Plasma pharmacokinetics and synovial concentrations of S-flurbiprofen plaster in humans.

PURPOSE: The purpose of this study is to investigate the pharmacokinetics and deep tissue penetration capability of the newly developed S-flurbiprofen plaster (SFPP) in humans. METHODS: Study 1: SFPP tape-type patch (2-60 mg) was applied to the lower back for 24 h in healthy adult volunteers. S-flurbiprofen (SFP) plasma concentration was measured over time to examine SFP pharmacokinetics. Study 2: SFPP (20 mg) was applied for 12 h to the affected knee of osteoarthritis (OA) patients who were scheduled for total knee arthroplasty. Deep tissues (synovial tissue and synovial fluid) were collected during surgery to compare SFP concentrations after application of SFPP or a commercially available flurbiprofen (FP) gel-type patch. RESULTS: Study 1: The plasma concentration of SFP was sustained during 24-h topical application of the SFPP, showing a high percutaneous absorption ratio of 51.4-72.2 %. Cmax and AUC0-∞ were dose-proportional. Study 2: After application of the SFPP for 12 h, SFP concentrations in the synovial tissue and synovial fluid were 14.8-fold (p = 0.002) and 32.7-fold (p < 0.001) higher, respectively, than those achieved by the FP patch. CONCLUSIONS: Sustained plasma concentration of SFP and high percutaneous absorption ratio was observed after 24-h topical application of the SFPP. Compared to the FP patch, the SFPP showed superior percutaneous absorption and greater tissue penetration of SFP into the synovial tissue. Greater tissue penetration of the SFPP seemed to be primarily due to its formulation. Thus, SFPP is expected to show higher efficacy for the treatment of knee OA.

Evaluation of systemic absorption and renal effects of topical ophthalmic flurbiprofen and diclofenac in healthy cats.

OBJECTIVE: To investigate systemic absorption and renal effects of topically applied ophthalmic flurbiprofen and diclofenac in healthy cats. ANIMALS STUDIED: Twelve domestic shorthair cats. PROCEDURES: Cats were randomly assigned to two treatment groups (n = 6) and administered one drop (approximately 40 µL) of either flurbiprofen 0.03% or diclofenac 0.1% in both eyes four times daily (6 am, 12 pm, 6 pm, and 12 am) for 14 days. Blood samples were collected on days 0, 4, 8, 14, 16, and 17 and analyzed by liquid chromatography and mass spectrometry for flurbiprofen and diclofenac plasma concentrations. A complete blood count (CBC), serum chemistry, and urinalysis were analyzed at the beginning of the study (Day 0) and at the end of topical drug administration (Day 15). RESULTS: Both drugs demonstrated systemic absorption. Flurbiprofen was detected (mean ± SD) at day 4 (237 ± 65 ng/mL), day 8 (396 ± 91 ng/mL), day 14 (423 ± 56 ng/mL), day 16 (350 ± 66 ng/mL), and day 17 (270 ± 62 ng/mL), and diclofenac was detected (mean ± SD) at day 4 (130 ± 44 ng/mL), day 8 (131 ± 25 ng/mL), day 14 (150 ± 36 ng/mL), and sporadically on day 16 [corrected]. Flurbiprofen plasma concentration decreased slowly over 48 h after the last dose. No clinically significant abnormalities were noted in the serum blood urea nitrogen, creatinine, or urine specific gravity at the end of topical drug administration compared to the beginning of the study. CONCLUSIONS: Flurbiprofen and diclofenac were systemically absorbed after topical administration four times daily to both eyes of healthy cats. Flurbiprofen reached higher plasma concentrations compared to diclofenac.

Influence of the oral dissolution time on the absorption rate of locally administered solid formulations for oromucosal use: the flurbiprofen lozenges paradigm.

Flurbiprofen is a nonsteroidal anti-inflammatory agent preferentially used for local oromucosal treatment of painful and/or inflammatory conditions of the oropharynx such as gingivitis, stomatitis, periodontitis, pharyngitis and laryngitis. In this study, we have investigated the bioavailability of a new generic formulation of flurbiprofen lozenges developed by Epifarma Srl, compared to the originator Benactiv Gola® taken as reference. Within the framework of a formal bioequivalence study, we investigated in particular the putative influence of oral dissolution time (i.e. the time spent suckling the lozenge from its intake to complete dissolution) on the absorption rate, and the contribution of this factor to the total variability of plasma flurbiprofen during absorption. We found that the amount of flurbiprofen absorbed into the systemic circulation is not significantly higher for the test drug compared to that of the reference product. We observed that the length of oral dissolution time is inversely correlated to 10-min flurbiprofen plasma levels in the test but not in the reference formulation. We estimated that oral dissolution time accounts for about 14% of overall variability in flurbiprofen plasma 10 min after test drug administration.

Determination of flurbiprofen in human plasma by gas chromatography with mass spectrometry and its pharmacokinetics.

This paper describes a GCIMS method for the determination of flurbiprofen in human plasma. Flurbiprofen and internal standard ibuprofen were extracted from plasma by using a liquid-liquid extraction method. Derivatization was carried out using N-Methyl-N-(trimethylsilyl)trifluoroacetamide. The calibration curve was linear between the concentration range of 0.10 and 5.0 microg/mL. Intraday and interday precision values for flurbiprofen in plasma were less than 5.49%, and accuracy (relative error) was better than 5.33%. The extraction recoveries of flurbiprofen from human plasma were between 93.6 and 98.6%. The LOD and LOQ of flurbiprofen were 0.03 and 0.10 microg/mL, respectively. This assay was applied to determine the pharmacokinetic parameters of flurbiprofen in healthy Turkish volunteers who had been given 100 mg of flurbiprofen.

Effect of protein binding on the pharmacokinetics of the six substrates in the Basel phenotyping cocktail in healthy subjects and patients with liver cirrhosis.

Phenotyping serves to estimate enzyme activities in healthy persons and patients in vivo. Low doses of enzyme-specific substrates are administered, and activities estimated using metabolic ratios (MR, calculated as AUCmetabolite/AUCparent). We administered the Basel phenotyping cocktail containing caffeine (CYP1A2 substrate), efavirenz (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6) and midazolam (CYP3A) to 36 patients with liver cirrhosis and 12 control subjects and determined free and total plasma concentrations over 24 h. Aims were to assess whether MRs reflect CYP activities in patients with liver cirrhosis and whether MRs calculated with free plasma concentrations (MRfree) provide better estimates than with total concentrations (MRtotal). The correlation of MRtotal with MRfree was excellent (R2 >0.910) for substrates with low (<30 %, caffeine and metoprolol) and intermediate protein binding (≥30 and <99 %, midazolam and omeprazole) but weak (R2 <0.30) for substrates with high protein binding (≥99 %, efavirenz and flurbiprofen). The correlations between MRtotal and MRfree with CYP activities were good (R2 >0.820) for CYP1A2, CYP2C19 and CYP2D6. CYP3A4 activity was reflected better by midazolam elimination than by midazolam MRtotal or MRfree. The correlation between MRtotal and MRfree with CYP activity was not significant or weak for CYP2B6 and CYP2C9. In conclusion, MRs of substrates with an extensive protein binding (>99 %) show high inter-patient variabilities and do not accurately reflect CYP activity in patients with liver cirrhosis. Protein binding of the probe drugs has a high impact on the precision of CYP activity estimates and probe drugs with low or intermediate protein binding should be preferred.

Pharmacokinetics and pharmacodynamics of CHF5074 after short-term administration in healthy subjects.

CHF5074 has been shown to inhibit brain ß-amyloid deposition and attenuate memory deficits in different transgenic mice models of Alzheimer disease. We evaluated the safety, pharmacokinetics, and pharmacodynamics of 3 ascending dose regimens of CHF5074 (200, 400, and 600 mg/d for 14 d) in a double-blind, placebo-controlled, parallel group study involving 48 healthy subjects. Plasma, urine, and cerebrospinal fluid (CSF) samples were collected for measuring drug and main metabolite concentrations and potential biomarkers of pharmacodynamic activity (ß-amyloid1-40, ß-amyloid1-42, soluble CD40 ligand, and tumor necrosis factor-α). All subjects completed the study, and no serious or severe adverse events were reported. The maximum tolerated dose was close to 600 mg/d with mild diarrhea being the most frequent adverse event at this dose. CHF5074 reached peak plasma levels 2 to 3 hours after drug administration and then was slowly eliminated (t(1/2z)=30 h) in the urine as glucoronide. Systemic exposure to the drug appeared to be dose-proportional with a 2-fold accumulation ratio at steady state. Metabolite plasma levels peaked at 4 to 5 hours and accounted for about 25% of the parent compound. Drug levels in the CSF were dose-proportional. The drug dose-dependently lowered the levels of the soluble CD40 ligand, a marker of microglia activation, in both plasma and CSF samples.

Alteration in plasma protein binding properties of propranolol and flurbiprofen during development of adjuvant-induced arthritis in rats.

Adjuvant-induced arthritis (AA) in the rat is used as a model for rheumatoid arthritis. In AA rats, the pharmacokinetics of various drugs is affected due to the alterations of plasma protein binding of drugs. We choose propranolol (PL) and flurbiprofen (FP) as model basic and acidic drugs, respectively, and investigated the effect of AA induction on their plasma protein binding at each developing stage of inflammation. The plasma protein binding of PL and FP was dramatically changed due to reduced albumin and increased α1-acid glycoprotein levels for at least 21 days after adjuvant treatment. Moreover, we illustrated the differences in protein binding in AA between both the drugs in each developing stage of inflammation. These results suggest that the changed plasma protein levels in AA rats accompanying the altered protein binding of drugs affect the pharmacokinetics of drugs which extensively bind to plasma protein under inflammatory condition.

Flurbiprofen-loaded nanoparticles prepared with polyvinylpyrrolidone using Shirasu porous glass membranes and a spray-drying technique: nano-sized formation and improved bioavailability.

A unique flurbiprofen-loaded nanoemulsion was listed earlier using a Shirasu porous glass (SPG) membrane emulsification technique, which gave constant emulsion droplets with a thin size distribution. In this study, a flurbiprofen-loaded nanoemulsion was developed further into a solid form using polyvinylpyrrolidone (PVP) as a carrier by a spray-drying technique. The flurbiprofen-loaded nanoparticles with a weight ratio of flurbiprofen/PVP/surfactant mixture of 1/8/2 were connected with about 130,000-fold enhanced drug solubility and had a mean size of about 70 nm. In these nanoparticles, flurbiprofen was found in an altered amorphous state. Additionally, the nanoparticles gave significantly shorter T(max), and greater AUC and C(max) compared to the commercially available product. Specially, the AUC of the drug from the nanoparticles was about 10-fold greater compared to the commercially available product. Therefore, these flurbiprofen-loaded nanoparticles can be convenient for distributing a poorly water-soluble flurbiprofen with improved bioavailability using uniform nano-sized particles.

The use of an artificial skin model to study transdermal absorption of drugs in inflamed skin.

Studies on drug disposition in inflamed skin are important for safe and effective application of topical drugs. Here, the absorption of flurbiprofen (FP) through inflamed skin was examined in vivo and in a skin-mimicking artificial model system. The model skin system consisted of a silicone membrane acting as a model stratum corneum, laminated dialysis membranes acting as a model of viable skin, and 2 microdialysis probes-one used for determination of FP concentration and one acting as a model vessel. This model system could be used for quantitative evaluation of complicated permeation processes. In the in vivo experiments, FP absorption was suppressed in rats with inflamed skin induced by an intracutaneous injection of a mixed solution of λ-carrageenan, zymosan, and casein. Bovine serum albumin solution was placed between the dialysis membranes in the model skin system to mimic protein leaching in skin; the results suggested that the delayed absorption of FP in inflamed skin was due to binding to serum proteins leaching in the tissue. Such a combination of in vivo experiments and a model skin system is useful for understanding complex phenomena in inflamed and damaged skin and reduces experimental animal use.

A randomised, two-period, cross-over, open-label study to evaluate the pharmacokinetic profiles of single doses of two different flurbiprofen 8.75-mg lozenges in healthy volunteers.

AIMS: To compare the bioavailability of a new oromucosal formulation of flurbiprofen 8.75-mg lozenges, developed by Alfa Wassermann S.p.A. (test drug) to that of marketed flurbiprofen 8.75-mg lozenges (Benactiv Gola®, reference drug). METHODS: This was an open, randomised, two-period, crossover, pharmacokinetic (PK) study in which flurbiprofen plasma levels were compared in 12 healthy volunteers after the administration of single doses (8.75 mg × 2) of two different oromucosal lozenges to be sucked and slowly dissolved in the mouth. A wash-out period of at least 7 days separated the two study periods. Blood samples were collected prior to dosing and at predefined intervals for 24 h after dose. Flurbiprofen plasma concentrations were determined by liquid chromatography/tandem mass spectrometry. PK parameters maximum plasma concentration (C(max)), time to maximum plasma concentration (T(max)), area under the plasma concentration-time curve from time zero to 24 hours (AUC(0-t)), area under the plasma concentration-time curve from time zero to infinity (AUC(0-)∞) and half-life were calculated and compared by analysis of variance using treatment, period and sequence as sources of variation. Bioequivalence between the two formulations was based on 90% confidence intervals of the ratio of the geometric means of C(max) and AUC falling within the 0.80-1.25 range as defined in bioequivalence guidelines by regulators. Tolerability of the two formulations was assessed by adverse event monitoring, routine laboratory tests, physical examination, electrocardiographic tracing and vital sign measurements. RESULTS: All enrolled subjects completed the study. Bioequivalence without significant treatment effect was demonstrated between the test drug/reference drug ratios of mean C(max) and AUCs. Moreover, mean T(max) was superimposable. No safety parameter presented a clinically relevant variation after administration of either formulation that were therefore well tolerated. CONCLUSION: The new formulation of flurbiprofen 8.75-mg compressed lozenges developed by Alfa Wassermann S.p.A. is bioequivalent to the reference product flurbiprofen 8.75-mg lozenges (Benactiv Gola) in healthy volunteers.

A Sensitive UPLC-MS/MS Method for the Determination of Flurbiprofen in Rat Plasma: Application to Real Sample.

For the quantification of flurbiprofen in rat plasma, a simple UPLC-MS/MS method with high sensitivity and short retention time for flurbiprofen was developed and validated using specific parameters. Etodolac was used as internal standard. The transitions (precursor to the product) of flurbiprofen and internal standard were obtained using the electrospray ionization in the negative ion multiple reaction monitoring mode, 243.2 â†’ 199.2, 286.2 â†’ 212.1, respectively. For chromatographic separation, C18 column was used for the stationary phase and gradient elution was used for the mobile phase. This mobile phase consisted of a methanol (A) and a 5 mM ammonium formate solution (B), which varied at a flow rate of 0.4 mL/min. For flurbiprofen, LLOQ was determined as 5 ng/mL. Quantification of flurbiprofen in the rat plasma with a linear calibration curve of 5-5000 ng/mL (r > 0.9991 for plasma) is possible with a retention time of 1.89 min. The total analysis time of the method was 3 min. The proposed method was validated. The intraday and inter-day precision (RSD%) and accuracy (RE%) were within 10% in all cases for flurbiprofen. The stability of flurbiprofen was evaluated under conditions such as short-term, long-term, autosampler and freeze/thaw. After method validation, flurbiprofen was succesfully quantified in real rat plasma samples.

Plasma and cerebrospinal fluid pharmacokinetics of flurbiprofen in children.

AIMS: This study was designed to characterize paediatric pharmacokinetics and central nervous system exposure of flurbiprofen. METHODS: The pharmacokinetics of flurbiprofen were studied in 64 healthy children aged 3 months to 13 years, undergoing surgery with spinal anaesthesia. Children were administered preoperatively a single dose of flurbiprofen intravenously as prodrug (n= 27) or by mouth as syrup (n= 37). A single cerebrospinal fluid (CSF) sample (n= 60) was collected at the induction of anaesthesia, and plasma samples (n= 304) before, during and after the operation (up to 20 h after administration). A population pharmacokinetic model was built using the NONMEM software package. RESULTS: Flurbiprofen concentrations in plasma were well described by a three compartment model. The apparent bioavailability of oral flurbiprofen syrup was 81%. The estimated clearance (CL) was 0.96l h(-1) 70 kg(-1) . Age did not affect the clearance after weight had been included as a covariate. The estimated volume of distribution at steady state (V(ss) ) was 8.1 l 70 kg(-1) . Flurbiprofen permeated into the CSF, reaching concentrations that were seven-fold higher compared with unbound plasma concentrations. CONCLUSIONS: Flurbiprofen pharmacokinetics can be described using only weight as a covariate in children above 6months, while more research is needed in neonates and in younger infants.

The disposition of free and niosomally encapsulated Rac-flurbiprofen in dairy bovines.

Pharmacokinetic parameters were established for flurbiprofen (FBP) after intravenous (i.v.) administration (0.5 mg/kg) of niosomal and nonniosomal formulations in dairy cattle. Niosomes of FBP showed a drug loading of 92.0 +/- 0.7% and the intravenous administration of the FBP niosomes to dairy cattle did not produce any immunological reaction associated to niosomal components. Niosomal FBP was slowly eliminated from plasma and mean residual time (MRT) and AUC(0-->t) and t (1/2) values were significantly higher than those for non niosomal FBP formulations. The results presented in this study indicate that the long circulation of FBP niosomes offers a potential application for improving the pharmacokinetic parameters of short half-life drugs for clinical use. Niosomes offer new promising perspectives of drug delivery modules in bovine therapeutics.

[Population pharmacokinetic modeling of flurbiprofen].

The paper is to report the establishment of a population pharmacokinetic model for flurbiprofen (FP), an active metabolite of flurbiprofen axetil (FA). 246 FP serum concentration and clinical data were perspectively collected from 23 general anaesthesia patients receiving FA intravenously before operation in Dentofacial Surgery and Otorhinolaryngology Department of the First Affiliated Hospital of Fujian Medical University. Population pharmacokinetic data analysis was performed using NONMEM software. The measure of Bootstrap was applied for internal validation, while Visual Predictive check was adopted for external validation. The data of FP correspond with two-compartment model. The body weight (WT) had conspicuous effect on clearance and volume of central compartment, while sex, age and daily dose of administration had no marked effect on pharmacokinetic parameter of FP. The basic model was described as follows: CL (L x h(-1)) = 1.28x EXP(ETA(1)), V1 (L) = 5.03x EXP(ETA(2)), Q (L x h(-1)) = 8.5 x EXP(ETA(3)), V2 (L) = 4.39 x EXP(ETA(4)). The final model was described as follows: CL (L x h(-1)) = 1.32 x (WT/60) x EXP(ETA(1)), V1 (L) = 5.23 x (WT/60) x EXP(ETA(2)), Q (L x h(-1)) = 8.45 x EXP(ETA(3)), V2 (L) = 4.37 x EXP(ETA(4)). The population typical value of CL, V1, Q and V2 were: 1.32 L x h(-1), 5.23 L, 8.45 L x h(-1) and 4.37 L, respectively. Bootstrap and visual predictive check show that the final model of FP is stable, effective and predictable. A novel population pharmacokinetic model is developed to estimate the individual pharmacokinetic parameter for patients intravenous injecting FA in terms of patients' characteristics and dosing history, and to design a prior dosage regimen.

Facile synthesis of magnetic carbon nanotubes derived from ZIF-67 and application to magnetic solid-phase extraction of profens from human serum.

Magnetic carbon nanotubes (CNTs) with encapsulated Co nanoparticles (Co@CNTs), was synthesized by exploiting the one-step pyrolysis strategy using ZIF-67 as template. The as-synthesized Co@CNTs is provided with the nanopores, a large specific surface area, and strong magnetic response. The obtained Co@CNTs was used as magnetic solid-phase extraction adsorbents to extract two profens including flurbiprofen and ketoprofen. The parameters of extraction efficiency, involving extraction time, sample solution volume, ionic strength, pH and the conditions of desorption efficiency, were optimized in detail. After determined by high-performance liquid chromatography-ultraviolet (HPLC-UV), the results evinced that Co@CNTs showed a high extraction efficiency with high enrichment factors of 832 and 672. The good linear range of both flurbiprofen and ketoprofen were all 5.0-1000 ng L-1, with the limit of detection were 0.60 ng L-1 and 0.70 ng L-1, respectively. Furthermore, a valid method for the extraction of flurbiprofen and ketoprofen from human serum was established. The spiking recoveries of two profens were between 86.74% and 97.22%, and the relative standard deviation was less than 6.55%. Co@CNTs can be repeatedly used at least 10 times, indicating its excellent regeneration and reusability. The results demonstrated that the Co@CNTs materials exhibits high enrichment ability and extraction efficiency, playing great promise in MSPE.

Lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in vitro, ex vivo and in vivo studies.

The aim of the study is to prepare aqueous dispersions of lipid nanoparticles--flurbiprofen solid lipid nanoparticles (FLUSLN) and flurbiprofen nanostructured lipid carriers (FLUNLC) by hot homogenization followed by sonication technique and then incorporated into the freshly prepared hydrogels for transdermal delivery. They are characterized for particle size, for all the formulations, more than 50% of the particles were below 300 nm after 90 days of storage at RT. DSC analyses were performed to characterize the state of drug and lipid modification. Shape and surface morphology were determined by TEM which revealed fairly spherical shape of the formulations. Further they were evaluated for in vitro drug release characteristics, rheological behaviour, pharmacokinetic and pharmacodynamic studies. The pharmacokinetics of flurbiprofen in rats following application of SLN gel (A1) and NLC gel (B1) for 24 h were evaluated. The Cmax of the B1 formulation was 38.67 +/- 2.77 microg/ml, which was significantly higher than the A1 formulation (Cmax = 21.79 +/- 2.96 microg/ml). The Cmax and AUC of the B1 formulation were 1.8 and 2.5 times higher than the A1 gel formulation respectively. The bioavailability of flurbiprofen with reference to oral administration was found to increase by 4.4 times when gel formulations were applied. Anti-inflammatory effect in the Carrageenan-induced paw edema in rat was significantly higher for B1 and A1 formulation than the orally administered flurbiprofen. Both the SLN and NLC dispersions and gels enriched with SLN and NLC possessed a sustained drug release over period of 24 h but the sustained effect was more pronounced with the SLN and NLC gel.

Cloud point extraction-HPLC method for determination and pharmacokinetic study of flurbiprofen in rat plasma after oral and transdermal administration.

A method based on cloud-point extraction (CPE) was developed for the determination of flurbiprofen (FP) in rat plasma after oral and transdermal administration by high-performance liquid chromatography coupled with UV detection (HPLC-UV). The non-ionic surfactant Genapol X-080 was chosen as the extract solvent. Variables parameter affecting the CPE efficiency were evaluated and optimized. Chromatography separation was performed on a Diamond C(18) column (4.6 mm i.d. x 250 mm, 10 microm particle size) by isocratic elution with UV detection at 254 nm. The assay was linear over the range of 0.2-50 and 0.1-10 microg/ml for oral and transdermal administration, respectively, and the lower limit of quantification (LLOQ) was 0.1 microg/ml. The extraction recoveries were more than 84.5%, the accuracies were within +/-3.8%, and the intra- and inter-day precisions were less than 10.1% in all cases. After strict validation, the method indicated good performance in terms of reproducibility, specificity, linearity, precision and accuracy, and it was successfully applied to the pharmacokinetic study of flurbiprofen in rats after oral and transdermal administration.

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug-protein binding in human plasma.

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid-liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent (S)-(-)-alpha-(1-naphthyl)ethylamine (S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C18 (4.6 mm x 250 mm, 5 microm) column with a mixture of acetonitrile-0.01 mol.L(-1) phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile-0.01 mol.L(-1) phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal-0.01 mol.L(-1) potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL.min(-1) and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 microg.mL(-1) for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 microg.mL(-1) (R.S.D.<10%, n=5). The method developed was used to study the drug-protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.

Dried blood spots and parallel artificial liquid membrane extraction-A simple combination of microsampling and microextraction.

In this paper, parallel artificial liquid membrane extraction (PALME) was used for the first time to clean-up dried blood spots (DBS) prior to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Fundamental studies exploring amongst others desorption from the DBS in alkaline or acidic aqueous conditions, total extraction time and absolute recoveries were executed. Desorption and PALME were performed using a set of two 96-well plates, one of them housing the sample and the other comprising the supported liquid membrane (SLM) and the acceptor solution. In one procedure, amitriptyline and quetiapine (basic model analytes) were desorbed from the DBS using 250 µL of 10 mM sodium hydroxide solution (aqueous), and subsequently extracted through the SLM consisting of 4 µL of 1% trioctylamine in dodecyl acetate, and further into an acceptor solution consisting of 50 µL of 20 mM formic acid. In a second procedure, ketoprofen, fenoprofen, flurbiprofen, and ibuprofen (acidic model analytes) were desorbed from the DBS into 20 mM formic acid, extracted through an SLM with dihexyl ether, and further into an acceptor solution of 25 mM ammonia. Within 60 min of PALME, both basic and acidic model analytes were effectively desorbed from the DBS and extracted into the acceptor solution, which was injected directly into the analytical instrument. Recoveries between 63 and 85% for the six model analytes were obtained. PALME provided excellent clean-up from the DBS samples, and acceptor solutions were free from phospholipids. Linearity was obtained with r2 > 0.99 for five of the six analytes. Accuracy, precision and UHPLC-MS/MS matrix effects were in accordance with the European Medicines Agency (EMA) guideline. Based on these experiments, PALME shows great potential for future processing of DBS in a short and simple way, and with the presented setup, up to 96 DBS can be processed within a total extraction time of 60 min.

Simultaneous analysis of cytochrome P450 probes-dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma.

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A "cocktail" approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4'-hydroxy-flurbiprofen and 1'-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify cartridges) extraction procedure from 200 microl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm Lx3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4'-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1'-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: -9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.