Patrick Bennett
, Wilmington

Pharmacology, Analytical Chemistry, Biochemistry

24.10

Publications

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    ABSTRACT: Flow cytometry is increasingly becoming an important technology for biomarkers used in drug discovery and development. Within clinical development flow cytometry is used for the determination of PD biomarkers, disease or efficacy biomarkers or patient stratification biomarkers. Significant differences exist between flow cytometry methodology and other widely used technologies measuring soluble biomarkers including ligand binding and mass spectrometry. These differences include the very heavy reliance on aspects of sample processing techniques as well as sample stabilization to ensure viable samples. These differences also require exploration of new approaches and wider discussion regarding method validation requirements. This paper provides a review of the current challenges, solutions, regulatory environment and recommendations for the application of flow cytometry to measure biomarkers in clinical development.
    No preview · Article · May 2015 · Bioanalysis

  • No preview · Chapter · Jan 2015
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    ABSTRACT: Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.
    Preview · Article · Jun 2014 · The AAPS Journal

  • No preview · Article · Oct 2013 · Bioanalysis
  • Min Meng · Spencer Carter · Patrick Bennett
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    ABSTRACT: This chapter details the current issues, the best practice, and recommendation for assessing and handling any analytical impact that may occur due to hemolysis and/or lipemia using LC-MS/MS technique. The extent of hemolysis and/or lipemia, the root cause, and the ultimate solution vary from analyte to analyte. In general, failure in regulated LC-MS/MS bioanalysis assay due to hemolysis or lipemia is uncommon. However, a hemolysis/lipemia testing failure is often an indication that with the assay being used, inaccurate results may be obtained when unknown samples are hemolyzed/lipemic. Therefore, the authors believe that the assessment of hemolysis/lipemic effects during method development (MD) and validation is necessary and important.
    No preview · Chapter · Aug 2013
  • Hongxia Wang · Patrick Bennett
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    ABSTRACT: Background: There continues to be consistent pressure for bioanalytical scientists to achieve lower limits of quantitation. The reasons range from smaller sample volumes available for analysis, to more potent analytes and the growth of biologics in drug development. This has led scientists to investigate alternative LC techniques, including microflow and nanoflow. These techniques have been shown to increase sensitivity of electrospray methods and reduce ionization matrix effects. Because high-resolution MS has significant benefits for the analysis of biologics, this type of mass spectrometer is becoming increasingly important in bioanalysis. Results: For microflow analysis, a new ion source and significant extra sample preparation or chromatographic separation are not required. However, increased sensitivity and reduced matrix effects were consistently demonstrated when compared with UHPLC flow rates. The extent of matrix effects observed were compound dependent. Discussion: This paper presents the utility of combining high-resolution/accurate mass with microflow LC from a quantitative standpoint. This includes evaluating the typical quantitative parameters of sensitivity, linearity/dynamic range, precision and accuracy. It also includes the evaluation of changes in signal suppression using microflow LC and microspray ionization. The benefits and disadvantages of using the combination of these two technologies for quantitative bioanalysis are also discussed.
    No preview · Article · May 2013 · Bioanalysis
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    ABSTRACT: Background: Quantification of bile acids using LC-MS has previously been very challenging on triple quadrupole MS systems due to the absence of a primary fragment ion for unconjugated bile acids. Results: A LC-high-resolution/accurate mass MS method for the analysis of six bile acids (cholic acid, chenodeoxycholic acid, taurocholic acid, deoxycholic acid, lithocholic acid and ursodeoxycholic acid) was developed and successfully validated. The method includes a single extraction and a single injection with all analytes separated using target-selected ion monitoring (SIM) mode in two periods with a resolution of 70,000 and 140,000, respectively. Conclusion: This is the first LC-high-resolution/accurate mass assay fully validated to quantify six bile acids for regulated bioanalysis.
    No preview · Article · May 2013 · Bioanalysis
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    ABSTRACT: In order to meet the drug discovery and development needs of pharmaceutical companies, CROs are constantly challenged by the requirements for rapid LC-MS/MS method development prior to method validation and sample analysis. In order to meet this challenge, a comprehensive method development program, nicknamed 'Amoeba™', which uses a series of written protocols for standardized and efficient method development was developed. In this paper, the genesis of the Amoeba method development program is elucidated in detail and a number of case studies are presented to showcase the execution of the Amoeba method development program. Using this program, the majority of the most critical information regarding the assay can be captured. While the Amoeba program has been proven to be effective, we recognize that the development of a robust bioanalytical method for use in pharmaceutical industry also requires the careful consideration of many critical parameters and the ability to identify and resolve potential issues. The refinement of the assay relies on further evaluation of several critical factors including, but not limited to, internal standard response, matrix effects (phospholipid or nonphospholipid related) and carryover.
    No preview · Article · Jan 2013 · Bioanalysis
  • Min Meng · Patrick K. Bennett
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    ABSTRACT: There are three primary stages of the regulated bioanalysis process using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS): method development, method validation, and sample analysis including incurred sample reanalysis (ISR). Robust and rugged LC-MS/MS methods are essential in support of drug discovery, toxicology studies, and clinical trials. The development of a robust bioanalytical method requires careful consideration of many critical parameters, such as accuracy and precision, linearity, matrix effect, sensitivity, selectivity, stability, throughput, and ruggedness (or reproducibility). Because bioanalytical data is critical for determining the safety and efficacy of a new drug, a bioanalytical method must be validated following the Food and Drug Administration’s (FDA) guidance for the industry, the recommendations from various white papers and Standard Operating Procedures (SOPs). A complete regulated method validation in biological matrix minimally requires three interday precision and accuracy runs, various short- and long-term solution and matrix stability assessments, extraction recovery, dilution capability and linearity, extract stability, reinjection reproducibility, selectivity and specificity, assessment of matrix effects, interference from concomitant medications and prodrug/metabolites, etc. The final evaluation of any high quality bioanalytical method is not complete until it passes the ultimate test of regulated sample analysis and incurred sample reanalysis (ISR) which are also conducted ­following the similar rules as validation.
    No preview · Chapter · Nov 2012
  • Keeley Murphy · Patrick K Bennett · Nicholas Duczak
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    ABSTRACT: High-throughput screening with LC-MS has been routinely implemented to various degrees throughout the entire drug-discovery process. One of the major advantages of utilizing LC-MS earlier at the lead discovery stage is reducing the cost of sample analysis while increasing assay selectivity. Avoiding labeling agents and other non-native species in an assay environment can reduce costly sample preparation, while chromatographic separation of the analyte of interest from interferences in the sample matrix has been shown to increase selectivity and sensitivity. In this paper, we utilize high-resolution MS-LC multiplexing to analyze phosphorylated peptides as part of a screening assay. Commonly used enzyme buffers were used to prepare phosphorylated peptide standards of varying concentrations and these were plated into a 96-well plate format for LC-MS analysis. The overall cycle time for analysis from sample to sample, LLOQ, Z' and coefficient of variance were determined. High-resolution MS coupled with LC multiplexing provides high-quality sample analysis at sampling rates of up to 18 s per sample. Samples analyzed in both simple and complex sample matrixes demonstrated an LOQ of 5 nM with linear response across the working range of the assay. Overall statistical analysis of the large samples produced Z' = 0.85 for sample sets in sodium citrate solution and Z' = 0.66 for sample sets in HEPES solution indicating a robust analytical method.
    No preview · Article · May 2012 · Bioanalysis
  • Patrick Bennett

    No preview · Article · Nov 2011 · Bioanalysis
  • Patrick Bennett

    No preview · Article · Apr 2011 · Bioanalysis
  • Min Meng · Scott Reuschel · Patrick Bennett
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    ABSTRACT: In 2007, the US FDA recommended that pharmaceutical companies and CROs conduct incurred sample reanalysis (ISR) following the analysis of study samples using validated methods. Between January 2008 and December 2009, over 250 separate analytes were tested for ISR in our laboratory (a bioanalytical CRO). Among these, nine analytes initially failed for ISR. While thorough investigations were conducted to identify the root cause of ISR failure for each study, these investigations were often painfully tedious and very costly, both financially and in terms of project timelines. In this paper, three representative studies are presented to showcase the detailed investigation processes, methodologies and final conclusions of the ISR investigations. Additionally, all nine ISR failures are analyzed to identify trends or common elements of the studies or methods that might help identify potential problems before they occur. Furthermore, suggestions and recommendations to minimize future ISR failures are provided.
    No preview · Article · Feb 2011 · Bioanalysis
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    Min Meng · Lisa Rohde · Vladimír Cápka · Spencer J Carter · Patrick K Bennett
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    ABSTRACT: Traditional chiral chromatographic separation method development is time consuming even for an experienced chromatographer. This paper describes the application of computer software ACD Lab to facilitate the development of chiral separation for the quantitation of eszopiclone using LC-MS/MS technology. Assisted by ACD/Chrom Manager and LC Simulator software, the optimal chiral chromatographic development was completed within hours. The baseline chiral separation was achieved with a total cycle time of 3 min. For sample extraction method development, a Waters Oasis Sorbent Selection Plate containing four different sorbents was utilized. Optimal conditions were determined using a single plate under various load, wash and elution conditions. This was followed by a GLP validation which demonstrated excellent intra- and inter-day accuracy and precision for the quantitation of eszopiclone in human plasma at 1.00-100 ng/mL range using LC/MS/MS technology. This method was utilized to support multiple clinic bioequivalence studies.
    Full-text · Article · Dec 2010 · Journal of pharmaceutical and biomedical analysis
  • Troy Voelker · Patrick Bennett · Min Meng · Lisa Rhode
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    ABSTRACT: Title Software Assisted Chiral Chromatographic Method Development for the Quantitation of Four Chiral Drugs in Monkey Plasma using LC/MS/MS Troy Voelker, Patrick Bennett, Min Meng, Lisa Rohde Tandem Labs, Salt Lake City, UT Introduction Traditional chiral chromatographic separation method development is time consuming even for an experienced chromatographer. This paper describes the application of computer software ACD Lab to facilitate the development of chiral separation for the quantitation of armodafinil, ramelteon, eszopiclone and dexlansoprazole using LC-MS/MS technology. Assisted by ACD/Chrom Manager and LC Simulator software, the optimal chiral chromatographic development was completed within hours. The baseline chiral separation for all four drugs was achieved less than four minutes. Except dexlansoprazole which is still in the method development stage, armodafinil, ramelteon, eszopiclone were validated under GLP guidance with excellent intra- and inter-day accuracy and precision. Methods The LC/MS/MS system was a Sciex API4000 or 5000 under positive ionization mode using turboionspray coupled with a Shimadzu LC-10AD high pressure pump and a CTC PAL autosampler. The SRM transitions for eszopiclone and eszopiclone-D8 (ISTD), armodafinil and modafinil-d5 (ISTD), ramelteon and ramelteon-d3 (ISTD), dexlansoprazole and lansoprazole-13C6 (ISTD) were monitored. ACD/Structure Designer, ChromManager and LC Simulator software (v. 10.0) were utilized for chiral chromatography development. AGP chiral analytical columns, 100x2 mm or 50x2 mm, were used to achieve chiral separations. Structure Designer was utilized for the calculation of physical chemical properties, i.e., structure, pKa value and solubility information. ChromManager software was utilized for data editing and processing. LC Simulator software was utilized for LC chromatography simulation and predictions. Results Using the ACD Lab software approach, two initial LC/MS/MS chromatographic conditions were acquired in the laboratory using a racemic mixture of the analytes. The same aqueous mobile phase i.e., 10 mM Ammonium acetate, pH unadjusted, and three organic solvents, i.e., isopropyl alcohol, methanol and pentanol, were utilized for all applications. Two injections were performed in the laboratory on a LC/MS/MS system based on semi-random selection of the LC program and composition. Under these conditions, the enantiomeric peaks either completely coeluted or were partially resolved. The Analyst wiff file from the two injections were then uploaded into the ACD/ChromManager software. After peak editing, i.e., peak picking and labeling, structure and LC condition assignment, these two chromatograms were imported into the ACD/LC Simulator software. The predicted separation conditions were obtained instantly by manipulating the column dimension, flow rate, mobile phase composition within the software. The computational results were then utilized as the guidance for further experimental work in the laboratory. This procedure was repeated until satisfactory chromatography was achieved within a few more injections. Following the success of the chiral chromatography development, full GLP validations were conducted for the quantitation of armodafinil, ramelteon and eszopiclone. The method validation of dexlansoprazole is in process. The validation demonstrated excellent intra- and inter-day accuracy and precision. In addition, extraction recovery, analyte stability in solution, matrix and processed extracts were conducted and reported per FDA GLP guidelines. These assays have been used to support clinic sample studies.
    No preview · Conference Paper · Oct 2009
  • Patrick Bennett · Laixin Wang · Brad Johnson
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    ABSTRACT: The primary causes of ionization matrix effects have been shown to be a result of endogenous phospholipids, plasticizers and dose formulations. However, matrix effects can still be observed after ensuring none of the above are at fault. Typically, the offending compound(s) is present in a very high concentration and/or may have surfactant qualities. During the method development of a method for a glucuronide metabolite in monkey plasma, bias was observed between different lots of spiked control plasma. Review of the data showed that the internal standard (5-bromo-4-chloro-3-indolyl -D-glucuronide) was variable and the bias could be attributed to this variation. Full scan MS data was obtained for the lots used to prepare both the calibration standards and QC samples. The spectra showed that there was a peak at the same retention time as the internal standard with very high intensity. The peak was observed at m/z 179. Other large peaks were also observed eluting earlier than the internal standard. These masses were monitored during further chromatographic method development to resolve the m/z 179 peak from the internal standard. The resulting method changes to the chromatography eliminated the bias. However, during the development of a rat plasma method for these compounds, the peak at m/z 179 was not observed. To be conservative, the monkey method was exactly transferred to the rat plasma and the m/z 179 mass was still monitored. During method validation of the rat plasma, a new lot of plasma used for the QC samples showed the peak at m/z 179 while the lot for calibration standard did not. When the same method was applied to human plasma method, the m/z 179 peak was not observed. However, full scan MS data showed a large peak at m/z 195 at the same retention time as the internal standard. This data shows that relatively low molecular weight anions may have: 1) variable presence and intensity within a species and matrix and 2) variable m/z between species for the same matrix. These peaks are very polar and are most likely to affect similarly small polar analytes or metabolites.
    No preview · Conference Paper · Jan 2006
  • H R Liang · T Takagaki · R L Foltz · P Bennett
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    ABSTRACT: Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0-10,000 ng/mL (correlation coefficient >0.999) for sorbitol-13C6 and 250-50000 ng/mL (correlation coefficient >0.999) for fructose-13C6 in human RBCs.
    No preview · Article · Oct 2005 · Journal of Chromatography B
  • H R Liang · T Takagaki · R L Foltz · P Bennett
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    ABSTRACT: Attachment of anions to sorbitol and fructose has been shown to enhance sensitivity in both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) mass spectrometry. The post-column addition of CHCl3 produced Cl-adducts of sorbitol and fructose but their signals were suppressed due to the elevated background. Different chlorinated compounds and different additive methods were systematically investigated to form more abundant Cl-adduct precursor ions and deprotonated product ions. The major causes of the high background were explored and effective methods were developed to improve the signal-to-noise ratios and reproducibility. The compositions of mobile phase, percentages of organic modifiers (MeCN, MeOH and water), columns, oven temperature, flow rates and different gradients were investigated to separate sorbitol from fructose along with their isomers including glucose, galactose, mannose, sorbose, mannitol, and dulcitol. The optimized separation was achieved on a Luna 5 mu NH2 100A column (150 x 4.6 mm) using a mobile phase containing MeCN with 0.1% of CH2Cl2 and 50% MeOH in water at a flow rate of 800 microL/min and an oven temperature of 40 degrees C using a gradient liquid chromatography (LC) system. Human nerve tissue samples were extracted by protein precipitation followed by mixed-mode solid-phase extraction. The LC/ESI-MS/MS method produced higher peak intensities than LC/APCI-MS/MS. However, there were matrix effects from extracted tissues in LC/ESI-MS/MS but not in LC/APCI-MS/MS. Consequently, APCI proved to be the more effective method of ionization. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with linear ranges of 0.2-80 ng/mg for sorbitol and 1-400 ng/mg for fructose in human nerve tissues.
    No preview · Article · Aug 2005 · Rapid Communications in Mass Spectrometry
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    Patrick Bennett · Min Meng · KC Van Horne

    Full-text · Article · Jan 2005
  • H R Liang · R L Foltz · M Meng · P Bennett
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    ABSTRACT: A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.
    No preview · Article · Jul 2004 · Journal of Chromatography B

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