Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.     

Rapid online-SPE-MS/MS method for ketoprofen determination in dermal interstitial fluid samples from rats obtained by microdialysis or open-flow microperfusion

Pharmacokinetic studies of topical ketoprofen formulations using continuous sampling techniques such as microdialysis (MD) or open-flow microperfusion (OFM) require sensitive assays due to small sample volumes. A simple and easy online-SPE-MS/MS method for ketoprofen analysis was developed for both MD and OFM samples obtained from rat dermal tissue. The quantification range is 25-5000 ng/ml with a limit of detection of 3 ng/ml using only 10 microl sample volume. The method is characterized by a simple setup using a short polymeric SPE column (OASIS HLB) for desalting with 1.5 min run times in combination with a sensitive MS detection in negative ESI MRM mode. An easy sample workup procedure was used which enables high throughput analysis of a large number of samples for pharmacokinetic studies. In addition, a commercial available (fenoprofen) as well as an isotopically labelled (deuterated ketoprofen) standard were investigated as potential internal standards. The method was validated according to FDA guidelines for bioanalytical validation in terms of accuracy, intra-batch and inter-batch precision, linearity, matrix effect, recovery and stability for both internal standards. Accuracies were 98-113% (fenoprofen) and 95-108% (deuterated ketoprofen), intra-batch precision was 2-3% R.S.D. (fenoprofen) and 2-6% R.S.D. (deuterated ketoprofen), and inter-batch precision was 2-6% R.S.D. (fenoprofen) and 3-6% R.S.D. (deuterated ketoprofen) over the entire quantification range. The presented method was applied to dermal interstitial fluid samples obtained in a topical administration study of ketoprofen in rats.     

Effect of chirality on PVP/drug interaction within binary physical mixtures of ibuprofen, ketoprofen, and naproxen: a DSC study

We report on the thermal behavior of freshly prepared binary drug/polymer physical mixtures that contained ibuprofen, ketoprofen, or naproxen as a drug, and polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), or methylcellulose (MC) as excipient. At 6-10 degrees C/min heating rates the DSC detected a sharp, single endotherm that corresponds to the melting of drug. On heating physical mixtures of PVP and racemic ibuprofen or ketoprofen at lower heating rates, another endotherm was registered in front of the original one. To observe the additional endotherm, specific minimal values of the heating rate and of PVP weight fraction were needed; for ibuprofen and ketoprofen they were 1.5 and 2.0 degrees C/min, and 5 and 15% (w/w), respectively. At greater PVP weight fractions the top temperatures, T(mp), of both peaks were reduced almost linearly indicating strong solid-state interfacial reaction between the drug particles and PVP matrix. The additional endotherm was abolished at greater heating rates (2 degrees C/min for ibuprofen, 3 degrees C/min for ketoprofen), by replacing the racemate with respective S+-enantiomer and by replacing PVP with HEC and MC. Hence, the possible inclusion of enantioselective component within the PVP/drug interaction, responsible for the amorphization of physical mixture over storage, is assumed.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensivity of 0.005 μg/ml for naproxen, and 0.05 μg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefanamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

Quantification of ketoprofen enantiomers in human plasma based on solid-phase extraction and enantioselective column chromatography

An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C₁₈ solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; (R)-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves (n = 14) were linear over the concentration range of 0.16 to 5.00 μg/ml per enantiomer [mean r² of 0.999 for both enantiomers, root mean square error were 0.015 for R(−) and 0.013 for S(+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the concentration range of 0.2 to 4.0 μg/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.     

Double-blind Cross-over Study of Ketoprofen and Ibuprofen in Management of Rheumatoid Arthritis

A double-blind cross-over study of ketoprofen (Orudis) 150 mg daily and ibuprofen (Brufen) 1,200 mg daily was carried out in 35 outpatients with rheumatoid arthritis. Results suggest that analgesic and anti-inflammatory activity of ketoprofen is superior to that of ibuprofen. Significantly greater pain relief (P < 0·05) and reduction in joint circumference (P < 0·01) was obtained with ketoprofen than with ibuprofen. Side effects of the drugs were comparable and not serious.     

Enantioselective pharmacodynamics of the nonsteroidal antiinflammatory drug ketoprofen: in vitro inhibition of human platelet cyclooxygenase activity.

The pharmacological activity of ketoprofen enantiomers was investigated in humans by an in vitro method. The antiplatelet effect of ketoprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood obtained from each of four healthy volunteers. Ketoprofen was added separately to whole blood as a range of concentrations of (1) predominantly (S)-ketoprofen, (2) racemic ketoprofen, and (3) predominantly (R)-ketoprofen. (S)-Ketoprofen was found to be solely active at inhibiting human platelet TXB2 production; (R)-ketoprofen was devoid of such activity and did not modify the potency of its optical antipode. A relationship between the percentage inhibition of TXB2 generation and the unbound concentration of (S)-ketoprofen in serum was modelled according to a sigmoidal Emax equation. The mean (+/- SD) serum unbound concentration of (S)-ketoprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 0.320 (+/- 0.062) ng/ml. This value for ketoprofen is considerably lower than previously reported values for (S)-ibuprofen and (S)-naproxen.     

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual periods, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay.The patients receiving placebo had high PGF levels 135 ± 27 ng/ml (Mean ± S.E.) which were significantly reduced by Ibuprofen to 24 ± 5 ng/ml (P<0.001). The PGE concentrations decreased from 5 ± 1 ng/ml to 2 ± 1 ng/ml (P<0.05). Ibuprofen also reduced the menstrual pain significantly (P<0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.     

Analysis of ibuprofen in serum by capillary electrophoresis

A rapid method for analysis of the analgesic drug ibuprofen in serum by capillary zone electrophoresis in a borate buffer 160 mmol/l pH 8.5 is described. The method involves deproteinization with acetonitrile to remove serum proteins followed by direct injection on the capillary. The recoveries of standards added to the serum were 84–92%. The method is suited for analysis of samples with concentrations >10 mg/l. Many other analgesics such as ketoprofen, daypro and salicylates can also be determined by this method.     

Determination of ibuprofen in serum by high-performance liquid chromatography and application to ibuprofen disposition

A high-performance liquid chromatographic method for quantitation of ibuprofen from serum and application of this method to ibuprofen disposition in the dog is described. The drug was extracted from acidified plasma with dichloromethane. The internal standard used was a methanolic solution of 4-n-butylphenylacetic acid. A μBondapak C₁ column was used for analysis; the mobile phase was methanol—water—glacial acetic acid (pH 3.4) (75:24:1, v/v). A wavelength of 272 nm was used to monitor ibuprofen and the internal standard.Method sensitivity was 0.5 μg/ml serum using either 0.5 or 1.0 ml of sample, and no interference was found from endogenous compounds or other commonly used anti-inflammatory agents. The coefficients of variation of the method were 4.2% and 6.0% for samples containing 50.0 and 6.25 μg/ml of ibuprofen, respectively, and the calibration curve was linear for the range of 0.5 to 100 μg/ml. This method was demonstrated to be suitable for pharmacokinetic and/or biopharmaceutical studies of ibuprofen in man and the dog.     

Enantiomeric Fraction Determination of 2‐Arylpropionic Acids in a Package Plant Membrane Bioreactor

Enantiomeric compositions of three 2‐arylpropionic acid (2‐APA) drugs, ibuprofen, naproxen, and ketoprofen, were monitored in a membrane bioreactor (MBR) treating municipal effluent in a small rural town in Australia. Specific enantiomers were determined as amide diastereomers using the chiral derivatizing reagent, (R)‐1‐phenylethylamine (PEA), followed by gas chromatography–tandem mass spectrometry (GC‐MS/MS). The six individual enantiomers were quantified by isotope dilution and the enantiomeric fractions (EFs) were determined. Over four separate sampling events, ibuprofen EF ranged from 0.88 to 0.94 (median 0.93) in the influent and 0.38 to 0.40 (median 0.39) in the effluent. However, no significant change in ketoprofen EF was observed, with influent EFs of 0.56–0.60 (median 0.58) and effluent EFs 0.54–0.68 (median 0.56). This is the first report of enantiospecific analysis of ketoprofen in municipal wastewater and it is not yet clear why such different behavior was observed compared to ibuprofen. Naproxen EF was consistently measured at 0.99 in the influent and ranged from 0.86 to 0.94 (median 0.91) in the effluent. This study demonstrates that EF is a relatively stable parameter and does not fluctuate according to concentration or other short‐term variables introduced by sampling limitations. The enantiospecific analysis of chiral chemicals presents a promising approach to elucidate a more thorough understanding of biological treatment processes and a potential tool for monitoring the performance of key biological pathways. Chirality 25:301–307, 2013. © 2013 Wiley Periodicals, Inc.     

The Safety and Pharmacokinetics of Carprofen,Flunixin and Phenylbutazone in the Cape Vulture (Gyps coprotheres) following Oral Exposure

The following study evaluates the overt toxic potential of carprofen (CRP), flunixin (FXN) and phenylbutazone (PBZ) in Old world vultures in relation to historic toxicity data for diclofenac and ketoprofen, with the Cape vulture (Gyps coprotheres) being the indicator species. The toxic potential of a single oral dose of CRP (11.5 mg/kg), FXN (1 mg/kg),PBZ (1.7 mg/kg) or water was evaluated by means of a four-way parallel study (n = 2), as means of ascertaining if these drugs were as toxic as diclofenac in the vulture. No unscheduled deaths or pathological lesions were noted following exposure. Clinical signs of lethargy and depression were, however, noted in one CRP, two FXN and one PBZ treated birds. Mild reversible inhibition of UA excretion was evident in all three groups, although UA remained within the population reference interval in contrast to the effects previously described for diclofenac and ketoprofen. All treatment groups had a drug concentration responsive increase in alanine transferase activity. CRP, FXN and PBZ were characterised by a maximum plasma concentration (Cmax) of 1051.8 ± 620.7 ng/ml, 335.9 ± 36.3 ng/ml and 11150 ± 2474.9 ng/ml at 4 ± 4.3, 0.45 ± 0.02 and 5.3 ± 5.2 hours (Tmax) respectively and a half-life of elimination of 13.3 ±5, 1.8±1 and 18.7 ±11.4 hours respectively. While we could not demonstrate a lethal effect of the tested substances, the presence of toxic clinical signs, clinical pathological changes and/or long half-lives of elimination suggests that all three drugs have a potential for toxicity in a larger population or on repeat administration. In conclusion while the studied substances were not as overtly toxic as diclofenac, they are of safety concern.     

High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected. The recovery of diclofenac was 92%, the analysis lasted 7 min with a sensitivity of 5 ng/ml and intra- and inter-day RSDs of 3 and 8%, respectively. The pharmacokinetics of diclofenac after oral and rectal administration in 10 healthy volunteers are reported.     

Plasma protein binding of ketoprofen enantiomers in man: method development and its application.

The protein binding of ketoprofen enantiomers was investigated in human plasma at physiological pH and temperature by ultrafiltration. 14C-labelled (RS)-ketoprofen was synthesized and purified by high-performance liquid chromatography and utilized as a means of quantifying the unbound species. In vitro studies were conducted with plasma obtained from six healthy volunteers. The plasma was spiked with (R)-ketoprofen alone, (S)-ketoprofen alone, and (RS)-ketoprofen in the enantiomeric concentration range of 1.0 to 19.0 micrograms/ml. The plasma protein binding of ketoprofen was nonenantioselective. At a racemic drug concentration of 2.0 micrograms/ml the mean (+/- SD) percentage unbound of (R)-ketoprofen was 0.80 (+/- 0.15)%. The corresponding value for (S)-ketoprofen, 0.78 (+/- 0.18)%, was not statistically different (P greater than 0.05). At this racemic drug concentration (2.0 micrograms/ml) the percentage unbound of each enantiomer was unaffected (P greater than 0.05) by the presence of the glucuronoconjugates of ketoprofen (10 micrograms/ml) in plasma. At clinically relevant concentrations, the plasma binding of ketoprofen did not exhibit enantioselectivity or concentration dependence nor was the binding of either enantiomer influenced by its optical antipode (P greater than 0.05).     

Acetylcholine's effect on vascular resistance and compliance in the pulmonary circulation

Acetylcholine's effect on the distribution of vascular resistance and compliance in the canine pulmonary circulation was determined under control and elevated vascular tone by the arterial, venous, and double occlusion techniques in isolated blood-perfused dog lungs at both constant flow and constant pressure. Large and small blood vessel resistances and compliances were studied in lungs given concentrations of acetylcholine ranging from 2.0 ng/ml to 200 micrograms/ml. The results of this study indicate that acetylcholine dilates large arteries at low concentrations (less than or equal to 20 ng/ml) and constricts small and large veins at concentrations of at least 2 micrograms/ml. Characterization of acetylcholine's effects at constant pulmonary blood flow indicates that 1) large artery vasodilation may be endothelial-derived relaxing factor-mediated because the dilation is blocked with methylene blue; 2) a vasodilator of the arachidonic acid cascade (blocked by ibuprofen), probably prostacyclin, lessens acetylcholine's pressor effects; 3) when vascular tone was increased, acetylcholine's hemodynamic effects were attenuated; and 4) acetylcholine decreased middle compartment and large vessle compliance under control but not elevated vascular tone. Under constant pressure at control vascular tone acetylcholine increases resistance in all segments except the large artery, and at elevated vascular tone the pressor effects were enhanced, and large artery resistance was increased.     

L-抗坏血酸洛芬酯非水相酶促合成的动力学与热力学

对酶法合成L-抗坏血酸洛芬酯(芬维C酯)的反应动力学与热力学进行研究,确定了最有效的酶促反应环境。合成布洛芬维C酯的最优条件:转速200r/min,温度65℃,加酶量5%(以底物的质量分数计),底物浓度1mol/L,平衡所需时间66h,平衡时产物质量分数为19.07%;合成酮洛芬维C酯的最优条件:200r/min,60℃,加酶量7.5%,底物浓度600mmol/L,平衡时间132h,产物质量分数为10.63%;合成氟比洛芬维C酯的最优条件:200r/min,65℃,加酶量5%,底物浓度400mmol/L,平衡时间144h,产物质量分数为6.76%。对底物进行了比较,得到了各自的动力学与热力学参数。布洛芬米氏常数为0.101μmol/L,vmax=32.68μmol/(min.g),热力学平衡常数为0.166;酮洛芬的分别为0.144μmol/L,12.97μmol/(min.g),0.091;氟比洛芬的分别为0.185μmol/L,9.35μmol/(min.g),0.055。     

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C₁₈ reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50–1000 ng/ml for fluvoxamine, 15–1000 ng/ml for fluoxetine, 25–500 ng/ml for norfluoxetine, 50–500 ng/ml for sertraline, 20–500 ng/ml for paroxetine, 25–550 ng/ml for citalopram, 25–750 ng/ml for desmethylcitalopram, 25–800 ng/ml for didesmethylcitalopram, 25–650 ng/ml for milnacipran, and 25–500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.     

Assay of 2-naphthol in human urine by high-performance liquid chromatography

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 μmol/mol creatinine) to 34.19 ng/ml (59.11 μmol/mol creatinine), and the mean±standard deviation was 5.08 ng/ml (6.60 μmol/mol creatinine)±5.75 ng/ml (9.22 μmol/mol creatinine). The mean±standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 μmol/mol creatinine)±6.16 ng/ml (10.23 μmol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 μmol/mol creatinine)±3.92 ng/ml (7.03 μmol/mol creatinine).     

High-performance thin-layer chromatographic determination of ibuprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for quantitation of ibuprofen from plasma is described. The drug was extracted from acidified plasma with hexane-isopropanol (85:15). The mobile phase composition was n-hexane-ethyl acetate-anhydrous acetic acid (75:25:2). Densitometric analysis of ibuprofen was carried out at 222 nm. The calibration curves of ibuprofen in chloroform and in plasma were linear over the range 2–20 μg. The mean values of intercept, slope and correlation coefficient were 0.0422±0.0018, 0.0356±0.0213 and 0.9976±0.0013 for standard curves in chloroform and 0.1044±0.003, 0.8759±0.0213 and 0.9939±0.001 for standard curves in plasma, respectively. The limit of detection of ibuprofen from human plasma (assay sensitivity) was 50 ng and no interference was found from endogenous compounds. The recovery of ibuprofen from human plasma using the described extraction procedure was about 85%. The mean relative standard deviations for within-day and between-day analyses were 2.24 and 2.6% for 5 μg and 3.67 and 3.2% for 15μg ibuprofen concentration, respectively. The method was utilized to monitor the plasma concentration of ibuprofen post administration of sustained release capsules in human patient volunteers.