Mobile Phase Of Hexane Research Articles

Simultaneous analysis of chlorzoxazone, diclofenac sodium and tramadol hydrochloride in presence of three potential impurities using validated HPLC-DAD and HPTLC methods

In recent years, a lot of single-pill combinations (SPC) are manufactured and are used as a promising choice in treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis. However, this trend made a serious challenge to drug analysts because of the difficulty in the analysis of two or more drugs in presence of each other. In this study, two chromatographic methods were developed for the simultaneous analysis of chlorzoxazone (CZ), diclofenac sodium (DIC) and tramadol hydrochloride (TRA) in presence of three of their related substances and potential impurities. Method I involved application of HPLC with diode array detection where Waters Symmetry C8 column was used as stationary phase. Mixture of ortho-phosphoric acid (0.03 M, pH 3) and acetonitrile was used as a mobile system with gradient elution and flow rate 1 mL/min. Peak areas were measured at the wavelengths 218 nm for TRA and 280 nm for CZ and DIC. Peaks eluted at retention times 2.30, 5.75 and 9.74 min for TRA, CZ and DIC respectively. Method II based on HPTLC separation. In this method, HPTLC silica gel 60 F254 plates were used with mobile phase of hexane: ethyl acetate: methanol: acetic acid (12: 6: 2: 0.1, by volume). Densitometry scanning was performed at 280 nm for all drugs. The retardation factor (Rf) values were 0.14, 0.40 and 0.58 for TRA, DIC and CZ respectively. Both methods were validated according to International Conference on Harmonization (ICH) Guidelines with respect to linearity, ranges, accuracy, precision, specificity, robustness and limits of detection and quantitation. Linearity ranges were 15–100 μg/mL for the three drugs in method I. In method II, the obtained data were linear in the ranges 300–1800 ng/spot for TRA and 25–150 ng/spot for DIC and CZ. Both methods showed good specificity by resolution of the three drugs from the related substances and potential impurities 2-amino-4-chlorophenol, 2.6-dichloroaniline and impurity A of DIC. Finally, both methods were successfully applied to the analysis of a real sample of combined tablets containing the three drugs. The suggested methods could be applied for the studied drugs in QC-lab.

Separation and quantification of olefins and diolefins in cracked petroleum fractions using silver-ion high performance liquid chromatography

A method using normal phase high performance liquid chromatography for the analysis of olefins and diolefins in cracked petroleum samples is proposed and validated in this work. A silver-impregnated silica column is used to separate olefin and diolefin species, resulting in accurate quantification of both group-types. Separations are completed using an isocratic hexane mobile phase, with no sample clean up or backflushing required. Results from the analysis of 11 model compounds are shown, as well as quantification data for four representative samples; figures of merit are also described.

Partial Validation of High Performance Liquid Chromatography for Analysis of Isoniazid in Rat Plasma

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Nicotinic acid derivatives such as isoniazid have the strongest anti-tuberculosis properties. For pharmacokinetics studying of isoniazid (INH), a method is needed to determine the levels of INH in plasma. Objective: The aim of this research is to partial validate of high performance liquid chromatography (HPLC) for analysis of INH in rat plasma. Method: For the preliminary study, rat plasma was used. The HPLC system used is a stationary phase C18 with length 250mm and temperature of 30°C, mobile phase hexane sulphonate acid 20mM pH 2.47–methanol (65:35). The analytical parameters in partial validated were linearity, lower limit of quantification (LLOQ), precision, accuracy, and recovery. Results: The results of linearity test of INH showed r value of 0.9996. LLOQ of this method was 0.1258μg/mL. The resulting accuracy and precision value met FDA requirements with a percent recovery ranging from 96.57–107.99%. Conclusion: The HPLC system was a valid method for analysis of INH in rat plasma.

DEVELOPMENT AND VALIDATION STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF VILDAGLIPTIN AND METFORMIN HYDROCHLORIDE IN THE PHARMACEUTICAL DOSAGE FORMS

Objective: A simple, precise, and accurate stability indicating high-performance thin layer chromatography method was developed and validated of vildagliptin (VIL) and metformin (MET) in pharmaceutical dosage forms.Methods: In the present study, system suitability test, stress study, alkali hydrolysis, acid hydrolysis, neutral hydrolysis, oxidative stress degradation, dry heat degradation, wet heat degradation, photodegradation study has been used. In this method, optimization by changing various parameters, such as organic solvent and the composition of the mobile phase, acid or base modifier used in the mobile phase; by varying one parameter and keeping all other conditions constant. 10 µl of the stock solution for MET (500 ng/band) and 2 µl of the stock solution for VIL (100 ng/band) were applied to TLC plates. The final solutions were applied on the HPTLC plates and these were developed as per the optimized densitometry conditions.Results: From the spectra, it was observed that MET and VIL exhibited good absorbance at about 217 nm. Both the drugs showed degradation with additional peaks at Rfvalues of 0.16 for MET and with Rfvalues 0.81 for VIL respectively. The method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, ruggedness, specificity, and robustness. Good separation was achieved by using the mobile phase Hexane: Methanol: Acetonitrile: Glacial Acetic Acid (2:3.5:2.5:0.2 v/v/v/v) with retardation factor (Rf) values of 0.22±0.01 for MET and 0.73±0.02 for VIL.Conclusion: A validated HPTLC method was developed for the determination of metformin hydrochloride and vildagliptin. The method is simple, quick, and can be applied routinely for the analysis of these drugs from marketed dosage forms.

Stability-Indicating TLC-Densitometric Assay for Methyltestosterone and Quantum Chemical Calculations.

Methyltestosterone is a synthetic testosterone derivative commonly used for the treatment of testosterone deficiency in males and one the anabolic steroids whose use is banned by World Anti-Doping Agency (WADA). This study presents a simple, cost-effective and rapid stability-indicating assay for densitometric quantification of methyltestosterone in pharmaceutical formulation. The developed method employed pre-coated TLC plates with mobile phase hexane:acetone (6.5:3.5 v/v). Limit of detection and limit of quantitation were found to be 2.06 and 6.24 ng/spot, respectively. Stress degradation study of methyltestosterone was conducted by applying various stress conditions such as hydrolysis under acidic, basic and neutral conditions, heating in anhydrous conditions and exposure to light. Methyltestosterone was found to be susceptible to photodegradation, acidic and basic hydrolysis. Degraded products were well resolved with significantly different Rf values. Acid degraded product was identified as 17,17-dimethyl-18-norandrosta-4,13(14)-dien-3-one through spectroscopic methods. The reactivity of methyltestosterone under applied stress conditions was also explained by quantum chemical calculations. The developed method is found to be repeatable, selective and accurate for quantification of methyltestosterone and can be employed for routine analysis.

Simultaneous Determination of Menthol and Eucalyptol by the Densitometric HPTLC Method in Some External Analgesic Formulations.

Several analgesic pharmaceutical formulations dispensed for external use contain menthol and eucalyptol. Simultaneous determination of the two monoterpenes was achieved utilizing high-performance thin layer chromatography (HPTLC) densitometric analysis. The stationary phase composed of silica gel 60 F254 (E-Merck, Germany) glass-backed plates coated with 0.2 mm layers. The used mobile phase hexane : ethyl acetate 8 : 2 (%, v/v) resulted in sharp, symmetrical and well-resolved peak at an Rf value of 0.34 ± 0.04 and 0.56 ± 0.04 for menthol and eucalyptol, respectively. Linearly range for menthol was 100-800 ng/spot (r(2) = 0.9979), whereas that for eucalyptol was 52.25-1107 ng/spot (r(2) = 0.9937). The developed method was validated following the International Conference on Harmonization guidelines. The method was found to be simple, accurate, time saving and enable analyses of many samples simultaneously.

Isolation of lupeol from white oak leaves and its anti-inflammatory activity

Lupeol [lup-20(29)-en-2-ol] is found mainly on the surface of plant barks, stems, leaves and fruits waxes. This research explored oaks leaves of several species (Quercus resinosa, Q. grisea, Q. laeta and Q. obtusata) as potential source of lupeol. It was extracted from Quercus leaves by maceration with CHCl3 at 35°C, followed by a purification in silica column (normal phase), and using as mobile phase hexane (100%), hexane:ethyl acetate (90:10) and hexane:ethyl acetate (80:20). Lupeol in oak leaves was identified by 13C NMR and quantified by GC–MS. Quercus obtusata leaves were an abundant source of lupeol (173.59μg/g of sample). Anti-cyclooxygenase activity has been used for determining bioactivity of lupeol in this research.

Retention behavior of isomeric triacylglycerols in silver‐ion HPLC: Effects of mobile phase composition and temperature

A systematic study of the retention behavior of isomeric triacylglycerols (TGs) in silver-ion HPLC on a ChromSpher Lipids column has been performed between 10 to 40°C using the most widespread hexane- and dichloromethane-based mobile phases. The randomization of mono-acyl TG standards and the random esterification of glycerol with fatty acids are employed to produce mixtures of TG isomers. The mobile phase composition has no influence on the general retention pattern, but significant differences in the retention order of double bond (DB) positional isomers in hexane and dichloromethane mobile phases are described and compared with the previous literature data. Saturated TGs with fatty acyl chain length from C7:0 to C22:0 are partially separated using the hexane mobile phase but not at all with the dichloromethane mobile phase. The hexane mobile phase enables at least partial resolution of TG regioisomers with up to seven DBs, while the resolution of only ALA/AAL and ALnA/AALn isomers is achieved with the dichloromethane mobile phase. The effect of temperature differs significantly depending on the mobile phase composition. Retention times of TGs increase with increasing temperature in the hexane mobile phase, while an opposite effect is observed for the dichloromethane mobile phase.

Hyphenated Flash Chromatographic Separation and Isolation of Coumarins and Polymethoxyflavones from Byproduct of Citrus Juice Processing Industry

The present study describes an efficient hyphenated flash chromatographic (FC) for the purification of coumarins and polymethoxy flavones (PMF's) from cold pressed grapefruit peel oil (GFO), a byproduct from citrus juice processing plant. GFO was subjected to silica column chromatography using sequential isocratic elution by hexane and chloroform to separate the waxes and phytochemicals enriched fractions, respectively. The chloroform extract was used for hyphenated FC separation using a gradient mobile phase of hexane and acetone. The peak separations were monitored at dual wavelengths of λ210 nm and λ340 nm. The individual fractions were analyzed by HPLC, pooled and concentrated resulting in isolation of two coumarins (auraptene and bergapten) and three PMF's (tangeretin, heptamethoxyflavone, and nobiletin). The purity and identity of the isolated compounds was confirmed by spectral analysis using high performance liquid chromatography, matrix-assisted laser desorption/Ionization-time of flight-mass spectrometry, and nuclear magnetic resonance. The hyphenated FC separation method was further validated by test of repeatability resulting in low RSD for the yield of the phytochemicals. Utilization of the developed separation method for isolation of value added products from grapefruit byproducts could be beneficial to the citrus processing industry. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.

Development of a rapid, high-throughput method for quantification of zeaxanthin in Chinese wolfberry using HPLC–DAD

A rapid, high-throughput method was developed to evaluate zeaxanthin levels in Chinese wolfberry fruit, based on the official AOAC (970.64) method. The sample preparation was optimized from the AOAC method with supporting material added to disperse the sample thoroughly. An HPLC method was adopted to replace the time-consuming column separation and spectrophotometric method used previously. Dried Chinese wolfberry fruit was extracted and saponified, simultaneously, in a volumetric flask with a four-solvent blend of hexane:ethanol:acetone:toluene (10:6:7:7); and 40% methanolic potassium hydroxide in order to convert the extracted zeaxanthin dipalmitate to free zeaxanthin in a single step. The flask was placed in a 56°C water bath and shaken at 200rpm for 20min. A normal-phase HPLC method was developed on a silica gel column with an isocratic mobile phase of hexane and ethyl acetate (70:30, v:v). Zeaxanthin was determined to be the dominant carotenoid in Chinese wolfberry fruit. This method was found to be rapid, sensitive and cost-effective for quantitating zeaxanthin in wolfberries compared to the column separation and the spectrophotometric methods traditionally used.Using the developed method, a total of 30 Chinese wolfberry samples from different growers/locations of China were investigated as potential sources of zeaxanthin. The fruit produced in the Ningxia Hui autonomous region had the highest level of zeaxanthin at greater than 1500mg/kg. The zeaxanthin content was not positively correlated to the size of fruit; suggesting that the low cost, smaller fruits may be an economical source for zexanthin extraction.

Chiral separation of cathinone derivatives used as recreational drugs by HPLC‐UV using a CHIRALPAK® AS‐H column as stationary phase

Cathinone derivatives gained high popularity on the recreational drugs market during the past 10 years. All these compounds are chiral, and the pharmacological potency of the enantiomers of these stimulants is supposed to differ. The goal of this research was to develop a reliable and easy-to-perform high-performance liquid chromatography ultraviolet method for the chiral separation of a set of 24 cathinone derivatives. A commercially available CHIRALPAK® AS-H column consisting of amylose tris [(S)-α-methylbenzylcarbamate] coated on 5-µm silica gel was found to be suitable to resolve a majority of the tested compounds. High-performance liquid chromatography measurements were performed in normal phase mode under isocratic conditions with a mobile phase consisting of hexane, isopropanol, and triethylamine at a flowrate of 1 ml/min. The ratio between hexane and isopropanol was optimized by means of three model substances. Under final conditions with a mobile phase of hexane, isopropanol, and triethylamine (97:3:0.1), 19 out of 24 compounds were successfully resolved into their enantiomers and detected at a wavelength of 254 nm. A correlation between the substituents of the nitrogen atom and the separation results are shown. Furthermore, enantiomer separation results of four cathinone derivatives were compared with the results of their amphetamine analogs.

Determination of the Content of Styrene-butadiene Rubber in Modified Asphalt Using High Performance Liquid Chromatography

A sensitive method was used to measure the content of styrene-butadiene rubber(SBR) in modified asphalt by HPLC.The temperature was set at 30 ℃ and a wavelength of 222 nm was used for the detector.A mobile phase of hexane,acetonitrile and THF with equal proportion was used.The RSD of this method is 1.5%,and a linear equation of y=800.94x+579.03 with a correlation coefficient of r=0.9998 was obtained.

High temperature normal phase liquid chromatography of aromatic hydrocarbons on bare zirconia

The normal phase HPLC behavior of a bare zirconia column was studied at temperatures up to 200 °C using a hexane mobile phase. The use of elevated column temperatures significantly decreased the retention of twenty five aromatic model compounds according to the van’t Hoff equation (>30-fold decrease for some compounds). Large improvements in peak shape, efficiency (>2.2-fold), aromatic group-type selectivity, and column re-equilibration times (>5-fold) were obtained at elevated temperatures. The thermal decomposition of two polar nitrogen compounds (indole and carbazole) was observed in a hexane/dichloromethane mobile phase at temperatures greater than 100 °C. The first order decomposition of carbazole was studied in further detail.

Characterization of warfarin unusual peak profiles on oligoproline chiral high performance liquid chromatography columns

Unusual peak profiles of warfarin were characterized on two oligoproline chiral stationary phases (CSPs). The pattern of 1st peak ( S(−)) broadening and the 2nd peak ( R(+)) compression was observed under mobile phase of hexane (0.1% TFA)/2-propanol (IPA) on a triproline CSP 1, and with other alcohol modifier such as ethanol, 1-propanol, 1-butanol, 2-butanol, and tert-butanol as well. Through analyzing system peak of additives, the unusual peak profile was interpreted by perturbation of TFA additive system peak. The unusual peak profile was also found in enantioseparation of coumachlor and on a covalently bonded doubly tethered diproline CSP 2. The pattern of 1st peak ( S(−)) broadening and the 2nd peak ( R(+)) compression can change to pattern of 1st peak compression and the 2nd peak broadening from 15 to 50 °C. Chiral separation of warfarin created nonlinear van’t Hoff plots on CSP. No peak broadening/compression were observed with methyl tertiary butyl ether or ethyl acetate as the modifier. The peak shapes of the two warfarin enantiomers can thus be tuned by varying alcohol concentration and column temperature. High separation factor and resolution may be carried out to tune the peak profiles into Langmuir/anti-Langmuir band-shape composition. Using none hydrogen donor modifier may avoid interference of the TFA system peak.

Enantioselective determination of cypermethrin in pig muscle tissue by immunoaffinity extraction and high performance liquid chromatography

SummaryA high performance liquid chromatography method was developed to detect enantiomers of cypermethrin in pork muscle. Eight isomers of racemic cypermethrin were separated using a normal mobile phase of hexane–isopropyl alcohol (99.3–0.7, v/v) on a chiral CD‐ph column in 25 min. The elution order of isomers was ascertained using enantiomer/diastereomer enriched cypermethrin standards. The extracts from pork samples were cleaned up by immunoaffinity column before detection. The recoveries from samples fortified levels of 0.1 and 1 mg kg−1 ranged from 67.3% to 113.5% with a relative standard deviation 6.7–16.8%; sufficient to effectively measure cypermethrin enantiomer concentrations in pig muscle tissue samples in the future.

Chemical composition of the fractions of leaf oil of Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. and antimicrobial activity

Leaf oil prepared by hydrodistillation of Alpinia zerumbet Pers.) B.L. Burtt & R.M. Sm. was analyzed by GC/FID and GC/MS to determine the major compounds and it was also evaluated for antimicrobial activity. The oil presented a high content of oxygenated monoterpenes (52.5%), terpinen-4-ol, 1,8 cineole and γ-terpinene as the major constituents. The antimicrobial activity of leaf oil was tested by drop diffusion and bioauthography methods. Through drop test, all bacteria and fungi tested were inhibited by leaf oil. Preparative TLC (thin-layer chromatography) plates were developed using the mobile phase hexane: ethyl acetate (70:20, v/v) and fractions of the leaf oil were separated in three zones, scraped, extracted from silica and identified by GC/MS. The bioauthography method permitted to verify pronounced inhibition of Cryptococcus neoformans by fractions F2 and F3, both rich in oxygenated monoterpenes. The fraction F2 comprised 1,8 cineole (9.6%), linalool (3.7%) and caryophyllene oxide (5.4%), while fraction F3 showed mainly terpinen-4-ol (43.6%) and an amount of 32.7% oxygenated sesquiterpenes. The obtained data may be used to suggest the constituents of A. zerumbet leaf oil involved in antimicrobial activity.

Development and Application of an Analytical Method for the Determination of Partition Coefficients of Tributyltin in the Forth and Clyde Canal, Glasgow, Scotland

Abstract Tributyltin (TBT) was widely used in the past as a biocide for boats. This has led to widespread environmental contamination. Most analytical methods are based on high resolution techniques with complicated sample preparation. For this study, a simple application of an analytical method was developed using HPLC/UV. The developed method was studied to determine TBT, dibutyltin, and monobutyltin in sediment and water samples. The separation was performed in isocratic mode on a cyanopropyl column with a mobile phase of hexane containing 5 % tetrahydrofuran and 0.03 % acetic acid. This method was confirmed using standard GC-MS techniques and verified by statistical paired t-test method. Under the experimental conditions used, the limit of detection of TBT and DBT were 0.70 and 0.50 ppm, respectively. An optimized extraction method for butyltins in water and sediment samples was developed by using hexane containing 0.05 %–0.5 % tropolone, and 0.2 % sodium chloride in water at pH1.7. The quantitative extraction of butyltin compounds in a certified reference material (BCR-646) and naturally contaminated samples was achieved with recoveries ranging from 95 % to 108 % and precision of 0.02 %–1.00 % relative standard deviation. This developed method was used to determine the contamination level of butyltins in environmental samples collected from the Forth and Clyde canal, Scotland, United Kingdom. The values obtained exceeded the Environmental Quality Standard values. Partition coefficients are major parameters influencing the behavior of pollutants, therefore the methodology developed was used to determine experimentally the partition coefficients Koc, Kd, and Kow. The comparison between values obtained experimentally and computational default model values (EPI Suite V 3.20) showed disparity. In terms of bioaccumulation, the implications of this finding may lead to greater biomagnification in the food chain.

Development of a low-cost method of analysis for the qualitative and quantitative analysis of butyltins in environmental samples

Most analytical methods for butyltins are based on high resolution techniques with complicated sample preparation. For this study, a simple application of an analytical method was developed using High Performance Liquid Chromatography (HPLC) with UV detection. The developed method was studied to determine tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT) in sediment and water samples. The separation was performed in isocratic mode on an ultra cyanopropyl column with a mobile phase of hexane containing 5% THF and 0.03% acetic acid. This method was confirmed using standard GC/MS techniques and verified by statistical paired t-test method. Under the experimental conditions used, the limit of detection (LOD) of TBT and DBT were 0.70 and 0.50 μg/mL, respectively. The optimised extraction method for butyltins in water and sediment samples involved using hexane containing 0.05–0.5% tropolone and 0.2% sodium chloride in water at pH 1.7. The quantitative extraction of butyltin compounds in a certified reference material (BCR-646) and naturally contaminated samples was achieved with recoveries ranging from 95 to 108% and at %RSD 0.02–1.00%. This HPLC method and optimum extraction conditions were used to determine the contamination level of butyltins in environmental samples collected from the Forth and Clyde canal, Scotland, UK. The values obtained severely exceeded the Environmental Quality Standard (EQS) values. Although high resolution methods are utilised extensively for this type of research, the developed method is cheaper in both terms of equipment and running costs, faster in analysis time and has comparable detection limits to the alternative methods. This is advantageous not just as a confirmatory technique but also to enable further research in this field.

TLC Determination of Betulinic Acid from Nymphodies macrospermum: A New Botanical Source for Tagara

The accepted botanical source of Tagara, an ayurvedic drug is Valeriana jatamansi Jones. In South India, a drug by the name of Granthika Tagara is used as Tagara in several therapeutic preparations. Currently, no analytical procedures appear to be available for quality control purposes. A sensitive, selective and precise thin-layer chromatographic method has been developed and validated for the analysis of betulinic acid in Nymphoides macrospermum. Separation and quantification was achieved by TLC using ternary mobile phase of hexane:ethyl acetate:acetic acid (7:3:0.03, v/v) (RF 0.60) on precoated silica gel 60F254 aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent in the reflection/absorption mode at 540 nm. The calibration curve was linear in the concentration range of 100–600 ng spot−1. The method was validated for precision, repeatability and accuracy. The proposed method was found to be simple, precise, specific, sensitive and accurate for the quantification of betulinic acid. This is the first TLC report for the identification and quantification of betulinic acid in N. macrospermum and may be useful for the routine quality control of Granthika Tagara.

Simultaneous determination and quantification of seven major phospholipid classes in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the application in diabetes nephropathy

A rapid and specific analytical method for simultaneous determination and quantification of seven major phospholipid classes in human blood was developed by normal-phase high-performance liquid chromatography tandem mass spectrometry. The optimal separation was achieved by using mobile phase hexane (A) and 2-propanol with water, formic acid and ammonia as modifiers (B) using an HPLC diol column. Isocratic elution method was used for better repeatability and no balance time. The seven major phospholipid classes in human blood that were detected including phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) phosphatidylcholine (PC), lysophosphatidylcholine (Lyso-PC), and sphingomyelin (SM). That can be separated in this condition. Every phospholipid class contains many molecular species which have similar structure. The structure of phospholipids molecular species was identified by ion-trap MS n which produced ion fragments. And the qualification was completed by TOF-MS which shows good accuracy. Through the accurate quantification of one representative phospholipids molecule in each class, a method for simultaneous estimation hundreds of molecular species in seven major classes was established. The intra-day and inter-day precision and recovery had been investigated in detail. The RSD of precision for most compound is below 8% and RE is below 10%. Recovery is almost over 80%. This method was applied to phospholipids disorder related with diabetes nephropathy successfully. The concentrations of most phospholipids for normal people are higher than that for diabetic nephropathy (DN) patients in three phases. For most of phospholipids, with the development of DN the concentration was decreasing.