Hybrid monoliths with metal-organic frameworks in spin columns for extraction of non-steroidal drugs prior to their quantitation by reversed-phase HPLC.

A (glycidyl methacrylate)-co-(ethylene glycol dimethacrylate) polymer (poly(GMA-co-EDMA)) was functionalized with metal-organic frameworks (MOF) and used as a sorbent for solid-phase extraction (SPE). The polymeric sorbent was prepared in-situ by photopolymerization in a previously wall-modified spin column, and then modified with an amino-modified MOF of type NH2-MIL-101(Cr). The sorbents were used for the extraction of nonsteroidal anti-inflammatory drugs (NSAIDs) from human urine samples. The sorbent was compared with the parent monolith and embedded approach, where the MOF particles are admixed in the polymerization mixture before the in-situ polymerization in the modified spin column. SPE is performed by percolating the sample solutions in a centrifuge, which streamlines the SPE steps. The hybrid composites were characterized by scanning electron microscopy and nitrogen intrusion porosimetry. Three NSAIDs (ketoprofen, flurbiprofen, and ibuprofen) were tested. They were eluted from the sorbent with acidified water-acetonitrile mixtures and subsequently analyzed by reversed-phase HPLC with UV detection. The detection limits varied in the range from 0.1 to 7 µg·L-1, and the precisions (relative standard deviation) were <14% in all the cases. The recoveries were between 71.0 and 78.0% in spiked urine samples. Graphical abstractA hybrid monolith modified with amino-modified MOF [named NH2-MIL-101(Cr)] in wall-modified spin columns was prepared. The resulting micro-extraction device was applied to the extraction and preconcentration of non-steroidal anti-inflammatory drugs.

Switchable-hydrophilicity solvent liquid-liquid microextraction of non-steroidal anti-inflammatory drugs from biological fluids prior to HPLC-DAD determination.

Switchable-hydrophilicity solvent liquid-liquid microextraction was used prior to high-performance liquid chromatography with diode-array detector (HPLC-DAD) for the determination of four non-steroidal anti-inflammatory drugs [i.e., ketoprofen, etodolac, flurbiprofen and ibuprofen] in human urine, saliva and milk. Optimum extraction conditions were as follows: 500 µL switched-on N,N-dimethylcyclohexylamine as the extraction solvent, 9.5 mL of the aqueous phase, 500 µL 20 M sodium hydroxide as a switching-off trigger, and within 30 s extraction time. A portion of the final extract was directly injected into HPLC. Under optimized extraction and chromatographic conditions, limits of detection ranged between 0.04 and 0.18 µg mL-1 in all matrices analyzed. Good linearity with coefficients of determination (R2) ranging between 0.9955 and 0.9998, and percentage relative standard deviations (%RSD) of 0.9-7.7% were obtained. The proposed method was efficiently used for the extraction of the analytes in the biological fluids with percentage relative recoveries (%RR) ranging between 95.7 and 109.2%.

Determination of non-steroidal anti-inflammatory drugs in urine by the combination of stir membrane liquid-liquid-liquid microextraction and liquid chromatography.

A stir membrane liquid phase microextraction procedure working under the three-phase mode is proposed for the first time for the determination of six anti-inflammatory drugs in human urine. The target compounds are isolated and preconcentrated using a special device that integrates the extractant and the stirring element. An alkaline aqueous solution is used as extractant phase while 1-octanol is selected as supported liquid membrane solvent. After the extraction, all the analytes are determined by liquid chromatography (LC) with ultraviolet detection (UV). The analytical method is optimized considering the main involved variables (e.g., pH of donor and acceptor phases, extraction time, stirring rate) and the results indicate that the determination of anti-inflammatory drugs at therapeutic and toxic levels is completely feasible. The limits of detection are in the range from 12.6 (indomethacin) to 30.7 µg/L (naproxen). The repeatability of the method, expressed as relative standard deviation (RSD, n = 5) varies between 3.4% (flurbiprofen) and 5.7% (ketoprofen), while the enrichment factors are in the range from 35.0 (naproxen) to 72.5 (indomethacin).

Oral flurbiprofen metabolic ratio assessment using a single-point dried blood spot.

We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.

Evaluation of flurbiprofen urinary ratios as in vivo indices for CYP2C9 activity.

AIMS: We investigated flurbiprofen pharmacokinetics in 12 volunteers to develop a phenotypic trait measure that correlates with the fractional clearance to 4'-hydroxyflurbiprofen. The effect of the CYP2C9 inhibitor fluconazole on flurbiprofen metabolism was also evaluated. METHODS: Flurbiprofen pharmacokinetics were evaluated before and after the first and seventh doses of fluconazole. The urinary recovery ratio was calculated as FLRR = 4'-OHF/[4'-OHF + F(tot)] and the urinary metabolic ratio was calculated as FLMR = 4'-OHF/F(tot), where 4'-OHF and F(tot) represent total (conjugated and unconjugated) amounts recovered in urine. RESULTS: There was a statistically significant relationship between the 4'-OHF formation clearance (4OHCLf) and both the 8-h FLRR and the 8-h FLMR with and without administration of fluconazole. The flurbiprofen apparent oral clearance (CL/F) was decreased by 53% [90% confidence interval (CI) -58, -48] and 64% (90% CI -69, -59), respectively, after administration of one and seven doses of fluconazole when compared with administration of flurbiprofen alone; similarly, the 4OHCLf decreased by 69% (90% CI -74, -64) and 78% (90% CI -83, -73), the 8-h FLRR decreased by 35% (90% CI -41, -29) and 40% (90% CI -46, -35) and the 8-h FLMR decreased by 61% (90% CI -65, -58) and 67% (90% CI -70, -63). The magnitude of decrease in CL/F and 4OHCLf was greater after seven doses compared with after one dose of fluconazole (P < 0.005). CONCLUSIONS: This study provides strong evidence that both the 8-h FLRR and the 8-h FLMR are suitable phenotypic indices for CYP2C9 activity.

The effects and metabolic fate of nitroflurbiprofen in healthy volunteers.

OBJECTIVE: Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are a new class of cyclooxygenase (COX) inhibitors. To investigate whether these drugs actually release nitric oxide (NO), we labeled the nitroxy group of nitroflurbiprofen with nitrogen 15 to determine the metabolic fate of this compound in humans. METHOD: Six healthy volunteers who fasted were given an oral dose of 15 N-nitroflurbiprofen (100 mg). Samples of blood, urine, and gastric headspace gas were taken over a 24-hour period to determine the levels of nitroflurbiprofen, flurbiprofen, total nitrate/nitrite, 15 N-nitrate/nitrite, COX activity, and gastric NO. In a crossover study (1 week apart), a further 6 healthy volunteers who fasted were given an oral dose of nitroflurbiprofen (100 mg) or flurbiprofen (65 mg) and levels of gastric NO were determined. RESULTS: Nitroflurbiprofen was undetectable in the systemic circulation. Levels of 15 N-nitrate/nitrite (5.2% +/- 1.5% enrichment) and flurbiprofen (2.4 +/- 0.7 microg/mL) peaked at 4 hours in the plasma and gradually decreased thereafter. In unstimulated blood, the plasma levels of thromboxane B 2 (COX-1 activity) were 2 to 3 ng/mL, and after calcium ionophore stimulation, large amounts of thromboxane B 2 were produced (112 +/- 31 ng/mL). Prostaglandin E 2 was undetectable in unstimulated blood. After lipopolysaccharide stimulation, the plasma levels of prostaglandin E 2 increased to 15 +/- 4 ng/mL. The metabolite flurbiprofen inhibited plasma COX-1 activity for the duration of the study period (maximum inhibition at 4 hours), whereas COX-2 activity recovered after 6 hours. In the crossover study, levels of gastric NO were higher in subjects given nitroflurbiprofen, when compared with those given flurbiprofen. (The area under the curve for gastric NO was 435 +/- 107 ppm . h versus 305 +/- 94 ppm . h [95% confidence interval of the difference, 89-172 ppm . h; P < .001]). CONCLUSION: Nitroflurbiprofen was undetectable in the systemic circulation, suggesting metabolism to 15 N-nitrate/nitrite and flurbiprofen in the presystemic circulation. Levels of gastric NO were significantly higher after ingestion of nitroflurbiprofen than flurbiprofen.

Enantioseparation of flurbiprofen and ketoprofen in patches and in urine excretions by achiral gas chromatography.

The enantiomeric composition tests on flurbiprofen and ketoprofen present in patch products and in urine excretions following patch applications were performed as diastereomeric (R)-(+)-1-phenylethylamides by achiral gas chromatography and by gas chromatography-mass spectrometry in selected ion monitoring mode. The method for determination of (R)- and (S)-enantiomers in the range from 0.1 to 5.0 microg was linear (r > or = 0.9996) with acceptable precision (% RSD < or = 5.2) and accuracy (% RE = 0.6 approximately -2.4). The enantiomeric compositions of flurbiprofen in one patch product and of ketoprofen in five different products were identified to be racemic with relatively good precision (< or = 6.4%). The urinary excretion level of (R)-flurbiprofen was two times higher than its antipode, while the comparable excretion levels of (R)- and (S)-enantiomers for ketoprofen were observed.

Differences in flurbiprofen pharmacokinetics between CYP2C9*1/*1, *1/*2, and *1/*3 genotypes.

OBJECTIVE: This study was conducted to examine differences in flurbiprofen metabolism among individuals with the CYP2C9*1/*1, *1/*2, and *1/*3 genotypes. METHODS: Fifteen individuals with the CYP2C9*1/*1 ( n=5), *1/*2 ( n=5), and *1/*3 ( n=5) genotypes received a single 50-mg oral dose of flurbiprofen. Plasma and urine samples were collected over 24 h, and flurbiprofen and 4'-hydroxyflurbiprofen pharmacokinetic data were compared across genotypes. RESULTS: CYP2C9 genotype was a significant predictor of flurbiprofen metabolism and accounted for 59% of the variability in flurbiprofen AUC(0- infinity ), and approximately 50% of the variability in flurbiprofen oral clearance, formation clearance to 4'-hydroxyflurbiprofen, and the 0 to 24-h urinary metabolic ratio of flurbiprofen to 4'-hydroxyflurbiprofen. Flurbiprofen AUC(0- infinity )was significantly higher and all measures of flurbiprofen clearance were significantly lower in the CYP2C9*1/*3 individuals than in those with *1/*1. Significant differences in these parameters were not detected between *1/*2 subjects and *1/*1 subjects. CONCLUSIONS: CYP2C9 genotype is a significant predictor of flurbiprofen disposition in humans by altering CYP2C9-mediated metabolism and reducing systemic clearance. The effects are most pronounced in individuals carrying the *3 allele.

Tolbutamide, flurbiprofen, and losartan as probes of CYP2C9 activity in humans.

The metabolic activity of CYP2C9 in 16 subjects expressing four different genotypes (CYP2C9*1/*1, *1/*2, *1/*3, and *2/*2) was evaluated. Single oral doses of tolbutamide, flurbiprofen, and losartan were administered in a randomized, crossover design. Plasma and urine were collected over 24 hours. The urinary metabolic ratio and amount of metabolite(s) excreted were correlated with formation clearance. The formation clearance of tolbutamide to its CYP2C9-mediated metabolites demonstrated a stronger association with genotype compared to flurbiprofen and losartan, respectively (r2 = 0.64 vs. 0.53 vs. 0.42). A statistically significant correlation was observed between formation clearance of tolbutamide and the 0- to 12-hour urinary amount of 4'-hydroxytolbutamide and carboxytolbutamide (r = 0.84). Compared to tolbutamide, the correlations observed between the respective measures of flurbiprofen and losartan metabolism were not as strong. Tolbutamide is a better CYP2C9 probe than flurbiprofen and losartan, and the 0- to 12-hour amount of 4'-hydroxytolbutamide and carboxytolbutamide is the best urinary measure of its metabolism.

Minimal in vivo activation of CYP2C9-mediated flurbiprofen metabolism by dapsone.

Dapsone has been shown to activate flurbiprofen 4'-hydroxylation by expressed CYP2C9 enzyme and in human liver microsomes. It has been suggested that this observation is due to substrate cooperativity on enzyme activity; however, the in vivo relevance of this observation is unknown. Thus, the purpose of this study was to evaluate whether dapsone can act cooperatively with flurbiprofen to activate the in vivo metabolism of flurbiprofen to 4'-hydroxyflurbiprofen. Twelve healthy subjects received single-dose flurbiprofen 50 mg on three occasions: alone (visit A); 2 h after a single dapsone 100-mg dose (visit B); and 2 h after the seventh daily dose of dapsone 100 mg (visit C). Concentrations of flurbiprofen and 4'-hydroxy flurbiprofen in plasma and urine and dapsone and N-acetyldapsone in plasma were determined by HPLC. Flurbiprofen pharmacokinetic parameters for the three visits were estimated by non-compartmental methods and compared in the absence and presence of dapsone. Flurbiprofen apparent oral clearance was increased by approximately 11% (P < 0.02) after dapsone 100 mg for 7 days. Dapsone plasma concentrations averaged 5 +/- 2 microM after a single dose and 11 +/- 4 microM after seven daily 100 mg doses. These dapsone plasma concentrations were within the range of concentrations producing activation of flurbiprofen metabolism by CYP2C9 in vitro. These results are consistent with the hypothesis that dapsone does influence flurbiprofen metabolism in vivo in a cooperative way to enhance metabolism. However, the magnitude of effect is substantially less than observed in vitro.

Sensitive and specific high-performance liquid chromatographic assay for 4'-hydroxyflurbiprofen and flurbiprofen in human urine and plasma.

A high-performance liquid chromatographic assay has been developed for the simultaneous quantitation of flurbiprofen and its major metabolite, 4'-hydroxyflurbiprofen, in urine and plasma. No extraction procedure was necessary for analysis of these compounds, which reduced time involved in sample preparation. The analytes were separated on a Brownlee Spheri-5 C18 column with a mobile phase of acetonitrile-20 mM dibasic potassium phosphate pH 3 buffer (40:60, v/v). Fluorescence detection was utilized with an excitation wavelength of 260 nm and an emission wavelength of 320 nm, providing excellent sensitivity. The limit of quantitation for 4'-hydroxyflurbiprofen and flurbiprofen was 0.25 microg/ml in urine and 0.05 microg/ml and 0.25 microg/ml, respectively, in plasma. All components were eluted within 16 min. Intra-day, inter-day, freeze-thaw, and in process stability were tested for both compounds and the coefficient of variation was less than 14% in all cases. This method provides a sensitive and specific assay for the detection of flurbiprofen and 4'-hydoxyflurbiprofen in urine and plasma and is suitable for use in in vivo studies evaluating the regulation of CYP2C9 activity.

Pharmacokinetic comparison of oral and local action transcutaneous flurbiprofen in healthy volunteers.

Flurbiprofen is a propionic acid-derived non-steroidal anti-inflammatory drug (NSAID) used widely in the treatment of rheumatism and non-arthritic pain. The pharmacokinetics of topically and orally administered flurbiprofen were compared in a two-part, open study involving healthy adult volunteers. In the first (cross-over) part of the study, 12 Caucasians were randomized to receive either a single oral dose of 50 mg flurbiprofen or a single topical application of a novel 40 mg flurbiprofen-containing patch on the right wrist for 12 h. In the second part of the study, each subject applied a flurbiprofen-containing patch twice daily to the same wrist for 7 days. Plasma concentrations of flurbiprofen and urinary concentrations of the NSAID and its metabolites were measured by high-performance liquid chromatography assay, to enable comparison of the pharmacokinetic parameters for delivery of the drug by both routes. Maximum concentrations of the NSAID in plasma (Cmax) were much lower after a single application of the topical 40 mg flurbiprofen patch than after a single oral dose of 50 mg of the NSAID (mean +/- SD: 43 +/- 16 ng/ml versus 5999 +/- 1300 ng/ml, respectively). After repeated application of the topical patch, Cmax increased only slightly to 103 +/- 57 ng/ml. The mean relative bioavailability of flurbiprofen from the patch was 3.5 +/- 1.7%, calculated from plasma area under the curve data and 4.4 +/- 2.8% from urinary excretion data.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of HPLC with 19F- and 1H-NMR spectroscopy to investigate the human urinary excretion of flurbiprofen metabolites.

Results of an on-line HPLC-NMR analysis of human urine from a volunteer administered the anti-inflammatory drug flurbiprofen are reported. The two major human urinary metabolites, namely the glucuronides of flurbiprofen and of 4'-hydroxyflurbiprofen, have been identified using 1H- and 19F-NMR spectroscopy. In vivo conjugation of the racemic drug and its metabolites with D-glucuronic acid results in diastereomeric molecules which give resolved NMR spectra thereby permitting the diastereomeric proportions to be evaluated. The cause of the observed deviation from equal proportions is discussed. This study represents the first use of both 19F- and 600 MHz 1H-NMR spectroscopy coupled to HPLC.

The pharmacokinetics of flurbiprofen in younger and elderly patients with rheumatoid arthritis.

The pharmacokinetics of flurbiprofen (Ansaid Tablets, Upjohn Company of Canada, Don Mills, Ontario) were evaluated in both younger (40 to 60 years) and elderly (65 to 83 years) rheumatoid arthritic patients after both a 100-mg single-dose administration and at steady state during a 100-mg twice-a-day dosage regimen. Both flurbiprofen plasma concentration-time profiles and the urinary excretion of flurbiprofen and its major metabolites were evaluated. The results indicate that the pharmacokinetics of flurbiprofen are linear in both age groups based on only minor changes between single-dose and steady-state parameter determinations and the agreement between calculated and predicted accumulation values in plasma concentrations. Only minor differences in the pharmacokinetic parameters were observed between the younger and elderly patients. Only free flurbiprofen clearance was found to have a significant but variable correlation to patient age. The effect of flurbiprofen on the urinary excretion of two prostaglandins were also evaluated throughout this study. In both age groups, the maximum decrease in urinary excretion was observed after the first dose, and this effect was maintained throughout the remainder of the study. Percent decreases from baseline in urinary excretion during drug administration were similar for both age groups. Similar side-effect profiles were observed between age groups.

Pharmacokinetic comparison of flurbiprofen in end-stage renal disease subjects and subjects with normal renal function.

This study compared the pharmacokinetics of flurbiprofen (F) and three major metabolites in patients with end-stage renal disease (ESRD) undergoing continuous ambulatory peritoneal dialysis (CAPD) with the pharmacokinetics of F in normal subjects. A single 100-mg dose of F was administered to each of nine normal subjects and eight ESRD subjects. Blood and urine samples were collected in both groups; serial and end of dwell dialysate samples were obtained from the ESRD subjects. Plasma was analyzed for both the R and S optical isomers of F and its major metabolite, 4'-hydroxy-flurbiprofen (HF). Urine and dialysate were analyzed for F and three known metabolites. Plasma concentrations of F in the ESRD subjects were approximately 50% of the values obtained from the normal subjects (P less than .05). Flurbiprofen half-life and Tmax were not different. Elimination of HF was reduced in ESRD subjects. Urinary data suggest that HF was the major metabolite excreted (36% of the dose) in normal subjects whereas 3',4'-dihydroxy-flurbiprofen was the major metabolite (9% of the dose) excreted in the ESRD group. Mean urinary recovery of the dose was 73% in the normal subjects, but only 16% in ESRD subjects. Neither F nor its metabolites were detected in dialysate. Small enantiomer differences were seen. This study suggests that ESRD subjects have lower plasma levels of F than normal subjects when administered equal size doses. Accumulation of metabolites may occur in ESRD subjects upon multiple dosing. Enantiomer differences are not clinically significant.

Influence of H2 receptor antagonists on the disposition of flurbiprofen enantiomers.

The effect of oral cimetidine or ranitidine on the pharmacokinetics of the R and S enantiomers of the nonsteroidal anti-inflammatory drug flurbiprofen and its major metabolite, 4'-hydroxyflurbiprofen, was evaluated. Nine healthy volunteers participated in a randomized crossover design study with the following treatments: (A) flurbiprofen 200 mg; (B) flurbiprofen 200 mg plus ranitidine 150 mg bid for 7 days before and for 2 days after receiving flurbiprofen and (C) flurbiprofen 200 mg plus cimetidine 300 mg qid for 7 days before and for 2 days after receiving flurbiprofen. Blood and urine samples were collected at various intervals during a 48-hour period. These samples were assayed stereospecifically for flurbiprofen and its metabolite. Small but statistically significant differences in the terminal elimination rate constant (K), maximum peak serum drug concentration (Cmax), time to reach peak concentration (tmax), oral clearance (Cl/F) and area under the curve (AUC) were noted for flurbiprofen enantiomers. No significant treatment*isomer interactions were observed, indicating that neither cimetidine nor ranitidine interacted stereospecifically with flurbiprofen. Cimetidine, but not ranitidine, resulted in small (less than or equal to 15%) but statistically significant changes in flurbiprofen pharmacokinetic parameters. The interaction between H2-antagonists and flurbiprofen is unlikely to be clinically important.

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid].

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.

Pharmacokinetics of flurbiprofen in man. I. Area/dose relationships.

Flurbiprofen pharmacokinetics were studied in 15 normal male subjects after four oral doses. Plasma levels of total (bound + free) drug were monitored for 48 h and urine was collected for 96 h after the doses. All subjects demonstrated linear relationships between administered dose and total flurbiprofen AUC, indicating that oral clearance is independent of dose for the dose range evaluated in this study. Urinary recovery data indicated that the efficacy of absorption was dose independent.