Methanol-water Mobile Phase Research Articles

Determination of chromatographic dissociation constants of some carbapenem group antibiotics and quantification of these compounds in human urine

The dissociation constant values (s (s) pKa ) of some carbapenem group drugs (ertapenem, meropenem, doripenem) in different percentages of methanol-water binary mixtures (18, 20 and 22%, v/v) were determined from the mobile phase pH dependence of their retention factor. Evaluation of these data was performed using the NLREG program. From calculated pKa values, the aqueous pKa values of these subtances were calculated by different approaches. Moreover, the correlation established between retention factor and the pH of the water-methanol mobile phase was used to determine the optimum separation conditions. In order to validate the optimized conditions, these drugs were studied in human urine. The chromatographic separation was realized using a Gemini NX C18 column (250 × 4.6 mm i.d., 5 µm particles) and UV detector set at 220 and 295 nm.

Determination of diacetyl in liquors by high performance liquid chromatography coupled with precolumn derivatization using 3,3'-diaminobenzidine

A method was developed for the determination of diacetyl in liquors by high performance liquid chromatography-ultraviolet (HPLC-UV) coupled with precolumn derivatization using derivatization reagent 3,3'-diaminobenzidine (DAB). Diacetyl reacted with DAB at room temperature for 10 min. Then the separation was carried out on a high performance liquid chromatograph equipped with a diode array detector (DAD). A Shim-pack VP-ODS (250 mm×4.6 mm, 4.6 μ m) column was employed using water-methanol mobile phases for gradient at a flow rate of 0.7 mL/min and detection wavelength of 254 nm. The method showed a good linearity at diacetyl concentrations from 0.20 μ mol/L to 180 μ mol/L with the correlation coefficient (R2) of 0.999. The limit of detection (S/N=3) and limit of quantification (S/N=10) were 0.09 μ mol/L and 0.20 μ mol/L, respectively. The intra-day precision (RSD) was 1.28% (n=6). The method was further evaluated in the analysis of liquor samples with the recoveries ranging from 92.0% to 103.6% and RSDs from 0.69% to 3.45% (n=3). The method is well suitable for the measurement of diacetyl in liquor samples.

High-Performance Liquid Chromatographic Determination of Memantine in Human Urine Following Solid-Phase Extraction and Precolumn Derivatization

A validated HPLC-UV method is presented for the quantification of urinary memantine hydrochloride, a novel medication approved to treat moderate and advanced cases of Alzheimer's disease. The drug and amantadine hydrochloride, the internal standard, were extracted from human urine using SPE. The extract was then buffered and derivatized at room temperature using o-phthalaldehyde in the presence of N-acetyl-L-cyteine. Chromatographic separation of the formed derivatives was achieved on a C18 column using methanol-water mobile phase adjusted to pH 7 and pumped isocratically at 1 mL/min. The UV detector was set at 340 nm. The chromatographic run time did not exceed 10 min. The LOD and LOQ were 8 and 20 ng/mL, respectively. The RSDs for intraday and interday precisions did not exceed 5.5%. The method was used to monitor memantine hydrochloride in human urine in order to determine an appropriate sampling interval for future noninvasive therapeutic drug monitoring. The assay could also be applied to the determination of amantadine. The described assay showed that a postdosing time interval of 25-75 h seems adequate for sampling and monitoring memantine in urine.

Determination of Methoxamine in Human Plasma and Urine by a Validated HPLC-Fluorescence Detection Method

A sensitive HPLC method for the determination of methoxamine in human plasma and urine is described. The method is based on derivatization of methoxamine with 4-chloro-7-nitrobenzofurazan in borate buffer of pH 9.5 to yield a yellow, fluorescent product. Isocratic HPLC separation was achieved on an Inertsil C18 column (250 x4.6 mm id, 5 microm particle size) using the mobile phase methanol-water (60+40, v/v) at a flow rate of 1.2 mL/min. Fluorescence detection was used at excitation and emission wavelengths of 458 and 521 nm, respectively. The assay was linear over the concentration ranges of 10-250 and 20-300 ng/mL for plasma and urine, respectively. The LOD values were 3.3 and 6.8 ng/mL and the LOQ values were 10 and 20 ng/mL for plasma and urine, respectively. The extraction recoveries were more than 97.10%. After strict validation, the method indicated good performance in terms of linearity, sensitivity, precision, accuracy (recovery), robustness, and system suitability, and it was successfully applied to the determination of methoxamine in human plasma and urine.

Stability-Indicating HPLC-UV for the Determination of 4-bromomethyl-3-nitrobenzoic acid, a Bioactive Nitrocompound

The nitroaromatic compound 4-bromomethyl-3-nitrobenzoic acid (ANB) is a promising antitumoral agent whose activity has recently been investigated. Forced degradation studies were conducted on ANB with a high-performance liquid chromatography-ultraviolet assay to establish its stability and selectivity. ANB was subjected to degradation studies under hydrolytic (acid and alkaline), oxidative and light exposition conditions. The compound showed greater lability only in acid and alkaline conditions by forming a major degradation product. The chromatographic separation of ANB and its degradation product was achieved on an octadecylsilane column using a mobile phase of methanol-water (80:20, v/v), pH 4.0 adjusted with formic acid, flow rate of 0.7 mL/min, ultraviolet diode array detection at λ 271 nm, injection volume of 20 µL and temperature of 30°C. The method was validated according to International Conference on Harmonization guidelines with respect to precision (average relative standard deviation 0.67%), accuracy (average 99.97%), linearity (y = 118730x + 12912; coefficient of determination > 0.999), specificity and robustness (p > 0.05). The degradation product formed as a result from the hydrolysis of nitrobenzyl bromide, corresponded to its benzyl alcohol, 4-hydroxymethyl-3-nitrobenzoic acid (ANOH) and was characterized by co-elution with a synthetic ANOH working standard.

Determination of Raddeanin A in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study

A simple, rapid and sensitive LC-MS/MS analysis method was developed and validated for the determination of Raddeanin A (RA) in rat plasma. Protein precipitation with three volumes of methanol as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Thermo Syncronis C18 column with the mobile phase of methanol-water (containing 5mM ammonium formate, pH 2.2) (85:15, v/v). RA and glycyrrhetinic acid (internal standard) were monitored under negative electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of RA and IS were 2.1 min and 3.5 min, respectively. The limit of detection was 5 ng/mL and the linear range was 50-50,000 ng/mL. The intra-day and inter-day precision was 1.87-2.94% and 3.25-5.36%, and the intra-day and inter-day accuracy ranged from 5.9% to 10.5% and 5.6% to 11.1%, respectively. The absolute recovery was above 90.3%. The method has been successfully translated to the pharmacokinetic study of RA in rats after intravenous and intraperitoneal administration (0.75 mg/kg).

An LC-MS/MS Method for Determination of Imperatorin and Isoimperatorin in Rat Plasma and Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the de-termination of imperatorin and isoimperatorin in rat plasma using psoralen as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column eluted with a mobile phase of methanol-water (25:75), containing 0.1% formic acid at a flow rate of 0.4 ml/min. The detection was performed by positive ion elec-trospray ionization in multiple reactions monitoring mode, monitoring the transitions m/z 203→147 and m/z 203→159 for imperatorin and isoimperatorin, respectively. The method was validated over the concentration range of 2.5 -1000.0 ng/mL for imperatorin and isoimperatorin. The limits of detection and quantification were 1.0 and 3.0 ng/mL, repectively for both analytes. The intra-day and inter-day accuracy and precision of the assay were less than 10.9%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of extract of Angelicae Dahuricae Radix to rats.

Evaluation of ACE inhibitors lipophilicity using in silico and chromatographically obtained hydrophobicity parameters

The aim of this study was to compare different calculation methods to determine lipophilicity, expressed as logP value, of seven ACE inhibitors (enalapril, quinapril, fosinopril, lisinopril, cilazapril, ramipril, and benazapril) with significantly different structure. Experimentally determined n-octanol/water partition coefficients, logPO/W values, were obtained from relevant literature. The correlations between all collected logP values were studied and the best agreements between calculated logP and experimentally determined logPO/W values, were observed for KOWWINlogP or MilogP values (r = 0.999 or r = 0.974, respectively). The correlations between all collected logP values and chromatographically (reversed-phase thin-layer chromatography) obtained hydrophobicity parameters, RM0 and C0, were established. The good correlations (r > 0.90) were obtained in the majority of relationships. The KOWWINlogP was established as the most suitable hydrophobicity parameter of investigated group of ACE inhibitors with r = 0.981 for correlation with RM0 and r = 0.977 for correlation with C0 parameters (water-methanol mobile phase). Using multiple linear regressions, it was established that application of two selected logP, calculated by different mathematical approaches, led to very good correlation due to the benefits of both calculation methods. The good relationships indicate that the computed logP, with careful selection of method calculation, can be useful in ACE inhibitors lipophilicity evaluation, as high-throughput screening technique.

Optimization Strategy for Isocratic Separation of Halogenated Phenols by Reversed Phase Liquid Chromatography with Methanol-Water Mobile Phase

Optimization Strategy for Isocratic Separation of Halogenated Phenols by Reversed Phase Liquid Chromatography with Methanol-Water Mobile Phase

Quantum mechanics study on a series of steroids relating separation with structure

A small series of steroid hormones and synthetic steroid anabolics, eluted from a sample by using classical methanol-water mobile phase, was designed at molecular level with in vitro geometrical modeling. Structure-based properties of the compounds were extracted for different strategies of geometry optimization and were related with the percent retardation factors observed in the experiment by using classical linear regression approach. The obtained models were assessed and the most accurate model was selected. The obtained relationship between the structure and retardation factor was interpreted in order to express the main factors driving liquid phase mobility of compounds as a function of their structure. The analysis allowed a series of general conclusions regarding the modeled property (percent of retardation factor), modeling strategy (with help of a family of molecular descriptors) as well as about the level of theory in geometrical modeling (Ab-initio 6-31G*, CLHF, core Hamiltonian guess, symmetr...

Quantitative Determination of Arbutin and Hydroquinone in Different Plant Materials by HPLC

A simple, fast method of high-performance liquid chromatography for the determination and quantification of arbutin and hydroquinone in many different raw materials was developed and validated. The optimum conditions for the separation and detection of these two constituents were achieved on a LiChro-CARD 125-4 Superspher®100 RP-18 column with the water-methanol (gradient elution) mobile phase and recorded at 289 nm. The purities of peaks were verified by PDA analysis of impurities. The results of validation have shown that the HPLC method is stable and accurate for the simultaneous determination of arbutin and hydroquinone in extracts from various plants. The developed method gave a good sensitivity (LOD 1µg/ml for arbutin and 0.49 µg/ml for hydroquinone) with linearity R2 >0.9993 (for both). The relative standard deviation of the method was less than 2.53% for intra-day assays and 3.23% for inter-day assay, the accuracy of the recovery test ranged from 98.96% to 106.4%. This method was used in comparative qualitative analysis of arbutin and hydroquinone in 16 different raw materials from families Lamiaceae, Ericacaeae, Saxifragaceae, Rosaceae. The content of arbutin in B. ciliata, B. cordifolia and Ledum palustre was examined for the first time.

Determination of β -Sitosterol with Chemical Course and Material Applications in Jatropha Seed Oil by High Performance Liquid Chromatography

A simple and rapid high performance liquid chromatography (HPLC) assay was developed to identify and measure theβ-sitosterol with chemical course and material applications in jatropha seed oil. The stigmasterol was isolated with a good selectivity by HPLC employing reversed phase C18 columns. The components were separated by mobile phase of methanol-water (99/1, v/v) and detected at 205nm. The quantitation of the stigmasterol was reproducible and the method relative standard deviation is 1.1%. The mean analytical recovery was 96.2%.

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse-phase liquid chromatography at different methanol-water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol-water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.

Influence of variation in mobile phase pH and solute pKa with the change of organic modifier fraction on QSRRs of hydrophobicity and RP-HPLC retention of weakly acidic compounds

The variation in mobile phase pH and ionizable solute dissociation constant (pKa) with the change of organic modifier fraction in hydroorganic mobile phase has seemingly been a troublesome problem in studies and applications of reversed phase high performance liquid chromatography (RP-HPLC). Most of the early studies regarding the RP-HPLC of acid–base compounds have to measure the actual pH of the mixed mobile phase rigorously, sometimes bringing difficulties in the practices of liquid chromatographic separation. In this paper, the effect of this variation on the apparent n-octanol/water partition coefficient (Kow″) and the related quantitative structure–retention relationship (QSRR) of logKow″ vs. logkw, the logarithm of retention factor of analytes in neat aqueous mobile phases, was investigated for weakly acidic compounds. This QSRR is commonly used as a classical method for Kow measurement by RP-HPLC. The theoretical and experimental derivation revealed that the variation in mobile phase pH and solute pKa will not affect the QSRRs of acidic compounds. This conclusion is proved to be suitable for various types of ion-suppressors, i.e., strong acid (perchloric acid), weak acid (acetic acid) and buffer salt (potassium dihydrogen phosphate/phosphoric acid, PBS). The QSRRs of logKow″ vs. logkw were modeled by 11 substituted benzoic acids using different types of ion-suppressors in a binary methanol-water mobile phase to confirm our deduction. Although different types of ion-suppressor all can be used as mobile phase pH modifiers, the QSRR model obtained by using perchloric acid as the ion-suppressor was found to have the best result, and the slightly inferior QSRRs were obtained by using acetic acid or PBS as the ion-suppressor.

Development of a stability-indicating LC method for determination of a synthetic chalcone derivative in a nanoemulsion dosage form and identification of the main photodegradation product by LC–MS

This study aimed to develop and validate a stability indicating LC for the determination of 5-(2-benzoylethenyl)-N-benzyl-2 methoxybenzenesulfonamide (SCD - a synthetic chalcone derivative), and evaluate the degradation profile of the compound under different stress conditions recommended by International Conference on Harmonization (ICH). Chromatographic separation was performed on a silica C18 column, methanol-water mobile phase 70:30 (v:v), pH 5.0 adjusted with the addition of trifluoroacetic acid (TFA) at a flow rate of 1.0 mL min(-1); detection by UV absorption at 330 nm. The method was validated for linearity, precision, accuracy, robustness and specificity. The SCD was subjected to forced degradation and peak photodegradation product was well separated from the pure substance, with retention times significantly different, indicating the specificity of the method. Second-order degradation kinetics of SCD was observed under photodegradation and base-induced degradation. In order to identify the product formed under photodegradation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed. These results indicate that the method can be successfully used in order to assay SCD in a nanoemulsion dosage form, and that this formulation has a protective effect over SCD degradation.

Determination of Descriptors for Plasticizers by Chromatography and Liquid–Liquid Partition

Retention factors on a minimum of eight stationary phases at various temperatures by gas–liquid chromatography and in acetonitrile–water and methanol–water mobile phases by reversed-phase liquid chromatography on a Synergi Polar-RP column were combined with further values taken from the literature and liquid–liquid partition coefficients for up to eight totally organic biphasic systems to estimate descriptors for 24 esters (phthalate, oleate, stearate, arbietate, adipate, succinate, sebacate, and diethylmalonate) widely used as plasticizers and solvents in industry. The descriptors facilitated the estimation of several properties of environmental interest (octanol–water, air–octanol, and air–water partition coefficients, and soil–water distribution coefficients, vapor pressure, and water solubility). The descriptors are suitable for use in the solvation parameter model and facilitate the estimation of a wide range of physicochemical, chromatographic, biological, and environmental properties using existing models.

Quantitative structure/eluent–retention relationships in reversed-phase high-performance liquid chromatography based on the solvatochromic method

Some predictive approaches aimed at modelling the combined effect of solute molecular structure and mobile phase composition on retention in reversed-phase high-performance chromatography (RP-HPLC) have been developed in the literature. These models are established for a given binary eluent (normally acetonitrile-water or methanol-water) by non-linear (curvilinear or artificial neural network) regression assuming as the mobile phase descriptor the volume fraction φ of the organic modifier. In the present investigation, we propose a model applicable simultaneously to acetonitrile-water and methanol-water eluents. To this end, the Kamlet-Taft solvatochromic descriptors of the eluent and the solvatochromic descriptors of the analytes are considered as the input variables of a multi-layer artificial neural network (ANN) providing the solute retention as the response. This approach is applied to a set of 31 molecules analyzed with five different columns in the φ range 20-70 % at 10 % steps for both acetonitrile- and methanol-containing mobile phases. For each column, an ANN-based model is built using retention data of 25 molecules selected by the Kennard-Stones algorithm while retention data of the unselected six solutes are considered in the final evaluation of predictive performance of the trained network. To test cross-eluent prediction, the network optimized for a given column was successively trained with data collected in eight out of 12 eluents and applied to deduce retention in the four remaining mobile phases. The results reveal that RP-HPLC behavior of external solutes is quite accurately modelled in the whole explored composition range of acetonitrile- and methanol-water mobile phases. Moreover, the model exhibits a promising capability of deducing retention of external solutes even in unknown eluents.

Determination of resveratrol content in resveratrol nanoemulsion gel by RP-HPLC method

Objective: To establish a RP-HPLC method for determination of resveratrol content in resveratrol nanoemulsion gel.Methods:The separation was performed on Diamond C18 column(250 mm×4.6 mm,5 μm) with the mobile phase of methanol-water(60∶40).The flow rate was 0.8 ml/min.The detection wavelength was 303 nm.Results:Resveratrol presented a good linearity in the range of 1.96-9.82 μg/ml(r=0.999 9).It had good precision,stability and reproducibility.The recovery rates of low,middle and high concentrations(4.91,6.88,8.84 μg/ml) of resveratrol were(102.45±1.59)%,(102.71±1.05)% and(100.05±1.22)%(n=5),respectively.The average contents of resveratrol in three batches of samples were(99.47±0.43)%,(99.05±0.84)% and(101.72±1.18)%(n=3).Conclusion:It is an accurate,sensitive,selective and reproducible method for determination of resveratrol content in resveratrol nanoemulsion gel.

HPLC Method for Determination of Atenolol in Human Plasma and Application to a Pharmacokinetic Study in Turkey

This paper describes a high-performance liquid chromatography method for the determination of atenolol in human plasma. Atenolol and the internal standard, metoprolol, were extracted from plasma by using a liquid-liquid extraction method. The method was developed on an Ace C18 reverse-phase column using a mobile phase of methanol-water (50:50, v/v) containing 0.1% trifluoroacetic acid. The calibration curve was linear within the concentration range of 5-150 ng/mL. Intra-day and inter-day precision values for atenolol in plasma were less than 6.1, and accuracy (relative error) was better than 5.5%. The mean recovery of atenolol was 98.4% for plasma. The limits of detection and quantification of atenolol were 1.5 and 5 ng/mL, respectively. Also, this assay was successfully applied to six patients with hypertension who had been given an oral tablet of 50 mg atenolol.

Evaluation of separation parameters for selected organophosphorus fungicides of forensic importance by RP-HPTLC

Summary Studies on the evaluation of separation efficacies are of primary interest to forensic analysts for newly available pesticides. In the present work, a method is described for the separation evaluation of three selected organophosphorus fungicides (OPFs) ditalimfos (D), edifenfos (E), and tolclofos-methyl (TM) by reversed-phase high-performance thin-layer chromatography (RP-HPTLC). The method is based on the separation of three OPFs on silica gel 60 RP-18WF254 plates with varying mobile phase compositions (10:0–6:4) of methanol-water. The relationship between the mobile phase composition and retention parameters (RF and RM) as well as separation parameters (ΔRF, RS, α, RFα) was analyzed. The three OPFs are completely separated from each other with ΔRF ≥ 0.04, except for the pair of compounds E-TM in the mobile phase composition 10:0, 7:3, 6.5:3.5, and 6:4. Under the chromatographic conditions used, TM was adsorbed the strongest, followed by E and D, which is indicated by the higher RM values obtained for TM. Peak resolution (RS) values greater than 1.5 were obtained over the entire range of mobile phase composition, except for the pair of compounds E-TM. The UV apex of maximum absorption was 200 nm for all the three OPFs as measured by multiwave-length scan in the UV range, and higher values of peak area and peak height were obtained at the same wavelength. The present study clearly indicates that the system of RPHPTLC with mobile phase methanol-water in a volume composition 8:2 (υ/υ) provides the optimum conditions for the separation of the three OPFs. However, the complete separation of E from TM was not achieved using this system.