Bondapak C18 Column Research Articles

Application of HPLC Method for Quality Control of Cefadroxil

Our previous study has shown that HPLC method is a suitable substitute for the microbiological method for potency assays and stability studies of cefadroxil preparations. The samples were analysed on u--Bondapak C18 column with a mobile phase consisting of acetonitrile and phosphate buffer. The assay was also applied successfully to five different commercial brands and proved to be free of interference from excipients normally used informulations. The contents of unifomity and dissolution rates in different brands were performed. The results have shown that this analytical method could be utilized readily for routine quality control of cefadroxil pharmaceuticals, since it offers a simple system and short analytical time coupled with reproducibility and accuracy.

Reversed-phase liquid chromatography with electrospray mass detection and 1H and 13C NMR characterization of new process-related impurities, including forced degradants of efavirenz: related substances correlated to the synthetic pathway.

In this study, a stability-indicating reversed-phase liquid chromatographic electrospray mass spectrometric method was developed and validated for the determination of process-related impurities and forced degradants of Efavirenz in bulk drugs. Efavirenz was subjected to acid, alkaline hydrolysis, H2O2 oxidation, photolysis, and thermal stress. Significant degradation was observed during alkaline hydrolysis, and the degradants were isolated on a mass-based purification system and characterized by high-resolution mass spectrometry, positive electrospray ionization tandem mass spectrometry, and (1)H and (13)C NMR spectroscopy. Accurate mass measurement and NMR spectroscopy revealed the possible structure of process-related impurities and degradant under stress conditions. The acceptable separation was accomplished on Waters bondapak C18 column (250 mm × 4.6 mm; 5 μm), using 5 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min. The eluents were monitored by diode array detector at 247 nm and quantitation limits were obtained in the range of 0.1-2.5 μg/mL for Efavirenz, degradants, and process-related impurities. The liquid chromatography method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantification as per International Conference on Harmonization guidelines.

Liquid chromatographic method for the simultaneous determination of captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulation, and human serum by programming the detector

A highly sensitive LC method with UV detection has been developed for the simultaneous determination of coadministered drugs captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulations, and human serum at the isosbestic point (235 nm) and at individual λmax (220, 255, and 238 nm, respectively) by programming the detector with time to match the individual analyte's chromophore, which enhanced the sensitivity with linear range. The assay involved an isocratic elution of analytes on a Bondapak C18 (10 μm, 25 × 0.46 cm) column at ambient temperature using a mobile phase of methanol/water 80:20 at pH 2.9 and a flow rate of 1.0 mL/min. Linearity was found to be 0.25-25, 0.10-6.0, and 0.20-13.0 μg/mL with correlation coefficient >0.998 and detection limits of 7.39, 3.90, and 9.38 ng/mL, respectively, whereas calibration curves for wavelength-programmed analysis were 0.10-6.0, 0.04-2.56, and 0.10-10.0 μg/mL with correlation coefficient >0.998 and detection limits of 5.79, 2.68, and 3.87 ng/mL, respectively. All the validated parameters were in the acceptable range. The recovery of drugs was 99.32-100.39 and 98.65-101.96% in pharmaceutical formulation and human serum, respectively, at the isosbestic point and at individual λmax . This method is applicable for the analysis of drugs in bulk drug, tablets, serum, and in clinical samples without interference of excipients or endogenous serum components.

Ultra Sensitive Liquid Chromatographic Method for the Simultaneous Determination of Carbamazepine with Nsaids in API, Pharmaceutical Formulation and Human Serum by Programming the Detector: Application to In Vitro Drug Interaction

Here we describe an ultra-sensitive reverse phase liquid chromatographic method for the simultaneous determination of carbamazepine with NSAIDs in API, pharmaceutical formulation and human serum with UV detection. The separation of these multiple drug components was made by a Bondapak C18 column (10 µm, 25 cm×0.46 cm) using a moderate acidic mixture of mobile phase i.e., methanol:water 80:20 with pH adjusted to 3.0 using 85% o-phosphoric acid. The flow rate of the mobile phase was affixed at 1.0 ml min -1 for the best minimum interval of elusion of all the analytes. The detector response was monitored at isobestic point of 220 nm at ambient temperature. Calibration curves were obtained in the range of 0.4-12 µg mL -1 for carbamazepine, 0.5-16 µg mL -1 for meloxicam and 0.25-8.0 µg mL -1 for ibuprofen and mefenamic acid. Limits of detection were found to be 4.0, 3.0, 1.0, and 13.0 ng mL-1 respectively. The method was compared by programming the detector at individual wavelength of components which showed more sensitivity with linear range 0.10-3.0, 0.15-5.0, 0.10-3.0 and 0.125-4.0 µg mL -1 and detection limits 2.0, 2.0, 1.0, and 3.0 ng mL-1 respectively. ICH guidelines were followed for validation study. The method showed good precision and accuracy within acceptable range and can be successfully applied for the determination of these drugs in pharmaceutical formulations, human serum and clinical laboratories without interference of excipients or endogenous components of serum. In vitro interaction studies have also been carried out at physiological temperature in buffers of various pH (4, 7.4 and 9.0) simulating human stomach environments and % availability has been calculated. Moreover, the solid charge transfer complexes of carbamazepine with interacting NSAIDs were synthesized and characterized by IR spectroscopy and verified by computational molecular modeling.

An Ultra-Sensitive LC Method for Simultaneous Determination of Rosuvastatin, Alprazolam and Diclofenac Sodium in API, Pharmaceutical Formulations and Human Serum by Programming the Detector

Here, we report a rapid and efficient liquid chromatographic method with UV detection for the simultaneous determination of rosuvastatin, alprazolam and diclofenac sodium in API, pharmaceutical formulations and human serum employing a reversed phase Bondapak C18 (25 cm, 0.46 cm, 10 μm) column at room temperature using methanol: water (80:20 v/v) and pH adjusted to 2.9 with 85% o-phosphoric acid. Separation was achieved within a time span of 6 min. The detector response was monitored at 240 nm and the flow rate was set at 1.0 mL.min-1. Various LC parameters were optimized and developed method was validated as per ICH (2006) guidelines. Linear calibration range was determined to be 0.02-0.64, 0.125-4.0 and 0.05-1.6 μg.mL-1 and detection and quantitation limits were found to be 4.0, 17.0, 7.0 and 12.0, 52.0, 22.0 ng.mL-1 for rosuvastatin, alprazolam and diclofenic sodium respectively. Method was linear for all analytes with correlation coefficients >0.998. The intra-day and inter-day accuracy and precision of the assay were in acceptable range of 98.21-101.91% recovery and 0.20-2.07% RSD. The sensitivity of the method was enhanced when the chromatograms were analyzed by programming the detector at individual λmax of 244, 222 and 284 nm for rosuvastatin, alprazolam and diclofenac sodium respectively. High peak intensity was observed on injecting same sample concentration after the detector was programmed. Linearity was obtained even at lower concentrations of 0.02-0.64, 0.05-1.60 and 0.02-0.64 μg.mL-1. LOD and LOQ values shifted down to 3.0, 6.0, 1.0 and 9.0, 19.0, 3.0 ng.mL-1 respectively. Recovery was found to be in the range of 98.32-101.87% and inter-day and intra-day precision was less than 2.10%. The proposed method was found to be robust and successfully employed for the determination of studied drugs in commercial formulations and human serum without interference of excipients or endogenous components of serum.

Determination of arsanilic acid and sulfanilic acid as adulterant in feed additives by reversed-phase high performance liquid chromatography

A reversed-phase high performance liquid chromatographic (RP-HPLC) method was established for the determination of arsanilic acid and sulfanilic acid as adulterant in the feed additives. The separation was carried out on a Waters Bondapak C18 column, and methanol-water (pH 2.9 adjusted by 0.01 mol/L phosphoric acid) (1 : 4, v/v) was used as the mobile phase with a flow rate of 1.0 mL/min. A diode array detector was used at 244 nm as the detection wavelength. Arsanilic acid and sulfanilic acid were separated within 3 min. The linear ranges all were 5 - 200 mg/L and the detection limits (S/N = 3) were 0.20 and 0.15 mg/L for arsanilic acid and sulfanilic acid, respectively. This method is simple and rapid, and suitable for the simultaneous determination of arsanilic acid and sulfanilic acid in feed additives.

Development and Validation of a Liquid Chromatographic Method for the Simultaneous Determination of Estradiol, Estriol, Estrone, and Progesterone in Pharmaceutical Preparations

Progesterone and estrogens are hormones produced in the human body that are essential for regulating many vital functions. The three major estrogens produced by women are estriol, estradiol, and estrone. Progesterone is a naturally occurring hormone in both men and women. Pharmaceuticals containing estrogens alone or estrogens in combination with progesterone are commonly used in therapy. Patients requiring unique combinations of the drugs rely on pharmacies to compound the ingredients. In order to assess the potency of drugs containing combinations of estrogens and progesterone, a method was developed to determine all four ingredients simultaneously. The liquid chromatographic method utilized a microBondapak C18 column with an isocratic mobile phase of acetonitrile-water (50 + 50, v/v) at a flow rate of 1.0 mL/min and temperature of 30 degrees C. Under these conditions, the order of elution was estriol, estradiol, and estrone, followed by progesterone. UV detection was at 205 nm to monitor elution of the estrogens, then switched to 270 nm to monitor progesterone. The method was applied to the analysis of pharmacy-compounded drugs containing combinations of the hormones. Validation studies demonstrated that the method is accurate and precise.

Validated HPLC Method for the Pharmacokinetic Study of Atenolol and Chlorthalidone Combination Therapy in Korean Subjects

A rapid, selective and sensitive reverse-phase HPLC methods for the determination of atenolol and chlorthalidone in human serum and whole blood were validated, and applied to the pharmacokinetic study of atenolol and chlorthalidone combination therapy. Atenolol and an internal standard, pindolol, were extracted from human serum by liquid-liquid extraction, and analyzed on a -Bondapak C18 column in a mobile phase of methanol-0.01 M potassium dihydrogenphosphate(30:70, v/v, adjusted to pH 3.5) and fluorescence detection(emission: 300 nm, excitation: 224 nm). Chlorthalidone and an internal standard, probenecid, were extracted form human whole blood by liquid-liquid extraction, and analyzed on a Luna C18 column in a mobile phase of acetonitrile containing 77% 0.01 M sodium acetate and UV detection at 214 nm. These analysis were performed at three different laboratories using the same quality control(QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum and whole blood, respectively. The methods showed linear responses over a concentration range of 10-1,000 ng/mL for atenolol and 0.05-20 for chlorthalidone, with correlation coefficients of greater than 0.999 at all the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. Stability studies(freeze-thaw, short-, long-term, extracted sample and stock solution) showed that atenolol and chlorthalidone were stable. The lower limit of quantitation of atenolol and chlorthalidone were 10 ng/mL and 0.05 , respectively, which was sensitive enough for pharmacokinetic studies. These methods were applied to the pharmacokinetic study of atenolol and chlorthalidone in human volunteers following a single oral administration of Hyundai tablet(atenolol 50 mg and chlorthalidone 12.5 mg) at three different laboratories.

HPLC Assay of Fluconazole and Its Application to Patients with Early Septic Shock

This work describes a precise, simple, and accurate HPLC method for the determination of fluconazole in plasma samples with enough sensitivity to monitor patients suffering from early septic shock for a month. The separation was done using an HPLC system with UV absorbance detector (set at 262 nm). It was performed using a μ Bondapak C18 column (3.9 × 150 mm2) packed with 10 µm spherical particles. The mobile phase consisted of acetonitrile (20%) in 0.05 M ammonium acetate containing 0.1% triethylamine; acetic acid was used to adjust the pH to 7. Sample run time was 15 min. Metoclopramide was used as the assay internal standard (IS). Using the chromatographic conditions described, fluconazole and metoclopramide were well resolved with mean retention times of 6.7 and 10.2 min, respectively. Linear response (r > 0.996) was observed over the range of 0.2–15 µg/mL of fluconazole. There was no significant difference (p > 0.05) between inter‐ and intra‐day studies for fluconazole. The mean relative standard deviation (RSD%) of the results of within‐day precision and accuracy of the drug was ≤7.6%, which confirmed the reproducibility of the assay method. The applicability of the assay was demonstrated in measuring fluconazole pharmacokinetics in human plasma after daily intravenous infusion of 200 mg fluconazole to patient with early septic shock. Re‐analysis of samples several weeks after the initial analysis showed no loss in fluconazole. Patient with early septic shock showed a mean t 1/2 of fluconazole of 12.3 ± 9 h after a single dose and 41.3 ± 20 h after multiple administrations.

Simple HPLC Method for the Determination of Enalaprilat in Human Plasma

A simple and reliable isocratic reversed phase high performance liquid chromatography (HPLC) method for the determination of enalaprilat in human plasma is described. After plasma extraction, enalaprilat is analyzed using a µ Bondapak C18 column kept at 50°C. The mobile phase consists of 60% A (0.01 M KH2PO4, pH 4.0) and 40% B (80% of 50:50 CH3CN:CH3OH + 20% A) v/v. Enalaprilat is monitored by UV detection at 205 nm. The method is quite simple and sufficiently sensitive, with a limit of quantitation of 5 ng/mL and limit of detection of 1 ng/mL; within day and between‐days assays showed a relative standard deviation (RSD) below 15%.

Stability-Indicating Assay of Secnidazole in the Presence of Its Degradation Products

Four new selective, precise, accurate and rapid first derivative spectrophotometric, RP-HPLC, TLC densitometric and colorimetric methods are described for the determination of secnidazole in the presence of its degradation products, 2 - methyl - 5- nitroimidazole and hydroxyl propanol. (Method A) is applying the first derivative spectrophotometry at 296 nm. (Method B) is RP- HPLC based on using 30% methanol as a mobile phase at a flow rate of 1.0 ml/min. and 5 μ Bondapak C18 column (5 micron, 150 × 4.6 mm) as a stationary phase Detection was earned out using a diode detector at 319 nm (Method C) is densitometry using ethyl acetate as mobile phase and the spot was scanned with a spectrodensitometry at 311 nm. (Method D) is colorimetry based on diazotization of sulfanilamide with the nitrite ions liberated by alkaline hydrolysis of secnidazole and subsequent coupling of the diazonium salt with N-1-(nantuhyl)- ethylenediamine dihydrochloride. Regression analysis of a Beer's plot showed good correlation in the concentration 4 - 30 μ g ml−1, 2-20 μg ml−1, 4-18 μg, 0.8-60 μg.ml−1 with mean percentage rccovcrics:99.87 ± 0.56%. 100.12 ± O.80 %, 99.33 ± 0.50 % and 99.68 ± 0.39 % for methods A,B,C and D, respectively. These methods are suitable for stability testing of secnidazole in bulk powder retaining their accuracy in the presence of up to 70 % for method A and up to 90 % for method B, C and D. The proposed methods were applied for the analysis of pharmaceutical formulations and the recoveries were 99.40 -100.52%. The results obtained compared statistically with those obtained with the reported method.

Studies on iodinated compounds. X. Isolation and purification of iodoglycyltyrosines

The methods for the isolation and purification of iodoglycyltyrosines [glycyl-3-iodotyrosine (Gly-MIT) and glycyl-3,5-diiodotyrosine (Gly-DIT)] from a reaction mixture were examined by the use of high-performance liquid chromatography (HPLC). Glycyltyrosine (Gly-Tyr) was iodinated with iodine monochloride (ICl) (the molar ratio of Gly-Tyr to ICl was 1:1.5) in 0.1 M NaOH. The synthesized Gly-MIT and Gly-DIT were separated on a mu Bondapak C18 column employing stepwise gradient systems of a 0.1% trifluoroacetic acid/acetonitrile mixture and a water/acetonitrile mixture. Chemically pure Gly-MIT and Gly-DIT were obtained in 30.2% and 28.2% yields, respectively.

Simultaneuous Determination of Enalpril Maleate and Hydrochlorothiazide in Tablets by Reversed Phase HPLC

A new simple, precise, rapid and selective RP-HPLC method has been developed for the simultaneous determination of Enalpril maleate and Hydrochlorothiazide from formulations, using 0.025M orthophosphoric acid [(adjusted to pH 3.0 with triethyl amine (TEA)]: Acetonitrile (84 16 V/V), as a mobile phase at a flow rate of 1.0 ml/min and μ Bondapak C18 column (10 micron, 30cm × 3.9 mm) as a stationary phase. Detection was carried out using a UV detector at 226 nm. p-hydroxy phenyl acetamide (PHPA) was used as an internal standard. Linearity range and percentage recoveries for Enalpril maleate and Hydrochlorothiazide were 40–200 μg/ml and 50–500 μg/ml, 99.74 and 99.69 respectively. Minimum detection limit obtained was 0.8 μg/ml and 0.4 μg/ml for Enalpril maleate and Hydrochlorothiazide respectively.

Determination of Flavopiridol (L 86 8275) in Rat Serum Using Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

A specific, sensitive, and simple high-performance liquid chromatographic method has been developed for assaying flavopiridol in rat serum for preliminary bioavailability and pharmacokinetic studies. The drug and internal standard were extracted with dichloromethane from serum. After evaporating the solvent, the extract was reconstituted with mobile phase. Chromatography was performed on a 10-μ. Bondapak C18 column (30 cm × 3.9 mm ID), using methanol:water (50:50, v/v) containing 0.05 M ion-pairing reagent PIC B-6 as a mobile phase at a flow rate of 1.1 ml/min, with ultraviolet detection at 278 nm. The drug and internal standard were well resolved from other serum constituents. Due to high sensitivity and reproducibility the method can be adapted very well for assaying flavopiridol in higher animals.

Development of a high-performance liquid chromatographic method for the analysis of staurosporine.

Staurosporine (Stsp), a protein kinase inhibitor, has been found to have a differential effect on the proliferation of normal and transformed cells in vitro. Hence, Stsp might be used in cancer therapy to arrest normal proliferating cells in G1, while permitting tumor cells to continue proliferation. The patient could then be treated with a therapeutic agent of maximum toxicity for actively proliferating tumor cells. To facilitate investigations of Stsp in vivo, we have developed an HPLC method for measuring the levels of Stsp in blood. Using a rat model, plasma containing Stsp is treated with acetone to precipitate proteins and extract the Stsp. The acetone extract is then subjected to reversed-phase HPLC on a mu Bondapak C18 column. Using a linear elution gradient of acetonitrile containing trifluoroacetic acid, Stsp elutes as a sharp peak at ca. 35 min which can be detected by UV absorption at 292 nm. No blood or reagent components interfere with its quantification. The calibration curve, ranging from 0.1 to 2.0 micrograms Stsp, demonstrated a linear response to Stsp concentration having a correlation coefficient (r2) of 0.990. Precision analysis demonstrated that the method will yield results that are +/- 11.6% from the mean 95% (two standard deviations) of the time. This method was used to measure Stsp levels in plasma after administering an injection of 0.2 mg Stsp into the jugular vein of rats. No Stsp could be detected in the plasma 5 min after injection, even though enough Stsp was administered to be easily detectable if it was simply contained in the plasma. Thus, it is concluded that some compartment other than the plasma must adsorb the Stsp from the plasma and sequester it in vivo.

Quantitative Determination of Oxaprozin and Several of Its Related Compounds by High-Performance Reversed-Phase Liquid Chromatography

Abstract An accurate, sensitive, and selective high-performance liquid chromatographic method for the quantitative determination and the separation of oxaprozin from several of its related compounds (including some precursors) has been developed. Reversed-phase ion-pair chromatography, with 1-decanesulfonic acid sodium salt as the ion pairing agent, was used. A μ Bondapak C18 column with a mobile phase consisting of 0.01 M KH2PO4, methanol, and acetonitrile (2:1:1), pH 4.2, was used at a flow rate of 1 mL/min. Oxaprozin and its related compounds were monitored at 254nm. The method is rapid because no sample extraction is involved. Precision and ruggedness (relative standard deviation) on oxaprozin and several of its related compounds were below 1.0%. Detection limits ranged from 0.05 μg/mL to 0.08 μg/mL depending on the compound. The method is stability-indicating, and has applications in bulk pharmaceutical manufacturing.

Determination of the polar drug dimiracetam in human plasma and serum by column-switching high-performance liquid chromatography.

A sensitive and fully automated column-switching HPLC method was developed for the determination of a novel cognition enhancer, dimiracetam, in human plasma and serum. A sample of plasma was ultrafiltered and then directly injected onto a first mu Bondapak C18 column. The sample was eluted with water. The portion of the eluate (heart-cut) from this column containing the drug was selected and loaded on a second mu Bondapak C18 column and eluted with water. During the elution of the second column, the first column was automatically rinsed with water-acetonitrile (50:50, v/v) and then equilibrated for a new injection. The total analysis time for each sample was 35 min. This corresponded to a 30% decrease in analysis time compared with the time that would have been needed with a simple run with two mu Bondapak C18 columns assembled in series. Ultraviolet detection at 200 nm was used to monitor the eluate. The method was fully validated over the quantitation range 27 ng/ml-15 micrograms/ml. The applicability of the method was demonstrated by analysis of serum samples from a study performed in human healthy volunteers.

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography.

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C18 Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and mu Bondapak C18 column (150 x 3.9 mm I.D., 10 microns particle size). The detection limit (A375 nm) for quercetin in plasma was 0.1 microgram/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.

Determination of baclofen in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

An isocratic reversed-phase column liquid chromatographic assay for baclofen was developed. Prior to analysis, derivatization of urine or deproteinized plasma with 4-chloro-7-nitrobenzofurazan (NBD-CI) reagent was employed. After solid-phase extraction on a small silica gel column, the mixture was chromatographed on a 10 microns Bondapak C18 column using methanol-water (45 + 55 v/v) as the mobile phase. The NBD-baclofen derivative was detected spectrofluorimetrically by monitoring the emission at 524 nm with excitation at 463 nm. The method was tested for suitability in routine analysis. After a single oral dose of 20 mg of baclofen, the plasma concentration and the cumulative urinary excretion of the drug were determined. The average recoveries were 99.5 and 98.5% for plasma and urine, respectively.

Determination of Ivermectin Residues in Meat and Liver by HPLC and Fluorometric Detection

Abstract The method describes the analysis of ivermectin in meat and liver at a quantification limit of 5 μg/kg. This level is lower than the maximum residue limit (MRL) imposed for EEC countries. The absolute detection limit was 250 pg. The recoveries ranged from 70 % to 88 %. Ivermectin was extracted by a mixture of acetonitnie and water, purified on Bond Elut C18 columns and converted to a fluorescent derivative using trifluoroacetic anhydride and Nmethylimidazol. The analysis was performed on a liquid chromatograph fitted with a μ- Bondapak C18 column and ivermectin was detected by fluorescence spectroscopy. The method has already been used for routine analysis.