Configuration, optimization and evaluation of a novel instrumental platform for automated SPE-LC-MS/MS analysis of drugs in whole blood (2013)

The thesis describes the configuration, optimization and evaluation of a novel instrumental platform for fully automated SPE-LC-MS/MS analysis of small molecules, such as drugs, in whole blood. The immunosuppressant Cyclosporine A was chosen as a model analyte, as this drug is predominantly bound to erythrocytes. First, anticoagulated blood is converted into so-called Cell-Disintegrated Blood (CDB) by heat-shock or cryogenic treatment. CDB represents a homogenous blood sample and consists of subcellular particles which do not sediment on standing and do not clog capillaries, sieves or HPLC column packings. For in-line treatment of anticoagulated whole blood, i.e. generation of CDB, a sample mixing unit, two special liquid handling units and two home-made sample processing modules were embedded into a XYZ-autosampler. The module for heat-shock treatment consists of a stainless-steel capillary jacketed with a heating sleeve. Under optimal conditions for sampling and in-line processing of 20 µL of whole blood, it takes 13 seconds at 75 °C to generate CDB. The latter is stored in a holding loop before further treatment. For cryogenic treatment of a blood sample, a stainless-steel processing needle with a large inner diameter was installed in one of the liquid handling units. The autosampler was programmed to introduce the processing needle containing the blood sample (40 µL) into a stand-pipe, which is located in a thermo-flask filled with liquid nitrogen. The processing needle therefore contacts liquid nitrogen and the blood sample is snap-frozen. Optimal conditions were found to be 10 seconds for snap-freezing at -196 °C and 60 seconds for thawing at room temperature. A CDB sample obtained either by heat-shock or cryogenic treatment is further processed by being pumped via a switching-valve through an in-line filter to retain cell nuclei and “cell debris”. It was found that a depth filter packed with spherical hydrophilic silica is optimal. This filter allows at least 200 analysis cycles before it has to be replaced. Next, the CDB sample is pumped on-line via another switching-valve through a SPE column (50 x 0.5 mm ID) at a high flow rate. Due to the special packing material and the very small inner diameter, a high linear flow velocity is achieved and turbulent flow is generated. By this, high-molecular matrix components such as proteins are eluted in the void volume to waste. The low-molecular weight target analyte Cyclosporine A and the Internal Standard Cyclosporine D are retained and extracted by reversed phase partitioning chromatography (RPC). After fractionation of CDB on the SPE column, the analyte and the IS are transferred to a series-connected analytical column and separated from residual matrix components by RPC. Finally, the analyte is detected by a tandem mass spectrometer applying electrospray ionization (ESI) and multiple reaction monitoring (MRM). The optimized method has a total analysis time of less than 11 minutes. The analytical procedure and the instrumental platform were validated for heat-shock treated blood samples with respect to linearity, range (10 - 1000 ng/mL), lower limit of quantitation (10 ng/mL), intra-day and inter-day accuracy and precision, as well as matrix-independent and matrix-dependent recovery (around 100 %). It was shown that the electrospray induced ionization is suppressed by approximately 25 %. These matrix effects, however, can be totally compensated for by the addition of an Internal Standard, i.e. Cyclosporine D. A comparison with a semi-automated SPE-LC-MS/MS method, established in the Institute, revealed a very good agreement. This was shown by Passing and Bablok plots. The robustness of the fully automated SPE-LC-MS/MS analysis platform was monitored during 500 consecutive analysis cycles with heat-shock treated blood samples. The relative standard deviation for the signal response was 15.6 % for Cyclosporine A and 15.2 % for Cyclosporine D. The back pressure of the total system rose only by 52 bar. These findings show that, despite its instrumental and chromatographic complexity, the described analysis platform fulfills the prerequisites to be used in routine clinical-chemical analysis.