CAMAG DBS-MS 500 DRIED BLOOD SPOT EXTRACTION SYSTEM FOR LC-MS

Transcription

1 CAMAG DBS-MS 500 DRIED BLOOD SPOT EXTRACTION SYSTEM FOR LC-MS Direct Analysis

2 FULLY AUTOMATED DBS EXTRACTION SYSTEM FOR MASS SPECTROMETRY Dried blood spot sampling was introduced around 40 years ago. It is an easy way of collecting, shipping and storing blood samples. In drug development the screening of a large number of blood samples is obligatory to determine the bioavailability, clearance, and safety margins of a certain drug substance in an organism. In recent years the usage of dried blood spots (DBS) has gained increasing importance since this method shows strong advantages compared to the conventional collection and analysis of blood or plasma samples. These advantages include the need for remarkably lower blood volumes and easier shipping and storage, often at ambient temperatures. This leads to a simplification of the blood collection process and a reduction of the costs involved. Furthermore, in preclinical studies the number of test animals can be reduced (due to the need for lower blood volumes) which is in accordance with the 3R requirement of animal studies (replacement, reduction, refinement). For DBS analysis, typically 15 μl of the blood sample are spotted onto filter paper (DBS cards), dried, shipped to the laboratory and analyzed by mass spectrometry (MS). Prior to MS analysis the blood sample needs to be extracted from the DBS card. This represents a major drawback of DBS since sample extraction from a high number of DBS cards is not yet automated and requires many processes to be performed manually. Hence, analysis of DBS is time-consuming and costly. CAMAG Switzerland has developed a system for the automated extraction of DBS cards that can be coupled directly to the MS or LC- MS system. The surge in interest in DBS techniques for supporting pharmaceutical exposure studies is due to the many advantages it offers over conventional plasma sampling Paul Abu-Rabie, GlaxoSmithKline, Bioanalytical Science and Development, UK ( 2

3 Features of the DBS-MS 500 Fully automated handling of up to 500 DBS cards Barcode reading device for card identification Automated internal standard application module Extraction module independent of LC mobile phase Wash station to eliminate carry-over Easy integration with any LC-MS system The fully automated CAMAG DBS-MS extraction system makes DBS analysis remarkably easy and has strong advantages compared to both the manual and partially automated punching techniques. With the CAMAG DBS-MS extraction procedure DBS analysis is an economical alternative for conventional blood sampling. It represents a time-, cost-, and animal-saving technique for analyzing pharmacological samples. Punch CAMAG DBS-MS 500 Add solvent Vortex mix Centrifuge card handling Read-in of identification data (optional) Application of internal standard (optional) Extraction of dried blood spot Transfer of sample extract to LC-MS Typical Chromatogram 1.4e6 1.2e6 1.0e6 Patents pending Supernatant transfer Intensity, cps 8.0e5 6.0e5 4.0e5 2.0e Time, min CAMAG DBS-MS 500 3

4 Features and Technical Details Pick up DBS cards Highly reliable robotics are used for handling of DBS cards. The cards (type: ID Biological Systems, or Whatman) are stored in up to 5 racks, each with a capacity of 100 cards. The robotics include a newly developed gripping tool specifically adapted to DBS cards. The gripping tool picks up cards and moves them to all of the subsequent processes in the corresponding modules Optical card recognition (OCR-module) A digital camera with integrated firmware and adapted sensors determines the card properties: spot number and position, checkbox status, and barcode information. The DBS card is only accepted for analysis if all parameters comply. CAMAG has incorporated its knowhow of more than a decade of optical HPTLC image analysis to build this precise and reliable OCR-module. 2 Technical data Dimensions Size: cm (w d h); net weight 120 kg Additional space of 10 cm for ventilation and height of 30 cm for solvent bottle placement required Requirements Pressurized air of 4 to 8 bar Line voltage V, 50 / 60 Hz, 100 W User interface Touch screen display for selecting and editing methods and setting parameters Communication with LC-MS Contact closure, optional Ethernet communication Tube connections to LC-MS 1/16 high pressure valve, coned ports (UPLC valve on request) i 4

5 Replace DBS cards Finally, the robotic gripping tool puts the cards back into their original position in the rack. Depending on user requirements there are additional options, e.g. verification of DBS cards for proper extraction or further tests depending on future requirements Wash station Requirements regarding carry-over limits are a challenge, particularly for direct sample loop filling without a trapping column. Therefore a designated wash station cleans all parts that are in contact with the DBS cards and additionally flushes the entire tubing to avoid any carry-over. Using this system, independent user tests confirm that carry-over is not an issue. 5 Extraction module The precise extraction process involves a sample loop which is independent from the LC-MS system, thus allowing for high flexibility for different fluid configurations. Whether you are using a standard sample loop of 20 μl or want to flush 200 μl of solvent through a trapping column the system can be configured according to your needs, just like your usual LC-MS system. 4 Internal standard application (ISA-module) Variability in the absolute response of LC-MS systems can now be corrected by applying an internal standard solution onto DBS cards directly before the analysis; this also allows correcting for extraction efficiencies. The application of the internal standard just before the extraction of the DBS card is much easier from a logistic point of view than adding the internal standard before the blood sample is spotted onto the DBS card. CAMAG has been manufacturing high precision application instruments for HPTLC for a long time and has used this experience to build the ISA-module. 3 CAMAG DBS-MS 500 5

6 Application examples The reproducibility of the peak area ratios was below 3 % which was beyond expectations and no significant carry-over was observed. (Dr. Neil Spooner, Director Bioanalytical Science and Development, PTS DMPK, GlaxoSmithKline, Ware, UK) Spraying the internal standard onto dried blood spots proved very suitable and is desired from a logistical point of view (Dr. Dieter Zimmer, Head of Bioanalytics and PK/TK, Harlan Laboratories Ltd., Itingen, Switzerland) Peak area ratio response over 560 consecutive DBS samples containing sitamaquine and [ 2 H 10 ]-sitamaquine (as an internal standard) using manual extraction and direct elution via the DBS-MS. Note each data series is normalised against the data point for sample#1, not against the other data series. The direct elution samples were analysed on two consecutive days, which explains the small gap around sample 170 but the data still compares very well with the manual extraction samples. % % Internal standard application (ISA-module), CAMAG DBS-MS system I am very convinced that the pre-requisite for a broad use of DBS is closely related to fully automatized online extraction of the spots reducing the workflow by two steps: punching and offline extraction. We have successfully evaluated a prototype of the CAMAG DBS-MS interface in our laboratories. The way of addition of the internal standard IS in case of DBS is still a matter of debate. I consider addition of the IS via the extraction solvent as non-adequate since the IS does not mimic the recovery of the analyte from the spot. Besides online analysis of spots, a further advantage of the DBS-MS interface is the capability of spraying the internal standard onto the spots. In several tests we were able to demonstrate a high reproducibility of the spray process and a similar recovery of IS and analyte (Poster, EBF Conference, Barcelona 2010). GlaxoSmithKline has performed extensive tests with the CAMAG DBS-MS system, evaluating robustness by analyzing large batches of 560 samples. The reproducibility of the analyses was good and the peak area ratio between analyte and internal standard was highly promising: across 560 samples the peak area ratio CV (or RSD) was well below 3 %. Carry-over data was assessed over time by extraction of a paper blank sample between DBS samples. Carry-over was impressively low: none of the peaks from the analysis of the blank samples was above background noise. In practice the installation of the CAMAG DBS-MS, and the development of direct elution extraction methods, proved to be very quick and easy. The wash system proved highly reliable and had no significant impact on the sample cycle times. Based on the initial tests the robustness of the automated DBS-MS method was good. We have also found that the DBS-MS offers on average an order of magnitude increase in assay sensitivity compared to manual DBS extraction, with no compromise in chromatographic performance. [Anal.Chem.81, (2009)] 6

7 In the future individualized drug therapy will play an important role. For Therapeutic Drug Monitoring (TDM) patients might do their sampling by finger prick and then directly send the DBS-cards to the laboratory for centralized analysis by automated DBS-MS (Dr. med Manuel Haschke, senior physician, Division of Clinical Pharmacology and Toxicology, University Hospital Basel, Switzerland) Concentration-time profile of efavirenz analyzed in dried blood spot samples extracted with the CAMAG DBS-MS system compared to manual punch / extraction and venous blood samples In a feasibility study for pharmacokinetic monitoring at the University Hospital Basel the HIV drug efavirenz was analyzed in dried blood spot samples using automated online-extraction with the CAMAG DBS-MS system. To optimize the therapeutic effect, efavirenz therapy has to be individualized for certain patients in order to account for drug-drug interactions or genetic differences influencing the metabolism of the drug. The concentration-time profile of efavirenz in DBS samples provides information on the pharmacokinetic behavior of the drug in a patient and thus enables monitoring of the therapeutic target concentration. The data obtained by the automated approach using the CAMAG DBS-MS system are in agreement with the data obtained by the former manual punch-and-elute sample preparation technique. Linear regression analysis of efavirenz concentrations obtained by either approach showed a highly significant correlation (R2=0.9966). Future applications of the CAMAG DBS-MS system at hospitals could be clinical pharmacokinetic studies or the centralized analysis of TDM samples from patients in individualized drug therapy. Linear correlation between concentrations in manual punch/extraction DBS samples and DBS samples extracted with the CAMAG DBS- MS system Please contact us for complete application examples and further details: dbs@camag.com i CAMAG DBS-MS 500 7

8 Ordering Information i DBS-MS 500 DBS-MS 500 base frame including fully automatic robotics for handling 500 cardboard cards and space for additional modules Extraction module including a high precision pump with a selector to choose between different solvents for optimized extraction conditions Wash station including a precision high pressure pump with a selector to choose between different solvents for optimized washing Modular design Slots for up to 3 modules protecting your investment and allowing easy extension and upgrade Master slave control Seamless integration with the LC-MS system Options Optical card recognition module (OCR-module) including spot localization, analysis and barcode reading Internal standard application module (ISA-module) including external application of IS to DBS cards before analysis * Patents pending CAMAG (Switzerland) Sonnenmattstrasse Muttenz Tel Fax info@camag.com CAMAG (Germany) Bismarckstrasse Berlin Tel Fax infoberlin@camag.com CAMAG Scientific (USA) 515 Cornelius Harnett Drive Wilmington, NC Phone (800) Fax (910) tlc@camag.com Direct analysis