Performance comparison between packed column supercritical fluid chromatography (SFC) and HPLC using cyclandelate as the model analyte

A reproducible and fast method has been developed for the assay of cyclandelate in bulk and drug forms using packed column supercritical fluid chromatography using dicyclohexyl phthalate (DCHP) as internal standard. The drug and the internal standard were resolved by elution with supercritical fluid carbon dioxide doped with 14.29% (v/v) methanol on an RP-C₁₈ column and detected spectrophotometrically at 228 nm. Chromatographic figures of merit using C₈, C₁₈, cyano and phenyl columns have been assessed. Parallel experiments have been performed by HPLC and the data have been compared. Supercritical fluid extraction using CO₂ modified with a small amount of methanol was found to give quantitative analytical recoveries of cyclandelate from a dosage form. SFC has been shown to be a viable, faster alternative technique to HPLC generating less disposable waste.     

High Performance Liquid Chromatographic Determination of Amiodarone in Plasma and Tissues

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of amiodarone (AD) in plasma and tissues was developed. The method involved deproteinization of plasma or homogenized tissue with acetonitrile containing an internal standard (N-Cetylpyridinium chloride) followed by reversed phase chromatography using μ bondapack C₁₈ column (10μm) with a mobile phase consisting of acetonitrile - methanol - sodium dihydrogen phosphate buffer (70:10:20%, v/v), the pH adjusted to 4.0 and pumped at flow rate of 1.0 ml/min. The column effluent was monitored at 242 nm. A linear relationship was obtained between peak height ratios (drug to internal standard) versus drug levels over the concentration range of 50–750 ng/ml. The detection limit of AD in plasma and tissues by this method was 20 ng/ml.     

Development of an Isocratic SFC Method for Four Centrally Active Muscle Relaxant Drugs

Abstract

A reproducible and selective supercritical fluid chromatography (SFC) method was developed for the estimation of four centrally active muscle relaxants, viz., chlorzoxazone, methocarbamot, tizanidine, and baclofen, using guaifenesin (expectorant, muscle relaxant) as the internal standard. The effect of temperature, pressure and the modifier concentration on retention of the five solutes has been explored and the parameters optimised. An arbitrary mixture of 5 components was base line resolved on a JASCO- RP- C₁₈ (150 × 4.6 mm) 5μ metaphase column with a tertiary mobile phase of 9.1 % modifier [methanol containing 2 % glacial acetic acid, v/v] in carbon dioxide at 1.5 ml/min, 50°C temperature, and 12.75 MPa outlet pressure. UV detection at 235 nm was employed. Without the acetic acid in the mobile phase it was not possible to elute tizanidine and baclofen. With the additive, all the analytes produced symmetrical peaks. Quantitative validation concentration ranges have been found, and the performance data evaluated.     

Packed column supercritical fluid chromatographic separation and estimation of acetaminophen, diclofenac sodium and methocarbamol in pharmaceutical dosage forms

A reproducible and fast method has been developed for the assay of acetaminophen, methocarbamol, and diclofenac sodium in bulk and drug forms using packed column supercritical fluid chromatography employing internal standard method. The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 11.1% (v/v) methanol on a Shendon-Phenyl (250x4.6 mm) 5 mum column with detection monitored spectrophotometrically at 225 nm. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on chromatograhic figures like retention time (t(R), min), retention factor (k(')) etc. Modifier concentration proved to be the most effective means for changing both retention and selectivity. Calibration data and recovery of the drug from spiked concentrations were determined to assess the viability of the method. The supercritical fluid chromatography (SFC) method was directly compared to an HPLC assay, developed in the laboratory, of the same analytes. With respect to speed and use of organic solvents SFC was found to be superior, while in all other aspects the results were similar to HPLC. The method has been successfully used for the assay of two formulations containing a combination of (A) acetaminophen and methocarbamol and (B) acetaminophen and diclofenac sodium. There was no interference from excipients. The present work validates the recent proposition that supercritical fluid chromatography using CO(2) and modifiers is a viable, faster alternative to reverse phase HPLC.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC determination of valproic acid in human plasma by derivatization with O-p-nitrobenzyl-N,N′-diisopropylisourea

A sensitive and specific method for the determination of valproic acid in plasma has been developed. After the proteins in the plasma have been precipitated with a saturated solution of ammonium sulfate in 1N HCl, the valproic acid, together with the internal standard, is extracted from the plasma with dichloromethane. An aliquot of the organic solution is taken for derivatization of the valproic acid and the internal standard with O-p-nitrobenzyl-N,N′-diisopropylisourea. Separation is carried out by HPLC using two chromatographic systems: an adsorption system with a μ Porasil column, hexane-chloroform (94:6) as mobile phase, and caproic acid as internal standard and a partition reverse phase system comprising a μ Bondapak T_M/C₁₈ column, acetonitrile/methanol/0.0035 M phosphate buffer (60:10:30), and caprylic acid as internal standard. UV detection is at 254 nm. This method, developed in both systems, permits the determination of plasma levels of valproic acid in the reported range of 50-100 μg/mL. With adequate sensitivity, specificity, precision, and accuracy. The plasma levels of valproic acid may be determined by this method without interference from the commonest antiepileptic drugs. Good correlation is obtained with the enzymatic immune analytic method: EMIT.     

Development and validation of a simple and efficient HPLC method for the determination of zonisamide in pharmaceuticals and human plasma

A simple and efficient liquid chromatographic method has been developed and validated for the determination of zonisamide in pharmaceuticals and human plasma. Plasma samples are analyzed after one step protein precipitation with methanol, and chromatographic separation of zonisamide and chloramphenicol (internal standard) is carried out using a C₁₈ column and the optimum mobile phase of acetonitrile/methanol/distilled water (20: 10: 70, v/v/v). The method is validated in both mobile phase and human plasma, and the obtained limits of quantification values are 0.099 and 0.12 μg/mL in mobile phase and human plasma, respectively. Fully validated method is reproducible and selective for the determination of zonisamide in pharmaceuticals and human plasma.     

High Performance Liquid Chromatograph 1C Method for Determination of Mifobate in Rat Feed

Abstract

A high performance liquid Chromatographie (HPLC) method is described for the quantitative determination of Mifobate (SR-202) in rat feed. Mifobate is extracted in acetone and isolated from other extractants on a Waters Sep-pak® C₁₈ disposable pre-column. The extracted drug and internal standard are chromatographed on a μBondapak? C₁₈ reverse phase column with a mobile phase consisting of water and acetonitrile (55:45, v/v). The eluent is monitored at 225 nm.

The method provided a 101.56 ± 5.1% mean recovery of Mifobate from spiked feed samples ranging in the 22.24 to 433.04 mg/kg concentration range. Standard curves bracketing this concentration range had linear coefficients greater than 0.9998. The average relative standard deviation (%) for the entire concentration range was 4.2%. The critical steps and precision of the method were also evaluated.     

Enantiospecific analysis of ibuprofen by high performance liquid chromatography: Determination of free and total drug enantiomer concentrations in serum and urine

Summary A reversed-phase high-performance liquid chromatographic (HPLC) assay, based on the indirect approach to enantiomeric analysis, for the determination of ibuprofen in human serum and urine has been developed. Following the addition of (R,S)-flurbiprofen, as internal standard, the enantiomers of ibuprofen were isolated from plasma and urine samples by liquid-liquid extraction at acidic pH. The enantiomers of flurbiprofen and ibuprofen were derivatized with (R)-1-(naphthen-1-yl)ethylamine, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole as coupling reagents, to yield the corresponding diastereoisomeric amides. Chromatographic resolution of the derivatives was achieved using a C₁₈ column (Waters Resolve C₁₈; 5 μm, 150×3.9 mm) using a mobile phase of phosphate buffer (pH 3.5, 0.01 M): acetonitrile (50∶50 v/v) at a flow rate of 1.5 mL min⁻¹ at ambient temperature. Quantification was carried out using a spectrofluorometer with excitation and emission wavelengths of 290 and 330 nm respectively. The use of a semimicrobore column (150×2.1 mm) containing the same stationary phase facilitated the analysis of the free drug enantiomer concentrations following equilibrium dialysis. The derivatization procedure was carried out as described above but with a reduction in the quantities of the reagents used in order to reduce the background noise in the chromatographic analysis. The HPLC methodology for the determination of free drug enantiomer concentrations was validated against a previously reported method employing the radiolabelled drug.     

Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study

A selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid‐phase extraction using 100 μL of human plasma. The separation was carried out on a BDS Hypersil C₁₈ (150 × 4.6 mm, 5 μm) column using a mixture of 0.2% formic acid in HPLC‐grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20–20 μg/mL with r² > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of an amide‐based stationary phase for supercritical fluid chromatography

A relatively new stationary phase containing a polar group embedded in a hydrophobic backbone (i.e., ACE ® C18‐amide) was evaluated for use in supercritical fluid chromatography. The amide‐based column was compared with columns packed with bare silica, C₁₈ silica, and a terminal‐amide silica phase. The system was held at supercritical pressure and temperature with a mobile phase composition of CO₂ and methanol as cosolvent. The linear solvation energy relationship model was used to evaluate the behavior of these stationary phases, relating the retention factor of selected probes to specific chromatographic interactions. A five‐component test mixture, consisting of a group of drug‐like molecules was separated isocratically. The results show that the C₁₈‐amide stationary phase provided a combination of interactions contributing to the retention of the probe compounds. The hydrophobic interactions are favorable; however, the electron donating ability of the embedded amide group shows a large positive interaction. Under the chromatographic conditions used, the C₁₈‐amide column was able to provide baseline resolution of all the drug‐like probe compounds in a text mixture, while the other columns tested did not.     

Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract

A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C₁₈, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C₁₈ column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C₁₈ insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine steady-state concentrations of the drug and its metabolites in paediatric patients.     

A new validated HPLC method for the determination of levodopa: Application to study the impact of ketogenic diet on the pharmacokinetics of levodopa in Parkinson's participants

A simple, accurate, and reproducible HPLC‐UV method has been developed and validated for the quantification of levodopa (l ‐Dopa) in human plasma. The method involves a simple protein precipitation procedure to extract both l ‐Dopa and methyldopa, the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20A HPLC system equipped with a Zorbax Eclipse XDB C₁₈ column and an isocratic mobile phase consisting of 20 mm KH₂PO₄ (pH 2.5) and methanol (95:5, v/v) run at a flow rate of 1 mL/min. The UV detection wavelength was set at 230 nm. The method exhibited good linearity (R² > 0.999) over the assayed concentration range (0.1–10 μg/mL) and demonstrated good intra‐ and inter‐day precision and accuracy (relative standard deviations and the deviation from predicted values were <15%). This method was also successfully applied for studying the potential effect of ketogenic diet on the pharmacokinetics of l ‐Dopa in Parkinson's participants. Our data analysis indicates that ketogenic diet does not significantly affect the pharmacokinetics of l ‐Dopa.     

A high performance liquid chromatography-tandem mass spectrometric method for the determination of mefenamic acid in human plasma: application to pharmacokinetic study

In the present study, the development and validation of an LC‐MS/MS method for quantifying mefenamic acid in human plasma is described. The method involves liquid–liquid extraction using diclofenac as an internal standard (IS). Chromatographic separation was achieved on a Thermo Hypurity C₁₈, 50 × 4.6 mm, 5 µm column with a mobile phase consisting of 2 m m ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) at a flow‐rate of 0.75 mL/min and the total run time was 1.75 min. Analyte was introduced to the LC‐MS/MS using an atmospheric pressure ionization source. Both the drug and IS were detected in negative‐ion mode using multiple reaction monitoring m/z 240.0 → 196.3 and m/z 294.0 → 250.2, respectively, with a dwell time of 200 ms for each of the transitions. The standard curve was linear from 20 to 6000 ng/mL. This assay allows quantification of mefenamic acid at a concentration as low as 20 ng/mL in human plasma. The observed mean recovery was 73% for the drug. The applicability of this method for pharmacokinetic studies has been established after successful application during a 12‐subject bioavailibity study. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 John Wiley & Sons, Ltd.     

High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

Determination of clarithromycin in rat plasma by HPLC-UV method with pre-column derivatization

A novel high-performance liquid chromatographic (HPLC) method using pre-column derivatization and UV detection at 275 nm for the determination of clarithromycin in rat plasma has been validated. Clarithromycin was extracted from plasma sample spiked with internal standard (erythromycin) under alkaline condition with ethyl ether and derivatizated with trimethylbromosilane. The analyses were run on a C₁₈ column, maintained at 40 °C during elution, using a mobile phase comprised of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), acetonitrile, and methanol (30:45:25, v/v/v). The standard calibration curve for clarithromycin was linear (r² = 0.9998) over the concentration range of 0.1-10 μg ml⁻¹ in rat plasma. The limit of detection (LOD) and limit of quantitation (LOQ) was 30 ng ml⁻¹ and 0.1 μg ml⁻¹ respectively. The intra- and inter-day assay variability range was 2.6-7.4% and 3.3-8.5%, respectively. This method has been successfully applied to a pharmacokinetic study of clarithromycin in rats.