Steven Hamilton Hoke

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.

Transformations in pharmaceutical research and development, driven by innovations in multidimensional mass spectrometry-based technologies

Abstract In the pharmaceutical industry, there is a tremendous need for qualitative and quantitative analysis of target analytes such as peptides, proteins, drugs, metabolites, biomarkers, impurities, and degradation products in various mixtures including synthetic reactions, in vitro cultures, biological fluids, drug substances, finished products, and many others. To provide adequate specificity for analysis in these complex mixtures, multidimensional analytical techniques are required. Mass spectrometry plays a central role in many of these multidimensional approaches to mixture analysis because it provides an unparalleled combination of sensitivity and specificity that is useful for both molecular identification and quantitative applications. Recent innovations in mass spectrometry and industrial implementation of these advances have transformed many aspects of pharmaceutical research and development. Data that were previously unattainable, or were not collected due to exorbitant cost or time constraints, can now be obtained using mass spectrometry-based technologies. The impact of these innovations has been most dramatically felt in early stages of discovery, as more data are available to make critical decisions, such as selecting compounds for advancement to costly preclinical and clinical trials. New MS technologies have also accelerated the progression of drug candidates through development and toward regulatory approval. Here, five major categories of pharmaceutical applications of mass spectrometry are reviewed. They are new chemical entity characterization, biomacromolecule characterization, bioanalytical quantitation, metabolite identification, and impurity and degradation product identification. A brief historical perspective and evolution of technologies for each application area are presented. Those discussions are followed with a description of the current strategies for implementation of the tremendous capabilities of the state-of-the-art approaches, along with representative applications. In addition, emerging technologies for each application area are presented to indicate the future directions of instrumentation for mixture analysis in the pharmaceutical industry.

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.

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography-tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography-tandem mass spectrometry-negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25-100 microl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-microl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100+/-10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 microg/kg) produced an approximately 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.

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%.