Methanol Buffer Research Articles

A new method for the HPLC determination of gamma-hydroxybutyric acid (GHB) following derivatization with a coumarin analogue and fluorescence detection: Application in the analysis of biological fluids

A new method of the determination of gamma-hydroxybutyric acid (GHB) in human biological fluids cerebrospinal fluid (CSF) and saliva after off-line derivatization is described. The proposed method was based on the reaction of 4-bromomethyl-7-methoxy coumarin (Br-MMC) with GHB in the presence of dibenzo-18-crown-6-ether (acting as reaction catalyst) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at λ ext. = 330 nm and λ em. = 390 nm. The effect of derivatization parameters such as the concentration of Br-MMC, reaction time and the temperature was investigated in order to achieve the maximum method's sensitivity. The separation was achieved by use of a C 18 analytical column (Kromasil ® 250 mm × 4 mm i.d., 5 μm) while the injected sample volume was set to 25 μL. A binary gradient elution program of methanol versus phosphate buffer (40 mM, pH 3) was selected for the quantitative analysis of GHB. The method showed satisfactory linearity ( R 2 = 0.9979) in a linear range from 2.4 × 10 −6 to 7.2 × 10 −5 M. Ultrafiltration method was employed for the pre-treatment of the cerebrospinal fluid (CSF) prior to the analysis of GHB. The limit of detection (LOD) of the method was 3 × 10 −7 M in saliva and 2 × 10 −7 M in CSF samples, respectively, while the limit of quantitation (LOQ) was 1 × 10 −6 M for both specimens. The proposed protocol offers sensitivity comparing with the existing HPLC analytical methods or the CE indirect UV methods and can function as an attractive alternative to be used in clinical and toxicological analysis.

Photolysis of Aqueous H2O2: Quantum Yield and Applications for Polychromatic UV Actinometry in Photoreactors

Methanol is used to measure the yield of *OH radicals produced in the photolysis of H2O2 in aqueous solutions. The UV photolysis of H202 generates *OH radicals, which in the presence of methanol, oxygen, and phosphate buffer form formaldehyde, namely, phi(HCHO) = phi(*OH). The quantum yield of *OH has been redetermined in view of literature inconsistencies resulting in phi(*OH) = 1.11 +/- 0.07 in the excitation range of 205-280 nm. The constancy of phi(*OH) and the ease and sensitivity of the formaldehyde product analysis makes the H2O2/CH3OH system suitable for polychromatic UV actinometry. In addition, the relatively low cost of the main components and the possibility of destroying the methanol before disposal qualify the system for both monochromatic and polychromatic actinometry in a large volume of water. The H2O2/CH3OH system was applied in different commercial UV photoreactors.

Validação de método por cromatografia líquida de alta eficiência para determinação da lamivudina e zidovudina em comprimidos

An HPLC method was validated to assay lamivudine and zidovudine combined in tablets. The chromatographic separation was carried out using methanol and acetate buffer pH 6.5 (50:50 v/v) and a RP-18 column, as mobile and stationary phase, respectively. The UV detection was at 270 nm. The method was linear in the range of 24 - 36 µg/mL (lamivudine) and 48 - 72 µg/mL (zidovudine). The recovery (accuracy) ranged from 101.35% to 103.04% and the precision (repeatability and intermediate precision) was less than 2%. The method can be also applied to the quantification of these drugs in the dissolution test of tablets containing both drugs.

Fatty acid composition of triacylglycerols from Wharton's jelly determined by high-performance liquid chromatography

The improved method for HPLC determination of fatty acids was proposed. The chromatographic separation of p-bromophenacyl derivatives of fatty acids under a gradient elution was achieved at 40 °C with an RP-18 LiChroCART 5 column and organic mobile phase containing methanol, acetonitrile, water and TEAP buffer pH 5.6. The quantitative determination of those derivatives was performed at 254 nm. Preeclampsia, the most common pregnancy complication, did not affect triacylglycerol content in the umbilical cord Wharton's jelly in comparison to the control material. However, it changed the composition of fatty acids, bound to that lipid class. The method allows the determination of almost all fatty acids forming the investigated neutral lipid class, contained in a solid tissue sample. The use of TEAP buffer excluded precipitation and flow stoppage in the HPLC system. The method reduced time and costs and might be useful for all other lipid classes and different tissues.

Rapid and sensitive bioanalytical method for measurement of fluvoxamine in human serum using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent: Application to a human pharmacokinetic study

A sensitive and rapid high-performance liquid chromatographic method for the analysis of fluvoxamine, a selective serotonin reuptake inhibitor in human serum, is described using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent. The drug and an internal standard (fluoxetine) were extracted from 0.25 mL of serum using ethyl acetate as extracting solvent and subjected to pre-column derivatization by the reagent. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.8) containing 1 mL/L triethylamine (72:28 v/v) was used and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm × 4.6 mm) column. The fluorescence derivatives of the drugs were monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. The calibration curve was linear over the concentration range of 0.5–240 ng/mL with a limit of quantification (LOQ) of 0.5 ng/mL using 0.25 mL serum sample. The method validation was performed for its selectivity, specificity, sensitivity, precision and accuracy. In this method, which was applied in a randomized cross-over bioequivalence study of two different fluvoxamine preparations in 24 healthy volunteers, the sensitivity and run time of analysis were significantly improved.

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-( trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 °C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD < 1.9%), linearity, lower limit of quantitation (LOQ < 0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.

Development and Validation of LC Method for the Determination of Famciclovir in Pharmaceutical Formulation Using an Experimental Design

A rapid and sensitive RP‐HPLC method with UV detection (242 nm) for routine analysis of famciclovir in pharmaceutical formulations was developed. Chromatography was performed with mobile phase containing a mixture of methanol and phosphate buffer (50:50, v/v) with flow rate 1.0 mL min−1. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient =0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered; percentage v/v of methanol in mobile phase, flow rate and pH; flow rate, the percentage of organic modifier and pH have considerable important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The RSD value (0.86%, n=24) indicated an acceptable precision of the analytical method. The proposed method was simple, sensitive, precise, accurate and quick and useful for routine quality control.

Development of an HPLC method for the determination of doripenem in human and mouse serum

A HPLC method utilizing solid phase extraction was developed to analyze doripenem (formerly S-4661) in human and mouse serum. A reversed-phase column was used with a UV detector set at 295nm. The mobile phase consisted of methanol and phosphate buffer at a flow rate of 1.5ml/min. Meropenem was used as the internal standard. The standard curve was linear over a range of 0.5–40μg/ml. The assay is simple, reproducible, and accurate and has been used successfully to analyze doripenem concentrations from a murine pharmacokinetic study.

Determination of phenolic acids in coffee by micellar electrokinetic chromatography

A seven-minute micellar electrokinetic chromatography analytical procedure capable of resolving the five principal phenolic acids in coffee, including the isomers of 5-caffeoylquinic acid (5-CQA), 4-CQA and 3-CQA, caffeic acid and ferulic acid is reported. The electrophoretic conditions consisted of an SDS (70 mM)-phosphate (17.6 mM)-methanol (5% v/v) buffer system, pH 2.5, 22.1 °C, −17 kV and detection at 325 nm. The Joule effect and the possible interactions between the buffer components and temperature with respect to peak quality, resolution and selectivity were assessed in the concentration range of 25–900 μg/mL. Performance evaluation of the system used a 3 3–7 factorial design at the 95% confidence level. The lowest correlation coefficient for linearity was 0.99888 for the 4-CQA. Limits of detection and quantification were 0.98 and 4 μg/mL. The method was tested with both green and roasted coffee beans using four systems of extraction.

Ontogenetic and Seasonal Variation in the Flavonoid Composition of Sophora japonica. Cultivated in Bulgaria

Quantitative changes in the accumulation of the flavonol glycosides rutin and kaempferol 3-rutinoside and the isoflavone glycosides genistin and sophorabioside have been investigated in flowers and fruits of cultivated in Bulgaria Sophora japonica. during ontogenetic and seasonal development. A method for extraction of flavonoids with good recovery was developed (98% for rutin and 97% for kaempferol 3-rutinoside). The subsequent high-performance liquid chromatography separation of flavonoid glycosides was achieved using Hypersil ODS C18 column and UV detection. The mobile phase comprised methanol and phosphate buffer, pH 3.23, and linear gradient elution mode within 25 min was applied. Accumulation of rutin started with initiation of the flower buds, having the maximal content up to 287 mg/g (dry weight) in mid-July during the earliest stages of flower development and tended to have high levels until mid-August. A further decrease in the amount of rutin up to 44 mg/g was found proportionally with the decline of flavonoids throughout and after the flowering period. Rutin was the predominant glycoside and can account for up to 90% of flavonoid glycoside mixture in young buds, whereas sophorabioside (64.7 mg/g, calculated as rutin equivalent) and kaempferol 3-rutinoside (46.8 mg/g) dominated at later development stages of the fruits. No significant differences in flavonoid quantity between the analysis periods of two successive years were observed. This indicated clearly the ontogenetic dependence of flavonoid formation.

Separation and Identification of Stereoisomers of Isomerized α-Acid Derivatives by HPLC/DAD and Electrospray HPLC/MS

Four classes of advanced isomerized hop products (advanced hop products) are commercially available. They are derivatives of α-acids that have undergone stereochemical transformations, resulting in iso-α-acids with cis and trans configurations. Their compositions play important roles in providing bitterness, foam, flavor, and flavor stability; each of these derivatives exhibits differences in its bitterness strength, foam enhancement, flavor, and flavor stability. The traditional IBU (international bitterness unit) method for measuring bitterness using UV absorbance neither distinguishes their compositions nor provides clues as to their corresponding sensory perception. A simple high-performance liquid chromatography (HPLC) method using a 100 × 4.6 mm C-18 column and an aqueous methanol buffer solution has been developed to rapidly resolve analogs and stereoisomers of iso-α-acids (class I), ρ-iso-α-acids (class II), tetrahydroiso-α-acids (class III), and hexahydroiso-α-acids (class IV). This method can also differentiate between these four classes of iso-α-acid derivatives. Additionally, we demonstrate that electrospray HPLC/MS (mass spectrometry) can be a versatile method for identifying these isomerized hop derivatives.

A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid–liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 × 4.6 mm) column. In this method the limit of quantification of 0.01 μg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 μg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from −3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.

Improving the in vivo predictability of an on-line HPLC stable free radical decoloration assay for antioxidant activity in methanol–buffer medium

The pivotal role of ROS (reactive oxygen species) in various (patho)physiological processes has stimulated research on the potential of intervening in these processes with antioxidants (AO). In vitro model systems to investigate AO activity against the various ROS are a valuable tool in classifying antioxidants. To improve the in vivo predictability of the results obtained, we have modified and characterized the widely used DPPH (2,2′-diphenyl-1-picrylhydrazyl) on-line decoloration assay. Previous investigations using the DPPH reaction in a pure methanolic medium exhibit slow kinetics and a reaction going to completion. In this study, a medium which includes an aqueous buffer at physiological pH has been applied, resulting in the rapid establishment of equilibrium. The results obtained in an aqueous medium at physiological pH are expected to be more relevant for extrapolation to in vivo circumstances than previously published findings. The antioxidants investigated are classified according to the results obtained and the relevance of their behavior to in vivo situations is discussed. Special emphasis is put on the significance of the results for prediction of redox-cycling characteristics and structure–activity relationships.

Photochemistry of 2,6-diisopropylphenol (propofol)

The photochemistry of the anaesthetic agent propofol (PPF) was investigated in three different solvents of quite different polarity (cyclohexane, methanol and phosphate buffer pH 7) by means of nanosecond laser flash photolysis and absorption spectroscopy. GC-MS spectrometry measurements of PPF in cyclohexane have revealed the formation of two major products upon low intensity UV continuous irradiation of PPF in aerated solution: the diphenol derivative of PPF and 2,6-diisopropyl-p-benzoquinone (PPFQ). Only the diphenol compound was obtained in anaerobic solution. PPF phenoxyl radical (PPF ) generation has been assigned as the original step leading to the formation of both the diphenol compound and PPFQ in cyclohexane as revealed by laser flash photolysis at 266 nm and by electron paramagnetic resonance spectroscopy as well. Investigation of PPF by nanosecond flash photolysis at 266 nm in the other solvents revealed the occurrence of different photochemical processes depending on the nature and the polarity of the solvent. A reaction scheme is proposed in order to discuss the mechanism of reaction of PPF in all media.

Simultaneous determination of sildenafil and N-desmethyl sildenafil in human plasma by high-performance liquid chromatography method using electrochemical detection with application to a pharmacokinetic study

A method which employed high-performance liquid chromatography coupled with electrochemical detection was developed for the simultaneous determination of sildenafil and its metabolite, N-desmethyl sildenafil, in human plasma has. The method was developed and validated for purposes of its application to a pharmacokinetic study in healthy volunteers after an oral dose of 50 mg/tablet under fasting conditions. High precision and accuracy were demonstrated. A one-step liquid–liquid extraction further provides a simple and practical way to process plasma samples containing sildenafil with good quantitative recovery. Sampling lasted for 24 h after dosing; consequently a limit of quantitation (LOQ) of 7.858 ng/mL was achieved for sildenafil whereas a LOQ of 8.675 ng/mL was obtained for N-desmethyl sildenafil. The mobile phase consisted of acetonitrile, methanol and phosphate buffer (0.05 M) (18.5:34.5:47.0, v/v/v) pH 7.68. The stationary phase was a C 8 (150 mm × 4.6 mm), 5 μm particle size operated at 27 °C. All analytes were stable at the pH of the supernatant, and during the analytical time window. At the applied potential of +1.20 V versus Ag/AgCl, no interferences from endogenous plasma compounds were recorded at the retention times of sildenafil, N-desmethyl sildenafil. High resolution was obtained between the analytes and the employed internal standards.

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N'-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of alpha-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20 degrees C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.

High-Performance Liquid Chromatographic-Mass Spectrometric Determination of Methamphetamine and Amphetamine Enantiomers, Desmethylselegiline and Selegiline, in Hair Samples of Long-Term Methamphetamine Abusers or Selegiline Users

We devised a highly sensitive method for simultaneously determining methamphetamine (MA) and amphetamine (AP) enantiomers, desmethylselegiline (DMSG) and selegiline (SG), in human hair using a derivatization technique and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). MA and AP enantiomers and DMSG were effectively converted to trifluoroacetic acid (TFA) derivatives, and the sensitivity of MA and DMSG increased five times over compared with that of free bases. The TFA derivatives of each compound were stable within one week in a stock solution of methanol or for 24 h in the HPLC mobile phase (mixture of methanol and ammonium formate buffer). Each compound was well separated, and calibration curves were linear in the concentration range 0.04-40 ng/mg for MA enantiomers, SG and DMSG, and 0.2-40 ng/mg for AP enantiomers. The accuracy and precision of the method were evaluated, and relative standard deviations were within 7%. Our method was successfully applied to hair samples obtained from long-term MA abusers and SG users. (+)-MA and (+)-AP were detected from three MA abusers at concentrations of 0.79-20.85 and 0.04-3.30 ng/mg, respectively. On the other hand, (-)-MA, (-)-AP, DMSG, and SG were detected in three SG users at concentrations of 2.48-9.05, 0.72-3.10, 0.12-0.59, and 0-0.04 ng/mg, respectively. Based on our obtained data, discrimination of MA abusers from SG users was considered to be possible by comparing optical isomers of MA and AP, the existence of DMSG and/or SG, and the concentration ratio of AP to MA in hair samples.

Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum require automated o-phthalaldehyde derivatization of the drug and immediate injection of the unstable derivatives formed. A new, very sensitive and simple high-performance liquid chromatographic method for quantitation of the drug in human serum using 4-chloro-7-nitrobenzofurazan (NBD-Cl) as a fluorescent labeling agent is presented. In this method the sensitivity was significantly improved and the limit of quantification of 0.002 microg/ml was obtained using 100 microl serum sample and 10 microl injection. However, the LOQ can be improved by increasing the sampling volume. The procedure involved protein precipitation of serum by acetonitrile followed by derivatization with NBD-Cl. Amlodipine was used as internal standard and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The fluorescence derivative of the drug was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.5) containing 1 ml/l triethylamine (65:35, v/v) was used. The calibration curve was linear over the concentration range of 0.002-15 microg/ml. No interferences were found from commonly co-administrated antiepileptic drugs. The method was applied in a randomized cross-over bioequivalence study of two different gabapentin preparations in 24 healthy volunteers.

Partition of polyhydroxy compounds of biological and pharmacological significance between AOT reverse microemulsions and aqueous salt solutions

AbstractThe partition behavior of shikimic acid, gallic acid, gallotannic acid, rutin and quercetin between reverse microemulsions of the surfactant bis(2‐ethylhexyl) sulfosuccinate (AOT) and aqueous salt solutions of LiCl, NaCl and KCl at different salt concentrations and pH was studied. Quantification of the extracted analytes was performed by HPLC on an octadecyl silica column using a methanol–buffer mobile phase at pH 3.0. Clean‐up before injection was not needed because the micellar organic phase was compatible with the HPLC mobile phase. A solvent gradient was used to elute all compounds and the surfactant in a reasonable time. The extraction from the aqueous media is fast and reproducible. Different solubilization behavior was observed by changing the salt concentration and the type of cation in the aqueous phase. By proper selection of the experimental conditions, it is observed that shikimic acid and gallic acid remain mostly in the aqueous phase, whereas significant amounts of gallotannic acid and rutin are transferred to the micellar phase in only one contact and quercetin is completely solubilized in this phase. Thus, the different solubilization behavior that these compounds exhibit in the AOT micellar media can be very useful for extraction and preconcentration purposes. Copyright © 2006 John Wiley &amp; Sons, Ltd.