Microsoft powerpoint - tamiflu_poster_revised.ppt
Determination of Tamiflu® and active metabolite in dried blood spots
using the SCAPTM DBS system and column-switching LC-MS/MS
Katja Heinig1, Thomas Wirz1, Franz Bucheli1, Werner Döbelin2
1 F. Hoffmann-La Roche, Basel, Switzerland
2 Prolab Instruments, Reinach, Switzerland
Recovery <70% → multi-elution+stacking on TC
Oseltamivir phosphate (OP, Tamiflu®) is an ethyl ester
prodrug, which is converted in vivo to the active
neuraminidase inhibitor oseltamivir carboxylate (OC).
It is widely used for treatment/prevention of influenza.
In rodent ex vivo
samples OP is degraded to OC by high
esterase levels → immediate stabilization at sampling.
OP and OC are usually analyzed in plasma by LC-MS/MS [i.e. 1].
Potential benefits of Dried Blood Spots
(DBS) for sample collection, storage and analysis of OP and OC:
• Human studies: only microliters collected by non-invasive sampling (i.e. finger prick); no need for processing;
shipping & storage at ambient conditions → infant studies, studies in remote areas, therapeutic monitoring
Run No. (multiple extraction of same spot)
Extraction time (min)
• Animal studies: low volume sampling for rodents and juveniles; logistic benefits.
⇒ Efficient extraction of 226 cards; recovery >85%
• Stability of OP may be enhanced (avoid addition of toxic esterase inhibitors at sampling) and biohazard risk
Response proportional to extracted DBS area?
eliminated (cards lyse cells, reduce enzyme activity).
Multiple Power- Elution from DMPK-B cards
→ matrix effect for OP with 5 mm; use 3.2 mm for LLOQ
(8 x 3 sec, 2 mL/min) increases recovery &
1 ng/mL; lower LLOQ would require improved clean-up
Concerns when using DBS
partially compensates hematocrit influence
• Relevance of (capillary) blood for drug exposure, correlation with plasma PK from previous studies
Clamp size 2 / 3.2 / 5 mm
• Spot quality issues, patient variability (hematocrit etc.)
• Bioanalytics: optimization parameters, assay quality, drug stability, throughput
Automation of DBS extraction
is needed to compete with the throughput of conventional liquid
sample analysis, i.e. parallel processing in the 96-well format. An elegant approach is the direct
elution of cards without tedious punching. A manual thin-layer chromatography – MS interface has
been reported for direct card analysis . A novel instrument for fully automated card feeding and
elution, the SCAPTM (Sample Card And Prep) DBS system, was recently introduced. It is based on a
device for injection and online cleanup of liquid blood  and was employed for the determination
hematocrit / elution type
of bosentan and metabolites . Its use for analysis of OP and OC in rat DBS is described here.
⇒ Recovery from DMPK-B still not optimal, hematocrit
Blood Aliquot (uL)
critical: 0.45 → 0.3 35% deviation, <20% with 226 cards
Precision/Accuracy [%] (Ahlstrom 226)
Sample Card and Prep
DBS system (Prolab) on
modified CTC PAL auto-
PK Profile in Rat 1 (226 type card)
2. Robotic gripper tool picks up card and
3. Clamp closes, DBS card integrated into
Degradation of OP to OC in rat blood and DBS
PK of OC in rat 1
, 15 mg/kg OP po
• Rat blood (optionally containing diclorvos) spiked with OP and OC in calibration range 1 to 2000 ng/mL,
• 15 μL spotted onto Ahlstrom 226 (untreated paper cards) or DMPK-B cards (containing thiocyanate),
• Operation with compressed air or N2 (3 bar), pressure limit ~200 bar (LC backpressure)
PK of OC in rat 2
, 15 mg/kg OP po DBS DMPK-B
• Clamp equipped with 3.2 mm diameter adapter (variable sizes of 2…5 mm available)
• Universal applicability for various DBS card materials from different manufacturers (Whatman FTA, FTA Elute,
1 Time (h)
• Controlled by Cycle Composer software (CTC) via macros (costumer programming possible)• Synchronization with MS software for complete automation of DBS elution and LC-MS/MS analysis
: OP stable for >3 weeks w/o inhibitor.
: Unstable! Esterase inhibitor needed.
data corrected for BPP, dichlorvos added to al but DMPK-B
LC-MS/MS instrumentation and analytical procedure
• The SCAP DBS system offers fully automated online DBS elution and hence reduces the sample
DBS extraction solvent: Water
Rinsing: EtOH/ACN/formic acid
preparation efforts by removing the punching and extraction procedure.
• SCAP was easily integrated into a standard column-switching system. Extraction efficiency was
+ solvent selector
increased and hematocrit influence reduced by multiple elution of same spot + “stacking” these extracts on the TC. Moisture expansion & clamp plugging was still an issue with DMPK-B cards.
• Autosampler used for internal standard injection (SCAP) or offline extracted samples (bypass).
• SCAP-LC-MS/MS with untreated 226 type cards achieved similar precision & accuracy as offline
extraction of DBS or plasma assays for OP & OC and sufficient sensitivity (1 ng/mL LLOQ).
• Chemically treated cards could stabilize OP without addition of esterase inhibitors, but showed
severe matrix effects (DMPK-A) or variable recovery depending on hematocrit (DMPK-B).
• Rat PK study data showed little deviation between DBS, blood & plasma and between offline and
Nucleosil C18, 4x8 mm
online extracted 226 cards. Limited data do not allow definite conclusion on further use of DBS.
• Further improvements: Spot detection, higher card loading capacity, MS software integration for
SCAP. Better standardization of card formats without batch-to-batch variability needed. Re-
Atlantis T3, 3.5
design of chemically treated material (DMPK-B) or modified clamps necessary for SCAP elution.
2.1 x 50 mm
2 x LC-10ADvp
A. Gajate-Perez, B. Lausecker, M.-S. Gruyer, J. Meyer, F. Schuler, G. Hoffmann
TSQ Quantum Ultra
A: Water-MeOH 80:20 (0.1% formic acid)
ESI, postive mode SRM
B: Water-MeOH 5:95 (0.1% formic acid)
1. Heinig K, Bucheli F. Sensitive determination of oseltamivir and oseltamivir carboxylate in plasma, urine, cerebrospinal fluid and
Sequence run was started in Cycle Composer; DBS card was inserted in clamp and waited for extraction. Next
brain by liquid chromatography tandem mass spectrometry. J. Chromatogr. B
876, 129-136 (2008).
the sequence (identical setup as in CC) was started in Xcalibur. Internal standard solution (10 μL of 10 ng/mL
2. Abu-Rabie P, Spooner N. Direct Quantitative Bioanalysis of Drugs in Dried Blood Spot Samples Using a Thin-Layer
deuterated OP & OC) was injected onto TC by rinsing water at 0.5 mL/min (pump C) through autosampler
Chromatography Mass Spectrometer Interface. Anal. Chem.
81, 10275–10284 (2009).
loop. Between 0 and 0.7 min, the bypass valve was switched to SCAP to let extraction solvent flow through
3. König S, Döbelin W. A Novel Concept for Sample Col ection and Sample Preparation. Poster WP 427, ASMS, Salt Lake City
DBS and further onto TC (2 cycles for 226, up to 8 cycles for DMPK-B). Extracted analytes were retained and
4. Glinski M, Döbelin W, Ganz N. Fully automated direct application of DBS combined with HPLC coupled to MS/MS for
polar matrix components washed out. Elution from TC between 0.8 and 1.6 min (trapping valve switched) and
simultaneous quantification of bosentan and its metabolites. Poster No. 58, LC-MS Symposium, Montreux (2010).
gradient separation within 1.5 min at 0.3 mL/min on AC with mobile phases A and B (pumps A & B). TC and SCAP (with clean card in clamp) rinsed with organic solvent from 1 to 2.5 min. Total run time was 3.6 min.
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