<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50&amp;page=2">Solid phase extraction</a> is routinely selected as a sample preparation technique prior to HPLC and GC analysis. It provides:<ul><li>Clean-up;</li><li>Analyte pre-concentration;</li><li>Solvent switching.</li></ul>This works well for any homogeneous liquid samples, but clearly a solid sample cannot be applied directly to an SPE device. Here we will consider how best to approach the handling of solid samples prior to SPE.The first stage of sample pre-treatment involves homogenising the solid sample with a suitable solvent. The solvent selected for this purpose will:<ul><li>Be effective at solubilising the analyte.</li><li>Be effective at solubilising the sample matrix, although this is not necessary providing the sample is blended well with the solvent.</li><li>Ideally it will be a weak solvent when it comes to the SPE stage. This may not be possible, and it may be necessary to dry down and reconstitute the sample prior to loading on to the SPE cartridge.</li></ul>Having formed a suspension of the sample in a suitable solvent the sample should be vortexed to promote thorough mixing, and then centrifuged to pellet the insoluble components of the matrix. The solvent should then be removed for further treatment.At this point the SPE mechanism needs to be selected:&nbsp;<h3>Reversed Phase SPE Method</h3>Mechanism / Weak Solvent / Strong Solvent<ul><li>Reversed phase – Water/buffer – Acetonitrile/Methanol (hexane strongest)</li><li>Normal phase – Hexane/Heptane – Mid polar organic such as DCM, MTBE, IPA (strongest is water)</li><li>Ion exchange – Aqueous buffer at a pH which promotes ion exchange interactions between analyte and SPE sorbent – Organic solvent, typically methanol with pH adjustment to disrupt the ion exchange interactions.</li></ul>Analyte properties and sample diluent both need to be considered at this point. Reversed phase extractions are effective for moderately polar to moderately hydrophobic analytes, but only if they are dissolved in a weak solvent such as water. If the sample is dissolved in a mid-polar solvent the sample will either need to be diluted, or alternatively dried down and reconstituted. The same is true for normal phase, where samples need to be dissolved in hexane/heptane for analytes to be successfully retained during sample loading onto the SPE device.These examples will hopefully illustrate this concept.<h4>The extraction of PCBs from soil.</h4>PCBs are hydrophobic and will dissolve readily in a non-polar solvent. Soil can be dried, weighed and then extracted using hexane (or heptane). Once thoroughly mixed and centrifuged the resulting solution can be applied directed to a conditioned silica SPE tube allowing for a normal phase extraction to be conducted.<h4>Tylosin from meat.</h4>Tylosin is an antibiotic, with a log P of 1.0. Extracting from meat with a mixture of acetonitrile and water (80:20) will break any hydrophobic interactions releasing the tylosin. After centrifugation the resulting liquid sample will require further dilution with water to reduce the loading solvent strength prior to a reversed phase extraction.<h4>Enilconazole from oranges.</h4>Enilconazole is a fungicide widely used in agriculture and is often used with citrus fruits. It can readily be extracted from oranges using acetonitrile. As enilconazole is basic, the addition of HCl to the acetonitrile extract will promote protonation of the analyte, allowing for it to be directly retained on a strong cation exchange SPE material. This greatly simplifies the extraction process as no dilution of the sample is required (in contrast to reversed phase, or normal phase extraction where 100% acetonitrile would cause the analyte to be unretained during loading).

Considerations when working with solid samples and SPE

Due to their nature, bioanalytical samples often require a pretreatment step prior to further cleanup by solid phase extraction (SPE). Each sample matrix poses its own unique challenges such as the removal of proteins from plasma and serum, the disruption of red blood cells in whole blood, hydrolysis of glucuronidated analytes in urine, and homogenization of tissue samples. This technical note outlines common sample pre-treatment procedures for bioanalytical samples.<h4>Plasma/Serum</h4>Plasma and serum pre-treatments are analyte dependent. If the analyte of interest is an acid, 2 % phosphoric acid can be used (20 µL 85 % H3PO4 to 1 mL of plasma or serum) to disrupt the drug-protein interaction. If the analyte of interest is basic, 0.1 M sodium hydroxide can be used to disrupt the drug-protein interaction. After addition of acid or base, the sample should be vortexed for 20-30 seconds followed by centrifugation. The supernatant is now ready for further analysis.<h4>Whole Blood</h4>There are several pre-treatment strategies that can be followed for whole blood. If the target analyte is present in red blood cells, a hemolysis step is necessary<ul><li>Hemolysis: To 0.2 mL whole blood (spiked with analytes and internal standard) in a 1.2 mL centrifuge tube, add 400 µL of 2 % zinc sulfate/80 % methanol. Vortex for 10-20 seconds followed by centrifugation at 14,000 rpm for 10 minutes. Collect the supernatant for further analysis. Preparation of zinc sulfate/methanol: Into a 100 mL volumetric flask add 20 mL water and 3.6 g ZnSO4, 7H2O. After the solution is clear and the salt crystals have dissolved, add 100 % methanol. Refrigerate the solution at 2-8 °C for 7 days.</li><li>Osmotic breakdown: To 1 mL of whole blood add internal standard and 4 mL of distilled water. Mix/vortex and let stand for 5 minutes. Centrifuge at 670 g for 10 minutes and discard the pellet. Adjust the pH of the supernatant accordingly with the addition of a buffer solution.</li><li>Sonication: Sonicate 1 mL whole blood for 15 minutes at room temperature. Add 3-6 mL of an appropriate pH buffer (such as potassium phosphate buffer). Mix/vortex. Let stand for 5 minutes. Centrifuge at 670 g for 15 minutes. Analyze supernatant.</li></ul>Note: A comparison of the above pre-treatment techniques for whole blood was performed for acidic, basic, and neutral drugs. Recoveries were generally the highest when the whole blood sample was diluted with buffer and subjected to physical denaturing (sonication) rather than chemical means. In fact, the sonication process disrupts the cell membranes to the extent that no clogging was observed when the procedure listed above was followed.1<h4>Saliva</h4>No hydrolysis is required for oral fluids and the generic protocol used for plasma/serum pre-treatment may be followed.<h4>Urine</h4>Enzymatic hydrolysis is necessary in case of conjugated forms (sulfated or glucuronide form) of the analyte present. Enzymatic hydrolysis requires specific pH (pH 4-5) and temperature ranges. An acid or base hydrolysis can be performed as well, depending on the stability of the compound.<ol><li>Enzymatic hydrolysis: To 500 µL sample (spiked with analyte and internal standard) add 100 µL acidic buffer (see below) and 20 µL beta-glucuronidase. Vortex for 5-6 seconds. Incubate in a water bath at 63 °C for 30 minutes. Transfer sample to a 96-well collection plate or autosampler vial. Seal and centrifuge for 10 minutes at 2,000 rpm. Preparation of acidic buffer (1.0 M acetate buffer, pH 4.0): Dissolve 3.0 g of glacial acetic acid and 4.1g of sodium acetate in a 1 L volumetric flask.</li><li>Base hydrolysis: To 1 mL urine (spiked with analyte and internal standard) add 100 µL 10 N KOH. Mix, vortex, and hydrolyze for 20 minutes at 60 ˚C. Cool and adjust pH to 3.5- 4.0 (by adding 200 µL glacial acetic acid).</li><li>Acid hydrolysis: To 1 mL urine add 0.25 mL HCl in a screw capped test tube. Screw the tube top on loosely and heat in a boiling water bath for 60 minutes. Adjust to pH 7 (or as needed) with 1.0 N NaOH.</li></ol><h4>Tissue</h4>Homogenize with organic or aqueous solvent depending upon analyte solubility. Settle, decant, centrifuge or filter supernatant. Perform direct Matrix Solid Phase Dispersion (MSPD) extraction on tissue.

Sample Pre-treatment Procedures for Bioanalytical Samples

This technical tip explores some issues encountered with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">Solid Phase Extraction</a> and in particular to Analyte Recovery. Recovery relates to the percentage of analyte captured and retrieved through SPE.&nbsp; A couple of other commonly used terms relating to recovery, which may be of interest are:Relative Recovery: Indirect determination of analyte capture and retrieval during the extraction process based on the recovery of an internal standard (which is a compound with a structure similar to the target analyte) that is added to the crude sample/matrix prior to the actual extraction procedure.Absolute Recovery: Quantitative method for the determination of the actual amount of analyte captured and retrieved during the extraction process. Analyte recovery is based on an external standard that is added to the eluant at the end of the extraction procedure (rather than being based on an internal standard that is co-extracted with the analyte).<h5>Symptom</h5><ul><li>Analyte recovery is less than 100%.</li></ul><h5>Clue</h5><ul><li>Analysis of the unretained fraction (void volume) indicates that the analyte is present in the “flow through”.</li></ul><h5>Problem</h5><ul><li>Analyte binding is not quantitative during the sample loading step.</li></ul><h5>Cause /&nbsp;Solutions / Suggestions</h5>A. Column conditioning is improper, incorrect, or not optimized.:<ol><li>Condition column with methanol or isopropanol.</li><li>Use sufficient methanol or isopropanol to wet the entire sorbent bed; allow more than two column volumes to percolate slowly under low vacuum.</li><li>Follow the methanol or isopropanol directly with one column volume of a solution with a composition similar to the actual sample (pH adjusted) but without the analyte(s).</li><li>Do not use too much of this second “conditioning solvent” in (3) above, or allow it to remain in the column for too long.</li><li>Do not over-dry the column during or after conditioning (use low vacuum for ~1 minute).</li></ol>B. Sample/matrix is in (or contains) a solvent which is “too strong”.:<ol><li>Dilute the sample in a “weaker” solvent/solution to promote binding (less polar for Normal Phase, more polar for Reversed Phase, less salt, buffered and/or pH adjusted for Ion-Exchange.</li><li>Increase the sample dilution with a “weak” solvent.</li><li>Load less sample.</li><li>Increase the sorbent mass.</li><li>Decrease the flow rate during sample loading.</li><li>Use a “stronger” sorbent with a higher affinity for the analyte.</li><li>Add an organic modifier (or adjust the pH) to enhance binding affinity for the analyte(s).</li><li>Adjust the sample pH so that the analyte is neutral for Reversed Phase or charged for Ion-Exchange.</li><li>Add salt (5 to 10% NaCl) to increase the solvent polarity and to enhance the retention of highly polar analytes in Reversed Phase.</li><li>Add an ion-pair reagent to enhance binding of charged analytes in Reversed Phase.</li></ol>C. Column “mass overload” (column is too small and/or the total mass of bound solutes/components is too large).:<ol><li>Decrease the volume of sample loaded.</li><li>Increase the sorbent mass.</li><li>Use a sorbent with higher surface area.</li><li>Use a “stronger” sorbent.</li><li>Decrease the flow rate during loading (to enhance “diffusion”).</li><li>Decrease the column inner diameter but use the same sorbent mass (to increase the pressure drop, reduce flow, and enhance “diffusion”).</li><li>Dilute the sample in a “weaker” solvent to improve capacity.</li></ol>D. Flow rate is too high during the sample loading step.:<ol><li>Decrease the flow rate during loading.</li><li>Increase the sorbent mass.</li><li>Decrease the column inner diameter to reduce flow.</li><li>Use a sorbent with a higher surface area.</li></ol>E. Sorbent is “too weak” (has poor or low affinity for the analyte relative to the sample/matrix and/or the dilution solvent).:<ol><li>Switch to a “stronger” sorbent, to one with a higher ligand density, from a non-endcapped to an endcapped sorbent (or vice versa).</li><li>See “Solutions/Suggestions” for Cause C (“Column mass overload..”), above.</li><li>See Solutions/Suggestions for Cause B (“Sample/matrix is in (or contains) a solvent which is “too strong””), above.</li></ol>F. Conditioning solvent is “washed away” with large sample volumes.:<ol><li>Add 2% methanol or isopropanol to the sample to prevent alkyl chain collapse during the sample loading step.</li></ol>

Troubleshooting SPE

When performing analyte extraction with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">SPE</a> we expect 100% recovery. If recovery is lower, this would suggest there is an issue with retrieval of the analyte from the sorbent. But what are some of the reasons you may see a recovery greater than what you were expecting (or over 100%)?<h3>Common problems</h3><ul><li>Co-eluting interferences</li><li>Contaminants from the solvent or sorbents selected</li><li>Internal standard is compromised making your reference point inaccurate</li><li>Recovery calculations are inaccurate</li></ul>Let's examine each of these common problems.<h4>Co-eluting interferences</h4>These are typically present in the matrix and the goal of the SPE step is to remove them. If you are experiencing recoveries greater than 100%, the first step would be to check the eluent using an orthogonal analytical technique to identify that the increased recovery is a result of a co-elution.&nbsp;Where a co-elution is identified, there are a few possible solutions.<ol><li>Modify the wash steps of the SPE method to selectively remove the impurity during washing</li><li>Select an alternative SPE sorbent which will better remove these impurities. This is easier if the analyte / impurity can be selectively ionized, as an ion-exchange sorbent will very often afford cleaner extracts than either reversed or normal phase solutions</li><li>Adjust your analytical method to resolve the 2 peaks analytically, negating the necessity to remove this impurity during the SPE step.</li><li>Run a blank plasma sample; this will allow you to ascertain the impurities present and use this blank in the calculations to subtract from your recovery</li></ol><h4>Contaminants from the solvent or sorbent</h4>These will typically be impurities present in the starting materials which are similar in nature to your compound of interest, and as a result co-elute with it during SPE and subsequent analytical analysis. You can follow the steps above to remove co-eluting impurities, or alternatively as this is not an impurity present in your matrix, you can identify the level present in the sorbent using blank runs and subtract this from your main peak during the calculation. Prior to addition of your sample if you equilibrate your sorbent with the elution solvent this should flush out potential contaminants from the sorbent. For impurities introduced from the solvent, typically switching the solvent should resolve this.<h4>Internal standard is compromised</h4>If your internal standard is not showing 100% recovery, your subsequent calculations will be inaccurate. It is always good practice to use an internal standard similar in nature to your compound of interest as this will give the more consistent reference point for your method.&nbsp;If there is concern your internal standard is not showing good recovery from the SPE material using your method, you can use an external standard which is added to the elution solvent to calculate absolute recovery.<h4>Recovery calculations are inaccurate</h4>It is important to follow this step wise to ascertain where the errors are appearing.<ol><li>Calculation Errors - Check all calculations and account for all dilution factors</li><li>Integration Errors - Reintegrate all peaks and use manual calculations to confirm</li><li>Analytical method errors – Ensure the analytical method used allows correct integration and calculation. Ensure no strong solvents are used for injection and run a calibration across a number of dilutions to ensure linearity. Troubleshoot your analytical method to determine any factors which may lead to poor reproducibility</li><li>Ensure detections selected are appropriate and working correctly.</li></ol>We hope this tip was useful! Check out our resources on <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/solid-phase-extraction">solid phase extraction</a>. See you next month for another tip!

Why are my recoveries greater than 100%?

<h3>Welcome to our new <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">SPE </a>technical tip!</h3>What is the cause when you encounter interferences post-extraction regardless of the analytical or detection method? Possible sources of the mystery peak(s) are outlined below.<h4>Possible Cause/Source &nbsp; / &nbsp; Suggested Remedy</h4><ul><li>Contaminated reagents: Analyze the purity of reagents and additives or try using these from a different supplier.</li><li>Sample pre-treatment: Compare chromatograms of pre-treated and non-pre-treated sample.</li><li>Mismatched injection solvent: Analytes that are dissolved in a strong injection solvent may retain poorly and thus appear as extra peaks. Using the initial mobile phase as the injection solvent is recommended.</li><li>Incorrect SPE method: The SPE sorbent might not have been properly conditioned and equilibrated. Typically, the strongest solvent used in the method should be used to condition the sorbent. Analyze the conditioning, loading and washing fractions to check if the same interferences appear.</li><li>SPE tube leaching: Before conditioning, clean the tube with elution solvent. If reagents or the nature of the sample are too harsh for the polypropylene SPE tube, a glass tube or PTFE tube could be used instead.</li><li>Degradation/side reaction: Check the reactivity and stability of all components in your sample. Degradation can happen if compounds are sensitive to factors such as heat, light, acids, bases, etc. Sample components may be reacting with each other or with reagents/additives.</li></ul>We hope this tip was useful. See you soon for another tip!

SPE Troubleshooting - Extra Peak

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SPE Troubleshooting - Extra Peak

Why are my recoveries greater than 100%?

Troubleshooting SPE

Sample Pre-treatment Procedures for Bioanalytical Samples

Considerations when working with solid samples and SPE

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