Methods for a drug class or one large panel – what’s the difference

Most clinical and forensic labs have a used a traditional approach for drug testing called screen with reflex to confirmation. This involves analyzing specimens (urine, blood, other body fluids, and solids like tissue and meconium) using an immunoassay technique that identifies a drug class. Positive immunoassay results are then analyzed by second, more specific method like GC-MS or LC-MS/MS to identify and quantitate specific drug analytes.  A quantitative result is reported.

Changes in reimbursement, a desire for increased throughput and more cost effective assays with a shorter turn-around-time have led many labs to skip the immunoassay screen and develop a single mass spectrometry method that identifies and quantitates a large panel of drugs and metabolites.  This can lead to lower cost and improved efficiency but it does come at a cost from a sample prep perspective. Continue reading Methods for a drug class or one large panel – what’s the difference

When should I choose SPE instead of SLE?

Supported liquid extraction is a simple, yet effective sample preparation technique. It works like liquid-liquid extraction (LLE) but is supported on a solid surface. Aqueous samples are dispersed as small droplets on a high surface area material. The entire sample remains on the SLE column. Samples are eluted with a water-immiscible solvent, like ethyl acetate, hexane, or dichloromethane. The compounds of interest partition into the organic phase, like an LLE, while salts, phospholipids and other impurities remain on the column. The simple load, wait, elute protocol is fast and easy and provides excellent sample cleanup that can be automated.

Solid phase extraction is more involved. Samples are loaded onto a SPE column, and the sample flows through and is discarded. Analytes of interest (and some undesirable compounds) are retained by hydrophobic interaction or ion exchange. The column is washed with aqueous and organic wash solvents to remove interfering compounds. Then, the analytes of interest are eluted based on their retention mechanism. Organic solvent is used for hydrophobic compounds, acid or base for ionized compounds.

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Why should I choose a protein precipitation 96-well plate for my method instead of doing a traditional protein crash?

Most clinical chemists have developed a blood, serum or plasma assay using a protein crash because it is inexpensive and generally removes proteins that interfere with detection or the analysis in some way.   This is usually done by adding a “crash” solvent like methanol or acetonitrile to the blood sample, vortex mixing, then centrifuging and removing the supernatant to remove the crashed proteins from the sample.  Sometimes the crash solvent is acidified to aid solubility and begin the denaturation process.  The crash solvent is usually added at a ratio of 3:1, but can sometimes require up to 10:1 solvent:specimen.  For more information on the process of doing a protein crash I recommend this article. Continue reading Why should I choose a protein precipitation 96-well plate for my method instead of doing a traditional protein crash?

When should I choose a mixed-mode SPE?

 

Mixed mode SPE phases have become very popular for sample clean-up prior to analysis using mass spectrometry.  Having the capability to retain compounds by two modes of interaction during solid phase extraction is useful when a large number of analytes with different properties are of interest.  Most mixed mode phases are bonded silica or polymeric reversed phase materials with an ion-exchange group bonded to it.  Continue reading When should I choose a mixed-mode SPE?

What’s the Best Way to do SLE?

SLE (supported liquid extraction) is a sample prep technique that has been in use for over ten years now, but many analytical chemists don’t know about, or understand the best way to do an SLE extraction.  In this post, I’m going to talk about how SLE works and the proper way to do an SLE extraction for sample clean up.

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Why is pH adjustment important for sample prep methods?

In my last blog post we talked about LogP and its role in sample prep.  Today we are going to discuss the acid dissociation constant, pKa, and how it affects method development.  Knowing and understanding the pKa of your compounds tells you if the compound can be ionized, and under what conditions, so you can use this property to develop better sample prep methods.

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What is the role of LogP in sample prep methods?

The goal of sample preparation is to create cleaner samples, collecting the compounds of interest and eliminating interferences – that “junk” we don’t care about the can cause ion suppression and matrix effects that affect sensitivity, accuracy and precision.  The ideal sample prep method removes all interfering compounds and produces 100% recovery of all analytes of interest.  The problem is that many interfering compounds have properties that are similar to the compounds we need to detect and quantitate.  It takes some skill and knowledge to develop a method that washes interfering compounds away and elutes the analytes of interest in a separate step.  You need to make sure your washes don’t elute the compounds you care about, and you don’t want interfering junk eluting with your analytes. 

Understanding the chemical properties of the compounds in your sample matrix, both the ones you want to detect and the ones you want to eliminate, is necessary for successful method development.  Is the molecule acidic or basic?  What functional groups are present?  Can they be ionized?  Is the molecule hydrophilic or hydrophobic? Polar or non-polar?  In this post, I am going to discuss the octanol-water partition coefficient, or LogP and its role in sample preparation using supported liquid extraction (SLE) and solid phase extraction (SPE).

Continue reading What is the role of LogP in sample prep methods?