High-throughput technique with preparation

Plastic microdevices for high-throughput screening with MS detection were also prepared for detection of aflatoxins and barbiturates. These devices incorporated concentration techniques interfaced with electrospray ionization MS (ESI-MS) through capillaries [2], The microfluidic device for aflatoxin detection employed an affinity dialysis technique, in which a poly (vinylidene fluoride) (PVDF) membrane was incorporated in the microchip between two channels. Small molecules were dialyzed from the aflatoxin/antibody complexes, which were then analyzed by MS. A similar device was used for concentrating barbiturate/antibody complexes using an affinity ultrafiltration technique. A barbiturate solution was mixed with antibodies and then flowed into the device, where uncomplexed barbiturates were removed by filtration. The antibody complex was then dissociated and electrokinetically mobilized for MS analysis. In each case, the affinity preconcentration improved the sensitivity by at least one to two orders of magnitude over previously reported detection limits. 

Single-enzyme systems, typically obtained from cDNA expression systems, are an effective technique for the study of individual enzyme reactions and their products. The incubation of enzyme, buffer, necessary cofactors, and drug produces extremely clean samples that are amenable to either simple cleanup procedures or direct injection into the LC/MS instrument (Chap. 6). These isolated enzymes can be stored at -80°C for extended periods of time, making them readily available whenever necessary. In addition, they are reasonably easy to work with either by hand or in a robotic environment where high-throughput incubation, sample preparation, and analysis are possible. 

Cell migration was assayed by automated time-lapse video microscopy. In this technique, cells are imaged on the surfaces and the individual cell tracks are reconstructed from the position of each cell at successive time points. The automation enables time lapse videos to be taken from multiple locations on the same surface at the same time. Thus, the entire gradient can be imaged at once. This high-throughput technique combined with the combinatorial sample preparation enables the exploration of a relatively large parameter space quickly. 

In real life, sample preparation techniques are utilized in tandem with HPLC/MS/MS. Due to the high selectivity of LC/MS/MS in the SRM mode, small quantities of impurities or interferences in the purified fractions from sample preparation protocols dealing with biological matrices can be tolerated. This is especially true for protein precipitation, which is widely used because of simplicity and rapidity, when high-throughput turn-around is required. High-throughput HPLC is discussed elsewhere in this book. 

HPLC has high-throughput capability when it can simultaneously determine multiple drugs and their metabolites or when coupled with a unique monolithic column or sample preparation technique. Some examples are summarized below. 

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