This is nice presentation given by Vishal Goyani, an Analytical student of National Institute Of Pharmaceutical education and research-INDIA. mail your query at - vngoyanii@gmail.com

1. Analysis of cocaine and its two
metabolites in dried blood spots
Vishal N. Goyani
M.S. (Pharmaceutical Analysis)
National Institute of Pharmaceutical Education and Research

2. Agenda
ØIntroduction
Bioanalysis
DBS
HPLC-Fluorescence
Cocaine and its metabolite
ØResearch paper
ØAim
ØMethod and Material
ØResult & Discussion
ØConclusion
ØFuture aspect

3. Introduction

4. Bioanalytical method
Ø Analytical methods for the quantitative
evaluation of drugs and their metabolites in
Biological matrix
Ø Use:
Ø Human clinical pharmacology
Ø Toxicokinetics
Ø BA and BE studies
Ø Therapeutic drug monitoring
Ø Forensic science

5. What are Dried Blood Spots? (DBS)
Ø Easy way of collecting, shipping & storing blood samples
Ø Newborns are screened for inherited metabolic
disorders (>95% in US)
Ø Therapeutic drug monitoring
Ø Mouse PK-PD study
Ø For trials in remote areas, e.g. anti-malarials
Ø Spot-punch-extract

6. Spot

7. Dry

8. Punch

9. Extract

10. Analysis
} LC-MS
} LC-MS-MS
} HPLC-Fluorescence detection

11. R3 : Reduce ,Refine ,Replace
Ø 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

13. Advantage
} Simplified blood sampling (finger prick)
} Less invasive
} Less solvent requirement
} Simplified process better quality
û Centrifugation
û Sub-aliquotting
û Freezing
û Defrosting

14. 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

15. 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

16. 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

17. Cocaine metabolism pathway
a. cocaine(coc), b.benzoylecgonine(beg), c. cocaethylene (cet)

18. Research paper

20. 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

21. 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

22. 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

23. } 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

24. Sample collection and preparation

25. 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

26. 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

27. 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

28. Accuracy
Extraction
Precision
yield
Bioanalytical
Method
Validation
Parameter
LOD
Selectivity
LOQ
Linearity of
Calibration
curve

29. Results and discussion

30. 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).

31. No interference

32. 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

33. Linearity, LOD and LOQ

34. 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

36. 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)

38. Analysis of samples from Cocaine users
(a) a DBS sample and (b) a plasma sample from a COC and ethanol user.

39. 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

40. Conclusion

41. 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.

42. 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

43. 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.

44. Thank
You ..

45. Now, Its
your turn!!!

46. stability
} Whole blood stability at 37°C for 4 hours
} On card stability
} Assay robustness to pipetting error (10 - 20 L)
} Processed sample stability

47. Table II: Specialty DBS cards commercially available

48. } 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

49. 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