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).
Octanol-water partition coefficient – LogP
The octanol-water partition coefficient or LogP is a measure of how hydrophilic or hydrophobic a molecule is. It indicates how readily an analyte will partition between an aqueous and organic phase. A more polar, hydrophilic compound will have a lower logP (the value can even be negative), and prefer to “reside” in the aqueous phase. More non-polar, hydrophobic compounds will have a higher logP, and will partition into an organic phase. Typical values range from -3 (polar) to 7 (non-polar). Liquid-liquid extraction methods work by partitioning compounds into combined immiscible organic and aqueous phases. These methods work best when the analytes of interest and the interfering compounds have different polarities and logP values. The logP is also an indication of how well the molecule will partition into the organic elution solvent used in SLE. The higher the logP, the more likely the compound will partition from the SLE phase into the organic elution solvent like dichloromethane, ethyl acetate or MTBE. Compounds with a low or negative logP will not partition into the organic elution solvent as well. SLE works best with more hydrophobic compounds, but more polar compounds can be recovered by modifying the elution solution with 2-10% of a more polar solvent like 2-propanol or acetonitrile. You have to make sure the elution solvent is still immiscible with water. Acid or base pretreatment of the sample can also influence the partitioning and improve elution of more hydrophilic compounds, or increase retention of interfering compounds.
The first thing I do when I need to develop a new sample extraction method is look up the logP and acid dissociation constant (pKa) of the analytes in the assay, and consider possible interfering compounds. Two good places to start are chemicalize.com and the human metabolome database. I base the choice of sample preparation product on this information. If the compounds can be ionized, ion exchange SPE is a good option. This will be discussed in a future blog post. If the compounds are more hydrophobic, SLE is a good choice that only requires loading the sample, waiting 5 minutes for it to absorb onto the SLE column, then eluting the analytes. Another good choice for hydrophobic compounds is reverse phase SPE. Retention of the analytes is based on logP. Aqueous and organic washes can be chosen to remove interferences. As long as you don’t use an organic wash solvent that is strong enough to elute your compounds you can get cleaner samples using SPE than with SLE. If you’re a chromatographer, think about where your compounds elute on an LC column. If they elute late in a reverse phase gradient separation where the organic mobile phase content is high, you can probably use a stronger organic wash and still retain your compounds for subsequent elution. If they elute from the LC column using a mobile phase with low organic content, you may only be able to use a wash with 5% or 10% organic in water (v/v) or no organic wash at all.
The hardest sample prep method development is when you have a large panel of drugs with different properties. Some urine and oral fluid assays I have work on recently have 50 or more drugs. Acidic, basic and neutral compounds are all present in a single assay. SLE works well for many of these assays, and so does reverse-phase SPE if the compounds are more hydrophobic.
The take home message is – KNOW YOUR COMPOUNDS! Look up their logP to understand their hydrophobic, reverse phase retention properties, and formulate your sample prep conditions from this information. In my next post, I’ll discuss the acid-dissociation constant, or pKa, and its role in analyte retention and elution by SPE.