Thomas H. Eichhold

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma.

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.

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.

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.

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.

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.

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 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.

On-line solid phase extraction using the Prospekt-2 coupled with a liquid chromatography/tandem mass spectrometer for the determination of dextromethorphan, dextrorphan and guaifenesin in human plasma.

An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt-2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on-line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL−1, respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05–50 ng mL−1 and was 5–5,000 ng mL−1 for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%.

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 μM,KI=0.35 μM), twoin vitro cellular systems: rat peritoneal macrophages (IC50=0.02 μM) and human whole blood (IC50=0.08 μM), andex vivo in man. In addition to PGE2 inhibition, which is common to all NSAIDs, higher concentrations of Tebufelone block thein vitro formation of products of the lipoxygenase pathway [leukotrienes (LTB4)] in rat macrophages (IC50=20 μM) and human whole blood (IC50=22 μM). 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 inhibitorin vitro. All three DTBP compounds were equipotent 5-LO inhibitors. It is likely that Tebufelones inhibitory effects on AA metabolism are, in part, responsible for itsin vivo efficacy and enhanced safety profile.