Waters iKey - Manual (Page 5)

5
[ CARE AND USE MANUAL ]
iKey
IV. iK EY USAGE
To ensure the continued high performance of your iKey Separation Device, please follow these guidelines:
a. Sample Preparation
1) Samples containing hundreds of nanograms of undigested protein (and higher) per injection have been shown to limit or shorten
iKey lifetimes.
Therefore, it is essential that proper sample preparation protocols are followed. For example, below are examples of recommended
protocols for preparing plasma:
Shaded green = Readily adaptable to ionKey/MS Shaded purple = Requires more careful consideration
Sample Preparation
Protein precipitation
>2:1 ACN:H
2
O
This method effectively removes the majority of protein from solution, pay attention to final organic content.
Liquid/liquid extraction
This method effectively extracts the analyte leaving the protein behind, pay attention to final
organic content.
Digestion Proteins are broken into smaller pieces that no longer present an issue.
Affinity
Small amounts of specific proteins are extracted from the sample for analysis This can be an effective way
of preparing samples by removing residual protein.
Solid-phase extraction
This is an issue when the analyte has similar properties to proteins. E.g. The methods used to isolate
larger peptides often leave significant protein content in the final extract. If solid-phase extraction is the
sole sample preparation technique, multidimensional chromatography (i.e., trap and back-flush elution)
is required.
Filter Filters will not remove protein that is in solution.
Centrifugation Centrifugation will not remove protein that’s in solution, unless it’s been crashed with PPT.
Dilution It depends on the dilution ratio, and the diluent. In general small aqueous dilutions will not remove protein.
Sample Type
Urine
Typically low protein content and does not require organic additive to crash proteins. Dilution and
centrifugation is recommended.
Bile
Typically low protein content and does not require organic additive to crash proteins. Dilution and
centrifugation is recommended.
Microsomes
Microsomal incubations contain proteins. Pay close attention to sample preparation to ensure that
minimal protein remains in the final sample extract.
Plasma
Plasma typically contains high concentrations of protein. Pay close attention to sample preparation
to ensure that minimal protein remains in the final sample extract. Injecting a large amount of undigested
proteins onto the iKey will result in compromised performance and shorter lifetimes.
Organic Extracts High amounts of organic affect the performance of any column, this effect is greater with small dimensions.
2) The use of multidimensional chromatography, specifically a trap and back-flush elution strategy, can provide further sample cleanup
and facilitate injections of higher organic strength injection solvents without experiencing analyte breakthrough. This multidimensional
chromatographic strategy is required when solid-phase extraction is the sole sample preparation technique (i.e., no additional
sample preparation technique is used to remove large amounts of undigested protein).
3) It is preferable to prepare the sample in the initial mobile phase conditions or a weaker solvent for the best peak shape and sensitivity.
4) If the sample is not prepared in the mobile phase, ensure that the sample, solvent, and mobile phases are miscible in order to avoid
sample and/or buffer precipitation.