ISOLUTE PLD+ plates deliver cleaner extracts than other phospholipid removal products, meaning lower detection limits can be achieved. In the example below, signal-to-noise using ISOLUTE PLD+ was more than double when compared with competitor products (figure 1), leading to an immediate enhancement in sensitivity.
Figure 1. Relative peak intensity, 20 pg/mL amitriptyline in plasma (Blue = ISOLUTE® PLD+, Orange = Competitor O, Red= Competitor P).
More than 99 % of phospholipids are removed as the sample passes through the multifunctional sorbent bed, leaving clean, purified extracts for analysis (figure 2). Removing phospholipids also improves analytical column lifetime, preventing the build-up of phospholipids over multiple injections, and maintaining analyte sensitivity over extended analytical runs.
Figure 2. Phospholipids in human plasma treated by a) protein precipitation and b) ISOLUTE® PLD+. Lysophospholipids and phospholipids present in plasma treated by protein precipitation (red and green respectively) are shown in 1b on an expanded scale for direct comparison with residual levels of lysophospholipids and phospholipids present in ISOLUTE® PLD+ treated plasma (purple and blue respectively).
Figure 3. Typical ISOLUTE PLD+ procedure
Using a simple solvent crash/filtration based procedure, proteins and phospholipids are simultaneously removed from plasma and other blood based samples while high, reproducible analyte recoveries are maintained. The optimised frit arrangement acts as a depth filter, efficiently trapping precipitated proteins, without blocking or plugging.
ISOLUTE® PLD+ plates have excellent flow characteristics, and do not plug or block during use. They can be processed easily using either positive pressure or vacuum based systems, and are compatible with most automated liquid handlers.
|918-0050-P01||ISOLUTE PLD+ Protein & Phospholipid Removal Plate, 50 mg||1 pc|
Phospholipid removal plate vs. SLE+
What is the advantage of a phospholipid removal plate over the ISOLUTE SLE+ plate?
There are a number of technical considerations, but one of the key things is that the extracts can be injected direct into the LC-MS system, no evaporation-recon required. Elimination of this time consuming step is an important factor for many chemists.
Improved sample prep with ISOLUTE® PLD+
How do ISOLUTE® PLD+ Protein and Phospholipid Removal Plates improve my sample preparation?
ISOLUTE® PLD+ Protein and Phospholipid Removal Plates combine effective protein and phospholipid removal in a single product providing a very effective but extremely simple sample clean-up for LC-MS/MS analysis. Requiring next to no method development, ISOLUTE PLD+ can be integrated quickly and easily into routine workflow,increasing productivity and reducing instrument downtime. ISOLUTE PLD+ plates remove >99 % of plasma proteins and phospholipids, the main causes of ion suppression, leading to cleaner extracts and increased sensitivity (signal-to-noise (S/N)) for a broad range of analytes.
ISOLUTE® PLD+ mechanism of action
How do ISOLUTE® PLD+ plates work?
In ISOLUTE® PLD+ plates, optimized frit components are combined with a novel proprietary sorbent to provide simultaneous protein and phospholipid removal. The procedure is very simple:
An appropriate volume of crash solvent is dispensed into the wells, and the proprietary treated top frit prevents solvent dripping. This allows for a more efficient ‘solvent first’ protein precipitation to occur when plasma samples are added.
An appropriate volume of plasma or other blood based matrix sample is added to the crash solvent. To ensure efficient protein precipitation, sample should be added directly into the crash solvent with force, and plates left for 5 minutes for protein precipitation to complete. A depth filter prevents precipitated proteins from blocking or plugging the wells. Alternatively, ISOLUTE PLD+ plates can be used with vortex mixing or aspirate/dispense protocols.
Low vacuum or positive pressure is applied to the plate, drawing the sample through the proprietary sorbent bed, for collection of purified extract in a pre-positioned collection plate. The optimized bottom frit controls flow rates to ensure sufficient contact time for efficient phospholipid removal by the ISOLUTE PLD+ sorbent.
ISOLUTE® PLD+ sorbent type
What is the ISOLUTE® PLD+ sorbent?
The ISOLUTE® PLD+ sorbent is a novel, proprietary multifunctional phase optimized to selectively retain phospholipids, but allow small molecule analytes (e.g. drugs and metabolites) with a broad range of chemical characteristics to pass through un-retained. Unlike some competitor products, phospholipid retention is not based on a purely hydrophobic retention mechanism.
The combination of efficient protein and phospholipid removal with high analyte recoveries leads to better sensitivity than other commercially available phospholipid removal products.
ISOLUTE® PLD+ analyte range
What analytes can be extracted using ISOLUTE® PLD+ plates?
ISOLUTE PLD+ plates can be used to extract wide range of acidic, basic and neutral analytes, with widely differing polarity/hydrophobicity. In most cases, a single simple generic methodology can be used, virtually eliminating method development time.
ISOLUTE® PLD+ detection limits
What detection limits should I expect when using ISOLUTE PLD+ plates for my sample preperation?
This is highly dependent on the analyte and analytical system employed, but increased sensitivity can be achieved using ISOLUTE® PLD+ plates compared with competitor products.
ISOLUTE® PLD+: Sample or solvent first?
Can I add sample matrix first rather than crash solvent first when using ISOLUTE PLD+ plates?
Yes, but we have found that a better protein precipitation (crash), and hence better sample purification, is achieved using the solvent first approach. The optimized top frit in ISOLUTE PLD+ plates can hold up acetonitrile for up to 24 hours, so there is no danger of the crash solvent dripping through before all samples have been loaded onto the plate.
ISOLUTE® PLD+ crash solvent
What crash solvent should I use with ISOLUTE® PLD+ plates?
We recommend acetonitrile, but methanol can be used as an alternative. Addition of 1 % formic acid to the crash solvent can lead to more efficient protein precipitation, and may increase recovery of certain analytes. For more viscous samples (e.g. whole blood), a crash solvent: sample of 4:1 (v/v) or higher may be required.
ISOLUTE® PLD+ processing conditions
What vacuum or pressure levels should I use to process ISOLUTE® PLD+ plates?
We recommend the use of vacuum (-0.2 bar) or low pressure (3 psi) for processing ISOLUTE PLD+ plates. Using these gentle processing conditions, processing of samples should be complete in 5 minutes or less. Due to the optimized depth filter frit arrangement and narrow sorbent particle size range, ISOLUTE PLD+ are less prone to plugging/blockage than competitor products, so do not require the high pressure/vacuum conditions recommended by other vendors. As a consequence ISOLUTE PLD+ plates are easier to use than some competitor products on automation systems utilizing vacuum processing. For particularly viscous samples, it may be necessary to increase processing pressure/vacuum. In this case, phospholipids will be removed, but removal efficiency may be reduced.
ISOLUTE® PLD+ sample load
How much sample can I load onto ISOLUTE PLD+ plates?
Most commonly, 100 μL of sample is loaded into 300–400 μL of crash solvent. However, 200 μL sample matrix with 600–800 μL of crash solvent can be loaded without loss of performance.
ISOLUTE PLD+: Sample volume recovery
What sample volume should I get back using ISOLUTE® PLD+ plates?
As with all protein precipitation based methods, a loss of sample volume can be expected. This can vary with sample type and the solvent used. Typically, ~ 75% of the original sample plus crash solvent volume is recovered following purification.
Using a partial ISOLUTE® PLD+ plate
What if I don’t have enough samples to utilize an entire ISOLUTE PLD+ 96-well plate?
You can process a partial ISOLUTE® PLD+ plate by sealing the unused wells with a piercable sealing cap (P/N 121-5204) or sealing tape.
ISOLUTE® PLD+ maximum fill volume
What’s the maximum total volume (sample + crash solvent) that I can load into each well of an ISOLUTE® PLD+ plate?
We would not recommend exceeding a total volume of ~1500 μL, to prevent well to well cross contamination on processing. If vortex mixing, this volume should be reduced to a maximum of ~1350 μL.
ISOLUTE® PLD+ sample matrix
Can I purify samples other than plasma using ISOLUTE PLD+ plates?
Yes, ISOLUTE PLD+ plates can be used to remove phospholipids from other sample matrices, including serum, whole blood and tissue homogenates.
Special considerations for whole blood
It is particularly important to ensure an efficient crash to prevent breakthrough of red blood cells/extract coloration.
Consider vortex mixing or aspirate/dispense cycles to improve crash efficiency.
Special considerations for tissue homogenates
Centrifugation of the sample prior to clean up on ISOLUTE PLD+
plates may improve performance.
ISOLUTE® PLD+ maximising analyte recoveries
How do I maximize analyte recoveries when using ISOLUTE PLD+ plates?
ISOLUTE® PLD+ eliminating cloudy extracts
What should I do if I get a cloudy extract when using ISOLUTE PLD+ plates?
A cloudy extract suggests an incomplete protein precipitation has occurred, due to insufficient mixing of the sample and crash solvent. To ensure good mixing, try the following practical tips.
ISOLUTE® PLD+ alternative sample prep
What if my analyte or sample type or volume is incompatible with ISOLUTE® PLD+ purification?
If your analytes or sample size requirements are not amenable to clean up using ISOLUTE PLD+ plates, we recommend evaluation of an alternative sample preparation technique such as supported liquid extraction (using ISOLUTE SLE+ plates or columns) or solid phase extraction (using EVOLUTE® or EVOLUTE EXPRESS SPE products).