Preconcentration of poorly volatile impurities in thin-layer chromatography by thermodesorption of a volatile matrix

A method for preconcentration of poorly volatile impurities by continuous thermodesorption of the major component on a TLC plate is proposed. The method provides multiple (200–500) concentration of the organic impurities present in the initial solution. A simple device for such concentration is suggested. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 2075–2076, October, 1998.     

Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.     

Selective spectrophotometric determination of phenolic β-lactam antibiotics in pure forms and in their pharmaceutical formulations

Four simple and selective spectrophotometric methods were developed for the quantitative determination of some phenolic β-lactam antibiotics (amoxicillin trihydrate, cefoperazone sodium, cefadroxil monohydrate, and cefprozil anhydrous) in pure forms as well as in their pharmaceutical formulations through their nitration and subsequent complexation with an nucleophilic reagent (method I), nitrosation and subsequent metal chelation (method II), coupling with diazo reagent (method III), and reaction with copper and extraction of the resulting chelate into chloroform (method IV). The reaction conditions were studied and optimized. Beer’s plots were obeyed in a general concentration range of 5-30 ug ml⁻¹ with correlation coefficients not less than 0.9997 for the four drugs. The methods are successfully applied to the analysis of pharmaceutical formulations containing amoxicillin, either alone or in combination with potassium clavulanate. They were also applied to the analysis of the other three studied drugs in vials, capsules, tablets, and suspensions with good recovery; percentage ranged from 99.0 (±1.42) to 100.2 (±1.25) in method I, 99.0 (±0.82) to 100.5 (±0.92) in method II, 99.5 (±0.09) to 100.8 (±0.98) in method III, and 99.3 (±0.01) to 100.2 (±0.05) in method IV. Interferences from other antibiotics and additives were investigated.     

Solubility of Solid 2,3-Dimethylbutane and Cyclopentane in Liquid Argon and Nitrogen at the Standard Boiling Points of the Solvents

The solubilities of solid 2,3-dimethylbutane and cyclopentene in liquid argon at a temperature of 87.3 K and in liquid nitrogen at 77.4 K have been measured by the filtration method. The hydrocarbon contents in solutions were determined using gas chromatography. GC–MS was used to identify impurities in solutes. The experimental value of the mole fraction solubility of solid 2,3-dimethyl-butane in liquid argon at 87.3 K is (8.26 ± 1.60) × 10^–6 and (2.77 ± 0.94) × 10^–8 in liquid nitrogen at 77.4 K. The experimental value of the mole fraction solubility of solid cyclopentene in liquid argon at 87.3 K is (5.11 ± 0.44) × 10^–6 and (4.60 ± 0.76) × 10^–8 in liquid nitrogen at 77.4 K. The Preston–Prausnitz method was used for calculation of the solubilities of solid hydrocarbons in liquid argon in the temperature range 84.0–110.0 K and in liquid nitrogen from 64.0 to 90.0 K. The solvent–solute interaction parameters l ₁₂ were also calculated. At 90.0 K liquid argon is a better solvent for investigated solid hydrocarbons than is liquid nitrogen.     

Production of radiochemically and radionuclidically pure153Sm for medical applications

A procedure is described for the large scale production of radiochemically and radionuclidically pure¹⁵³Sm economically, from neutron irradiated natural Sm targets. Cation-exchange chromatography using 200–400 mesh Dowex-50×8 resin in the elution mode was used with 0.2M -hydroxyisobutyric acid (-HIBA) as the eluent at pH 4.7±1 and at 26±1 °C for this purpose. Typical decontaminations of carrier-free^(155+156)Eu radioactive impurities, of the order of 5×10², could be achieved in individual fractions, from millicurie amounts of¹⁵³Sm activity produced in milligram quantities of heavily neutron-irradiated natural Sm targets. Even though the¹⁵³Sm yields were at about 3% at these decontamination levels of impurities, the yields increased to an average of about 50% at about 95% level of decontamination of these impurities. It has been shown that the method is successful for producing radiochemically and radionuclidically pure¹⁵³Sm in sufficiently high radioactive concentrations for its probable conversion into¹⁵³SmEDTMP complex, a potential radiopharmaceutical useful in the palliative therapy of metastatic bone cancer.     

Solubility of Solid 1-Hexyne in Liquid Argon and Nitrogen at the Standard Boiling Points of the Solvents

The solubilities of solid 1-hexyne in liquid argon at 87.3 and in liquid nitrogen at 77.4 K have been measured by the filtration method. The hydrocarbon contents in solutions were determined using gas chromatography. GC–MS was used to identify impurities in 1-hexyne. The experimental value of the mole fraction solubility of solid 1-hexyne in liquid argon at 87.3 K is (0.85 ± 0.19) × 10^–7 and (1.25 ± 0.08) × 10^–8 in liquid nitrogen at 77.4 K. The Preston–Prausnitz method was used for calculation of the solubilities of solid hydrocarbon in liquid argon in the temperature range 84.0–110.0 K and in liquid nitrogen from 64.0 to 90.0 K. The solvent–solute interaction parameters l ₁₂ were also calculated. At 90.0 K liquid argon is a better solvent for solid 1-hexyne than is liquid nitrogen.     

A gravimetric and an X-ray fluorescence method for the determination of rubidium in Rb2U(SO4)3

Chemical characterization of rubidium uranium(IV) trisulfate, Rb₂U(SO₄)₃, a new chemical assay standard for uranium requires accurate analysis of rubidium. A gravimetric and an X-ray fluorescence method (XRF) for the determination of rubidium in this compound are described. In the gravimetric method, rubidium is determined as Rb₂Na[Co(NO₂)₆].H₂O without separating uranium with a precision of the order of ±0.5%. In the XRF method, the concentration ratio of rubidium to uranium, C_Rb/C_U, is determined in the solid samples by the binary ratio method using calibration between intensity ratios (I_Rb/I_U) and concentration ratios (C_Rb/C_U). The concentration of rubidium is derived using the uranium value which is known with a precision better than ±0.05%. The XRF method has a precision better than ±0.8% for rubidium determination.     

Solubility of Solid 2-Methyl-1,3-butadiene (Isoprene) in Liquid Argon and Nitrogen at the Standard Boiling Points of the Solvents

The solubility of solid 2-methyl-1,3-butadiene (isoprene) in liquid argon at a temperature of 87.3 K and in liquid nitrogen at 77.4 K has been measured by the filtration method. The hydrocarbon contents in solutions were determined using gas chromatography. GC–MS was used to identify impurities in the solute. The experimental value of the mole fraction solubility of solid isoprene in liquid argon at 87.3 K is (1.41 ± 0.27) × 10^–6 and (1.56 ± 0.36) × 10^–7 in liquid nitrogen at 77.4 K. The Preston–Prausnitz method was used for calculation of the solubilities of solid hydrocarbon in liquid argon in the temperature range 84.0–110.0 K and in liquid nitrogen from 64.0 to 90.0 K. The solvent–solute interaction parameters l ₁₂ were also calculated. At 90.0 K liquid argon is a better solvent for isoprene than is liquid nitrogen. The experimental values of the solubilities of isoprene in liquid argon and nitrogen were compared with results obtained for selected unsaturated and aromatic hydrocarbons.     

Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).     

Selective ion exchange separation of uranium from concomitant impurities in uranium materials and subsequent determination of the impurities by ICP-OES

An ion exchange method has been developed for the separation of uranium from trace level metallic impurities prior to their determination by inductively coupled plasma optical emission spectrometry (ICP-OES) in uranium materials. Selective separation of uranium from trace level metallic impurities consisting Cr, Co, Cu, Fe, Mn, Cd, Gd, Dy, Ni, and Ca was achieved on anion exchange resin Dowex 1 × 8 in sulphate medium. The resin (100–200 mesh, in chloride form) was packed in a small Teflon column (7.8 cm × 0.8 cm I.D.) and brought into sulphate form by passing 0.2 N ammonium sulphate solution. Optimum experimental conditions including pH and concentration of sulphate in the liquid phase were investigated for the effective uptake of uranium by the column. Uranium was selectively retained on the column as anionic complex with sulphate, while impurities were passed through the column. Post column solution was collected and analyzed by ICP-OES for the determination of metallic impurities. Up to 2,500 μg/mL of uranium was retained with >99% efficiency after passing 25 mL sample through the column at pH 3. Percentage recoveries obtained for most of the metallic impurities were >95% with relative standard deviations <5%. The method established was applied for the determination of gadolinium in urania–gadolinia (UO₂–Gd₂O₃) ceramic nuclear fuel and excellent results were achieved. Solvent extraction method using tributylphosphate (TBP) as extractant was also applied for the separation of uranium in urania–gadolinia nuclear fuel samples prior to the determination of gadolinium by ICP-OES. The results obtained with the present method were found very comparable with those of the solvent extraction method.     

Development and validation of an HPLC method for the determination of process-related impurities in pridinol mesylate, employing experimental designs

A simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound pridinol mesylate, has been developed and validated. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C₁₈ column with a mobile phase containing 50 mM potassium phosphate buffer (pH 6.4), MeOH and 2-propanol (20:69:11, v/v/v), delivered at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 245 nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r² > 0.99) over the range 0.05-2.5% (5-250% with regards to the 1% specification limit for both process-related impurities); it is also precise, regarding repeatability (RSD ≤ 1.5% for all of the analytes) and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1-102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.     

Composition of structural carbohydrates in biomass: precision of a liquid chromatography method using a neutral detergent extraction and a charged aerosol detector

We adapted and optimized a method to quantify the cellulose, hemicellulose, xylan, arabinan, mannan, galactan contents in lignocellulosic biomass. This method is based on a neutral detergent extraction (NDE) of the interfering biomass components, followed by a sulfuric acid hydrolysis (SAH) of the structural polysaccharides, and a liquid chromatography with charged aerosol detection (LC-CAD) to analyze the released monosaccharides. The first step of this NDE-SAH-LC-CAD method aims at removing all compounds that interfere with the subsequent sulphuric acid hydrolysis or with the subsequent chromatographic quantification of the cellulosic and hemicellulosic monosaccharides. This step includes starch hydrolysis with an analytical thermostable α-amylase followed by an extraction of soluble compounds by a Van Soest neutral detergent solution (NDE). The aim of this paper was to assess the precision of this method when choosing fiber sorghum (Sorghum bicolor (L.) Moench), tall fescue (Festuca arundinacea Schreb.) and fiber hemp (Cannabis sativa L.) as representative lignocellulosic biomass. The cellulose content of fiber sorghum, tall fescue and fiber hemp determined by the NDE-SAH-LC-CAD method were 28.7 ± 1.0, 29.7 ± 1.0 and 43.6 ± 1.2 g/100 g dry matter, respectively, and their hemicellulose content were 18.6 ± 0.5, 16.5 ± 0.5 and 14.5 ± 0.2 g/100 g dry matter, respectively. Cellulose, mannan and galactan contents were higher in fiber hemp (dicotyledon) as compared to tall fescue and fiber sorghum (monocotyledons). The xylan, arabinan and total hemicellulose contents were higher in tall fescue and fiber sorghum as compared to fiber hemp. The precision of the NDE-SAH-LC-CAD method was better for polysaccharide concentration levels above 1 g/100 g dry matter. Galactan analysis offered a lower precision, due to a lower CAD response intensity to galactose as compared to the other monosaccharides. The dispersions of the results (expanded uncertainty) of the NDE-SAH-LC-CAD method were smaller as compared to the Van Soest (VS) method. In addition, the NDE-SAH-LC-CAD method was able to provide additional information on the composition of the hemicellulose (xylan, arabinan, mannan and galactan content) that is not provided by the Van Soest method. The NDE-SAH-LC-CAD method offers also the advantage of a better specificity for hemicellulose and cellulose, as compared to the NREL and Uppsala methods.     

Simultaneous square-wave voltammetric determination of riboflavin and folic acid in pharmaceutical preparations

The square-wave voltammetric (SWV) behaviour of riboflavin and folic acid was studied at a static mercury drop electrode by square wave voltammetry. In 0.05M KCl (pH 5.89) a cathodic scan gave peaks at — 0.56 and — 0.87 V vs. Ag/AgCl for riboflavin and folic acid, respectively. The reduction peak currents are linearly dependent on the concentration of vitamins. Both vitamins can be simultaneously determined from the same voltammogram. The method proposed for the determination of riboflavin and folic acid in multivitamin tablets is very simple, rapid and does not involve time-consuming separation steps. The average contents of riboflavin and folic acid were found to be 14.8 ± 1.26% and 1.46 ± 2.66%, for tablet A and 9.86 ±1.40% and 1.47 ± 2.0% for tablet B, respectively.     

A novel foam based separation strategy for extracting minute target impurities

The purpose of this study was to extract impurities from compounds using a simple separatory bottle to purify target compounds with a foam column and allow for the further characterization of impurities. Charged dyes were used as target compounds due to the ease of detection of dyes and isolated impurities. Foaming agents were used in a glass bottle with a modified cap to separate a target impurity using an appropriately charged ligand. By passing N₂ gas through the solution, the surfactants sodium dodecyl sulfate and cetylpyridinium chloride generated foams that separated the dyes, Methylene blue and Orange G, respectively, from a solution containing both dyes. Sodium dodecyl sulfate condensed Methylene blue from the solution with high purity while cetylpyridinium chloride condensed Orange G with less purity. A range of concentrations (0.01–0.5 mmol/L) of dyes were used for separation. The condensability (volume and/or concentration) of the target compound increased as its concentration decreased. This novel separation method is a simple, rapid, inexpensive, and effective way to prepare samples and allows for the characterization of these impurities using sensitive analytical detection techniques.     

Determination of traces of boron and cadmium in manganese sulphate solutions

Summary A well established method to calibrate ²⁵²Cf neutron sources makes use of a manganese bath technique based on the measurement of the induced ⁵⁶Mn-,-radioactivity. The knowledge of the impurities present in the bath is required because they may affect the results of the source strength measurements by their neutron absorption properties. Some specific impurities which may introduce a significant correction must be quantified and selective methods have to be used for their determination. The determination of two of these specific impurities, having a high capture cross section for neutrons, the boron and the cadmium, is described; it is based on spectrophotometry with curcumin (for B) and on pulse polarography (for Cd). Analytical results obtained on baths prepared in different laboratories are given. Errors are ±4–7% for B and ±3% for Cd.

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Bestimmung von Bor- und Cadmiumspuren in Mangansulfatlösungen

Zusammenfassung Eine übliche Methode zur Eichung von ²⁵²Cf-Neutronenquellen basiert auf der Manganbad-Technik, wobei die induzierte ⁵⁶Mn-,-Aktivität gemessen wird. Die Ergebnisse können durch Verunreinigungen des Bades beeinflußt werden. Methoden zu deren selektiver Erfassung sind daher notwendig. Die Bestimmung von Bor und Cadmium, die einen besonders hohen Einfangquerschnitt für Neutronen haben, wird beschrieben. Sie beruht auf der spektralphotometrischen Bestimmung mit Curcumin (für B) bzw. auf der pulspolarographischen Bestimmung (für Cd). Analysenwerte für verschiedene MnSO₄-Bäder werden verglichen. Die Fehler liegen im Bereich von ±4–7% (für B) und ±3% (für Cd).

     

Sampling 4-chlorophenol in water by DGT technique with molecularly imprinted polymer as binding agent and nylon membrane as diffusive layer

For the first time, a diffusive gradients in thin films (DGT) device using molecularly imprinted polymer (MIP) as the binding agent and nylon membrane (NM) as the diffusive layer (NM-MIP-DGT) has been developed for sampling 4-chlorophenol (4-CP) in water. The MIP was prepared by precipitation polymerization with methacrylic acid as monomer and ethyleneglycoldimethacrylate as cross-linker. The diffusion coefficient of 4-CP through NM was obtained to be 0.788 ± 0.040 μ cm² s⁻¹ by diffusion cell method. The ratio was 1.01 ± 0.05 (mean ± standard deviation) for the concentration of 4-CP sampled by NM-MIP-DGT and analyzed by HPLC method to the total concentration of 4-CP in the synthetic solution where free 4-CP species dominated. The results showed that NM-MIP-DGT could sample 4-CP in synthetic solution accurately. The performance of NM-MIP-DGT for sampling 4-CP was independent of pH in the range of 3–7 and ionic strength in the range of 0.0001–0.1 mol L⁻¹ NaCl solution. The concentration of free form of 4-CP sampled by NM-MIP-DGT decreased with the increasing concentration of dissolved organic carbon in different water samples due to the electrostatic interaction of natural organic compounds with 4-CP. 1.8 mg L⁻¹ of the free form of 4-CP was determined by HPLC which was sampled by NM-MIP-DGT in an intermediate untreated industrial effluent. The NM-MIP-DGT can be a potential passive tool for sampling the free form of 4-CP in water.     

Solid phase extraction of copper (II) by sorption on octadecyl silica membrane disk modified with a new Schiff base and determination with atomic absorption spectrometry

A simple, selective and reliable method for rapid extraction and determination of trace amounts of Cu (II) ions from aqueous samples using octadecyl-bonded silica membrane disks modified with bis-(3-methoxy salicylaldehyde)-1,6-diaminohexane and flame atomic absorption spectrometry (FAAS) is presented. Extraction efficiency, the influence of pH, flow rates, amount of ligand, and type and least amount of eluant were investigated. The linear dynamic range of the proposed method for Cu (II) ions was found in a wide concentration range of 1.0 (± 0.2)–150 (± 2) μg l^− 1. The detection limit and preconcentration factor of this method were found 30.0 (± 0.7) ng l^− 1 and 100 respectively. The reproducibility of the procedure is at the most 2.0%. The effects of various cationic interferences on the percent recovery of copper ion were studied. The method was used to the recovery of copper ion from different synthetic, alloys and biological samples.     

Species selective preconcentration and quantification of gold nanoparticles using cloud point extraction and electrothermal atomic absorption spectrometry

The determination of metallic nanoparticles in environmental samples requires sample pretreatment that ideally combines pre-concentration and species selectivity. With cloud point extraction (CPE) using the surfactant Triton X-114 we present a simple and cost effective separation technique that meets both criteria. Effective separation of ionic gold species and Au nanoparticles (Au-NPs) is achieved by using sodium thiosulphate as a complexing agent. The extraction efficiency for Au-NP ranged from 1.01 ± 0.06 (particle size 2 nm) to 0.52 ± 0.16 (particle size 150 nm). An enrichment factor of 80 and a low limit of detection of 5 ng L⁻¹ is achieved using electrothermal atomic absorption spectrometry (ET-AAS) for quantification. TEM measurements showed that the particle size is not affected by the CPE process. Natural organic matter (NOM) is tolerated up to a concentration of 10 mg L⁻¹. The precision of the method expressed as the standard deviation of 12 replicates at an Au-NP concentration of 100 ng L⁻¹ is 9.5%. A relation between particle concentration and the extraction efficiency was not observed. Spiking experiments showed a recovery higher than 91% for environmental water samples.     

Spectrophotometric and spectrodensitometric determination of Clopidogrel Bisulfate with kinetic study of its alkaline degradation

Four sensitive, selective and precise stability-indicating methods for the determination of Clopidogrel Bisulfate (CLP) in presence of its alkaline degradate and in pharmaceutical formulations were developed and validated. Method A is a second derivative (D²) spectrophotometric one, which allows the determination of CLP in presence of its alkaline degradate at 219.6, 270.6, 274.2 and 278.4 nm (corresponding to zero-crossing of the degradate) over a concentration range of 4-37 μg mL⁻¹ with mean percentage recoveries 99.81 ± 0.893, 99.72 ± 0.668, 99.88 ± 0.526 and 100.46 ± 0.646, respectively. CLP can be determined in the presence of up to 65% of its degradate. D² method was used to study the kinetic of CLP alkaline degradation that was found to follow a first-order reaction. The t_1/2 was 6.42 h while K (reaction rate constant) was 0.1080 mol/h. Method B is the first derivative of the ratio spectra (DD¹) spectrophotometric method, by measuring the peak amplitude at 217.6 and 229.4 nm using acetonitrile and CLP can be determined in the presence of up to 70% of its degradate. The linearity range was 5-38 μg mL⁻¹ with mean percentage recoveries 99.88 ± 0.909 and 99.70 ± 0.952, respectively. Method C based on the determination of CLP by the bivariate calibration depending on simple mathematic algorithm which provides simplicity and rapidity. The method depends on quantitative evaluation of the absorbance at 210 and 225 nm over a concentration range 5-38 μg mL⁻¹ with mean percentage recovery 99.27 ± 1.115. CLP can be determined in the presence of up to 70% of its degradate. Method D is a TLC-densitometric one, where CLP was separated from its degradate on silica gel plates using hexane:methanol:ethyl acetate (8.7:1:0.3, v/v/v) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of CLP at 248 nm over a concentration range of 0.6-3 μg/band with mean percentage recovery 99.97 ± 1.161. CLP can be determined in the presence of up to 90% of its alkaline degradate. The selectivity of the proposed methods was checked using laboratory prepared mixtures. The proposed methods have been successfully applied to the analysis of CLP in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with the official method.     

Stability indicating high-performance thin-layer chromatographic determination of nelfinavir mesylate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method of analysis of nelfinavir mesylate both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-methanol-acetone (7:1.5:1.5, v/v/v). This system was found to give compact spots for nelfinavir mesylate (R_f value of 0.45±0.02). Nelfinavir mesylate was subjected to acid and alkali hydrolysis, oxidation, dry heat treatment and photodegradation. Also the peaks of degraded products were well resolved from the pure drug with significantly different R_f values. Densitometric analysis of nelfinavir mesylate was carried out in the absorbance mode at 250 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r²=0.999±0.002 in the concentration range of 1000-6000 ng per spot. The mean value of correlation coefficient, slope and intercept were 0.999±0.002, 0.014±0.001 and 21.73±1.26, respectively. The method was validated for precision, robustness and recovery. The limits of detection and quantitation were 60 and 140 ng per spot, respectively. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.