Assay of ibuprofen in human plasma by rapid and sensitive reversed-phase high-performance liquid chromatography:application to a single dose pharmacokinetic study

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method is developed to determine the concentration of ibuprofen in human plasma. Ibuprofen is isolated from plasma by adding 0.50 mL of acetonitrile to 1.0 mL of plasma. The endogenous substances precipitated by acetonitrile are separated by centrifugation. The supernatant is saturated with ammonium sulfate to salt-out the acetonitrile. The salted-out acetonitrile is injected directly into the HPLC system. A 150-mm x 4.6-mm column packed with 3-microns reversed-phase octadecylsilane particles (C18) is used for the method finally developed. The mobile phase is a 1:1 ratio of acetonitrile-phosphoric acid (pH 2.2). Ibuprofen is monitored with a UV-visible detector at 220 nm and 0.10-0.002 absorbance units full scale (A.U.F.S.). The mean percent of relative standard deviations for within-day and between-day analyses are less than 3. The limit of detection for ibuprofen (in human plasma) is 25 ng/mL for a 100-microL injection volume. Quantitation of ibuprofen in human plasma at 100 ng/mL can be achieved with a relative standard deviation of less than 5%. The completion time of assay is less than 20 minutes.     

Development and validation of an UPLC method for rapid determination of ibuprofen and diphenhydramine citrate in the presence of impurities in combined dosage form

A novel, stability-indicating gradient reverse-phase ultra-performance liquid chromatographic method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in the presence of degradation products and process related impurities in combined dosage form. The method was developed using C18 column with mobile phase containing a gradient mixture of solvent A and B. The eluted compounds were monitored at 220 nm. Ibuprofen and diphenhydramine citrate were subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Major unknown impurity formed under oxidative degradation was identified using LC-MS-MS study. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantitation, accuracy, precision and robustness. The described method was linear over the range of 0.20-6.00 μg/mL (r>0.998) for Ibuprofen and 0.084-1.14 μg/mL for diphenhydramine citrate (r>0.998). The limit of detection results were ranged from 0.200-0.320 μg/mL for ibuprofen impurities and 0.084-0.099 μg/mL for diphenhydramine citrate impurities. The limit of quantitation results were ranged from 0.440 to 0.880 μg/mL for ibuprofen impurities and 0.258 to 0.372 μg/mL for diphenhydramine citrate impurities. The recovery of ibuprofen impurities were ranged from 98.1% to 100.5% and the recovery of diphenhydramine citrate impurities were ranged from 97.5% to 102.1%. This method is also suitable for the simultaneous assay determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms.     

Sensitive High-Performance Liquid Chromatographic Determination of Mebeverine in Plasma Using Fluorescence Detection

Abstract

A sensitive high-performance liquid chromatographic (HPLC) method for mebeverine (MB) determination in plasma is described. Sample preparation involves extraction of MB and Ibuprofen (internal standard) from 0.5 ml plasma. The analysis is carried out on reversed-phase chromatographic system using U-Bondapack C₁₈ column with a mobile phase consisting of water: acetonitrile:acetic acid (59:40:1) mixture. The effluent was monitored using a fluoremetric detection at excitation and emission wave lengths 270 and 362 nm, respectively. The method gave accurate, precise and reproducible results with high sensitivity. The within-day coefficients of variation ranged from 2.5 to 6.1% and between-days from 7.5 to 13.5% at four different concentrations. Injection-volumes containing as small amount of MB as 0.5 ng in plasma was detected. This method was applied to a bioavailability study with a single 10 mg/kg oral dose in two rabbits.     

Tandem column for the simultaneous determination of arginine, ibuprofen and related impurities by liquid chromatography

Ibuprofen arginate is a rapidly absorbed salt designed to promote more rapid onset of analgesia than commercially available forms of ibuprofen. Ibuprofen and arginine have very different polarities and this becomes in a chromatographic problem, further complicated with the determination of related compounds, which is necessary in stability assays of the pharmaceutical forms. The common solution is the employment of two separate methods, but this is time consuming. A LC method has been developed to determinate both compounds and related impurities in one run. Ibuprofen, arginine and three ibuprofen related impurities (B, E and J) have been baseline separated with isocratic conditions at pH 3.0 and run time under 20 min by employing a tandem combination of two different stationary phases: first a ZORBAX SB-C18 column from Agilent (250 mm x 4.6 mm and 5 microm) and downstream a SUPELCOSIL LC-NH2 column from Supelco (150 mm x 4.6 mm and 3 microm). The octadecyldiisobutylsilane column provides the separation of ibuprofen and its impurities by a hydrophobic mechanism, whereas aminopropyl column offers selective retention of arginine by dipolar interaction mechanism. Method has been successfully validated following ICH guidelines and it has been demonstrated to be reliable for arginine, ibuprofen and related impurities determination in sachets of two different dosages as pharmaceutical forms. Moreover, stress test has proved the selectivity of the method for degradation products, such as those that can emerge throughout long-term stability assays.     

High-performance liquid chromatography and pharmacokinetics of aceclofenac in rats

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for quantification of aceclofenac in rat plasma. Ibuprofen was used as an internal standard (IS). The present method used protein precipitation for extraction of aceclofenac from rat plasma. Separation was carried out on reversed-phase C₁₈ column (250 mm × 4.6 mm, 5 μ) and the column effluent was monitored by UV detector at 282 nm. The mobile phase used was methanol-triethylamine (pH 7.0; 0.3% v/v in Milli-Q water) (60:40%, v/v) at a flow rate of 1.0 mL min⁻¹. This method was linear over the range of 50.0-3500.0 ng mL⁻¹ with regression coefficient greater than 0.99. The mean recovery of aceclofenac and IS were 84.62 ± 3.23 and 89.19 ± 1.57%, respectively and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of aceclofenac in rats.     

Ibuprofen-loaded PCL meshes manufactured using rapid tooling for ocular orbital repair

This study investigated the manufacture of resorbable polycaprolactone/ibuprofen (PCL/IBP) meshes by injection molding for application in ocular orbital repair. The pore dimension sizes used demonstrate that micro-porous meshes can be manufactured by injection molding using a prototype mold. The mechanical properties were observed to be dependent on the material composition and morphology. Lower stiffness, strength and elongation at failure were observed for the 8 mm pore sized samples. The PCL/Ibuprofen meshes initially showed a fast drug release but after 3 days the release was slow and controlled. The cytotoxicity test results of the PCL/Ibuprofen meshes indicated that the large initial quantity of Ibuprofen released was too high and resulted in cell toxicity. However, after this initial release, the PCL/Ibuprofen meshes showed a good interaction with the cells seeded on their surface. The presence of a low concentration of Ibuprofen does not negatively influence cell viability in culture.     

Determination of 3-n-butylphthalide in rabbit plasma by HPLC with fluorescence detection and its application in pharmacokinetic study

A rapid, sensitive and specific reversed-phase high-performance liquid chromatographic method was developed for the determination of 3-n-butylphthalide, a drug currently being developed for treatment of stroke, in rabbit plasma. Fluorescence detection at an excitation wavelength of 280 nm and an emission wavelength of 304 nm was used for quantification of 3-n-butylphthalide. Ibuprofen was used as internal standard. Plasma samples were extracted with diethyl ether under acidic conditions. After evaporation of the organic phase, the extract was dissolved in mobile phase and injected into the chromatograph with C(18) column and a mobile phase of 0.05 mol/L sodium acetate buffer (pH 4.5)-acetonitrile (400:600). The peak area ratio vs concentration in plasma was linear over the range of 0.0212-4.24 microg/mL (correlation coefficient r = 0.9984) and the limit of quantification was 0.0212 microg/mL. Mean recovery was determined as 101.0% by analysis of plasma standard samples containing 0.0424, 0.424, 2.12 and 4.24 microg/mL of 3-n-butylphthalide. The intra-day relative standard deviations (RSDs) ranged from 3.6 to 8.9% and inter-day RSDs were within 8.0%. Pharmacokinetics of a single intravenous dose of 3-n-butylphthalide to the rabbits was presented to illustrate the applicability of this method. 3-n-Butylphthalide exhibited linear pharmacokinetics after intravenous administration to rabbits over the dose range 1-10 mg/kg.     

Determination of the plasticizer N-butylbenzenesulfonamide and the pharmaceutical Ibuprofen in wastewater using solid phase microextraction (SPME)

A rapid, inexpensive and solvent-free method for the simultaneous determination of the polyamide plasticizer N-butylbenzenesulfonamide (NBBS) and the widely used pharmaceutical Ibuprofen by solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MSD) in wastewater samples was developed. Besides the optimized analytical conditions, results of investigations with varying analytical parameters are reported. Problems, which may occur during the analytical procedure (e.g. salt deposits, adsorption phenomena, carry-over), are discussed. For the determination of Ibuprofen, it is important to carry out the extraction under acidic conditions with sufficiently buffered samples; the GC/MSD system must be very clean and well maintained. SPME allows an extraction of Ibuprofen without derivatization of its carboxylic group. For quantification in complex matrices, the standard addition technique is necessary. Limit of detection and limit of determination are 0.1 μg/L for both analytes. NBBS and Ibuprofen were detected in several raw and treated wastewater samples from municipal wastewater treatment plants in the range from < 0.1 to 3.5 μg/L.     

Automated Solid Phase Extraction and Hplc Analysis of Ibuprofen in Plasma

Abstract

Ibuprofen is a non-steroidal anti-inflammatory drug, widely used in arthritis and other disorders. We describe a high pressure liquid chromatographic (HPLC) method for the analysis of ibuprofen in plasma, using an automated solid phase extraction technique (the Varian AASP^R). In this method ibuprofen was extracted from 0.5 ml of plasma by application to a C₂ extraction cartridge followed by “on line” elution with the HPLC mobile phase (55% acetonitrile / 45% 0.02 H phosphate buffer; pH 3.0), at a flow rate of 1.5 ml/min. The analytical column was a Nucleosil C₁₈ column and the fluorescence detector was set at 253 nm (excitation wavelength) and 300 nm (emission wavelength). Chromatography was complete in less than 10 mins and the limit of detection was 1.3 /μg/ml. The method is linear through the range of 1.0 to 100.0 /μg/ml with a mean correlation coefficient of 0.9964. Absolute recovery of ibuprofen from the spiked plasma samples ranged from 77.8% to 86.5%. The method was shown to be precise within 11% C.V. and accurate to within 8% over the concentration range studied.     

Determination of the plasticizer N-butylbenzenesulfonamide and the pharmaceutical Ibuprofen in wastewater using solid phase microextraction (SPME)

A rapid, inexpensive and solvent-free method for the simultaneous determination of the polyamide plasticizer N-butylbenzenesulfonamide (NBBS) and the widely used pharmaceutical Ibuprofen by solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MSD) in wastewater samples was developed. Besides the optimized analytical conditions, results of investigations with varying analytical parameters are reported. Problems, which may occur during the analytical procedure (e.g. salt deposits, adsorption phenomena, carry-over), are discussed. For the determination of Ibuprofen, it is important to carry out the extraction under acidic conditions with sufficiently buffered samples; the GC/MSD system must be very clean and well maintained. SPME allows an extraction of Ibuprofen without derivatization of its carboxylic group. For quantification in complex matrices, the standard addition technique is necessary. Limit of detection and limit of determination are 0.1 μg/L for both analytes. NBBS and Ibuprofen were detected in several raw and treated wastewater samples from municipal wastewater treatment plants in the range from < 0.1 to 3.5 μg/L. Received: 13 March 1998 / Revised: 16 June 1998 / Accepted: 19 June 1998     

Screening out Potential Cardio-Toxic Components of Chinese Herb Radix Aconiti Lateralis in Rat Dosed Plasma by High Performance Liquid Chromatography/Electrospray Ionization Quadrupole Time-Of-Flight Mass Spectrometry

This study established a new, rapid, and accurate method for bioactive compound screening by the combination of serum pharmacochemistry and serum pharmacology based on a spectrum-effect relationship model. HPLC/ESI-Q-TOF-MS was used for the chemical profiling of samples of dosed plasma, control plasma, and Radix Aconiti Lateralis decoction. A comparison was made of the resulting chemical profiles, which totaled 20 compounds including 16 prototype compounds and 4 metabolites derived from SF decoction that were detected as potential bioactive components. Using MS/MS analysis and accurate molecular weight assessments of TOF, 17 components in 20 potential bioactive compounds were structurally identified. Moreover, the pretreated plasma samples were injected into venous sinus of isolated toad hearts to investigate the cardio-toxicity according to heart ratio (HR) situation and electrocardiograph (ECG) parameters, including PR interval, QRS interval, and QT interval. Finally, 4 compounds in these 20 potential bioactive components that had closest correlation with 4 cardio-toxic experimental indexes respectively were investigated by spectrum-effect relationship model for the first time. The 4 compounds were considered as the real cardio-toxic components. This study proposed a series of potential bioactive components in Radix Aconiti Lateralis and provided a general research pattern to identify the potential bioactivity compounds in Chinese herbs or natural pharmacy.     

Analysis of the constituents in rat plasma after oral administration of Shexiang Baoxin pill by HPLC‐ESI‐MS/MS

A valid method using liquid chromatography coupled with electrospray ionization (ESI) and ion trap mass spectrometry was established for the study of the absorbed components in rat plasma after oral administration of a traditional Chinese medicine (TCM) Shexiang Baoxin pill. The plasma was deproteinated by adding methanol prior to liquid chromatography, in which separation was carried out on a Symmetry C₁₈ column (5 µm, 250 × 4.6 mm). A linear gradient with 0.5% formic acid–water–acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. Twenty‐one components including 17 components from Shexiang Baoxin pill and four metabolites were observed from a comprehensive analysis of the chromatography of Shexiang Baoxin pill, controlled plasma and dosed plasma. All of the 17 prototype compounds and three of the metabolites were identified by comparing their retention behaviors and MS and MS/MS spectra with reference compounds and literature data. This study developed an integrated method for screening the bioactive constituents in plasma after oral adminstration of Chinese herbal medicine and provided helpful chemical information for further pharmacology and active mechanism research on TCM. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of hydrophilic interaction and reversed-phase liquid chromatography coupled with tandem mass spectrometric detection for the determination of three pharmaceuticals in human plasma

In the present study, hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) combined with tandem mass spectrometric detection (MS/MS) were evaluated and compared for the determination of donepezil, cetirizine and loratadine in human plasma, in terms of sensitivity and sample preparation procedure. A retention study for the above compounds of various polarities was performed, using both C(18) and silica columns, with several aqueous-organic mobile phase ratios, in order to investigate their retention mechanism profile under HILIC and RPLC. Both chromatographic conditions were compared for chromatographic analysis of plasma samples processed with a liquid-liquid extraction (LLE) method for donepezil determination, resulting in significantly higher sensitivity under HILIC. Furthermore, HILIC and RPLC were compared for direct injection, and novel methods including LLE, solid-phase extraction and protein precipitation protocols were developed. Direct injection technique significantly reduced sample preparation time, increasing at the same time method sensitivity. The current study contributes to broadening the range of analyzable compounds by HILIC-MS/MS to molecules of medium polarity.     

Nanosizing drug particles in supercritical fluid processing

The supercritical fluid-processing technique, rapid expansion of a supercritical solution into a liquid solvent (RESOLV), was applied to the nanosizing of water-insoluble drug particles. Selected for demonstration were antiinflammatory drugs Ibuprofen and Naproxen, for which CO2 and CO2-cosolvent systems were used. The RESOLV process produces exclusively nanoscale (less than 100 nm) Ibuprofen and Naproxen particles suspended in aqueous solutions, and the aqueous suspensions of the drug nanoparticles are protected from particle agglomeration and precipitation by using common polymeric and oligomeric stabilization agents.     

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC.This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work.The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.     

Incorporation of biotransformed silver nanoparticles in plant polysaccarides resin and their effect on sustained drug release

The intension of current study was to determine antibacterial and drug releasing capacity of green synthesized silver nanoparticles (AgNps) with Moringa oleifera resin in the presence of Montelukast sodium and Ibuprofen. This plant gum is economic, easily available, biodegradable, safe and potential tablet binder. There was no significant study reported on the incorporation of green synthesized silver nanoparticle with plant resin in drug release. The aqueous extract of Clerodendron phlomoides was used for the bioreduction of silver nanoparticles as well as a capping agent. This green synthesized AgNps was observed in UV at 489 nm due to the SPR (Surface Plasmon Effect) effect, and the presence of protein and polyol compounds was identified by FTIR. The crystalline structure of AgNps was analyzed by XRD, elemental silver composition was measured by EDAX, morphological structure and size was revealed by SEM and TEM analysis. The antibacterial effect of green synthesized AgNps was analyzed by zone of inhibition method. Silver nanoparticles incorporated in M. oleifera plant resin and its functional groups and thermal degradation properties were characterized by FTIR and TGA, respectively. The drug release properties of the AgNps incorporated with plant resin were evaluated for the sustained release and compared with raw plant gum without AgNps consistency.     

A new metabonomics method for simultaneous determination of EFAs and NEFAs in plasma using GC-MS and its application

A new metabonomics method was developed for simultaneous qualitative and quantitative analysis on esterified and nonesterified fatty acids(EFAs and NEFAs) in plasma.Merely 10μL of plasma was required.The pretreatment of the sample was simple without disposing the protein.After simple extraction and derivation,15 FAs in plasma were precisely quantified.Gas chromatography tandem mass spectrometry(GC-MS) was used in the study and the quantities of the analytes,which varied in abundance over three orders of ...     

Simultaneous determination of allantoin, hypoxanthine, xanthine, and uric acid in serum/plasma by CE

Allantoin (All) is an oxidative end product of purines in mammals. The small amount of All present in human plasma or serum results from free radical action on urate and may provide a stable marker of in vivo free radical activity. Because free radicals have been implicated in the development and progression of atherosclerosis, this study focused on the metabolic compounds of the All pathway. We propose a new fast CE (CE/UV) method for the simultaneous determination of All, uric acid (UA), hypoxanthine (HX), and xanthine (X) in human plasma. These products were quantified in the plasma of patients with chronic renal failure before hemodialysis (n = 6), patients with chronic heart failure (n = 6) and controls (n = 6). The filtered plasma were diluted ten-fold before the direct injection in CE/UV (195 nm), which allows separating the four compounds in less than 13 min. The metabolites were detectable at concentrations of 0.3-0.6 micromol/L. The method was linear over the range 0.5-150 micromol/L for All, HX, and X and 10-1500 micromol/L for UA (r > 0.99). The analytical performance of this method is satisfactory with intra-assay CV < 3.4%, inter-assay CV < 5% (HX and X < 7%), and recovery (93-101%). The proposed CE-UV method appears to be a useful tool for studying physiological and pathological changes of HX, UA, and All levels in plasma samples, the latter being a possible indicator of free radical damage in vivo.     

Quantitative determination of alpha-cyclodextrin in human plasma by liquid chromatography/positive ion electrospray mass spectrometry

A sensitive and selective method for the determination of alpha-cyclodextrin in human plasma is described using beta-cyclodextrin as an internal standard. After protein precipitation with perchloric acid, the analytes were isolated from human plasma by solid-phase extraction on Bond Elut C18 cartridges. The compounds were chromatographed on a narrow-bore aminopropyl column (125 x 2 mm i.d., 5 microm) and analyzed by electrospray ionization mass spectrometry in the positive selected-ion mode using the [M+NH4]+ ion. The lower limit of quantitation was 5 ng ml(-1) of human plasma. Linear calibration curves were obtained over the concentration range 5-1000 ng ml(-1) of human plasma. The intra- and inter-assay precisions were <18% and the accuracy was <10.5% over the entire concentration range. During the method development, the ionization efficiencies of the analytes in plasma samples originating from different sources were examined to overcome the matrix effect problems caused by co-eluting endogenous compounds. The method was successfully applied to pharmacokinetic studies in human volunteers.