Rapid bioanalytical determination of dextromethorphan in canine plasma by dilute-and-shoot preparation combined with one minute per sample LC-MS/MS analysis to optimize formulations for drug delivery.

The determination of dextromethorphan in canine plasma is used to demonstrate the high throughput bioanalytical approach of automated dilute-and-shoot (DAS) sample preparation followed by a 1 min isocratic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Dilute-and-shoot preparation is commonly used for the determination of drugs in several biological matrices such as urine and saliva, but is not typically used with plasma samples because the amount of protein present in plasma can lead to a variety of problems including column failure. As a result, plasma sample preparation usually removes protein by precipitation, extraction or filtration; however, the dilute-and-shoot approach solubilizes proteins throughout the chromatographic portion of the assay. The attributes of this approach are compared with a previously validated liquid/liquid extraction procedure for determination of dextromethorphan in plasma. Accuracy and precision of both methods are similar. The lower limit of quantitation (LLOQ) of the dilute-and-shoot approach is much higher at 2 ng/ml versus 5 pg/ml with the liquid/liquid extraction; however, the sample throughput of the preparation portion of the dilute-and-shoot approach is more than 50-fold greater. The ruggedness of the dilute-and-shoot method was thoroughly investigated because of the problems traditionally associated with the direct injection of diluted plasma onto an LC-MS/MS instrument. With the optimal conditions, greater than 1,000 injections of diluted plasma have been successfully performed on a single column in less than 19 h making this technique an excellent approach for the rapid preparation and high throughput of plasma samples containing drug levels in the ng/ml range or higher. Application of this methodology to measure the levels of dextromethorphan in canine plasma to evaluate drug delivery from various formulations is also presented.

Comparison of packed-column supercritical fluid chromatography--tandem mass spectrometry with liquid chromatography--tandem mass spectrometry for bioanalytical determination of (R)- and (S)-ketoprofen in human plasma following automated 96-well solid-phase extraction.

The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma. After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC.

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma.

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1x10 mm) afforded rapid isocratic elution of fluprostenol (t(R)=40 s) but still provided a relatively large k' value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml(-1) fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.

Determination of (R)- and (S)-ketoprofen in human plasma by liquid chromatography/tandem mass spectrometry following automated solid-phase extraction in the 96-well format.

A sensitive and selective method was developed for the determination of (R)-ketoprofen ((R)-kt) and (S)-ketoprofen ((S)-kt) in human plasma using chiral liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope-labeled [(13)C(1), (2)H(3)]-(R and S)-ketoprofen, for use as the internal standards, were prepared for analysis using automated solid-phase extraction (SPE) in the 96-well microtiter format. The enantiomers were separated on an (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid (Chirex 3005) 250x2.0 mm i.d. analytical column, equipped with a 30x2.0 mm i.d. guard column using isocratic mobile phase conditions. The (R)- and (S)-kt levels were quantifiable from 0.05 to 2500 ng ml(-1) by constructing two separate curves from calibration standards covering the same range. The first curve ranged from 0.05 to 100 and the second from 100 to 2500 ng ml(-1). A concentration of 0.05 ng ml(-1) of either enantiomer was easily detected using a 1 ml plasma sample volume. The average method accuracy, evaluated at four levels over an extended period, was better than +/-3% over the entire range. The precision for the same set of quality control samples ranged from 4.0 to 7.0 % RSD (n = 24). The method was applied to the evaluation of pharmacokinetic parameters in human plasma obtained from volunteers who received 25 mg of kt by peroral administration of Actron caplets or by topical administration of Oruvail gel.

Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes.

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 3. 7-tert-butyl-2, 3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations at the 5 position.

We report an expansion of the scope of our initial discovery that 5-keto-substituted 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofurans (DHDMBFs) are antiinflammatory and analgesic agents. Several other functional groups have been introduced at the 5 position: amides, amidines, ureas, guanidines, amines, heterocycles, heteroaromatics, and heteroaryl ethenyl substituents in the 5 position all provide active compounds. These compounds are dual cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibitors. They inhibit both COX-1 and COX-2 with up to 33-fold selectivity for COX-2.

Comparison of p-fluoroketorolac and [18O3]ketorolac for use as internal standards for the determination of ketorolac by gas chromatography/mass spectrometry (GC/MS).

A chemical and a stable-isotope analog, p-fluoroketorolac and [18O3]ketorolac respectively, were directly compared for applicability as internal standards for the determination of ketorolac in plasma samples using gas chromatography/mass spectrometry (GC/MS) with selective-ion-monitoring detection, following derivatization to form the methyl esters. This comparison involved analyzing ketorolac calibration standards and spiked plasma samples that contained both internal standard candidates. The response for ketorolac and each internal standard was monitored simultaneously and electronically integrated peak heights were obtained. Thus, for each analysis performed, a response ratio was obtained for each internal standard relative to an identical ketorolac response. Linearity of response for ketorolac calibration standards and accuracy for spiked plasma sample analysis were compared using each internal standard. The use of [18O3]ketorolac as the internal standard provided superior accuracy data for the analysis of ketorolac in plasma samples.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 1. 7-tert-buty1-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: discovery and variation of the 5-keto substituent.

A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were prepared and evaluated as potential nonsteroidal antiinflammatory and analgesic agents. Interest in this class of compounds arose when a DHDMBF was found to be an active metabolite of the di-tert-butylphenol antiinflammatory agent tebufelone. We have now found that a variety of 5-keto-substituted DHDMBFs have good in vivo antiinflammatory and analgesic activity after oral administration. These compounds inhibit both cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) in vitro. The cyclooxygenase inhibition was found to be selective for the cyclooxygenase-2 isoform, and this combination of COX-2/5-LOX inhibition may be responsible for the gastrointestinal safety of compounds such as 30.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 2. 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations of the dihydrobenzofuran ring.

A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were found to be nonsteroidal antiinflammatory and analgesic agents. These compounds are inhibitors of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) with selectivity for the COX-2 isoform. A series of analogues were prepared to investigate the scope of this lead. Five ketone side chains from active DHDMBFs were used to investigate the effects of changes in the DHDMBF "core": the size and identity of the heterocycle and the substituent requirements of the heterocycle and phenyl ring. Biological testing showed that a variety of structural changes can be accommodated, but no structure was clearly superior to the DHDMBF structure.

Highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method for the analysis of dextromethorphan in human plasma.

A stable-isotope-dilution HPLC-tandem mass spectrometry-based method was developed for the determination of dextromethorphan in human plasma. Plasma samples were prepared for analysis by solid-phase extraction on octadecylsilane extraction cartridges. Dextromethorphan and the deuterium-labeled dextromethorphan internal standard were chromatographed on a short reversed-phase column and detected by a selected-reaction-monitoring scheme. Linear standard curves were obtained over three orders of magnitude and the limit of quantitation for dextromethorphan was 50 pg/ml, using a 1-ml plasma sample. The combination of HPLC and electrospray tandem mass spectrometry resulted in a rapid, selective and sensitive method for the analysis of dextromethorphan in plasma. The method was applied for the evaluation of the pharmacokinetic profile of dextromethorphan in human volunteers following peroral administration.

Determination of dextromethorphan and dextrorphan in human plasma by liquid chromatography/tandem mass spectrometry.

Rapid, sensitive and selective methods were developed for the determination of dextromethorphan and its major metabolite, dextrorphan, in human plasma using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope internal standards were prepared for analysis by a liquid-liquid back-extraction procedure. Dextromethorphan and dextrorphan were chromatographed on a short reversed-phase column, using separate isocratic mobile phase conditions optimized to elute each compound in approximately 1.1 min. For both analytes, calibration curves were obtained over four orders of magnitude and the limit of quantitation was 5 pg ml-1 using a 1 ml plasma sample volume. The accuracy across the entire range of spiked DEX and DOR concentrations was, in general, within 10% of the spiked value. The precision was generally better than 6% for replicate sample preparations at levels of 50 pg ml-1 or higher and typically better than 12% at levels below 50 pg ml-1. The method was applied for the evaluation of the pharmacokinetic profiles of dextromethorphan and dextrorphan in a human volunteer following peroral administration of a commercially available cough formulation.

Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor.

Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degradation of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. A highly selective and sensitive separation and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extracting FAC from tissue and spiking the extract with [18O]2-fluoroacetate (18O-FAC) as an internal standard. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extraction. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatographic separation. Two-dimensional gas chromatography coupled with selected-ion-monitoring detection provided the final measurement. The assay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a S/N ratio of 40:1. Linearity was established over two orders of magnitude, from 2-500 ng ml-1, with 5 microliters injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor.

Evaluation of ketorolac concentrations in plasma and gingival crevicular fluid following topical treatment with oral rinses and dentifrices.

Two clinical studies were conducted to determine the relative amounts of ketorolac detectable locally in the gingival crevicular fluid (GCF) and systemically in plasma after oral, topical drug administration. The rinse study compared topical administration of three concentrations of ketorolac tromethamine (0.1%, 0.5%, and 0.01%) in oral rinse formulations administered topically and a perorally administered capsule (10 mg), and the dentifrice study compared two concentrations of ketorolac in dentifrice formulations (0.15% and 1.0%) with a 0.1% oral rinse, all treatments administered topically. The dose-corrected systemic availability of the three oral rinses evaluated in the rinse study relative to the peroral capsule was about 15%. However, the ratios of the observed maximum GCF ketorolac concentration to maximum plasma ketorolac concentration ranged from 22 to 49, compared to less than 1 for the peroral ketorolac capsule. Using this ratio as an estimate of the ability of a treatment to target the drug to the gingival tissue, these data indicate that the ketorolac oral rinses achieved greater delivery of drug to the gingival tissue (presumed site of action for periodontitis) with a lower systemic drug load than peroral administration of a ketorolac capsule. The dose-corrected relative systemic bioavailabilities for the dentifrice treatments with respect to the 0.1% rinse in the dentifrice study were 59.2% and 86.4% for the 1.0% and 0.15% dentifrices, respectively, indicating that significantly less ketorolac was systemically available from the two dentifrices relative to the oral rinse. The relative bioavailabilities of ketorolac in the GCF after dosing with the dentifrice formulations with respect to the rinse were 89.1% for the 1.0% dentifrice and 19.7% for the 0.15% dentifrice. Thus, the 1.0% dentifrice appears to provide statistically equivalent levels of ketorolac to the gingival tissue as the 0.1% oral rinse with significantly less systemic exposure. The T1/2 of ketorolac in the GCF was about 0.5 h for all three treatments, which is significantly less than the plasma half-life of about 5.3 h. These data suggest that GCF levels of ketorolac should remain above the IC50 for PGE2-stimulated IL-1 bone resorption for about 7 h following treatment, assuming continuation of the first-order elimination observed over the first two postdosing hours. We conclude that oral rinses and dentifrices are effective and preferred vehicles for administration of ketorolac for use in treatment of periodontitis.

Effects of tebufelone (NE-11740), a new anti-inflammatory drug, on arachidonic acid metabolism.

Tebufelone is a novel nonsteroidal anti-inflammatory drug (NSAID), of the di-tert-butylphenol (DTBP) class, which displays potent anti-inflammatory, analgesic and anti-pyretic properties in a variety of animal models. In this report, the effects of Tebufelone on arachidonic acid (AA) metabolism are reviewed. Tebufelone potently inhibits the formation of prostaglandins (PGE2) a key mediator of pain and inflammation, in isolated enzyme preparations (IC50 = 1.5 microM, KI = 0.35 microM), two in vitro cellular systems: rat peritoneal macrophages (IC50 = 0.02 microM) and human whole blood (IC50 = 0.08 microM), and ex vivo in man. In addition to PGE2 inhibition, which is common to all NSAIDs, higher concentrations of Tebufelone block the in vitro formation of products of the lipoxygenase pathway [leukotrienes (LTB4)] in rat macrophages (IC50 = 20 microM) and human whole blood (IC50 = 22 microM). Substrate incorporation studies (14C-AA) indicate that Tebufelone reversibly inhibits cyclooxygenase (CO) and 5-lipoxygenase (5-LO) enzymes rather than regulating the release of AA. Tebufelone was shown to be a more potent CO inhibitor than indomethacin and a less potent 5-LO inhibitor than RG-5901. Comparisons to structurally related compounds under development (E-5110, Esai; KME-4, Kanagafuchi), found Tebufelone to be the most potent CO inhibitor in vitro. All three DTBP compounds were equipotent 5-LO inhibitors. It is likely that Tebufelone's inhibitory effects on AA metabolism are, in part, responsible for its in vivo efficacy and enhanced safety profile.

Comparison of tebufelone distribution in rat blood plasma and inflamed and noninflamed tissues following peroral and intravenous administration.

It is recognized that some acidic nonsteroidal antiinflammatory drugs (NSAIDs) accumulate in the synovial fluids of inflamed joints. The distribution of tebufelone, a member of the di-tert-butylphenol class of NSAIDs, between rat plasma and paw tissues (inflamed and noninflamed) was determined. Tebufelone was administered (doses providing 80% maximal effectiveness) perorally (po, 10 mg/kg) or intravenously (i.v., 0.5 mg/kg) to Sprague-Dawley rats just prior to injection of an inflammatory adjuvant (carrageenan) into one of the hind paws. Levels of the drug were measured in plasma, inflamed paws, and noninflamed paws at prescribed times postdosing by capillary gas chromatography/stable isotope dilution mass spectrometry. Tebufelone levels, as determined from areas under the curves of concentration versus time were sevenfold (po) and 11-fold (i.v.) higher in paw tissue than in plasma. Inflamed and noninflamed tissues have equal affinity for the drug. Calculated terminal-phase half-lives of tebufelone in paw tissue were similar following i.v. and po dosing (approximately 14 h) and equivalent to the plasma half-life of the drug after po dosing (approximately 10 h). The plasma half-life determined following i.v. dosing was considerably lower (2.8 h). The anti-inflammatory activity of tebufelone appears to correlate more closely with tissue distribution profiles than plasma drug levels at the dose levels examined.

Absorption, bioavailability, and pharmacokinetics of tebufelone in the rat.

Tebufelone (NE-11740) is a member of the new di-tert-butylphenol class of anti-inflammatory agents. It exhibits good inhibitory activity against cyclooxygenase and 5-lipoxygenase in vitro. It also shows excellent anti-inflammatory activity and inhibits bone resorption in vivo in the rat adjuvant arthritis model at an oral dose level of 1 to 2 mg/kg. The absorption, bioavailability, and pharmacokinetics of tebufelone were investigated in male Sprague-Dawley rats. Tebufelone labeled with carbon-14 was administered intravenously at doses of 0.5 and 2 mg/kg and perorally at doses of 2 and 10 mg/kg to fasted rats. Plasma samples taken from the rats at timed intervals were analyzed for total radiolabel by scintillation counting and for tebufelone by a mass spectrometric method. Comparison of the total radiolabel and tebufelone areas under the curves (AUCs) of concentration of tebufelone versus time from the 2-mg/kg intravenous and 2-mg/kg oral doses indicates that tebufelone is completely absorbed and 100% bioavailable at this dose level in the rat. The AUCs are a linear function of dose at the 0.5- and 2-mg/kg dose levels, but the AUC of the 10-mg/kg dose exhibits a nonproportional increase, suggesting saturation of elimination processes at this higher dose.

Determination of tebufelone (a new anti-inflammatory drug) strength and stability in bulk drug, dosage formulations and feed admixtures by reversed-phase high-performance liquid chromatography.

A rugged reversed-phase high-performance liquid chromatographic method suitable for the quantitative determination of tebufelone, a new anti-inflammatory drug, in bulk drug, various pharmaceutical formulations and animal feed admixtures is described. Tebufelone was easily separated from synthetic by-products and detected by ultraviolet absorption (280 nm). Standard curves were linear (r2 greater than 0.999) over 2 orders of magnitude with a detection limit of 0.1 microgram/ml at a signal-to-noise ratio of 2 (0.05 ml injected). Recovery of tebufelone from bulk drug and dosage formulations was greater than 99% with a coefficient of variation of 1.8% throughout the range of the standard curve. Recovery of tebufelone from feed admixtures was 96-102% with a less than 5% relative standard deviation at the levels assayed.

Determination of tebufelone, a new anti-inflammatory drug, in plasma and tissue using capillary gas chromatography/stable isotope dilution mass spectrometry.

A capillary gas chromatographic/mass spectrometric method for the determination of tebufelone, a new anti-inflammatory drug, in plasma and rat paw tissue is described. Trideuteriated tubufelone was employed as an internal standard and the drug quantified by selected ion monitoring using a mass-selective detector following positive electron ionization (70 eV). Tebufelone was recovered from plasma (greater than 93%) and pulverized paw tissue (greater than 88%) by ethereal extraction. Standard curves were linear between 5 ng ml-1 and 5 micrograms ml-1 with a detection limit (S/N = 2) of 2 ng ml-1. The coefficient of variation for successive injections of standards (200 ng ml-1) or samples (10-1500 ng ml-1) was 7.3% and 8.5%, respectively.

Determination of leukotriene B4 in human plasma by gas chromatography using a mass selective detector and a stable isotope labelled internal standard. Effect of NE-11740 on arachidonic acid metabolism.

A highly selective gas chromatographic method, coupled with selected ion monitoring using a mass selective detector and positive electron ionization, was developed for the determination of leukotriene B4 (LTB4) in human plasma. Plasma was separated from whole human blood via centrifugation, proteins precipitated with acetonitrile and LTB4 recovered (approximately 82.0%) by ethyl acetate extraction. The methyl ester, bis-t-butyldimethylsilyl ether derivative of LTB4 was formed prior to analysis and determined quantitatively using [18O]2-LTB4 as an internal standard. The limit of detection (S/N = 2) was 425 pg on column (m/z 335/339) using a 1-microliter injection. Standard curves were linear over two orders of magnitude with an RSD of less than 5.0% (n = 10). NE-11740, a new anti-inflammatory drug, was shown to inhibit, in a dose-dependent manner (ED50 = 22 microM) ionophore-stimulated LTB4 biosynthesis by human whole blood in vitro.

Biotransformation products of 3,4,4'-trichlorocarbanilide in rat, monkey, and man.

3,4,4'-Trichlorocarbanilide (TCC), uniformly labeled with 14C in the monochloro ring, was administered to rats, rhesus monkeys, and humans. Radioactive materials in the plasma and urine of all three species and in the bile of rats and monkeys were separated by high performance liquid chromatography. The chromatography showed great similarity between the monkey and the human. Principal metabolites common to all species were the sulfate and glucuronide conjugates of 2'-, 3'-, and 6-hydroxy-TCC. The rat also produced the glucuronide and sulfate conjugates of 2',6-dihydroxy-TCC. The major urinary excretion products found in humans and monkeys were the N- and N'-TCC glucuronides.