Optimization of the extraction conditions of amino acid dabsyl derivatives

Summary Moist buffered paper chromatography is proposed for the selection of the optimum conditions for the extraction of dabsyl derivatives of amino acids. It is shown for each individual acid and optimum pH value exists at which the distribution coefficient reaches a maximum. The optimum pH values can be easily determined from chromatographic data. The linear correlation between the static distribution coefficients and the R_M values permit the direct calculation of the distribution coefficient from the chromatographic data.     

Quantitative determination of phenol derivatives fromOleum thymi

Summary The thymol and carvacrol content inOleum Thymi was determined by densitometry. Phenols were separated on a thin—layer of silica gel using two-step gradient elution. The dependence of measurement time on the amount of phenols was investigated. For comparison, the total phenolic content of the essential oil was also determined by colorimetry, the results indicating that the optimal measurment time at which the following measurements should be performed, is 5 min after the end of heating.     

Non-radioactive determination of PTH and dabsyl phosphoamino acids by capillary electrophoresis

Summary Capillary electrophoresis is a novel technique in the non-radioactive determination of phosphoamino acids. The main advantage of the method presented is the high selectivity and the ability to separate all phosphoamino acid derivatives. Non-radioactive determination of PTH or dabsyl phosphoamino acids by capillary electrophoresis provides a fast and simple screening procedure for all O-phosphorylated amino acids in protein and peptides in the low picomolar range.     

Separation of DABS derivatives of amino acids by multiple gradient development (MGD) in thin-layer chromatography

Summary A mixture of 13 DABS-amino acids has been chromatographed on high-performance silica gel layers developed with eluents containing increasing concentrations of ethyl acetate in heptane + chloroform, using a modification of stepwise multiple development MGD described in a earlier paper. Densitograms were obtained at 485 nm. The MGD method was very efficient, separating all 13 DABS-amino acids, and rapid, owing to the use of a non-aqueous mobile phase.     

Lipophilicity determination of some nitrostyrene derivatives on RP-2, PR-8 and RP-18 layers

Summary The lipophilicity of 33 nitrostyrene derivatives was determined by reversed-phase thin-layer chromatography on RP-2, RP-8 and RP-18 layers using methanol as the organic phase. The R_M value of each compound linearly decreased with increasing concentration of the organic solvent. The retention strength of RP-2 layer was lower than that of RP-8 and RP-18 layers. Between the retention strength of RP-8 and RP-18 layers no significant difference was detected. Minor differences among the selectivities of the layers were also observed. The impact of the change of methanol concentration on the retention was higher on RP-2 than on RP-18 layers probably due to the sterically favourable interaction of methanol with the shorter alkyl groups on the silica surface.     

A sensitive and selective spectrophotometric estimation of catechol derivatives in pharmaceutical preparations

A sensitive and simple spectrophotometric method for the estimation of catechol and its derivatives like dopamine hydrochloride (DPH), levodopa (LDP), methyldopa (MDP) and adrenaline hydrochloride (ADH) in both pure form and in pharmaceutical formulation, is described. The method is based on the interaction of diazotised sulphanilamide (DSA) with catechol derivatives in the presence of molybdate ions in acidic medium. Absorbance of the resulting red coloured product is measured at 490 nm for pyrocatechol (PCL) and at 500 nm for other catechol derivatives. The colour reaction is stable for 24–30 h. Under optimal conditions, Beer's Law range for pyrocatechol was found to be between 0.04 and 2.4 (R.S.D.=0.78%), for DPH was 0.02–2.8 (R.S.D.=0.98%) for LDP was 0.1–2.8 (R.S.D.=1.21%) for MDP was 0.5–7 (R.S.D.=1.41%) and for ADH was 0.5–7 (R.S.D.=1.58%). The method is highly reproducible and specific for these selected catechol derivatives. The common excipients used as additives do not interfere in the proposed method. Analytical data for the determination of the pure compound is presented together with the application of the proposed method to the analysis of some pharmaceutical formulations. The results compare favourably with those of official and reported methods.     

Simultaneous determination of serum propafenone and its metabolites using high‐performance liquid chromatography

Propafenone, a class Ic antiarrhythmic agent, is metabolized to 5‐hydroxypropafeone (5‐OHP) and N‐depropylpropafenone (NDPP). Simultaneous determination of serum propafenone and its metabolites was performed using HPLC equipped with a conventional octadecylsilyl silica column and ultraviolet detector. The wavelength was set at 250 nm. Propafenone and its metabolites in the serum were extracted using diethyl ether. The mobile phase solution, comprising 1‐pentanesulfonic acid sodium salt (0.1 m ), acetonitrile and acetic acid (280:185:2.5, v/v/v), was pumped at a flow rate of 1 mL/min. The recoveries of propafenone, 5‐OHP and NDPP were greater than 85, 82 and 60%, respectively, with the coefficients of variation (CVs) less than 5.4, 1.9 and 2.9%, respectively. The calibration curves were linear for a concentration range of 12.5–1500 ng/mL for propafenone and 2–500 ng/mL for 5‐OHP and NDPP (r > 0.999). CVs in the intraday assays were 1.0–3.8% for propafenone, 0.6–2.0% for 5‐OHP and 0.6–1.7% for NDPP. CVs in interday assays were 1.3–7.7% for propafenone, 1.1–6.5% for 5‐OHP and 5.4–8.0% for NDPP. The present HPLC method can be used to assess the disposition of propafenone and its metabolites for pharmacokinetic studies and therapeutic drug monitoring of propafenone.     

Diantipyrilmethane as complex-forming reagents in the thin-layer chromatographic determination of metals

Summary The efficiency of diantipyrilmethane used as a reagent for the chromatographic separation of metals, including titanium, zirconium and hafnium, rare earth elements, transition and platinum metals is shown. The peculiarities of the chromatographic behaviour of metal diantipyrilmethanates and the mechanism of their retention in TLC are discussed. Methods were developed for the determination of metals based on complex formation directly in the sorbent layer or by liquid extraction. The chromatographic separation takes place in silica gel thin layers with elution by organic solvent-mineral acid mixtures. The metals are determined by densitometric or spectrophotometric methods. After the complexes are isolated from the layer. The procedures are characterized by simplicity, efficiency, and a rather high selectivity. They were used to analyze steels, alloys, industrial solutions and other samples.     

Rapid thin-layer chromatography of homocysteine-cysteine mixtures as their thiolyte-derivatives

Summary Fluorescent labelling of cysteine and its homologue homocysteine with monobromobimane followed by TLC on silica gel allows rapid identification of both compounds through their intense yellow fluorescence when excited at 366 nm; a visual detection limit of about 2ng is reached for both thiols.     

High performance thin layer chromatographic determination of erythromycin in pharmaceutical preparations

Summary A high performance thin layer chromatographic method was developed for the determination of erythromycin. The drug was separated on a silica gel 60 plate and developed in methanol by means of an automatic multiple development. The chromatogram was sprayed with 10% sulphuric acid solution and heated at 100°C for 10–15 minutes. The area of the spot was quantified by a TLC scanner at a wavelength of 410 nm. A linear calibration curve was established over the range of 4–6 μg in 10μl of erythromycin. The relative standard deviation for five replicate determinations was found to be 1.45% for 5 μg in 10 μl of erythromycin standard. The average percentage recovery was found to be 99.87. The method has been applied to the determination of erythromycin in various pharmaceutical dosage forms. Common excipients in formulations do not interfere. After optimizing the solvent system, it was found that the use of silica gel 60 F₂₅₄ TLC plate with a DVS composed of ethyl acetate, ethanol and 10% sodium acetate pH 9.5 (9:7:8) led to the differentiation and quantitation of erythromycins A, B and C with an R.S.D. of less than 2.0%. The method is simple, precise and inexpensive. It should be used for routine analysis.     

Stability-indicating high performance thin layer chromatography determination of Paroxetine hydrochloride in bulk drug and pharmaceutical formulations

A simple selective precise and stability-indicating high performance thin layer chromatographic method of analysis of Paroxetine hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed TLC (Thin Layer Chromatography) aluminum precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of butanol:acetic acid:water (8:2:0.5, v/v/v). This system was found to give compact spots for Paroxetine HCl (Rf, retardation factor, value-0.48 ± 0.02). Paroxteine HCl was subjected to acid and alkali hydrolysis, oxidation and photodegradation, where the degraded product was well separated from the pure drug. Densitometric analysis of Paroxetine hydrochloride was carried out in the absorbance mode at 295 nm. The linear regression analysis data for the calibration spots showed good relationship with (regression) r² = 0.9903 in the amount range of 300-1500 ng (nanogram) per spot. The mean value of co-relation co-efficient, slope and intercept were 0.9903 ± 0.001, 5.38 ± 0.058 and 182.5 ± 2.16 respectively. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 50 and 150 ng, respectively. The drug doesnot undergo degradation with oxidation, but gets affected in acidic and alkaline conditions. The acid and alkali degradation showed extra peaks at 0.4 and 0.08 Rf, respectively. This indicates that the drug is susceptible to acidic and alkaline medium. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one.     

Liquid chromatographic determination of aniline and derivatives in environmental waters at nanogram per litre levels using fluorescamine pre-column derivatization

Summary Fluorescamine (fluram) has been used as a fluorogenic compound for pre-column derivatization of aniline and some derivatives. Anilines were derivatized with fluram in citrate buffer media (pH 5.5) to form pyrrolinones. The highly fluorescence pyrrolinones were isolated and pre-concentrated by solid phase extraction. A reversed phase, Spherisorb RP-8 column and tetrahydrofuran: water:formic acid (42:56:2) mobile phase was used for separation. Detection method was by a sensitive fluorimetric method and quantitation was at 395 and 495 nm. The various parameters such as reaction conditions between anilines and fluram, solid phase extraction and chromatographic separation were optimized. Calibrations were linear over the range considered with excellent correlation coefficients (r>0.999). Relative standard deviations are less than 2.5 % and detection limits for aniline,p-toluidine, 4-chloroaniline and 4-bromoaniline were 6, 30, 6 and 8 ng L⁻¹, respectively. This method has been used successfully for the determination of anilines in environmental waters.     

Parameters influencing theoretical determination of RM-values of naphthalene derivatives in thin-layer chromatography by using binary mobile phase

The purpose of the studies being carried out is to find some regularities of a general character which determine the relationship between the structures of the chromatographed substances and the composition of the mobile phase, i.e., between the log of the partition coefficient of the sample (A_z), the K₁-values which characterize the adsorption equilibrium of component “1” in a given chromatographic system, the structures of the mobile phase components and the structures of the substances being chromatographed. This paper deals with the relationship between the A_z and K₁ parameters and the structure of naphthalene and a number of its derivatives. It has been shown that there is a close relationship between the kind, size, frequency and site of the substituent on the one hand and the A_z and K₁ values on the other. In order to eliminate the possibility of unwanted side effects, the investigations were carried exclusively using non-active binary mobile phases of the type N + N or N + /B/. From these investigations, a comparison between the parameter A_z and the surface of adsorbed molecules (A_s) was also possible.     

Quantitative determination of phospholipids in mitochondria using HPTLC and fluorimetric assay in situ

A method is described for quantitative determination of phospholipids of mitochondria following one-dimensional thin-layer chromatography without previous elution. Using high performance thin-layer chromatography (HPTLC) and an in situ fluorescence technique, the time needed for quantitative determination is relatively short. As the method is rather sensitive, small amounts of the extracts can be applied (about 200 ng of total phospholipids per spot). The reproducibility for the phospholipid fractions determined was in the range of 8–17%. The procedure was tested with lipid extracts of rat liver mitochondria. The method allows the determination of cardiolipin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl choline, and sphingomyelin.     

Sequential injection chromatographic determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations

A new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations in this contribution.The coupling of short monolith with SIA system results in an implementation of separation step to until no-separation low-pressure method.A Chromolith^® Flash RP-18e, 25-4.6 mm column (Merck, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-water (20:90, v/v), pH 2.5 adjusted with 98% phosphoric acid, flow rate 0.48 ml min⁻¹, UV detection was at 213 nm.The validation parameters have shown good results: linearity of determination for both compounds including internal standard (ethylparaben) >0.999; repeatability of determination (R.S.D.) in the range 0.5-5.4% at three different concentration levels, detection limits in the range 0.5-2.0 μg ml⁻¹, and recovery from the pharmaceutical preparation in the range 99.3-99.9%. The chromatographic resolution between peak compounds was >5.0 and analysis time was <9 min under the optimal conditions. The method was found to be applicable for routine analysis of the active compounds ambroxol hydrochloride and doxycycline in various pharmaceutical preparations.     

The GC determination of cisplatin as platinum(II) from pharmaceutical preparations and blood sample of cancer patients

Summary A capillary gas chromatographic method is described for the determination of cisplatin based on the complexation of platinum(II) with bis(isovalerylacetone) ethylenediimine (H₂IVA₂en) and extraction in chloroform. The chromatography was carried out on a BP1 or a BP5 column with an FID. Copper(II), nickel (II) and palladium(II) separated completely and did not affect the determination of platinum(II). The method was applied of the determination of cisplatin in a pharmaceutical preparation and blood samples of cancer patients after infusion of cisplatin. The amounts of cisplatin in blood were found to be within 246–283 ng mL⁻¹ with a C.V. of 2.35–4.26%.     

Determination of fluoroquinolone antibacterials as N-acyl derivatives

Summary A simple and quantitative method for the preparation and high performance liquid chromatography (HPLC) of N-acylated fluoroquinolones has been developed. The acylation procedure was performed with four different acid anhydrides and found to be applicable to several fluoroquinolones. The acylated derivatives were chromatographed directly on a Nucleosil C₁₈ column without the necessity of further sample manipulation. Detector response for the N-acetyl derivatives of norfloxacin, ciprofloxacin, sarafloxacin, and temafloxacin was linear to at least 50 g/mL. The retention behavior of the N-acyl derivatives can be predicted by the length of the carbon chain of the amide.