Purospher RP-18 Research Articles

Stability Indicating RP-HPLC for Quantification Mangiferin in Extract of Three Species Mango Leaves

The stability indication of Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was validated for quantitative determination of mangiferin on three species mango leaves (Mangifera odorata Griff, Mangifera foetida Lour, and Mangifera indica L.). The samples were extracted by maseration method using methanol and concentrated using rotary evaporator. The method carried out on stationary phase a purospher RP-18 endcapped (25 cm × 4.6 mm i.d., 5 µm) column with a mobile phase consisting of methanol: phosphoric acid 0.1% (v/v) (31:69); flow rate:0.8 mL/min; solvent methanol, detection was carried out at 258 nm. The analytical performace this measurement is good with the value of linearity (r2=0.998), precision (%RSD=0.649%), and accuration (10.67%). The forced degradation studies were carried out according to the International Conference on Harmonization (ICH) guidelines. The results indicating that the complete separation between degradation products and mangiferin peak occured. The degradation limit of mangiferin 5–20% (according to the guideline of ICH) except in basic condition (100%). The method was succesful applied to determine of the mangiferin in pakel (Mangifera foetida), kweni (Mangifera indica) and kopyor (Mangifera odorata) extract. The mangiferin content was obtained are pakel (9.95%), kopyor (7.40%) and kweni (Mangifera odorata) (2.49%) respectively.

In Vitro Investigation of D- and Lenantiomer Synergistic Efects of Some Amino Acids

Abstract D-amino acids can arise from endogenous microbial flora, from ingestion with the diet or from spontaneous racemization of L-amino acids during ageing. In this work, the behavior of methionine, homocysteine and cysteine enantiomers was investigated in human serum in vitro during 0-72 h at incubation temperature 37°C. The separation of enantiomers was realized in two dimensional on-line system (the connection of an achiral column Purospher RP-18 endcapped and a chiral column Chirobiotic TAG). This system allowed simultaneous monitoring all tested amino acids and their enantiomers. The possible effect D-enantiomer on the behavior of its L-enantiomer (the synergistic effect) was evaluated during incubation time. The first results have showed that no synergistic effect of D-enantiomer on its Lisomer has been observed in our experimental conditions in vitro.

Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum

A two-dimensional HPLC system with electrochemical detection was used for determination of homocysteine, methionine and cysteine enantiomers in biological samples. The amino acid separations were not possible only by using a chiral column. The compounds were separated from each other on an achiral column (Purospher RP-18 endcapped 250–4mm, 5μm) and their enantiomers were separated on Chirobiotic TAG (250–4.6mm, 5μm) column in an on-line system. The mobile phase composition and a choice of electrode potentials for detection were investigated. The l-enantiomers always eluted before the d-enantiomers. The proposed method was applied to the analysis of human serum of healthy volunteers and patients with multiple sclerosis. The limit of detection (LOD) and quantitation (LOQ) were defined as the concentration that produced a signal-to-noise ratio (S/N) of 3 and 10. The method LOD values were found to be between 0.05 and 0.50μgmL−1. The range of LOQ values were between 0.17 and 1.67μgmL−1, respectively.

P3: Hair and whole blood matrix effect in identification of a model analyte and determination of its limit of detection

Any kind of biological material matrix may represent a serious problem during real samples analyses, especially using LCMS method. It causes both signal intensity loss and/or signal to noise ratio (S/N) decrease. Unfortunately, this phenomena touches selected ions sometime in a different way. It means that even deuterated standards in some cases cannot protect from precision disturbance or even substance misidentification. The study aim was to elaborate a control method of a hair or whole blood matrix influence on a limit of detection (LOD) in identification and quantitation aspects. Waters Alliance 2695 Separations Module with Quattro Micro Micromass were applied in the research. Analyses were performed using LiChroCART 55x4 Purospher RP-18 column from Merck as well as by direct injection without separation in flow injection analysis (FIA). The mobile phase consisted of 0.1 % (v/v) formic acid in acetonitrile and water. Flow rate was set to 0.5 ml/ min. Hair samples free of drugs were used in testing. Hair were powdered in ball-mill and 50 mg weighed samples were macerated in 1 ml of methanol during 72 hours in 37°C. Results were compared to extract of 0,5 ml whole blood sample (also free of drugs) obtained by ethyl acetate at pH 9. Short segments of mass range were set (25 amu) to improve sensitivity in scan mode. SIR and MRM modes were applied in LOD testing. Morphine was engaged as a model drug for matrix influence investigation. Ions map for m/z 50 to m/z 450 was created both for blood and hair matrix. That allowed to compare possible interferences from those matrices. Signal intensity and S/N were used for calculations. Plots of S/N versus analyte concentration appeared to better show LOD than signals intensity. Some ions (not the weakest) or MRM transitions disappeared from chromatograms of morphine causing lack of all parameters for appropriate identification although analyte was present in higher than LOD concentration. Standard infusion during real sample analysis a few seconds after analyte retention time helped to solve problem of analyte presence in a sample. Matrix effect very often cannot be avoided but always should be controlled.

A rapid and simple HPLC method for the analysis of propofol in biological fluids

A selective and sensitive high-performance liquid chromatographic method for the analysis of propofol in biological samples was developed. Propofol and thymol (internal standard) were analysed on a Purospher RP-18 endcapped (75 mm × 4 mm, 3 μm) stationary phase using acetonitrile and water (65:35, v/v) as eluents at a flow rate of 0.6 mL/min. The excitation and emission wavelengths were 276 and 310 nm, respectively. Sample treatment consisted of deproteinization by acetonitrile containing the internal standard and direct injection of the supernatant. Mean analytical recovery were 105% (CV 2.0%) at concentrations ranging from 0.05 to 10 mg/L. The quantification limit was 3 ng/mL for a 500 μL sample plasma volume and 5 ng/mL for a 500 μL blood sample. The intra-day and inter-day precisions were lower than 5.5% for three concentrations assessed (0.05, 1.0 and 10.0 mg/L). Considering the column size and the flow rate, the separation was achieved with an analysis time less than 6 min with a reduced consumption of solvent. This rapid HPLC method using a simple treatment procedure is sensitive enough for monitoring propofol in human biological samples.

High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma

Mesalazine (5-aminosalicylic acid, 5-ASA), an anti-inflammatory agent for the treatment of inflammatory bowel diseases, is metabolized in organism to the principal biotransformation product, N-acetyl-5-ASA. Some other phase II metabolites ( N-formyl-5-ASA, N-butyryl-5-ASA, N-β- d-glucopyranosyl-5-ASA) have also been described. 5-ASA is a polar compound and besides it exhibits amphoteric properties. The extraction of this compound from biomatrices and its chromatographic analysis is complicated. In order to improve the reliability of the determination of parent 5-ASA, a derivatization of 5-ASA together with 4-ASA (added to samples as a precursor of I.S.-2) was involved into the method. More lipophilic N-propionyl-5-ASA and N-propionyl-4-ASA (I.S.-2) were obtained using propionic anhydride. These derivatives were well extractable together with N-acyl-5-ASAs (metabolites) and N-acetyl-4-ASA (I.S.-1). As the first internal standard (I.S.-1) was used for the evaluation of extracted N-acyl-metabolites, the second internal standard (I.S.-2) served for the evaluation of both derivatization and extraction steps of parent drug 5-ASA. Based on these reasonings, new HPLC bioanalytical method for the determination of 5-ASA and its metabolites in blood plasma was developed and validated. The sample preparation step consists of the deproteination of plasma by HClO 4 and the above-mentioned derivatization of ASAs followed by liquid–liquid extraction of all N-acyl-ASA-derivatives. Chromatographic analyses were performed on a 250-4 mm column containing Purospher RP-18 e, 5 μm (Merck, Darmstadt, Germany) with a precolumn (4-4 mm). The column effluent was monitored using both UV photodiode-array ( λ = 313 nm) and fluorescence detectors ( λ exc. = 300 nm/ λ emiss. = 406 nm) in tandem. The identity of individual N-acyl-ASAs in the extracts from biomatrices was verified by characteristic UV-spectra and by HPLC/MS experiments. The whole analysis lasted 23 min at the flow rate of 1 ml min −1. LLOQ (LOD) was estimated 126 (20) pmol ml −1 of plasma for N-acetyl-5-ASA and 318 (50) pmol ml −1 of plasma for N-propionyl-5-ASA. The validated HPLC method was applied to pharmacokinetic studies of mesalazine in humans and animals.

Determination of Nifedipine in Serum ofWomen in Preterm Labor by High-Performance Liquid Chromatography with Diode Array Detection

Nifedipine (Nif) is widely used in treating cardiovascular disorders (especially hypertension) and for inhibiting preterm labor. A fully validated selective high-performance liquid chromatographic method with diode array detection, using solid-phase extraction, was developed for the determination of Nif in human serum. To assess specificity, Nif and its degradation products were separated on a Purospher RP-18 (5 microm, 125 x 4 mm) column plus a LiChrospher 100 RP-18 (5 microm, 4 x 4 mm) precolumn with a mobile phase of methanol-10 mM aqueous trifluoroacetic acid, pH 7.3 (57 + 43, v/v); chromatographic separation was followed by UV detection at 238 nm. For toxicological analysis, Nif in the presence of other calcium-channel antagonist drugs was identified under optimum chromatographic conditions. The calibration graph was constructed over the concentration range of 12.5-400 ng/mL in serum with good correlation (r = 0.9956). This method was not subject to interference by other plasma components and was successfully applied to the assay of Nif in spiked human serum and in serum of women in preterm labor after sublingual administration of 30 mg Nif per day divided into 3 equal doses. The mean recovery based on the ratio of the slopes of serum and mobile phase standard curves was 96.5%. The detection and quantification limits of the drug in spiked human serum were found to be 6 and 17.5 ng/mL, respectively. Validation of the method demonstrated good intraday and interday precision, which ranged from 2.18 to 6.67% and from 6.52 to 11.93%, respectively.

High-performance liquid chromatographic determination of pesticides in tomatoes using laboratory-made NH 2 and C 18 solid-phase extraction materials

A method for high-performance liquid chromatographic (HPLC) multiresidue determination of six pesticides in tomatoes was developed and validated. Silica-based C 18 (octadecyl) and NH 2 (aminopropyl) solid-phase extraction (SPE) sorbents, made in our laboratory, were used for sample preparation. The SPE materials were obtained by thermal immobilization of appropriate polysiloxanes onto 40 μm silica surfaces and were used in sample preparation for multiresidue analysis of the following pesticides: tebuthiuron and diuron (urea herbicides), simazine, atrazine and ametryn (triazines herbicides) and benomyl (benzimidazol fungicide). The results were compared with similar commercial materials. Reversed-phase high-performance liquid chromatography (RP-HPLC) using a Purospher RP-18 5 μm column and ACN: 0.01% aqueous NH 4OH, pH 8.4 (35:65, v/v) as mobile phase, at 0.7 ml min −1, with 235 nm UV detection, was used for separation and quantification of the pesticides. Method validation was performed at three fortification levels (100, 200, 1000 μg l −1). Limits of detection and quantification show that the methods developed can be used to detect the pesticides in concentrations below the maximum residue levels (MRL) established by the Codex Alimentarius, USA, European Union and Brazilian legislations. The results showed that aminopropyl materials have a better performance than the octadecyl sorbents. Laboratory-made materials give results similar to commercial sorbents, with recoveries and precisions in agreement with directives for method validation in residue analysis.

Development of an HPLC method for the identification and dosage of non-allowed substances in cosmetic products. Part I: local anaesthetics and antihistaminics

An HPLC method with ultraviolet detection coupled with a solid-phase extraction sample clean up was developed for the analysis of five local anaesthetics and four antihistaminics in cosmetic products. The presence of these compounds in commercial cosmetic samples is fordbidden. Extracts from real samples were applied to a solid-phase extraction C18 cartridge, and the analytes were eluted with 8:2 (v/v) acetonitrile/water containing 1% trifluoroacetic acid. HPLC separation was then performed for the identification and determination of the analytes using a Purospher RP-18 column, two gradient eluting systems and a photodiode-array detector. The accuracy of the method was verified by spiking experiments on home-made cosmetic samples. The analytical recoveries were satisfactory.

Determination of total oncopterin, neopterin and biopterin in human urine by high performance liquid chromatography with solid phase extraction

AbstractA new, sensitive method for the determination of oncopterin, biopterin, and neopterin in human urine has been developed using SPE with 6,7‐dimethylpterin as internal standard and gradient HPLC with fluorescence detection. SPE was tested for the pre‐treatment of urine samples on different types of sorbents (strong ion exchange resins, polar adsorbents, and reversed‐phase sorbents). RP‐SPE with subsequent evaporation of eluate has been found to be the most appropriate. The extraction efficiency exceeded 95% for all selected pterins. The extracted pterins were subsequently analyzed on a Purospher RP‐18 RP column. The LOD of oncopterin was 1.43 nmol/L of urine. The intra‐day and inter‐day imprecision at a physiological oncopterin concentration never exceeded 10%. The potential of this method was tested using urine samples of healthy volunteers and cancer patients without methotrexate therapy.

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL−1. A demethoxy analogue of cisapride was used as internal standard.

Development of a tandem thin-layer chromatography–high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products

A solid-phase extraction clean-up and and a liquid chromatographic method with ultraviolet detection were developed for the analysis of 51 corticosteroids in cosmetic samples in order to screen commercial samples for the presence of undeclared synthetic corticosteroids. A thin-layer chromatographic analysis was carried out on silica gel plates, using different eluants and detection reagents. When such a preliminary chromatographic separation gave some indications about the presence of steroid compounds, the methanol extracts from real samples were applied to a solid-phase extraction C 18 cartridge, and the analytes eluted with ethyl ether. The high-performance liquid chromatographic separation was then carried out for the identification and determination of the analytes using a Purospher RP-18 column, an isocratic or a gradient elution with a mixture acetonitrile–water and a photodiode-array detector. The accuracy of the method was determined by spiking experiments on home-made cosmetic samples. The analytical recoveries were satisfactory.

Determination of the N-methyl- d-aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- d-aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.

HPLC determination of ketamine, norketamine, and dehydronorketamine in plasma with a high-purity reversed-phase sorbent

We developed an isocratic, selective, and very sensitive HPLC method for the determination of ketamine and its two main metabolites in plasma. The compounds were extracted from plasma by a liquid-liquid extraction with a dichloromethane:ethyl acetate mixture followed by an acidic back-extraction. Separation was achieved on a new stationary phase, Purospher RP-18 endcapped, with a mobile phase containing acetonitrile:0.03 mol/L phosphate buffer (23:77 by vol) adjusted to pH 7.2. Because of the high column efficiency and the significant improvement of peak symmetry, the quantification limit could be down to 5 micrograms/L for ketamine and norketamine (NK). The intraday and interday CVs ranged from 1.7% to 5.8% and 3.1% to 10.2% for all compounds respectively. The method is sensitive enough for monitoring ketamine, NK, and dehydroketamine in plasma during pharmacokinetic studies after an intravenous bolus of a low dose of ketamine.

Determination of several N-methyl-d-aspartate receptor blockers in plasma and brain by a selective high-performance liquid chromatographic method with column switching

A general procedure is presented for the determination of several N-methyl-d-aspartate (NMDA) receptor open-channel and subtype-selective blockers, which have been evaluated and developed as neuroprotective drugs for the treatment of brain stroke and trauma. The method involves deproteination of plasma with ethanol, or homogenization of brain samples in ethanol, dilution of the supernatant with ammonium acetate and direct injection into an HPLC column-switching system. Although the investigated NMDA receptor blockers are all tertiary amines, they have quite different structures. However, they are all concentrated on the first column (Purospher RP-18, 125×4 mm), whereas polar interfering compounds are washed out with 1% ammonium acetate–acetic acid–acetonitrile (100:1:5, v/v/v). Due to the special selectivity of the Purospher RP-18 material, the analytes and the internal standard are then selectively eluted with 25% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column (Superspher 60 RP-select B, 250×4 mm), where they are separated by gradient elution and detected by UV or fluorescence detection. The low degree of interference allowed the development of sensitive methods with quantification limits of 5 ng/ml for animal plasma (0.4 ml used), 0.5 ng/ml for human plasma (1 ml used) and 50 ng/g for brain tissue (200 mg used).

Interactions of basic compounds in reversed-phase high-performance liquid chromatography influence of sorbent character, mobile phase composition, and pH on retention of basic compounds

Differences in a degree of base deactivation of selected reversed-phase sorbents were evaluated. Five silica-based sorbents (SGX C 18, LiChrosorb RP-18, LiChrospher RP-select B, Purospher RP-18, Symmetry C 18), a polybutadiene-coated alumina (Aluspher RP-select B) and 2-hydroxyethylmethacrylate-based stationary phase (HEMA-BIO 1000 C 18) were compared. The best results were obtained for Symmetry C 18 and HEMA-BIO 1000 C 18. For testing purposes a set of nineteen basic compounds differing in p K a constants and hydrophobicity was used. The bases possessing high p K a values (p K a>9.0) and/or low hydrophobicity proved to be the best indicators of sorbent surface deactivation. Dependencies of capacity factor on pH of the mobile phase were measured. Shape deviations between the sigmoidal theoretically predicted and experimentally obtained curves evidence for a complex retention mechanism in which not only hydrophobic but also other interactions, for example, ion-exchange, participate. The effect of methanol content in the mobile phase on the dissociation of buffer and a basic analyte used was studied. The characteristic shifts of k versus pH of the mobile phase dependencies caused by the addition of methanol were found to be in a good agreement with theory.

Comparison of ODS-modified silica gels as stationary phases for electrochromatography in packed capillaries

The electroosmotic mobilities of the eluent generated during electrochromatography with a range of commercial ODS-bonded silica gel stationary phases packed into fused-silica capillary columns of 50 μm I.D. have been determined using thiourea as an unretained marker compound. The electrochromatography was carried out using acetonitrile-25 m M 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid buffer (pH 9.53) (70:30, v/v) as the mobile phase with electric field strengths in the range of 0.2 to 1.5 kV/cm. The experiments were carried out without pressurisation of the column, but the temperature of the column was controlled with a liquid coolant. Nucleosil 5 C 18 and LiChrospher RP-18 showed the highest electroosmotic mobilities. However, these stationary phases had low chemical stabilities in the alkaline medium and only retained their properties for 2–10 days. Spherisorb S5 ODS2, Zorbax BP-ODS and Hypersil ODS all demonstrated high chemical stability but showed only moderate or low electroosmotic mobilities. No electroosmotic flow was detected with Partisil 5 ODS3 or with Purospher RP-18, highly base-deactivated column materials. The electroosmotic mobilities have been compared with the carbon loading and surface areas of the stationary phases and with the silanol activities determined chromatographically on the same stationary phases packed into polyether ether ketone (PEEK) micro columns (0.5 mm I.D.) using an aniline/phenol test mixture and elution with methanol-water (45:55, v/v). However, there was little correlation between the electroosmotic flow-rate and the silanol activity or carbon loading of the stationary phases. It appeared that materials specially suited for good liquid chromatography, such as Purospher, may not be useful in electrochromatography because they may create only a limited electroosmotic flow-rate.

Automated quantitative determination of a new polyamine biosynthesis inhibitor (CGP 48 664) and a potential metabolite in human and animal plasma by high-performance liquid chromatography

A specific and sensitive liquid chromatographic assay for the determination of 4-amidino-1-indanone-2′-amidinohydrazone (CGP 48 664, I) and a potential metabolite, 2-(4-carbamoyl-2,3-dihydro-1H-inden-1-yliden) hydrazine carboximidamide (CGP 53 391, II), in human and animal plasma was developed. CGP 51 467, a structural analog, was added to the plasma samples (up to 1 ml) as an internal standard. After mixing, the samples were processed automatically using an ASPEC solid-phase extraction system. The final extracts were chromatographed on a 5 μm Purospher RP-18 HPLC column. Chromatography was performed using a gradient system and UV detection. The described HPLC method is suitable for specific and quantitative measurement of concentrations of I, as well as its potential metabolite II down to 5–10 ng/ml in human and animal (dog, rat) plasma with acceptable reproducibility and accuracy.