Dried blood spots as a sampling technique for the quantitative determination of guanfacine in clinical studies.

BACKGROUND Dried blood spot (DBS) technology was evaluated for the quantitative determination of guanfacine in human blood in clinical studies. A very sensitive DBS assay has been developed using HPLC coupled with an AB Sciex 5500 QTRAP® (Applied Biosystems/MDS Sciex, ON, Canada) MS system (LC-MS/MS) with a linear calibration range of 0.05 to 25 ng/ml. High-resolution MS using an Exactive Orbitrap® (ThermoFisher, LLC., CA, USA) was compared with the QTRAP using extracted exact mass ion current profiles for guanfacine and its stable-isotope-incorporated internal standard. The sample preparation employed liquid-liquid extraction with methyl t-butyl ether of 5 mm punched DBS card disks, followed by reversed-phase HPLC separation coupled with either MS/MS or high-resolution MS. Routine experiments were performed to establish the robustness of the DBS assay, including precision, accuracy, linearity, selectivity, sensitivity and long-term stability of up to 76 days. In addition, several factors that potentially affect quantitation were investigated, including blood volume for DBS spotting, punch size and punch location. RESULTS A sensitive research assay with a LLOQ of 0.05 ng/ml was developed and subjected to several components of a method validation common to a regulated bioanalysis procedure employing DBS. This method development and partial validation study determined that spot volume, punch size or punch location do not affect assay accuracy and precision. The DBS approach was successfully applied to a clinical study (a Phase I, randomized, double-blind, placebo-controlled, crossover study to assess the effect of varying multiple oral doses of guanfacine on the pharmacokinetic, pharmacodynamic, safety, and tolerability profiles in healthy adult subjects). The pharmacokinetic profiles for 12 volunteers generated from the DBS assay and from a previously validated plasma assay were compared and were found to be comparable. DBS incurred samples collected from finger prick blood and directly applied to the DBS cards were also analyzed for comparison. CONCLUSION From a bioanalytical perspective, DBS sample collection and analysis is a potentially viable alternative for guanfacine determination in clinical studies, utilizing approximately 100 µl of blood per subject profile compared with a few millilitres of blood drawn for conventional plasma bioanalysis.

[1]  Fumin Li,et al.  LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots. , 2011, Bioanalysis.

[2]  P. Timmerman,et al.  EBF recommendation on the validation of bioanalytical methods for dried blood spots. , 2011, Bioanalysis.

[3]  M. Barfield,et al.  Use of dried plasma spots in the determination of pharmacokinetics in clinical studies: validation of a quantitative bioanalytical method. , 2011, Analytical chemistry.

[4]  K. Heinig,et al.  Sensitive determination of a drug candidate in dried blood spots using a TLC-MS interface integrated into a column-switching LC-MS/MS system. , 2010, Bioanalysis.

[5]  N. Spooner.  Dried blood spot sampling for quantitative bioanalysis: time for a revolution? , 2010, Bioanalysis.

[6]  G. Corso,et al.  A powerful couple in the future of clinical biochemistry: in situ analysis of dried blood spots by ambient mass spectrometry. , 2010, Bioanalysis.

[7]  Neil Spooner,et al.  The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. , 2010, Bioanalysis.

[8]  M. Rowland,et al.  Use of Dried Blood Spots in Drug Development: Pharmacokinetic Considerations , 2010, The AAPS Journal.

[9]  D. Raynie.  Modern extraction techniques. , 2010, Analytical chemistry.

[10]  S. Cheetham,et al.  The neuropharmacology of ADHD drugs in vivo: Insights on efficacy and safety , 2009, Neuropharmacology.

[11]  M. Barfield,et al.  Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method. , 2009, Analytical chemistry.

[12]  Neil Spooner,et al.  Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies. , 2008, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[13]  Surendra Bansal,et al.  Key elements of bioanalytical method validation for small molecules , 2007, The AAPS Journal.

[14]  A. Lyne,et al.  Pharmacokinetics of a Guanfacine Extended‐Release Formulation in Children and Adolescents with Attention‐Deficit–Hyperactivity Disorder , 2007, Pharmacotherapy.

[15]  Walter A. Korfmacher.  Using Mass Spectrometry for Drug Metabolism Studies , 2007 .

[16]  A. Lyne,et al.  A phase I, randomized, open-label, crossover study of the single-dose pharmacokinetic properties of guanfacine extended-release 1-, 2-, and 4-mg tablets in healthy adults. , 2007, Clinical therapeutics.

[17]  R. Majors.  Advanced topics in solid-phase extraction : Chemistries , 2007 .

[18]  R. Ramanathan,et al.  Using mass spectrometry for drug metabolism studies , 2006, Journal of the American Society for Mass Spectrometry.

[19]  R. Oballa,et al.  A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry. , 2005, Rapid communications in mass spectrometry : RCM.

[20]  Kevin P Bateman,et al.  Discovery stage pharmacokinetics using dried blood spots. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[21]  Vinod P. Shah,et al.  Bioanalytical Method Validation—A Revisit with a Decade of Progress , 2000, Pharmaceutical Research.

[22]  B. Incledon,et al.  Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. , 2003, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[23]  Brian K. Kobilka,et al.  Mutation of the α2A-Adrenoceptor Impairs Working Memory Performance and Annuls Cognitive Enhancement by Guanfacine , 2002, The Journal of Neuroscience.

[24]  J. Henion,et al.  Liquid-liquid extraction in the 96-well plate format with SRM LC/MS quantitative determination of methotrexate and its major metabolite in human plasma. , 1999, Analytical chemistry.

[25]  F. Klink.  New sample preparation approaches to biological matrices for LC-MS , 1999 .

[26]  T. Wehr,et al.  Basic HPLC and CE of Biomolecules , 1998 .

[27]  J. Henion,et al.  Immunoaffinity Ultrafiltration with Ion Spray HPLC/MS for Screening Small-Molecule Libraries. , 1997, Analytical chemistry.

[28]  J. T. Stewart,et al.  Supercritical fluid extraction of selected pharmaceuticals from water and serum. , 1997, Journal of chromatography. B, Biomedical sciences and applications.

[29]  P. Jerie.  LOW, SINGLE DAILY DOSES OF GUANFACINE IN THE AMBULATORY TREATMENT OF HYPERTENSION , 1983 .

[30]  J. Doxey,et al.  Studies on the pre- and postjunctional activities of alpha-adrenoreceptor agonists and their cardiovascular effects in the anaesthetized rat. , 1981, Journal of autonomic pharmacology.

[31]  J. Kiechel.  Pharmacokinetics and metabolism of guanfacine in man: a review. , 1980, British journal of clinical pharmacology.

[32]  M. Safar,et al.  Guanfacine kinetics in patients with hypertension , 1979, Clinical pharmacology and therapeutics.

[33]  K. Thimann,et al.  The paper chromatography of indole compounds and some indole-containing auxins of plant tissues. , 1954, Archives of biochemistry and biophysics.