C18 High-performance Liquid Chromatography Column Research Articles

Green method for determination of four anti-viral drugs using micellar liquid chromatography: Application to dosage form analysis

A micellar liquid chromatographic (MLC) method was developed to separate and analyze four anti-viral drugs. The method utilized a Thermo Hypersil ODS C18 HPLC column with an isocratic elution mode employing a mobile phase consisting of sodium dodecyl sulfate (0.15 M, pH 3), isopropanol and triethylamine (79.7:20:0.3, v/v/v). The Design Expert 8® software was used to optimize four factors simultaneously and the developed quadratic D-optimal model could achieve maximum resolution between peaks while minimizing the run time. The method was found linear over the concentration ranges of 10–100 μg/mL for sofosbuvir, daclatasvir, and 20–200 μg/mL for ledipasvir and velpatasvir, and successfully applied to the analysis of sofosbuvir and velpatasvir in tablets. The MLC method was compared with a previously reported high-performance liquid chromatography (HPLC) method using the analytical GREEnness metric approach (AGREE), and was found to exhibit greener performance due to the use of safer reagents. The results indicate the high potential of micellar liquid chromatography in the separation of quaternary mixtures with the aid of response surface methodologies.

Multivariate Optimization of Chromatographic Conditions for Rapid Simultaneous Quantification of Antidiarrheal Drugs in Formulation Using Surface Response Methodology

A combination of antibiotics and antiprotozoal and antisecretory medicines has been prescribed for the treatment of diarrhea. A rapid, reproducible liquid chromatographic procedure was established for the concurrent analysis of metronidazole (MET), ofloxacin (OFL), and racecadotril (RAC) in suspension. The Box–Behnken design, a full factorial multivariate optimization technique, was utilized to optimize chromatographic parameters with fewer runs. The separation of MET, OFL, and RAC was accomplished within 3.2 min, using a Zorbax C18 high-performance liquid chromatography column with a simple mobile phase comprising acetonitrile (55 vol.%): methanol (10 vol.%):20 mM phosphate buffer (35 vol.%, pH 6, regulated with ortho-phosphoric acid). The mobile phase was pumped in the isocratic mode at a rate of 1.4 mL/min at ambient temperature. Analytes were monitored by adjusting the wavelength at 295 nm for MET and OFL and 231 nm for RAC. Validation of the proposed HPLC method exhibited linearity in the concentration of 20–250 µg/mL, 10–150 µg/mL, and 5–80 µg/mL for MET, OFL, and RAC respectively, along with an excellent regression coefficient (r2 > 0.999). The accuracy and precision of the chromatographic procedure were also evidenced by the low percent relative error and relative standard deviation. A Pareto chart developed by the two-factor interaction (2FI) study confirmed that the method was robust, as the slight variation in a single factor had no significant influence on the assay outcomes. Lastly, the developed HPLC process was utilized for the concurrent quantification of MET, OFL, and RAC in liquid oral preparation. Furthermore, when the assay results were compared to the described techniques, it was discovered that there was no significant difference in the accuracy and precision of the results. Hence, the developed rapid HPLC method could be employed for the quality control study of a preparation comprising of MET, OFL, and RAC in industries and regulatory authority laboratories.

Regional Differences in Bile Acid Composition in Gallbladder Bile

Background: Chemical and structural analyses of gallstones (GS) from the Indian subcontinent has shown that the formation of GS type is dependent on regional and dietary factors. Aim of the Study: The aim is to determine the proportion of primary and secondary bile acids in gallbladder (GB) bile in patients with GS from South and North India using high-performance liquid chromatography (HPLC). Materials and Methods: Standards for primary and secondary bile acids were prepared and concentrations were determined by reversed-phase C18 HPLC column. Thirty-three GB bile samples from southern India and 28 samples from northern states of India were analyzed for differences in the proportion of primary and secondary bile acids. Ethics Committee of Gleneagles Global Health City, Chennai, approved the study. Statistical Analysis: concentration of bile acids (in mmol/L) were expressed as median and range. Chi-square test and Mann–Whitney U-test were applied. A P < 0.05 was considered as significant. Results: The median concentrations of cholic acid (CA) (P = 0.005) and its derivative deoxycholic acid (DCA) (P < 0.006) were significantly high in GB bile samples from South India with no differences in the concentration of chenodeoxycholic acid between the two samples. Furthermore, samples from North India had a significantly higher proportion of lithocholic acid (LCA) and low DCA compared to samples from South India. Conclusion: Primary bile acid CA and its derivative is high in GB bile from South; the proportion of hepatotoxic LCA is significantly high with low concentrations of DCA in bile samples from North India.

Determination of dehydroepiandrosterone in dietary supplements by reversed-phase HPLC


 
 
 
 Purpose: To develop a reversed phase high performance liquid chromatography (HPLC) method for the determination of dehydroepiandrosterone (DHEA) in dietary supplements.
 Methods: A reversed-phase high performance liquid chromatography (HPLC) method was developed for the determination of DHEA in dietary supplements. An isocratic system consisting of methanol and water (70:30 v/v) was run at a flow rate of 1 mL/min on a C18 HPLC column to achieve the separation. The method was validated with regard to linearity, intra-day and inter-day precision, and limits of both detection and quantification.
 Results: The method achieved a retention time of 10.8 min, a resolution of 4.12, a detection limit (LOD) of 50 ng/μL, a quantification limit (LOQ) of 166.7 ng/μL and a label claim of 108.6 % with a relative standard deviation (RSD) of 0.38 % over a range of 0.0625 – 0.50 mg/mL with a correlation coefficient of 0.9997.
 Conclusion: The method is simple, cost effective, time-saving and reliable for determining DHEA when compared to other reported methods in literature. Thus, it will be of benefit to manufacturers of this dietary supplement to adopt the method for quantitative laboratory analysis.
 
 
 

In Vitro and In Vivo Evaluation of Two Hydroxychloroquine Tablet Formulations: HPLC Assay Development.

The relative in vitro and in vivo evaluation of two hydroxychloroquine (HCQ) products was conducted. In vitro studies involved assay, content uniformity and dissolution test, and a two-way crossover fashion were used for in vivo studies. Blood samples were collected at appropriate intervals and HCQ levels were measured using a validated reversed-phase high-performance liquid chromatography (HPLC) method. The drug and the internal standard, chloroquine (CQ), were extracted from blood with diethyl ether, separated and dried under nitrogen gas. Residues were reconstituted in the mobile phase and analyzed at 340nm on a μ-bondapack C18 (250×4.6mm) HPLC column with acetonitrile:methanol:KH2PO4 (10:10:80) mixture containing 0.01% triethylamine. The standard curve was linear within 50-1,500ng/mL HCQ (R2=0.9996), relative errors were 1.6 to 5%, and the CV% ranged from 7 to 15.4. The resolution factor and RSD were 1.62 and 0.35% and in vitro data of both products met the USP requirements. The 90% confidence intervals for the ratios of the AUC0-96, Cmax and Tmax and their corresponding logarithmically transformed values of generic product over those of Plaquenil® were within the acceptable limit of 0.80-1.20 and 0.80-1.25, respectively. Therefore, the generic HCQ was bioequivalent to the innovator formulation.

Simultaneous Determination of Moxifloxacin and Flavoxate by RP-HPLC and Ecofriendly Derivative Spectrophotometry Methods in Formulations.

Simple, fast, and precise reversed-phase (RP)-high-performance liquid chromatography (HPLC) and two ecofriendly spectrophotometric methods were established and validated for the simultaneous determination of moxifloxacin HCl (MOX) and flavoxate HCl (FLX) in formulations. Chromatographic methods involve the separation of two analytes using an Agilent Zorbax SB C18 HPLC column (150 mm × 4.6 mm; 5 µm) and a mobile phase consisting of phosphate buffer (50 mM; pH 5): methanol: acetonitrile in a proportion of 50:20:30 v/v, respectively. Valsartan was used as an internal standard. Analytes were monitored by measuring the absorbance of elute at 299 nm for MOX and 250 nm for FLX and valsartan. Two environmentally friendly spectrophotometric (first derivative and ratio first derivative) methods were also developed using water as a solvent. For the derivative spectrophotometric determination of MOX and FLX, a zero-crossing technique was adopted. The wavelengths selected for MOX and FLX were −304.0 nm and −331.8 nm for the first derivative spectrophotometric method and 358.4 nm and −334.1 nm for the ratio first-derivative spectrophotometric method, respectively. All methods were successfully validated, as per the International Conference on Harmonization(ICH) guidelines, and all parameters were well within acceptable ranges. The proposed analytical methods were successfully utilized for the simultaneous estimation of MOX and FLX in formulations.

Development of stability-indicating HPLC method and accelerated stability studies for osmotic and pulsatile tablet formulations of Clopidogrel Bisulfate

The purpose of the present study is to develop a simple, rapid and sensitive stability-indicating high-performance liquid chromatography (HPLC) method for Clopidogrel Bisulfate (CBS) and further extending it for assessment of CBS stability in osmotic and pulsatile tablet formulations tested under accelerated conditions. A stability-indicating HPLC method for quantitative determination of CBS in gastro-retentive formulations is developed by using a C18 HPLC column, acetonitrile and 0.1% formic acid (60:40 v/v) as mobile phase, with a flow rate of 0.9 mL/min, UV detection at 222 nm and subsequently validated. The key objective was to analyze the stability profile of formulations under accelerated conditions. The retention time (Rt) of CBS was observed as 5.9 min with the linearity range between 0.06–1.95 μg/mL. Forced degradation studies were performed on bulk samples of CBS using acidic, basic, oxidative, thermal (80 °C) and photolytic (under sunlight) conditions. The resulting method was validated as per ICH Q2(R1) guidelines. Moreover, an attempt has been made to identify the degradation products by Liquid chromatography-mass spectrometry (LC–MS) analysis. The proposed method was successfully applied to novel gastro-retentive tablet formulations (osmotic tablet and pulsatile tablet) for assessment of stability under accelerated conditions.

Development of a Rapid and Precise Reversed-phase High-performance Liquid Chromatography Method for Analysis of Docetaxel in Rat Plasma: Application in Single-dose Pharmacokinetic Studies of Folate-targeted Micelles Containing Docetaxel.

Background:A simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method based on liquid-liquid extraction was established and validated for determination of docetaxel (DTX) in plasma of rat.Materials and Methods:Samples were spiked with paclitaxel as the internal standard and the chromatographic separation was carried out using C18 HPLC column. The mobile phase consisted of a mixture of acetonitrile/water with the ratio of 60/40 v/v. The ultraviolet detector was operated at 230 nm, and the flow rate of mobile phase was 1 ml/min. The method was validated for linearity, precision, accuracy, recovery, and limit of quantification (LOQ). Then the method was applied to quantify DTX in the rat plasma after intravenous (IV) administration of the self-assembled micelles of folate-targeted Synpronic F127/cholesterol (FA-PF127-Chol) loaded with DTX and Taxotere® as the reference marketed solution of DTX. The blood samples were taken from the ophthalmic vein at predetermined time intervals after treatment.Results:Calibration curve was linear between the concentration ranges of 0.1–7.5 μg/ml with the relative standard deviation % and evaluating error % ranged from 2.263 to 15.53 and −12.75 to 12.7 for intra- and inter-day validity, respectively. The mean recovery of the drug after plasma extraction was 95.67 ± 0.99% for the concentration of 1 μg/ml. The LOQ and the limit of detection for DTX in serum were 100 ng/ml and 30 ng/ml, respectively.Conclusions:The results indicated that the developed method could be adopted for pharmacokinetic studies of DTX-loaded FA-PF127-Chol micelles and Taxotere® in rat.

Simultaneous determination of kasugamycin and validamycin-A residues in cereals by consecutive solid-phase extraction combined with liquid chromatography–tandem mass spectrometry

ABSTRACTTwo polar aminoglycosides, kasugamycin and validamycin-A, were determined in cereals (brown rice, wheat and corn) by high-performance liquid chromatography–tandem mass spectrometry. The analytes were extracted from samples using methanol and water (70:30, v/v) at pH 5.5, purified using both a hydrophilic–hydrophobic-balanced cartridge and a strong cation-exchange cartridge, and then analysed using multiple reaction monitoring in positive electrospray ionisation mode with a special ReproSil 100 C18 high-performance liquid chromatography column. This newly proposed method yielded good sensitivity and excellent chromatographic performance. The limits of quantification for kasugamycin and validamycin-A were 4.1 µg/kg and 1.0 µg/kg, respectively. The recoveries for both compounds at three fortification levels (4, 100 and 500 µg/kg for kasugamycin; 1, 10 and 100 µg/kg for validamycin-A) ranged from 75% to 110%, and the relative standard deviations were below 15%.

A Direct Method for the Measurement of Everolimus and Sirolimus in Whole Blood by LC–MS/MS Using an Isotopic Everolimus Internal Standard

Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. Whole-blood samples (20 μL) were treated with ammonium bicarbonate (20 μL), zinc sulfate (20 μL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 μL). After centrifugation, 20 μL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 μg/L for sirolimus, 1-41 μg/L for everolimus), and a lower limit of quantitation of 1 μg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

DEVELOPMENT OF A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR QUANTIFICATION OF FLUAZURON IN CATTLE TISSUES

A high-performance liquid chromatography (HPLC) method with UV detection was developed for determination of fluazuron in cattle tissues. Tissue samples extracted with acetonitrile using an ultrasonic bath were partially purified using solid-phase extraction (SPE) Florisil cartridges. Separation was achieved in an Agilent C18 HPLC column using an isocratic system eluting with acetonitrile and water (68:32, v/v) at a flow rate of 1.2 mL min−1, with UV detection at 260 nm. The recovery of fluazuron from different tissues, spiked at levels of 0.02, 1.0, and 10.0 mg kg−1 ranged from 82.0–94.59% in muscle, 81.86–95.35% in kidney, 81.89–95.26% in liver, and 80.84–95.24% in fats; the inter-day and intra-day relative standard deviation (RSD) values were less than 6.0%. The limit of detection for fluazuron was 0.005 mg kg−1 and the limit of quantification was 0.01 mg kg−1. This simple and rapid method may be applicable to the determination of fluazuron in animal tissues.

Quantitative determination of imatinib stability under various stress conditions

Objective:A simple, rapid, reverse phase and stability-indicating high-performance liquid chromatography (HPLC) method for the estimation of imatinib in solution and in plasma under forced degradation conditions was developed.Materials and Methods:The method employed isocratic elution using a Waters Atlantis C18 (5 μ, 4.6 mm × 150 mm) HPLC column. The mobile phase consisted of acetonitrile and 10 mM KH2PO4 buffer in the ratio of 35:65 (v/v, pH = 4.6), which was delivered using isocratic flow at a rate of 1 mL/min. The injection volume of 50 μL and imatinib was monitored using UV detection 270 nm after a clean-up step with diethyl ether and with a total run time of 6 min.Results:The method was validated in solution as well as in plasma, and the response was found to be linear in the concentration range of 0.5-20 μg/mL. The coefficient of correlation was found to be >0.99. Forced degradation studies revealed that imatinib undergoes degradation under different stress conditions.Discussion:The developed HPLC method could effectively resolve degradation product peaks from imatinib except at neutral pH. Further, no interference was found at the retention time of imatinib from any plasma components, indicating selectivity of the developed method. The limits of detection and quantitation of the method were 0.025 and 0.5 μg/mL, respectively.

Stability-Indicating HPLC Method for the Determination of Darunavir Ethanolate

A novel stability-indicating reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the quantitative determination of darunavir ethanolate, an HIV-1 protease inhibitor. The chromatographic separation was achieved using an X-Bridge C18 (150 × 4.6 mm × 3.5 µm) HPLC column in isocratic mode employing 0.01M ammonium formate (pH.3.0) buffer and acetonitrile in the ratio of 55:45 (v/v) with a flow rate of 1.0 mL/min. The detector wavelength was monitored at 265 nm and the column temperature was maintained at 30°C. Darunavir ethanolate was exposed to thermal, photolytic, acid, base and oxidative stress conditions. Considerable degradation of the drug substance was found to occur under acid, base and oxidative stress conditions. The peak homogeneity data of darunavir ethanolate obtained by photodiode array detection demonstrated the specificity of the method in the presence of degradants. The degradation products were well resolved from primary peak of darunavir, indicating that the method is specific and stability-indicating. The HPLC method was validated as per International Conference on Harmonization guidelines with respect to specificity, precision, linearity, accuracy and robustness. Regression analysis showed a correlation coefficient value greater than 0.999. The accuracy of the method was established based on the recovery obtained for darunavir ethanolate.

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu 2SO 4. The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min −1, and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g −1 ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0–2.0 μg g −1. This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.

Prediction of pesticides chromatographic lipophilicity from the computational molecular descriptors

Quantitative structure-property relationship models were developed for the prediction of pesticides and some PAH compounds lipophilicity based on a wide set of computational molecular descriptors and a set of experimental chromatographic data. The chromatographic lipophilicity of pesticides has been evaluated by high-performance liquid chromatography (HPLC) using different chemically bonded (C18, C8, CN and Phenyl HPLC columns) stationary phases and two different organic modifiers (methanol and acetonitrile, respectively) in the mobile phase composition. Through a systematic study, by using the classic multivariate analysis, several quantitative structure-property/lipophilicity multi-dimensional models were established. Multiple linear regression and genetic algorithm for the variable subset selection were used. The internal and external statistical evaluation procedures revealed some appropriate models for the chromatographic lipophilicity prediction of pesticides. Moreover, the statistical parameters of regression and those obtained by applying t-test for the intercept (a(0)) and for the slope (a(1)) in order to evaluate relationship between experimental and predicted octanol-water partition coefficients in case of the test set compounds, revealed two statistically valid models that can be successfully used in lipophilicity prediction of pesticides.

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5 μm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.

Quantitative Determination of ABT-925 in Human Plasma by On-Line SPE and LC-MS/MS: Validation and Sample Analysis in Phase II Studies

A fully automated 96-well On-Line Solid Phase Extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometric (MS/MS) method for the determination of ABT-925 (2-{3-[4-(2-tert-Butyl-6-trifluoromethyl-pyrimidin-4-yl)-piperazin-1-yl)-propyl-sulfanyl}-3H-pyrimidin-4-one fumarate) in human plasma was developed, validated and utilized in Phase II clinical studies. 50 µL of plasma sample was fortified with internal standard (IS, d8-ABT-925) and extracted on-line with Cohesive Turbo Flow Cyclone P HTLC column. The chromatographic separation was performed on Aquasil C18 (3 μm 50 × 3 mm) HPLC column with a mobile phase consisting of 50/50/0.1 (v/v/v) ACN/H2O/formic acid. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 457.4 → 329.4 for analyte and m/z 465.5 → 337.5 for IS. The peak area ratio (analyte/IS) was used to quantitate ABT-925. A dynamic range of 0.0102 μg/mL to 5.24 μg/mL was established after the validation. The validated method was then used for two Phase II studies. To demonstrate the method reproducibility, approximately 10% of the incurred samples from one study were repeated in singlet. The repeated values were compared to the initial values. All repeated values agreed within ±15% of the mean values.

The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

Probing the interaction of solvents with the stationary phase of C18 high-performance liquid chromatographic column material by variable-temperature dependent 129Xe nuclear magnetic resonance spectroscopy

VT 129Xe NMR was applied to probe the interactions of solvents having different polarity indices with the stationary phase of a RP-C18 HPLC column material. It was observed that the highly polar ethylene glycol molecules do not mix with the alkyl chains of the RP-C18 stationary phase and the solvent is unable to enter the pores and the spaces between the particles. Three phases in this sample are defined as stationary/xenon phase, xenon gas phase (in the pores and the spaces between the particles) and ethylene glycol/xenon phase. In contrast to ethylene glycol, the nonpolar solvent cyclohexane was observed to be well mixed with the RP-C18 stationary phase. The capillary rise effect allows the solvent to enter the pores and the spaces between the particles. Two phases in this sample are defined as stationary/cyclohexane/xenon phase and cyclohexane/xenon phases. The properties of ethyl acetate are between those of ethylene glycol and cyclohexane. The 129Xe NMR results show that the rational reversed phases should be conditioned from highly solvating to more polar solvents to remove the trapped air. The 129Xe NMR results also show that pure stationary phase exists only when a highly polar solvent is used in reversed-phase chromatography. For a solvent with lower polarity, a stationary/solvent phase actually forms. This, together with the mobile phase, determines the selective factor for separating mixtures.

A simplified analytical method for a phenotyping cocktail of major CYP450 biotransformation routes

An efficient, fast and reliable analytical method was developed for the simultaneous evaluation of the activities of five major human drug metabolising cytochrome P450 (1A2, 2C9, 2C19, 2D6 and 3A4) with a cocktail approach including five probe substances, namely caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam. All substances were administered simultaneously and a single plasma sample was obtained 2 h after the administration. Plasma samples were handled by liquid-liquid extraction and analysed by gradient high performance liquid chromatography (HPLC) coupled to UV and fluorescence detectors. The chromatographic separation was achieved using a Discovery semi-micro HS C18 HPLC column (5 μm particle size, 150 mm×2.1 mm i.d.) protected by a guard column (5 μm particle size, 20 mm×2.1 mm i.d.) The mobile phase was constituted of a methanol, acetonitrile and 20 mM ammonium acetate (pH 4.5) with 0.1% triethylamine mixture and was delivered at a flow rate of 0.3 mL min −1. All substances were separated simultaneously in a single run lasting less than 22 min. The HPLC method was formally validated and showed good performances in terms of linearity, sensitivity, precision and accuracy. Finally, the method was found suitable for the screening of these compounds in plasma samples.