Publications by authors named "Douglas Fast"

28 Publications

  • Page 1 of 1

Quantification of fumarate and investigation of endogenous and exogenous fumarate stability in rat plasma by LC-MS/MS.

Bioanalysis 2016 Apr 15;8(7):661-75. Epub 2016 Mar 15.

Covance Laboratories Inc., 3301 Kinsman Blvd, Madison, WI 53704, USA.

Background: Fumaric acid is a commonly used excipient in pharmaceutical products. It is not known if its presence may lead to fluctuation of endogenous fumarate levels. An LC-MS/MS method was developed and validated to quantify fumarate in support of a toxicokinetics study.

Results: Stability evaluation showed that endogenous fumarate was stable for 6 h at room temperature, while exogenously added fumaric acid was converted to malate within 1 h due to the presence of fumarase. Citric acid, a fumarase inhibitor, prevented the conversion of added fumaric acid in rat plasma.

Conclusion: The method was validated in citric acid stabilized rat plasma using a surrogate matrix approach. A discrepancy in stability was observed between endogenous fumarate and exogenously added fumaric acid.
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http://dx.doi.org/10.4155/bio-2015-0026DOI Listing
April 2016

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

Bioanalysis 2014 ;6(22):2957-63

Covance Laboratories, Chantilly, VA, USA.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
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http://dx.doi.org/10.4155/bio.14.287DOI Listing
July 2015

Recommendations on incurred sample stability (ISS) by GCC.

Bioanalysis 2014 Sep;6(18):2385-90

Quintiles Bioanalytical & ADME Labs, Ithaca, NY, USA.

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.
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http://dx.doi.org/10.4155/bio.14.155DOI Listing
September 2014

Small molecule specific run acceptance, specific assay operation, and chromatographic run quality assessment: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization teams.

AAPS J 2014 Sep 25;16(5):885-93. Epub 2014 Jun 25.

Merck Research Laboratories, WP 75B-300, West Point, Pennsylvania, 19486, USA,

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.
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http://dx.doi.org/10.1208/s12248-014-9633-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147057PMC
September 2014

Accurate weighing and dilution-assisted plasma microsampling (AWADA-PM): an easy-to-implement and rugged strategy.

Bioanalysis 2014 Mar 12;6(6):805-17. Epub 2014 Mar 12.

Covance Laboratories, Inc., Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, WI 53704, USA.

Background: Liquid microsampling can realize ethical benefits through reduced animal usage. It inherently deals with a minute amount of sample; for example, <30 µl of plasma, which is generally insufficient for multiple analyses.

Results: We report accurate weighing and dilution-assisted plasma microsampling (AWADA-PM) that substantially increases sample sizes (e.g., by tenfold). Plasma samples are harvested from blood samples (~70 µl) in capillaries. The plasma samples are weighed with a calibrated balance. The weights are converted to volumes using a standard plasma density of 1.025 g/cm(3). Diluent is added with a calibrated pipette to the harvested plasma to achieve a tenfold dilution.

Conclusion: The proof-of-concept for AWADA-PM was successfully demonstrated for the quantitation of acetaminophen in rat plasma (K2EDTA) using liquid chromatography-tandem mass spectrometry employing water and blank rat plasma as diluents. The results clearly demonstrate that the AWADA-PM strategy is an easy-to-implement and reliable microsampling technology that has potential for full regulatory compliance.
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http://dx.doi.org/10.4155/bio.14.22DOI Listing
March 2014

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

Bioanalysis 2012 Sep;4(17):2117-26

Advion Bioanalytical Laboratories, Quintiles, NY, USA.

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.
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http://dx.doi.org/10.4155/bio.12.192DOI Listing
September 2012

Perforated dried blood spot accurate microsampling: the concept and its applications in toxicokinetic sample collection.

J Mass Spectrom 2012 May;47(5):655-67

Covance Laboratories, Inc, Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, Wisconsin 53704, USA.

Dried blood spot (DBS) sampling has gained considerable interest as a microsampling technique to support drug discovery and development owing to its enormous ethical and practical benefits. Quantitative determinations of drugs and/or their metabolites collected in DBS matrix in its current format, however, have encountered technical challenges and regulatory uncertainty. The challenges of DBS bioanalysis are largely ascribed to the way how samples are collected and analyzed. Currently, an uncontrolled amount of a blood sample, e.g. 20 µl, is collected per time point per sample and spotted onto cellulose paper. Quantitation is based on removal of a fixed area of the DBS sample, resulting in sample waste, a need for mechanical punching and concomitant potential punching carryover, uncertainty in recovery assessment and the adverse impact of hematocrit on accurate quantitation. Here, we describe the concept and applications of a novel concept, namely perforated dried blood spot (PDBS), for accurate microsampling that addresses previous challenges. Advantages of PDBS are enumerated and compared with conventional DBS in the context of microsampling and liquid chromatography tandem mass spectrometry bioanalysis. Two approaches for accurate microsampling of a small volume of blood (5 µl) are proposed and demonstrated, i.e. Microsafe® pipettes and the Drummond incremental pipette. Two online sample enrichment techniques to enhance liquid chromatography tandem mass spectrometry sensitivity for microsampling bioanalysis are discussed. The PDBS concept was successfully applied for accurate sample collection (5 µl) in a toxicokinetic study in rats given a single oral gavage dose of acetaminophen. Perspectives on bioanalytical method validation for regulated DBS/PDBS microsampling are also presented.
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http://dx.doi.org/10.1002/jms.3015DOI Listing
May 2012

Systematic evaluation of supported liquid extraction in reducing matrix effect and improving extraction efficiency in LC-MS/MS based bioanalysis for 10 model pharmaceutical compounds.

J Chromatogr B Analyt Technol Biomed Life Sci 2012 Apr 23;891-892:71-80. Epub 2012 Feb 23.

Huazhong University of Science & Technology, Tongji School of Pharmacy, Wuhan, China.

In past a few years, there has been a large increase in the application of supported liquid extraction (SLE) for LC-MS/MS based bioanalysis due to its distinct practical advantage in reduced time cost, ease of operation and the feasibility for automation. The main purpose of this study was to systematically evaluate supported liquid extraction in reducing matrix effect and improving extraction efficiency/recovery under various extraction conditions with 10 model pharmaceutical compounds in liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) analysis. Selected compounds have diverse physicochemical properties where logP ranges from 0.1 to 6.24 and pK(a) ranges from 4.0 to 11.1. The factors that may have the impact on the recovery of analytes and phospholipids (PL) were assessed. Over 75% recovery was achieved for every analyte under its respectively optimized extraction conditions where the selection of the polarity of extraction solvent and buffered pH can be critical for efficient recovery. Furthermore, the matrix effect was assessed by postextraction spike and postcolumn infusion method. The matrix effect was considerably reduced for all analytes under most extraction conditions evaluated for SLE, compared with protein precipitation (PPT) method. The correlation between matrix effect and residual phospholipids in sample extract was clearly shown. Although analyte-dependent matrix effect was observed prominently in sample extract prepared by PPT, it was minimized by SLE sample preparation process that effectively removes the majority of phospholipids. Sample extracted by ethyl acetate contained more phospholipids and demonstrated stronger matrix effect than by other organic solvents. Water-miscible organic content, such as methanol and acetonitrile in samples prior to loading has significant impact on PL recovery when eluting with methyl tert-butyl ether. However, isopropanol does not enhance the recovery of PL when adding to dichloromethane for elution. In addition, the compromise between improved extraction efficiency by SLE and reduced matrix effect is sometimes necessary to yield clean extract with acceptable recovery. The effective removal of phospholipids and reduction of matrix effect, while achieving good recovery for all pharmaceutical compounds with diverse physicochemical properties, demonstrated that SLE is a valuable alternative technique to liquid-liquid extraction (LLE) in high throughput LC-MS/MS based bioanalysis.
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http://dx.doi.org/10.1016/j.jchromb.2012.02.031DOI Listing
April 2012

Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS.

Bioanalysis 2011 Nov;3(21):2459-80

Covance Laboratories, Inc., Bioanalytical Chemistry, Madison, WI 53704, USA.

The advancement of biotechnology has led to an increase in biotherapeutic drugs, especially recombinant proteins and monoclonal antibodies. Ligand-binding assays or immunoassays are the standard methods of choice in pharmacokinetic studies in support of drug discovery and development for protein therapeutics. LC-MS-based methodologies are increasingly used as alternatives to immunoassays for absolute protein quantitation in biological samples. We review recent advancements in absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS.
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http://dx.doi.org/10.4155/bio.11.237DOI Listing
November 2011

Perforated dried blood spots: a novel format for accurate microsampling.

Bioanalysis 2011 Oct;3(20):2321-33

Covance Laboratories, Inc., Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, WI 53704, USA.

Dried blood spots (DBS) in their current format encounter challenges in bioanalysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for analysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5-10 µl), was deposited, dried and stored on the PDBS discs. Upon sample analysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof-of-concept study was carried out on a PDBS LC-MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K(3)EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels.
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http://dx.doi.org/10.4155/bio.11.219DOI Listing
October 2011

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2011 Sep 3;879(25):2663-8. Epub 2011 Jul 3.

Tongji School of Pharmacy, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei, China.

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.
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http://dx.doi.org/10.1016/j.jchromb.2011.06.039DOI Listing
September 2011

Determination of carboplatin in human plasma using HybridSPE-precipitation along with liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2011 Jul 13;879(22):2162-70. Epub 2011 Jun 13.

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei, China.

The main purpose of this study was to develop and validate a rapid, specific, sensitive, and reliable LC-MS/MS-based bioanalytical method for the determination of carboplatin in human plasma. The optimal chromatographic behavior of carboplatin was achieved on a Biobasic SCX column (50 mm × 2.1 mm, 5 μm) using ion exchange chromatography. The total LC analysis time per injection was 2.6 min with a flow rate of 1.5 mL/min with a gradient elution. Optimization with regard to improving recovery and minimizing matrix effects using HybridSPE-precipitation (HybridSPE-PPT) has been evaluated under various extraction conditions. As a result, sample preparation via HybridSPE-PPT with 1% formic acid in acetonitrile in a 96-well format was applied for method validation and sample analysis and showed acceptable recovery of greater than 25% and negligible matrix effects. The method validation was conducted over the curve range of 2.00-2000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.8% relative standard deviation (RSD) and -13.2 to -3.6% relative errors (RE). The method was successfully applied to determine carboplatin in human plasma samples.
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http://dx.doi.org/10.1016/j.jchromb.2011.05.057DOI Listing
July 2011

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay.

Anal Biochem 2011 Sep 6;416(1):45-52. Epub 2011 May 6.

Biomarker Research, Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Pfizer, Groton, CT 06340, USA.

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC-MS/MS (2D-LC-MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00-250 pg/ml for human plasma and 50.0-10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.
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http://dx.doi.org/10.1016/j.ab.2011.04.041DOI Listing
September 2011

2009 White Paper on recent issues in regulated bioanalysis from the 3rd Calibration and Validation Group Workshop.

Bioanalysis 2010 Jan;2(1):53-68

Algorithme Pharma Inc., Laval (Montreal) QC, Canada.

The 3rd Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis was organized by the Calibration and Validation Group as a 1.5-day full immersion workshop for contract research organizations, pharmaceutical companies and regulatory agencies to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges. A consensus was reached among panelists and attendees on many points regarding method validation of small molecules.
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http://dx.doi.org/10.4155/bio.09.134DOI Listing
January 2010

Comparison of fused-core and conventional particle size columns by LC-MS/MS and UV: application to pharmacokinetic study.

J Pharm Biomed Anal 2009 Oct 22;50(3):491-500. Epub 2009 May 22.

Pharmacokinetics, Dynamics and Metabolism, Pfizer Global R&D, Groton, CT 06340, USA.

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC-MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C(18) column (5 microm, 2.0 mm x 30 mm) and a fused-core C(18) column (2.7 microm, 2.1 mm x 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2-3-fold increased efficiency (N). Loading capacity up to 25 microl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2-5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 microm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC-MS/MS. The calibration curves were linear in the concentration range of 0.5-1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within +/-7.8% and +/-7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.
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http://dx.doi.org/10.1016/j.jpba.2009.05.011DOI Listing
October 2009

Workshop report and follow-up--AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples--implications of Crystal City recommendations.

AAPS J 2009 Jun 21;11(2):238-41. Epub 2009 Apr 21.

Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., MS-8118D-2061, Eastern Point Road, Groton, Connecticut 06340, USA.

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR. These recommendations from the Workshop, along with the subsequent evolution of approaches leading to a robust ISR program, may be used by scientists performing bioanalytical assays for regulated studies to provide additional confirmation of assay reproducibility for incurred samples.
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http://dx.doi.org/10.1208/s12248-009-9100-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691460PMC
June 2009

Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2009 Mar 11;877(8-9):765-72. Epub 2009 Feb 11.

Department of Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.

A highly selective, sensitive and robust LC-MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M+2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 microm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within +/-15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.
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http://dx.doi.org/10.1016/j.jchromb.2009.02.019DOI Listing
March 2009

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Nov;875(1):148-53

Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research and Development, Pfizer Inc., Ann Arbor, MI 48105, USA.

Pregabalin (Lyrica) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a gamma-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.
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http://dx.doi.org/10.1016/j.jchromb.2008.07.042DOI Listing
November 2008

Beyond pass/fail: a procedure for evaluating the effect of carryover in bioanalytical LC/MS/MS methods.

J Pharm Biomed Anal 2008 May 23;47(1):146-55. Epub 2007 Dec 23.

Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, San Diego, CA, USA.

Eliminating carryover from bioanalytical methods can be a time and resource consuming process. While it is necessary to investigate root causes of the carryover and reduce problem areas, complete elimination of carryover may not be practical or even possible. The purpose of this paper is to suggest an avenue to investigate the effect of carryover within an analytical run rather than employ a simple pass/fail criterion. With more robust carryover information a risk threshold level can be established for individual injections based on the peak response of the previous injection. It is then possible to quickly evaluate the risk that any value in an analytical run has been adversely affected by a previous injection. Those samples which are identified as "at risk" can be reanalyzed to obtain a value that is not affected.
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http://dx.doi.org/10.1016/j.jpba.2007.12.019DOI Listing
May 2008

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma.

J Pharm Biomed Anal 2003 Sep;33(1):61-72

Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., 4901 Searle Parkway, Skokie, IL 60077, USA.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.
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http://dx.doi.org/10.1016/s0731-7085(03)00349-2DOI Listing
September 2003

Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2003 Apr;787(2):333-44

Global Drug Metabolism, Pharmacia, 4901 Searle Parkway, Skokie, IL 60077, USA.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.
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http://dx.doi.org/10.1016/s1570-0232(02)00964-9DOI Listing
April 2003

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine.

J Pharm Biomed Anal 2003 Feb;31(1):103-15

Global Drug Metabolism, Pharmacia, 4901 Searle parkway, Skokie, IL 60077, USA.

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.
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http://dx.doi.org/10.1016/s0731-7085(02)00595-2DOI Listing
February 2003

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2003 Feb;785(1):123-34

Global Drug Metabolism, Pharmacia, Skokie, IL 60077, USA.

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.
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http://dx.doi.org/10.1016/s1570-0232(02)00863-2DOI Listing
February 2003