The LDTD technology has been around for quite some time and yet because it is so far removed from the typical HPLC-MS systems people are used to, everyone keeps comparing it to a standard LC-MS system. Therefore, in order to clarify how similar and different LDTD Technology (paired with an MS) is to traditional LC-MS, we decided to answer the most common questions we get on the technology.
Topic #3: How does multiplexing work with LDTD?
First of all, why would users want to use this multiplexing technique? To save time! Multiplexing reduces the analysis time of the mass spectrometer. So, how does it work?
Multiplexing with different technologies can be quite … well, different! If you compare the multiplexing technique with liquid chromatography to the multiplexing technique used with immunoassays, you will find that they are not the same at all and multiplexing with the LDTD can be mind-boggling at first but is, in fact, very simple.
Let’s break it down!
Liquid chromatography uses multiple LC channels to inject into a single mass spectrometer. Users determine the window when their peaks appear and time their multiple units so that these detections do not overlap. This way, they can reduce a 10 minute/sample run to a 5 minute/sample run.
Immunoassays can be multiplexed by adding microbeads of different fluorescence colors coated with a specific antibody. With flow cytometry, the number of analytes can be calculated by measuring the number of microbeads of each color by their fluorescence. In this case, the plate must be read twice. This technique is mostly used in research rather than in production environments. A second approach is to spatially separate captured antibodies on a shared solid surface. The captured antibodies are placed in an array, which is read once and the coordinates of the spot analyzed can be used to determine which protein is detected.
With LDTD, multiplexing requires the user to add the extracted samples one on top of the other in the same well of the same LazWell™ plate. The plate is only analyzed once and the analytes are detected by the MS in the same time frame. Multiplexing with LDTD is mostly used for pharmaceutical in vitro assays. So, if for example, three different reactions are needed for three interactions with enzymes or antibodies, the samples can react separately and added into the same well when it is time to analyze them.
Multiplexing with the LDTD is not used in toxicology, diagnostics or other fields with heavily loaded matrices. Adding one sample on top of the other in these cases would overload the plate with interferents found in the matrix, and the peak shape and sensitivity would suffer. The samples could be diluted to lower the interferents, but sensitivity would again be lowered. In these fields, a single extraction approach containing multiple analytes is preferred.
Topic #4: Do I need an automated liquid handler to deposit samples into LazWell™ plates?
No, manual deposition can be used. Of course, automated liquid handlers can significantly reduce manipulation errors and variability, so they are recommended when using LDTD regularly. Also, the LazWell1536 can be quite a pain to spot manually!
Keep in mind that if you choose to spot by hand, your pipette should be calibrated regularly to maintain the reproducibility of your analyses. For the LazWell96 and LazWell384, a micropipette with a range of 0.5 to 10 µL is sufficient. For LazWell1536, a pipette with a range of 0.1 to 1.0 µL can be quite useful.