for sulfanilamide and 100-10,000 μg/L for sulfadiazine, sulfamerazine, sulfamethazine. Average recoveries of four SAs (spiked 0.5, 1.0 and 1.5 μg/mL) ranged from 80.1% to 87.6%, with relative standard deviations between 3.4% and 5.8%. The total time and solvent required for the analysis of one sample were <15 min and <1.0 mL of ethanol and 0.6 mL of acetonitrile, respectively. The developed procedure was nearly harmless to the human and environment.     

A novel approach for the synthesis of alkyl and aryl sulfonamides

A novel approach for the synthesis of alkyl and aryl sulfonamides by the reaction of sulfonic acids, isocyanides and water in dichloromethane is reported at ambient temperature in excellent yields within 20 min. To the best of our knowledge this is the first report on the synthesis of this biologically important family using easily available sulfonic acids and isocyanides.     

High-performance liquid chromatography of sulfonamides and quinolones on p-tert-butyl-calix[6]arene-bonded silica gel stationary phase

The high-performance liquid chromatographic behavior of some sulfonamides and quinolones was studied on a p-tert-butyl-calix[6]arene-bonded silica gel stationary phase. The effect of mobile phase variables such as methanol content, ionic strength and pH on their chromatographic behavior was investigated. The retention behavior of sulfonamides on the stationary phase was compared with that on both Zorbax C₁₈-bonded silica gel and γ-(ethylenediamino)propyltriethoxylsilane-bonded silica gel (diamino-bonded phase). The retention mechanism of sulfonamides and quinolones on the stationary phase was also discussed. The results indicate that the stationary phase behaves as a reversed-phase packing and its separation selectivity is much better than that of not only Zorbax C₁₈ phase but also diamino-bonded phase. Some sulfonamides and quinolones were separated on the stationary phase, but the separation of sulfonamides is far more successful.     

Hollow fiber supported ionic liquid membrane microextraction for determination of sulfonamides in environmental water samples by high-performance liquid chromatography

By using ionic liquid as membrane liquid and tri-n-octylphosphine oxide (TOPO) as additive, hollow fiber supported liquid phase microextraction (HF-LPME) was developed for the determination of five sulfonamides in environmental water samples by high-performance liquid chromatography with ultraviolet detection The extraction solvent and the parameters affecting the extraction enrichment factor such as the type and amount of carrier, pH and volume ratio of donor phase and acceptor phase, extraction time, salt-out effect and matrix effect were optimized. Under the optimal extraction conditions (organic liquid membrane phase: [C₈MIM][PF₆] with 14% TOPO (w/v); donor phase: 4 mL, pH 4.5 KH₂PO₄ with 2 M Na₂SO₄; acceptor phase: 25 μL, pH 13 NaOH; extraction time: 8 h), low detection limits (0.1–0.4 μg/L, RSD ≤ 5%) and good linear range (1–2000 ng/mL, R² ≥ 0.999) were obtained for all the analytes. The presence of humic acid (0–25 mg/L dissolved organic carbon) and bovine serum albumin (0–100 μg/mL) had no significant effect on the extraction efficiency. Good spike recoveries over the range of 82.2–103.2% were obtained when applying the proposed method on five real environmental water samples. These results indicated that this present method was very sensitive and reliable with good repeatabilities and excellent clean-up in water samples. The proposed method confirmed hollow fiber supported ionic liquid membrane based LPME to be robust to monitoring trace levels of sulfadiazine, sulfamerazine, sulfamethazine, sulfadimethoxine and sulfamethoxazole in aqueous samples.     

An HPTLC method for quantification of cholesteryl esters from human plasma and rat liver microsomes

Cholesteryl oleate present as a neutral lipid in low‐density lipoprotein has been speculated to be a biomarker for atherosclerosis. Methods which are at hand for the quantification of cholesteryl oleate are either costly or entail the use of radioactive compounds. Charring of TLC plates has been used to identify cholesteryl esters for a long time but has never been applied to quantification of cholesteryl esters in biological matrices. Here, we report a novel method based on planar chromatography for the analysis of the products of the acyl CoA–cholesterol acyltransferase (ACAT) assay, viz. cholesteryl esters. Using silica gel 60 F₂₅₄ as stationary phase, compounds were spotted on the plate and run using a solvent system comprising n‐hexane–diethyl ether–glacial acetic acid (90:10:1, v/v/v). The plates were developed by dipping in anisaldehyde–sulfuric acid reagent and were scanned at 546 nm for quantification. The developed method shows good linear relationship in the concentration range of 100–500 ng/band with a correlation coefficient (r) value of 0.9996. The method was validated for accuracy, precision and robustness. Percentage recovery of the method was found to be in the range 96.88–103.01% with intra‐ and inter‐day precision analysis yielding <2% relative standard deviation at nominal concentrations for analysis. The limits of detection and quantification were found to be 6.45 and 19.54 ng, respectively. The method was validated for robustness by making deliberate changes in mobile phase composition, volume and temperature of analysis, and the standard deviations of peak areas for these intentional changes were found to be 1.07, 1.02 and 1.30 respectively. The method was applied to the estimation of cholesteryl esters in plasma samples from patients diagnosed with hypercholesterolemia. No interferences were found from the biological matrices used in the assay. The proposed method could be of immense potential for estimation of cholesteryl oleate as a marker of ACAT activity, for screening of ACAT inhibitors in drug discovery process and in the prognosis of atherosclerosis. Copyright © 2013 John Wiley & Sons, Ltd.     

Quasi-column chromatography of barbituric acid derivatives II. Separation on Non-polar adsorbents using binary water-organic solvent mixtures as the mobile phase

Summary Twenty barbituric acid derivatives having four different types of substitution were separated in TLC-S chambers. Using non-polar adsorbents (silanized silica gel or silica gel coated with paraffin oil) and binary water-organic solvent mixtures as the mobile phase. Linear relationships between R_M and the concentration of the organic solvent were observed for the majority of the investigated compounds. The non-polar absorbents ensure a better separation than untreated silica gel especially for the therapeutically useful C₅ disubstituted barbiturates. The results can be used for the optimization of the systems for the chromatography of barbiturates. The R_f values were correlated with the number of carbon atoms of the substitutions, molecular connectivity and a parameter associated with the molecular volume. The best correlations were obtained for this last parameter.     

Prediction of chromatographic parameters for some anilines by molecular connectivity

Summary The possible relation existing between R_F values obtained by thin-layer chromatography for a group of anilines with connectivity indices proposed by Kier and Hall has been studied. Using multivariable regression the corresponding connectivity functions, selected for their respective correlation coefficients, standard deviations, Snedecor's F and Student's t were obtained. Regression analysis of the connectivity functions gives a correct prediction of the experimental elution sequence for this group of substances on silica gel stationary phases and various mobile phases of different polarity. The corresponding random and stability studies of the different prediction models selected were carried out, showing good stability and null randomness in all cases.     

Prediction of chromatographic parameters for some anilines by molecular connectivity

Summary The possible relation existing between R_F values obtained by thin-layer chromatography for a group of anilines with connectivity indices proposed by Kier and Hall has been studied. Using multivariable regression the corresponding connectivity functions, selected for their respective correlation coefficients, standard deviations, Snedecor’s F and Student’s t were obtained. Regression analysis of the connectivity functions gives a correct prediction of the experimental elution sequence for this group of substances on silica gel stationary phases and various mobile phases of different polarity. The corresponding random and stability studies of the different prediction models selected were carried out, showing good stability and null randomness in all cases.     

A rapid densitometric method for simultaneous quantification of gallic acid and ellagic acid in herbal raw materials using HPTLC

Gallic acid and ellagic acid are two widely occurring phenolic compounds of plant origin, to which many biological activities including anticancer and antiviral activity have been attributed. A simple HPTLC method has been developed for the simultaneous quantification of gallic acid and ellagic acid. The method was validated for precision, repeatability, and accuracy. Instrumental precision was found to be 0.083 and 0.78, and the repeatability of the method was found to be 1.07 and 1.50 (% CV) for gallic acid and ellagic acid, respectively. The accuracy of the method was checked by a recovery study conducted at two different levels and the average percentage recovery was found to be 101.02% for gallic acid and 102.42% for ellagic acid. The above method was used for the quantification of gallic acid and ellagic acid content in seeds of Abrus precatorius Linn., whole plant of Phyllanthus maderaspatensis Linn., and flowers of Nymphaea alba Linn. The proposed HPTLC method for the simultaneous quantification of gallic acid and ellagic acid was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of herbal raw materials and for the quantification of these compounds in plant materials.     

A normal phase HPTLC method for the quantitative determination of ochratoxin A in rice

Summary A previously published [1] liquid chromatographic method for the determination of ochratoxin A in corn, barley and kidney has been modified for application to parboiled rice with quantification by high performance thin layer chromatography (HPTLC). The method has been validated by spiking uncontaminated extracts of rice with ochratoxin A over the range 0 to 198 g kg^–1. The proposed method has some significant advantages over the current AOAC method [2], especially for determining low levels of ochratoxin A in parboiled rice.     

An improved rapid method for the determination of actinides in water

A new rapid method has been developed for the determination of Th, Pu, Np, U, Am and Cm isotopes in water samples of about 1 L. Actinides are pre-concentrated by co-precipitation with Ca phosphate, sequentially separated on stacked TEVA and TK221 cartridges and measured by alpha spectrometry. The TK221 extraction chromatographic resin contains i.e. CMPO and DGA extractants. It has been characterized by measuring the weight distribution ratios (D_w) of actinides which are higher than 1000 for all actinides in 3 M HNO₃. The method has been optimized, applied for the analysis of tap and seawater samples and validated by participating in an IAEA proficiency test. Chemical recoveries for all actinides are better than 50%. The method can be performed within one day.

     

An accurate and precise high-performance liquid chromatography method for the rapid quantification of the novel HIV integrase inhibitor raltegravir in human blood plasma after solid phase extraction

The quantification of the HIV integrase inhibitor raltegravir in blood plasma is described using solid phase extraction (SPE) coupled with an accurate high-performance liquid chromatography assay with ultraviolet (UV) detection. The method was validated over the range of 20-10,000 ng/mL using simple sample preparation and chromatography. The SPE method was optimized to be selective and highly efficient. The buffer’s ionic strength and pH were optimized for retaining RAL and the internal standard on the column, the percentage of methanol was optimized in the cleaning step to remove unwanted plasma contaminants, and the type and amount of acid was optimized for complete elution of the compounds. This method has no interference with other potentially co-administered antiretrovirals or common drugs. Average recoveries for the extraction method were consistently high: 90% for raltegravir and 90% for the internal standard diazepam. This method was found to be accurate and precise. Within day (n = 6) and between day (n = 18) accuracies ranged from 97.5 to 104.4%. Within-day (n = 6) and between-day (n = 18) precision ranged from 1.4 to 3.8%, and from 2.4 to 7.9%, respectively. This is the first published method to use simple UV technology and reliable SPE extraction methodology for the quantification of raltegravir in human plasma. This method can be easily implemented in most bioanalytical laboratories.     

An improved SPE method for fractionation and identification of phospholipids

This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel‐aminopropyl‐silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high‐resolution LC‐MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP‐HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP‐HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC‐MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified.