High-performance liquid chromatographic determination of ibuprofen, its metabolites and enantiomers in biological fluids

An isocratic high-performance liquid chromatographic method to determine racemic ibuprofen (assay I) and its major metabolites (assay II) in biological fluids (plasma, urine, bile) using a conventional reversed-phase column is described. A third assay using beta-cyclodextrin as stationary phase (Cyclobond I) for the separation of the ibuprofen enantiomers is also described. A wavelength of 220 nm was used to monitor the substances. The sensitivity of the method was 0.1 microgram/ml for all three assays. The method was demonstrated to be suitable for stereoselective pharmacokinetic studies of ibuprofen in humans and animals.     

手性HPLC法测定布洛芬立体选择性酯化的转化率和对映体过量

以脂肪酶在正己烷中催化消旋布洛芬与正丁醇的立体选择性酯化反应为例,研究了用手性ＨＰＬＣ法进行分析时产物及底物的峰面积变化与转化率的关系,确定了布洛芬丁酯与未反应布洛芬的峰面积－浓度响应因子的比值,从而可通过一次进样用峰面积直接测定布洛芬的转化率和对映体过量。     

Metabolites from the biodegradation of pharmaceutical residues of ibuprofen in biofilm reactors and batch experiments

The three metabolites hydroxyibuprofen (OH-Ibu), carboxyibuprofen (CA-Ibu), and carboxyhydratropic acid (CA-HA), also known from human metabolism of ibuprofen, could be identified in biodegradation experiments. Identification was based on EI mass spectra and comparison with literature data. Detection was performed by selective MS-MS measurements by GC-ion-trap MS and online methylation. Ibuprofen (Ibu), OH-Ibu, and CA-Ibu could be detected with a signal-to-noise ratio of 10:1 at a concentration of 2 nmol L(-1), CA-HA at 0.5 nmol L(-1). Degradation experiments in both biofilm reactors (BFR) and batch experiments with activated sludge (BAS) reveal OH-Ibu as the major metabolite under oxic conditions, and CA-HA under anoxic conditions. CA-Ibu was found under oxic and anoxic conditions almost only in the BAS. The metabolites together do not account for more than 10% of the initial concentration of Ibu.     

Stereoselective high-performance liquid chromatographic determination of ketoprofen, ibuprofen and fenoprofen in plasma using a chiral alpha 1-acid glycoprotein column

The effect of mobile phase composition, pH and temperature on the chiral resolution and retention of some 2-arylpropionic acids using the chiral alpha 1-acid glycoprotein column EnantioPac is described. Furthermore, a direct stereoselective high-performance liquid chromatographic assay to determine the enantiomers of ketoprofen, ibuprofen and fenoprofen in plasma is presented. Detection was at 260, 220 and 220 nm for ketoprofen, ibuprofen and fenoprofen, respectively. The limit of detection was 0.1 micrograms/ml for the enantiomers of ketoprofen and ibuprofen, and 0.25 micrograms/ml for the enantiomers of fenoprofen. The method was demonstrated to be applicable for stereoselective pharmacokinetic studies of ketoprofen, ibuprofen and fenoprofen after administration under clinical conditions.     

A throughput method using the quick easy cheap effective rugged safe method for the quantification of ibuprofen and its main metabolites in soils

Quick, easy, cheap, effective, rugged, and safe extraction is a modern sample preparation method that involves a number of steps with a low susceptibility to error. The efficiency of a laboratory can be improved not only through labor reduction and consumable savings but also through the use of high‐throughput methods and the reduction of wastes. In commercially available kits, different salts and buffers are mixed in anhydrous packages. The composition of these kits are optimized and fixed for particular applications by the suppliers. In this work three model compounds (ibuprofen and two of its main metabolites: hydroxyibuprofen and carboxyibuprofen) were chosen and the amount of each salt (magnesium sulfate, sodium chloride, sodium citrate, and disodium citrate sesquihydrate) in the quick, easy, cheap, effective, rugged, and safe content was optimized for three different soil samples (soils A, B, and C) with different organic carbon contents, using a 2⁴ factorial design. The optimized extraction procedure was applied to 12 soil samples; ranging from river sediments to agricultural soils. Based on the analysis 100 samples, a price reduction of 5.1‐ (soil B), 5.7‐ (Soil C), and 6.1‐fold (soil A) was achieved without compromising the performance of the method when compared to commercial kits.     

基于鞘脂组学技术探究布洛芬对斑马鱼脑组织的立体选择性影响

以斑马鱼成鱼为研究对象,借助于鞘脂组学技术,探究了一种新型手性环境污染物布洛芬对斑马鱼脑组织中鞘脂含量的影响。建立了超高效液相色谱-三重四极杆-线性离子阱复合质谱检测斑马鱼成鱼脑组织中46种鞘脂的定量方法。借助于该方法对经布洛芬外消旋体及对映体暴露后的斑马鱼脑组织中的46种鞘脂进行了定量分析,通过考察主成分分析（PCA）、正交偏最小二乘法分析（OPLS-DA）及显著性差异分析（P）筛选出差异代谢物。同时,结合组间差异分析的结果可知,环境水平的布洛芬会导致斑马鱼脑组织中鞘脂代谢发生紊乱,且布洛芬的对映体之间存在立体选择性毒性。     

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94-100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 micrograms/ml free ibuprofen in 100 microliters urine.     

The application of high performance liquid chromatography, coupled to nuclear magnetic resonance spectroscopy and mass spectrometry (HPLC-NMR-MS), to the characterisation of ibuprofen metabolites from human urine

Summary The use of HPLC-NMR-MS for the detection and identification of the metabolites of ibuprofen present in a solid phase extract of human urine is described. Gradient reversed-phase HPLC was used to separate the components present in the extract, which were then characterised by a combination of stopped-flow¹H NMR and on line electrospray-MS. This approach led to the rapid identification of the known phase 1 human metabolites of ibuprofen, including hydroxy- and carboxy- metabolites, together with their respective glucuronide conjugates. In addition a probable artefact resulting from the dehydration of one of the side chainhydroxylated glucuronides was also identified.     

HPLC determination of ibuprofen, diclofenac and salicylic acid using hollow fiber-based liquid phase microextraction (HF-LPME)

This paper describes an extraction method using a polypropylene membrane supporting dihexyl ether (three-phase hollow fiber-based liquid phase microextraction (HF-LPME)) for the analysis of several pharmaceuticals (salicylic acid (SAC), ibuprofen (IBU) and diclofenac (DIC)) followed by a HPLC determination using a monolithic silica type HPLC column, that allows lower retention times than the usual packed columns with adequate resolution. Detection was realized by means of a coupled in series diode array (DAD) and fluorescence (FLD) detectors. HF-LPME is a relatively new technique employed in analytical chemistry for sample pretreatment which offers more selectivity and sensitivity than any traditional extraction technique. Detection limits by DAD are 12, 53 and 40 ng mL⁻¹ for salicylic acid, diclofenac and ibuprofen, respectively and by FLD 7 and 2 ng mL⁻¹ for salicylic acid, and ibuprofen. The method has been successfully applied to their direct determination in human urine and the results obtained demonstrated that could be also applied to the determination of the corresponding metabolites.     

Direct chiral resolution of cloquintocet-mexyl and its application to in vitro degradation combined with clodinafop-propargyl

A simple chiral high‐performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for measuring Cloquintocet‐mexyl (ClM) enantiomers and clodinafop‐propargyl (CP) using cellulose tris‐(3,5‐dimethylphenylcarbamate) (CDMPC) as chiral stationary phase (CSP). The effects of mobile phase composition and column temperature on the ClM enantiomer separation were investigated. Good separation was achieved by using a mixture of n‐hexane and n‐propanol as mobile phase. Based on the chiral HPLC method, enantioselective quantitative determination analysis methods for this herbicide combined with CP in diluted plasma were developed and validated. The assay method was linear over a range of concentrations (0.5–100 µg/mL) in diluted plasma and the mean recovery was greater than 80% for both enantiomers and CP. The limits of quantification and detection for both ClM enantiomers and CP were 0.5 and 0.2 µg/mL, respectively. Intra‐ and interday relative standard deviations did not exceed 10% for three tested concentrations. The result suggested that the degradation of ClM enantiomers was stereoselective in rabbit plasma, and both rac‐ClM and CP degraded quickly in plasma, showing that the main existing forms with biological effect in animals are their metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

毛细管区带电泳法快速测定复方布洛芬片的有效成分

 研究了用毛细管区带电泳法快速测定复方布洛芬片中布洛芬和伪麻黄碱含量的方法。在０．０２５ｍｏｌ／Ｌ的磷酸盐缓冲液（ｐＨ８．１）中，上述两组分可在３ｍｉｎ内得以完全分离，用紫外检测器在２１０ｎｍ处检测，并以外标法定量。１１次测定含有９．５ｍｇ／Ｌ盐酸伪麻黄碱和６６．７ｍｇ／Ｌ布洛芬的试样溶液，相对标准偏差为２．９％（伪麻黄碱）和１．９％（布洛芬），回收率为１０３．１％（伪麻黄碱）和９７．６％（布洛芬）。应用毛细管区带电泳法测定复方布洛芬片剂的含量，所得结果与ＨＰＬＣ法一致。     

An HPLC‐UV method for the simultaneous quantification of curcumin and its metabolites in plasma and lung tissue: Potential for preclinical applications

Curcumin, derived from turmeric, has been extensively investigated for its broad spectrum of biological activities. Previously reported HPLC‐UV methods have focussed on analysis of the parent compound. Here, a sensitive HPLC‐UV method was developed and partially validated, then used for the simultaneous determination of curcumin and its glucuronide and sulfate metabolites in plasma and lung tissue from mice. The assay was applied to an in vivo pharmacokinetic study comparing formulated curcumin (Meriva™) with standard curcumin. Plasma levels of glucuronide and sulfate metabolites were 5‐ and 2‐fold higher after Meriva™ administration compared with standard curcumin. In lung tissue, free curcumin was 4‐fold higher following Meriva™ administration vs standard curcumin. This assay represents a rapid, cheap method for simultaneous detection of curcumin and its major metabolites that has applicability in pre‐clinical settings.     

毛细管区带电泳法快速测定复方布洛芬片的有效成分

研究了用毛细管区带电泳法快速测定复方布洛芬片中布洛芬和伪麻黄碱含量的方法。在０．０２５ｍｏｌ／Ｌ的磷酸盐缓冲液（ｐＨ８．１）中，上述两组分可在３ｍｉｎ内得以完全分离，用紫外检测器在２１０ｎｍ处检测，并以外标法定量。１１次测定含有９．５ｍｇ／Ｌ盐酸伪麻黄碱和６６．７ｍｇ／Ｌ布洛芬的试样溶液，相对标准偏差为２．９％（伪麻黄碱）和１．９％（布洛芬），回收率为１０３．１％（伪麻黄碱）和９７．６％（布洛芬）。应用毛细管区带电泳法测定复方布洛芬片剂的含量，所得结果与ＨＰＬＣ法一致。     

High-performance liquid chromatographic evaluation of 2-(alpha-thenoylthio)propionylglycine and its two metabolites in biological fluids

A high-performance liquid chromatographic (HPLC) method for determining 2-(alpha-thenoylthio)propionylglycine (TTPG) and its two main metabolites, thiophenecarboxylic acid and thiopronine, in biological samples was developed. TTPG and its metabolites were extracted by solvent partition and then determined by reversed-phase HPLC with UV detection at 245, 295 and 360 nm. This procedure was validated in order to allow the assay of these compounds in plasma and urine samples with sufficiently low detection limits (50 ng/ml for TTPG and TCA and 100 ng/ml for thiopronine) and with good linearity within the concentration range investigated. It was applied to a comprehensive pharmacokinetic investigation of TTPG in healthy volunteers.     

Ultralow concentrations of ibuprofen activate cell prostaglandin synthesis

he interest in the prostaglandin (PG) synthesis by animal cells today grows steadily because of the difficulties in obtaining them by any other way. Murine peritoneal macrophages can under certain con ditions synthesize large amounts of PGs. The effect of well-known nonsteroidal anti-inflammatory drug ibuprofen on PG synthesis by the cells using a high-performance liquid chromatography (HPLC) method with fluorescence detection of 4-bromomethyl-7-methoxy coumarin (BrMMC) derivatives was studied. In our case, the main metabolites were PGE₂ and PGF_2a. The PG synthesis activation effect was shown by ibuprofen concentrations in the 10^-10-10^-14M range with the maximum effect at the 10^-12M. In this case, the ibupro fen effect was comparable in value with the effect of the well-known cell PG synthesis activator—calcium ionophore A₂₃₁₈₇. Although the exact mechanism of such an effect is not clear at the moment, at low concentration, ibuprofen itself is able to activiate PG synthesis in murine peritoneal macrophages.     

Simultaneous determination of phenylglyoxylic acid, mandelic acid, styrene glycol and hippuric acid in primary culture of rat hepatocytes incubate by high-performance liquid chromatography

A simple HPLC method for the simultaneous determination of phenylglyoxylic acid (PGA), mandelic acid (MA), styrene glycol (SG) and hippuric acid (HA) in cell culture medium was developed. Analysis was performed on a C(18) column with a mobile phase composed of methanol-potassium dihydrogen phosphate (pH 2.5; 10 mM; 10:90, v/v) at 220 nm. The flow-rate of mobile phase was set at 0.5 mL/min. The mean absolute recoveries of PGA, MA, SG and HA were 95.9, 98.4, 98.0 and 97.1%, respectively. The inter-day and intra-day precisions, determined at three concentration levels, were less than 10% of RSD. The limits of quantification for PGA, MA, SG and HA were 13.2, 13.1, 14.5 and 11.2 microM with RSD less than 20%. The limits of detection for PGA, MA, SG and HA were 4.6, 4.6, 5.1 and 3.9 microM, respectively. The method was successfully applied to study the stereoselective metabolism of SG and MA in primary culture of rat hepatocytes. The results show that there is stereoselective metabolism for both of MA and SG in primary culture of rat hepatocytes. The extent of biotransformation from S-MA to PGA is significantly greater than that from the R enantiomer and the main metabolites are PGA and HA for S-SG and R-SG, respectively.     

A validated high‐performance liquid chromatography–tandem mass spectrometry method for quantification of gefitinib and its main metabolites in xenograft mouse tumor: Application to a pharmacokinetics study

Monitoring gefitinib and its metabolites may help to explore the underlying mechanisms of gefitinib resistance. The concentration of gefitinib and its metabolites in tumor tissues could influence its anticancer activities more than that in the plasma. In the present study, a rapid and specific HPLC–MS/MS method was developed and validated to simultaneously determine gefitinib, M387783, M523595, M537194 and M608236 in tumor tissues of H1975 human lung cancer xenografts of nude mice. The established HPLC–MS/MS method was validated for specificity, linearity, accuracy and precision, matrix effect and recovery, carryover and dilution integrity, and analyte stability. The standard curves were linear (r² ≥ 0.99) over the range of 0.5–100 ng/mL for M608236 and 1–200 ng/mL for gefitinib, M523595 and M537194 as well as M387783. The accuracy ranged from ?8.35 to 6.03% relative error; and the precision was <15% relative standard deviation. Recoveries (87.74–99.96%) and matrix effects (86.60–106.40%) were satisfactory in the biological matrix examined. Stability studies showed that the analytes were stable during the assay procedure and storage. Finally, the validated method was successfully applied to study the pharmacokinetics profiles for gefitinib and its metabolites in nonsmall cell lung cancer (NSCLC) xenograft mouse tumors. Meanwhile, MTT assay showed that gefitinib had a more powerful inhibitory effect than its four major metabolites in H1975 NSCLC cells. This validated HPLC–MS/MS method may be applied to help understand the mechanisms of gefitinib resistance in EGFR‐mutant nonsmall cell lung cancer.     

Synthesis and Molecular Recognition of Molecularly Imprinted Polymer with Ibuprofen as Template

Ibuprofen and ketoprofen are chemically similar non‐steroidal anti‐inflammatory drugs widely used in the treatment of arthritis. Using a molecular imprinting technique, a simple and rapid method was developed for the simultaneous separation and determination of ibuprofen and ketoprofen. Molecular imprinting introduces artificial binding sites into a synthetic polymer matrix, allowing it to exhibit selective rebinding of template molecules. Imprinted polymers can be regarded as an HPLC stationary phase, important for pharmaceutical analysis. Most molecularly imprinted polymers (MIPs) are synthesized by free radical polymerization of functional monomers, resulting in an excess of crosslinking monomers. In this study, MIPs have been prepared with a ibuprofen template, which can form intramolecular hydrogen bonds. Methacrylic acid (MAA) and ethyleneglycol dimethacrylate (EGDMA) were used as the functional monomer and cross‐linker, respectively. Bulk polymerization was carried out at 4 °C under UV radiation. The resulting MIP was ground into 25?44 μm particles, which were slurry‐packed into analytical columns. Template molecules were removed by methanol‐acetic acid (9:1, v/v). We evaluated the template binding performance of the MIP using HPLC, with ultraviolet (UV) detection at 234 nm. Chromatographic resolution of ibuprofen and ketoprofen on the MIPs were appraised using buffer/acetonitrile (45/55, v/v) as the mobile phase. Results show that the MIPs prepared using ibuprofen as the template had a significant molecular imprinting effect. The method was successfully applied to the separation and analysis of ibuprofen and ketoprofen in pharmaceuticals.     

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.