Bioanalytical methodØ Analytical methods for the quantitative evaluation of drugs and their metabolites in Biological matrixØ Use: Ø Human clinical pharmacology Ø Toxicokinetics Ø BA and BE studies Ø Therapeutic drug monitoring Ø Forensic science
What are Dried Blood Spots? (DBS)Ø Easy way of collecting, shipping & storing blood samplesØ Newborns are screened for inherited metabolic disorders (>95% in US)Ø Therapeutic drug monitoringØ Mouse PK-PD studyØ For trials in remote areas, e.g. anti-malarialsØ Spot-punch-extract
R3 : Reduce ,Refine ,ReplaceØ Lower blood volumes Ø Fewer experimental animals Ø Eliminating satellite colonies, so improved data quality Ø Juvenile animals and humans can be studied Ø Mice in place of Rat 100-200 l per sample 3 sample time point/mice 10 to 20 l per sample 6-10 sample time point /mice
Advantage} Simplified blood sampling (finger prick)} Less invasive} Less solvent requirement} Simplified process better quality û Centrifugation û Sub-aliquotting û Freezing û Defrosting
Advantage} Stability: Due to moisture removal greater analyte stability, especially for enzyme-sensitive compounds} Easier storage and transportation: } Room temperature stability saves on cost of dry ice shipments } Less bulky compare to liquid sample } Remote sampling, particularly important for human clinical trail samples} Cost savings } Animal numbers } Procedures } storage
HPLC with Fluorescence detector} Based on principle of fluorescence - light absorbed by a molecule causes one or more electrons to be excited to higher energy state then molecule may return to ground state by emitting energy as electromagnetic radiation} The wavelength (energy) required is specific to the molecule} Advantages } Sensitivity } Selectivity } Insensitivity to pressure and flow rate fluctuations
Cocaine} Cocaine (benzoylmethylecgonine) is a crystalline tropane alkaloid that is obtained from the leaves of the caco plant.} Use: } Stimulant of CNS } Appetite suppressant } Topical anesthetic} illicit drug in Western countries} Need to analysis from blood to confirm abuse
Cocaine metabolism pathway a. cocaine(coc), b.benzoylecgonine(beg), c. cocaethylene (cet)
Aim:} Analysis of cocaine and two metabolites in dried blood spots by HPLC with uorescence detection: A novel test for cocaine and alcohol intake} To carry out comparison between DBS & Plasma sampling} Analysis of samples from cocaine abusers
Chemicals and solutions} Methanolic stock solutions of COC, BEG,CET and Venlafaxine (1 mg/mL) were purchased from LGC Standards (Teddington, UK)} Acetonitrile and methanol HPLC grade (>99.8%), 85% (w/w) phosphoric acid, TEA and monobasic potassium phosphate pure for analysis (>99%) were purchased from Sigma Aldrich (Milan, Italy)} Ultrapure wate was obtained by means of a MilliQ apparatus} Standard solutions were obtained by diluting stock solutions with the mobile phase
Instrumentation and HPLC conditions} The chromatographic system with spectro uorimetric detector (Jasco)} exc = 230 nm, em = 315 nm} Varian (Walnut Creek, USA) Microsorb MV C8 column (250 mm × 4.6 mm I.D., 4 m)} Mobile phase was composed of acetonitrile potassium phosphate buffer (pH 3.0; 50 mM)(15:85,v/v)} The ow rate was 1.5 mL/min.} Injections Volume was 50 l} Data processing by Jasco Borwin 3.0 software
} Solid-phase extraction (SPE) was carried out on a Varian Vac Elut apparatus using C8 cartridges (50 mg, 1 mL)} Whatman (Maidstone, UK) 903 Protein Saver cards were used for DBS sample collection} Harris Uni-Core Punch 3.0 mm} Lancet
DBS sample pretreatment} Each subject was punctured on a nger and 10µL of blood apply with micropipette onto card} The blood spot was left to dry for 2 h} Circle with 10 mm diameter DBS, was punched out from the card and placed into a vial with 500 L of methanol} The vial was then vortexed for 30 s and centrifuged at 1400 × g for 10 min} The supernatant was brought to dryness under vacuum, re-dissolved with 100 L of mobile phase and injected into the HPLC
Plasma sample pretreatment} For plasma analysis, blood was sampled from the same subject into a vial containing EDTA as the anticoagulant. The blood was centrifuged at 1400 × g for 10 min to separate plasma} The SPE of analytes from 400µl plasma was carried out on C8 Cartridges & washed twice with 1 mL of ultrapure water and once with 1 mLof water/methanol (90:10, v/v) Then eluted with 1mL of methanol} The eluate was dried under vacuum, re-dissolved with 100 L of mobile phase and injected into the HPLC system
Spiking for method validation} DBS: Aliquots of 5µL of analyte standard solutions at seven different concentrations, containing the IS at a constant concentration, were added to 10µL DBS} Plasma sample: 20µLof analyte standard solutions at seven different concentrations, containing the IS at a constant concentration, was added to 400 µLof blank plasma} The resulting spiked DBS or plasma samples were subjected to the previously described pretreatment procedures and injected into the HPLC
Analysis of standard solutions(Without biological matrix)} Good linearity (r2 > 0.9998) was obtained over the 2 2000 ng/mL concentration range for COC and BEG and over the 1.2 2000 ng/mL concentration range for CET.} The LOQs and the LODs, respectively, were 2 ng/mL and 0.7 ng/mL for COC and BEG and 1.2 ng/mL and 0.4 ng/mL for CET.} Precision assays were carried out at three different levels and gave good results: the RSD values of repeatability} Intraday precision < 2.2% (2.0% for the IS).} Intermediate < 2.9% (3.3% for the IS).
Linearity} Seven different concentrations, containing the IS at a constant concentration spiked in DBS or plasma samples were analyzed} The procedure was carried out in triplicate for each concentration} The analyte/IS peak area ratios obtained were plotted against the corresponding concentrations of the analytes & the calibration curves prepared} The values of LOQ and LOD were the analyte concentrations, which give rise to peaks whose heights are 10 and 3 times the baseline noise, respectively
Extraction yield, precision} Extraction yield (absolute recovery) and precision assays were carried out on blank DBS and plasma spiked with analyte concentrations corresponding to the lower limit, an intermediate point and a high value of the respective calibration curves} Above three sample repeated six times within the same day to obtain repeatability (intraday precision)} Six times over six different days to obtain intermediate precision (interday precision)} Both expressed as relative standard deviation (RSD%) values
Selectivity} Interference from endogenous compounds :Blank DBS or plasma samples from six different volunteers were subjected to the sample pre-treatment procedure and injected into the HPLC} The acceptance criterion was no interfering peak higher than an analyte peak corresponding to its LOD} Interference from xenobiotics: Standard solutions of several different compounds active on the central nervous system were injected into the HPLC system. The concentration of each standard solution was equal to 5 times the upper limit of linearity for the analyte standard solutions (i.e., 10 g/mL)
Analysis of samples from Cocaine users (a) a DBS sample and (b) a plasma sample from a COC and ethanol user.
Comparison between DBS & Plasma} The analyte amounts found in DBS and those found in plasma are always in good agreement, taking into account the presence of hematocrit in DBS and its absence from plasma} Since the mean normal hematocrit is 38% (v/v) in women and 48% (v/v) in men, the concentrations found in DBS samples were multiplied by a corresponding correction factor (1.62 for women and 1.92 for men) Concentration cocaine benzoylecgonine cocaethylene (ng/mL) DBS 37 113 34 DBS x 1.62 60 183 55 Plasma 62 180 56
Conclusion} The HPLC method with uorescence detection presented here for the determination of cocaine, benzoylecgonine, cocaethylene in DBS and plasma is fast, sensitive and selective} The proposed method is suitable for the monitoring of cocaine use & of cocaine -ethanol simultaneous intake, through DBS testing and plasma} DBS sampling can be considered a good candidate to be applied, in the near future, for the testing of subjects suspected of driving under the in uence of drugs and alcohol, hopefully becoming a useful and reliable tool to contain number and severity of car accidents.
Outlook (In future )} Direct analysis off the spot to enable routine application of DBS Analysis by surface MS e.g. TLC-MS (CAMAG)} DBS cards to become less expensive for routine use
Reference} Analysis of cocaine and two metabolites in dried blood spots by liquid chromatography with uorescence detection: A novel test for cocaine and alcohol intake Laura Mercolini et.al} Dried blood spot microvolume sampling for DMPK manual by GE Healthcare} Guidance for Industry, Bioanalytical Method Validation, U.S. Food and Drug Administration} A.J. Jenkins, B.A. Goldberger, On-site Drug Testing, Humana Press, Totowa, 2002.
stability} Whole blood stability at 37°C for 4 hours} On card stability} Assay robustness to pipetting error (10 - 20 L)} Processed sample stability
Table II: Specialty DBS cards commercially available
} Fluorescence detectors } Based on principle of fluorescence - light absorbed by a molecule causes one or more electrons to be excited to higher energy state. The wavelength (energy) required is specific to the molecule. The molecule may return to ground state by emitting energy as electromagnetic radiation.} Advantages } Sensitivity } Selectivity } Relative insensitivity to pressure and flow rate fluctuations
Future aspect} Direct analysis off the spot to enable routine application of DBS} - Analysis by surface MS} - e.g. TLC-MS (CAMAG), DESI, other?} - MS vendors encouraged to provide the tools