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

Considerations when working with solid samples and SPE

<p><strong>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.</strong></p><h4><strong>Plasma/Serum</strong></h4><p>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 % H<sub>3</sub>PO<sub>4</sub> 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.</p><h4><strong>Whole Blood</strong></h4><p>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</p><ul><li><strong>Hemolysis:</strong> 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.<br>Preparation of zinc sulfate/methanol: Into a 100 mL volumetric flask add 20 mL water and 3.6 g ZnSO<sub>4</sub>, 7H<sub>2</sub>O. 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><strong>Osmotic breakdown: </strong>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><strong>Sonication: </strong>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><p><strong>Note: </strong>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</p><h4><strong>Saliva</strong></h4><p>No hydrolysis is required for oral fluids and the generic protocol used for plasma/serum pre-treatment may be followed.</p><h4><strong>Urine</strong></h4><p>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.</p><ol><li><strong>Enzymatic hydrolysis: </strong>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.<br>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><strong>Base hydrolysis:</strong> 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><strong>Acid hydrolysis:</strong> 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><strong>Tissue</strong></h4><p>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.</p>

Sample Pre-treatment Procedures for Bioanalytical Samples

<p><strong>This technical tip explores some issues encountered with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>Solid Phase Extraction</strong></a><strong> and in particular to Analyte Recovery. Recovery relates to the percentage of analyte captured and retrieved through SPE.&nbsp;</strong><br><strong>A couple of other commonly used terms relating to recovery, which may be of interest are:</strong></p><p><strong>Relative Recovery:</strong> 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.</p><p><strong>Absolute Recovery:</strong> 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).</p><h5><strong>Symptom</strong></h5><ul><li>Analyte recovery is less than 100%.</li></ul><h5><strong>Clue</strong></h5><ul><li>Analysis of the unretained fraction (void volume) indicates that the analyte is present in the “flow through”.</li></ul><h5><strong>Problem</strong></h5><ul><li>Analyte binding is not quantitative during the sample loading step.</li></ul><h5><strong>Cause /&nbsp;Solutions / Suggestions</strong></h5><p><strong>A. Column conditioning is improper, incorrect, or not optimized.</strong>:</p><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><p><strong>B. Sample/matrix is in (or contains) a solvent which is “too strong”.</strong>:</p><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><p><strong>C. Column “mass overload” (column is too small and/or the total mass of bound solutes/components is too large).</strong>:</p><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><p><strong>D. Flow rate is too high during the sample loading step.</strong>:</p><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><p><strong>E. Sorbent is “too weak” (has poor or low affinity for the analyte relative to the sample/matrix and/or the dilution solvent).</strong>:</p><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><p><strong>F. Conditioning solvent is “washed away” with large sample volumes.</strong>:</p><ol><li>Add 2% methanol or isopropanol to the sample to prevent alkyl chain collapse during the sample loading step.</li></ol>

Troubleshooting SPE

<p><strong>When performing analyte extraction with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>SPE</strong></a><strong> 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%)?</strong></p><h3><strong>Common problems</strong></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><p><strong>Let's examine each of these common problems.</strong></p><h4><strong>Co-eluting interferences</strong></h4><p>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 <strong>check the eluent using an orthogonal analytical technique </strong>to identify that the increased recovery is a result of a co-elution.&nbsp;</p><p><strong>Where a co-elution is identified, there are a few possible solutions.</strong></p><ol><li><strong>Modify the wash steps of the SPE method </strong>to selectively remove the impurity during washing</li><li><strong>Select an alternative SPE sorbent </strong>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><strong>Adjust your analytical method</strong> to resolve the 2 peaks analytically, negating the necessity to remove this impurity during the SPE step.</li><li><strong>Run a blank plasma sample</strong>; this will allow you to ascertain the impurities present and use this blank in the calculations to subtract from your recovery</li></ol><h4><strong>Contaminants from the solvent or sorbent</strong></h4><p>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 <strong>follow the steps above to remove co-eluting impurities, or alternatively</strong> 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.</p><h4><strong>Internal standard is compromised</strong></h4><p>If your internal standard is not showing 100% recovery, your subsequent calculations will be inaccurate. It is always good practice to <strong>use an internal standard similar in nature to your compound of interest</strong> as this will give the more consistent reference point for your method.&nbsp;</p><p>If there is concern your internal standard is not showing good recovery from the SPE material using your method, you can use an <strong>external standard</strong> which is added to the elution solvent to calculate absolute recovery.</p><h4><strong>Recovery calculations are inaccurate</strong></h4><p>It is important to follow this step wise to ascertain where the errors are appearing.</p><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><p>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"><strong>solid phase extraction</strong></a>. See you next month for another tip!</p>

Why are my recoveries greater than 100%?

<h3><strong>Welcome to our new </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>SPE </strong></a><strong>technical tip!</strong></h3><p><strong>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.</strong></p><h4><strong>Possible Cause/Source &nbsp; / &nbsp; Suggested Remedy</strong></h4><ul><li><strong>Contaminated reagents</strong>: Analyze the purity of reagents and additives or try using these from a different supplier.</li><li><strong>Sample pre-treatment</strong>: Compare chromatograms of pre-treated and non-pre-treated sample.</li><li><strong>Mismatched injection solvent</strong>: 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><strong>Incorrect SPE method</strong>: 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><strong>SPE tube leaching</strong>: 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><strong>Degradation/side reaction</strong>: 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><p><strong>We hope this tip was useful. See you soon for another tip!</strong></p>

SPE Troubleshooting - Extra Peak

<p><strong>Solid Phase Extraction (SPE) is one of the most highly selective sample preparation methods, meaning that the targeted analytes and their matrices must be considered when choosing the correct SPE sorbent and format. Drying down the sorbent before elution is an important step.</strong></p><ul><li>Wash solvents are used to flush out unwanted compounds (i.e., contaminants). If the wash solvent is not completely removed, then residual wash solvent could contaminate the final elution.</li><li>If you do not dry the SPE sufficiently, it could make the final solution less concentrated and result in a reduction in sensitivity.</li><li>Drying helps physically break down the interactions between the sorbent and analyte for a more concentrated extract.</li></ul><p>After the wash solvent has been passed through the cartridge, continue to apply negative pressure (vacuum) or positive pressure. Use this pressure to dry the cartridge for 2 to 5 minutes (or until dry). Turn off the pressure and wipe the tips of the manifold needles to remove any residual solvent.</p><h5><strong>Notes</strong></h5><ol><li><strong>Reproducibility:</strong> Drying removes traces of residual sample/matrix and wash solvent from the sorbent. This produces a more concentrated final extract with a consistent volume and composition and yields better reproducibility and higher recoveries.</li><li><strong>Immiscibility:</strong> Drying is especially critical when using elution solvents that are immiscible with water (e.g. hexane, ethyl acetate, and methylene chloride). Residual water in the sorbent bed may prevent dissolution of the analyte in the elution solvent, resulting in low recoveries and irreproducibility.</li><li><strong>Over-drying: </strong>Avoid over-drying the sorbent, since in some cases, analyte recovery may decrease with excessive vacuum or drying times above 5 minutes. Dry cartridges look distinctly different from moist ones. Make a note of the cartridge appearance before and after conditioning. Also, you could touch the cartridge while it is drying; wet cartridges feel colder than dry cartridges.&nbsp;</li></ol>

Why Is It Necessary to Dry the SPE Sorbent Before Elution?

<p><strong>Low analyte recovery is one of the most common challenges encountered during Solid Phase Extraction (SPE) sample preparation. In this Phenomenex Video Tip, Genevie Hodson, Senior Manager of Technical Support and Applications at Phenomenex, explains a practical troubleshooting approach for situations where analyte recovery after an </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=6&amp;manufacturerItems=50"><strong>SPE </strong></a><strong>procedure is lower than expected.</strong></p><p>The key objective of troubleshooting is not simply to identify that recovery is low, but to determine exactly where the analyte was lost during the extraction process. Once the point of loss has been identified, corrective actions can be implemented systematically.</p><h3><strong>Fundamental Principle of SPE Recovery Troubleshooting</strong></h3><h4><strong>Determine Where the Analyte Is Going</strong></h4><p>The most important step when troubleshooting low recovery is to identify the location of the missing analyte.</p><p>The process begins with the assumption that the sample originally contained 100% of the target analyte. If the final recovery is lower than expected, some portion of the analyte has been lost during SPE processing.</p><p>To identify the loss point, each fraction generated during the SPE procedure must be collected and analyzed separately.</p><h5><strong>Fractions to Collect</strong></h5><p>During the SPE workflow, the following fractions should be collected individually:</p><ul><li><strong>Load fraction (flow-through)</strong></li><li><strong>Wash fraction(s)</strong></li><li><strong>Elution fraction</strong></li></ul><p>Each collected fraction is transferred into a vial and analyzed using the laboratory’s analytical method.</p><p><strong>The analytical results reveal whether the analyte is:</strong></p><ul><li>Present in the load fraction</li><li>Present in one of the wash fractions</li><li>Present in the elution fraction</li><li>Missing from all collected fractions</li></ul><p>The location of the analyte provides direct information about the root cause of the recovery problem and guides the next troubleshooting steps.</p><h4><strong>Scenario 1: Analyte Detected in the Load Fraction</strong></h4><h5><strong>What It Means</strong></h5><ul><li>If the analyte appears in the load fraction, it was not retained by the SPE sorbent during sample loading.</li><li>Instead of binding to the stationary phase, the analyte passed directly through the cartridge.</li></ul><h5><strong>Possible Causes</strong></h5><ol><li><strong>Incorrect Phase Selection</strong></li></ol><ul><li>The selected SPE sorbent may not be appropriate for the analyte being extracted.</li><li>If the analyte cannot interact effectively with the sorbent chemistry, retention will not occur.</li></ul><ol start="2"><li><strong>Excessively Strong Sample Solvent</strong></li></ol><ul><li>The sample may be dissolved in a solvent that is too strong.</li><li>A strong sample solvent can prevent proper interaction between the analyte and the sorbent, causing breakthrough during loading.</li></ul><ol start="3"><li><strong>Incorrect pH Conditions</strong></li></ol><ul><li>Retention often depends on maintaining the analyte in the correct ionization state.</li><li>If the sample pH is not appropriate, the analyte may not interact correctly with the sorbent and therefore will not be retained.</li></ul><ol start="4"><li><strong>Sorbent Overload</strong></li></ol><ul><li>The SPE cartridge may have received:<ul><li>Too much analyte</li><li>Too much sample matrix</li></ul></li><li>When sorbent capacity is exceeded, analytes can break through the cartridge and appear in the load fraction.</li></ul><h5><strong>Corrective Action</strong></h5><p>When overload is suspected, increasing the amount of sorbent can improve analyte retention and prevent breakthrough.</p><h4><strong>Scenario 2: Analyte Detected in the Wash Fraction</strong></h4><h5><strong>What It Means</strong></h5><p>If the analyte appears in the wash fraction, it was initially retained but subsequently removed during the washing step.</p><h5><strong>Possible Causes</strong></h5><ol><li><strong>Wash Solvent Too Strong</strong></li></ol><ul><li>The most common cause is an overly aggressive wash solvent.</li><li>A wash solution with excessive elution strength can remove the analyte before the intended elution step.</li></ul><ol start="2"><li><strong>Incorrect pH During Washing</strong></li></ol><ul><li>When pH control is required for retention, the same conditions must be maintained throughout the process.</li><li>A pH change during washing may alter analyte retention and cause premature elution.</li></ul><h5><strong>Corrective Action</strong></h5><p><strong>Review:</strong></p><ul><li>Wash solvent composition</li><li>Wash solvent strength</li><li>pH conditions during washing</li></ul><p>Adjustments should ensure that the analyte remains retained until the elution step.</p><h4><strong>Scenario 3: Analyte Missing from All Fractions</strong></h4><h5><strong>What It Means</strong></h5><ul><li>If the analyte is not found in the load fraction, wash fractions, or elution fraction, it is likely still retained on the SPE sorbent.</li><li>In this situation, the analyte has not been effectively eluted from the cartridge.</li></ul><h5><strong>Possible Cause</strong></h5><p><strong>Elution Solvent Not Strong Enough</strong></p><ul><li>The final elution solvent may lack sufficient strength to remove the analyte from the sorbent.</li><li>As a result, the analyte remains bound to the stationary phase and never reaches the collection vial.</li></ul><h4><strong>Corrective Action</strong></h4><ul><li>Increase the strength of the elution solvent during the final SPE elution step.</li><li>A stronger solvent can improve analyte desorption and recovery.</li></ul><h3><strong>Summary of Low Recovery Troubleshooting Strategy</strong></h3><p>The recommended troubleshooting workflow consists of four steps:</p><h4><strong>1. Collect All SPE Fractions</strong></h4><p><strong>Gather:</strong></p><ul><li>Load fraction</li><li>Wash fraction(s)</li><li>Elution fraction</li></ul><h4><strong>2. Analyze Each Fraction Separately</strong></h4><p>Use the analytical method to determine where the analyte is located.</p><h4><strong>3. Identify the Root Cause</strong></h4><div><div><h5><strong>Analyte Location&nbsp;&nbsp;/&nbsp;&nbsp;Likely Cause</strong></h5><ul><li><strong>Load fraction:</strong> Incorrect phase, strong sample solvent, incorrect pH, sorbent overload</li><li><strong>Wash fraction: </strong>Wash solvent too strong, incorrect pH</li><li><strong>Not found in any fraction:</strong> Incomplete elution from sorbent</li></ul></div></div><h4><strong>4. Apply Corrective Measures</strong></h4><p><strong>Modify:</strong></p><ul><li>Sorbent selection</li><li>Sample solvent composition</li><li>pH conditions</li><li>Sorbent amount</li><li>Wash solvent strength</li><li>Elution solvent strength</li></ul><h3><strong>Other Common SPE Issues</strong></h3><p>In addition to low recovery, several other SPE-related problems are commonly encountered.</p><p><strong>These include:</strong></p><ul><li>Contamination</li><li>Reproducibility Problems</li><li>Flow-Related Issues</li></ul><h3><strong>Conclusion</strong></h3><p>Successful troubleshooting of low SPE recovery depends on identifying where the analyte is lost during the extraction process. Collecting and analyzing individual SPE fractions provides a systematic method for locating the analyte and determining the underlying cause of poor recovery.</p><p>Whether the analyte passes through during loading, elutes during washing, or remains bound to the sorbent after extraction, fraction-based investigation enables targeted corrective actions. This structured approach allows SPE methods to be optimized efficiently and helps restore acceptable analyte recovery.</p>

SPE Troubleshooting: Understanding and Resolving Low Recovery in Solid Phase Extraction

<p><strong>In this troubleshooting tip, Genevie Hodson, Senior Manager of Technical Support and Applications at Phenomenex, addresses a common solid phase extraction (SPE) question:</strong></p><blockquote><p><strong>"My sample won't flow through my SPE cartridge. Why is this happening?"</strong></p></blockquote><p>Several factors can contribute to flow problems during SPE. A systematic troubleshooting approach can help identify the root cause and restore proper cartridge performance.</p><h3><strong>Identifying When the Flow Problem Occurs</strong></h3><p>The first step in troubleshooting is determining <strong>when</strong> the flow issue appears.</p><p>Ask the following question:</p><ul><li>Does the problem occur during the <strong>sample loading step</strong>?</li><li>Or does it occur <strong>before sample loading</strong>, during cartridge conditioning or when running blanks?</li></ul><p>The timing of the problem can help identify its underlying cause.</p><h3><strong>Cause 1: Viscous or Cloudy Samples</strong></h3><h4><strong>Description</strong></h4><p>Samples that are thick, highly viscous, or contain large amounts of particulate matter can restrict flow through the SPE sorbent.</p><h4><strong>Recommended Actions</strong></h4><p>Before SPE, the sample should be:</p><ul><li>Diluted</li><li>Centrifuged</li><li>Filtered</li></ul><p>Another option is selecting a sorbent with a larger particle size.</p><h4><strong>Example</strong></h4><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=6&amp;manufacturerItems=50&amp;search=x"><strong>Strata-X</strong></a> – 33 µm particle size</li><li><strong>Strata XL</strong> – 100 µm particle size</li></ul><p>The larger particle size of Strata XL is better suited for viscous samples and can improve flow performance.</p><h4><strong>When This Problem Appears</strong></h4><p>If sample viscosity or particulate content is the cause, the flow issue will typically occur during the <strong>sample loading step</strong>.</p><h3><strong>Cause 2: Insufficient Manifold Pressure</strong></h3><h4><strong>Description</strong></h4><ul><li>Flow problems may originate from the SPE manifold or instrumentation.</li><li>The applied pressure must be sufficient to generate proper flow through the cartridge.</li></ul><h4><strong>Recommended Actions</strong></h4><p>Check:</p><ul><li>The SPE manifold</li><li>Associated instrumentation</li><li>Individual manifold positions</li></ul><p>In some cases, a specific position within the manifold may be responsible for the problem.</p><h4><strong>When This Problem Appears</strong></h4><p>If manifold pressure is the issue, poor flow will typically be observed during:</p><ul><li>Conditioning steps</li><li>Blank runs</li></ul><h3><strong>Cause 3: Immiscible Solvents</strong></h3><h4><strong>Description</strong></h4><p>Using solvents that are not mutually miscible can interfere with flow through the SPE cartridge.</p><h4><strong>Recommended Actions</strong></h4><p>Always use solvents that are miscible with each other.</p><h4><strong>Example</strong></h4><ul><li>Hexane</li><li>Water</li></ul><p>These solvents are immiscible and may cause flow problems if used together.</p><h4><strong>When This Problem Appears</strong></h4><p>Flow issues caused by solvent incompatibility are generally observed during:</p><ul><li>Conditioning steps</li><li>Blank runs</li></ul><h3><strong>Cause 4: Precipitation and Protein Build-Up</strong></h3><h4><strong>Description</strong></h4><ul><li>Sample constituents or proteins may precipitate and clog the sorbent bed.</li><li>This blockage can significantly restrict or stop flow through the cartridge.</li></ul><h4><strong>Recommended Actions</strong></h4><p>Possible corrective actions include:</p><ul><li>Diluting the sample</li><li>Reducing sample volume</li><li>Avoiding denaturing solvents</li><li>Washing the sorbent with a low ionic strength buffer before the regular wash step</li></ul><h4><strong>When This Problem Appears</strong></h4><p>If protein precipitation or sample-derived precipitates are responsible, the flow problem will typically occur <strong>after the sample has been loaded</strong>.</p><h3><strong>Systematic Troubleshooting Approach</strong></h3><p>When investigating SPE flow issues, use a systematic elimination strategy.</p><h3><strong>Key Principle</strong></h3><ul><li>Evaluate one variable at a time and isolate potential causes individually.</li><li>This approach helps identify the true root cause rather than changing multiple parameters simultaneously.</li></ul><h3><strong>Summary of Common SPE Flow Problems</strong></h3><h5><strong>Cause &nbsp; / &nbsp; Typical Observation Point &nbsp; / &nbsp; Suggested Solution</strong></h5><ul><li><strong>Viscous or cloudy sample: </strong>During sample loading - Dilute, centrifuge, filter, or use larger particle size sorbent</li><li><strong>Insufficient manifold pressure: </strong>Conditioning or blanks - Check manifold and instrumentation</li><li><strong>Immiscible solvents:</strong> Conditioning or blanks - Use only miscible solvents</li><li><strong>Precipitation or proteins: </strong>After sample loading - Dilute sample, reduce volume, avoid denaturing solvents, use low ionic strength buffer wash</li></ul><h3><strong>Conclusion</strong></h3><p>Flow problems in SPE can arise from several common sources, including viscous samples, insufficient manifold pressure, incompatible solvents, and precipitation of sample components or proteins. Determining exactly when the problem occurs during the SPE workflow is a critical first step in troubleshooting. A systematic, one-variable-at-a-time approach allows the root cause to be identified efficiently and corrective actions to be implemented.</p>

SPE Troubleshooting: Why Won't My Sample Flow Through the SPE Cartridge?

<div><p><strong>Per- and polyfluoroalkyl substances (PFAS) represent a vast class of more than 3,000 synthetic compounds containing perfluorinated chains (with the general formula CnF₂n+₁COOH), commonly utilized in a wide range of consumer products and industrial processes.</strong></p><p>The unique structure of PFAS, characterized by multiple carbon-fluorine (C-F) bonds, contributes to their <strong>exceptional chemical stability and resistance</strong> to degradation through environmental or biological processes. This durability makes them some of the <strong>most persistent pollutants in the environment</strong>. Their resistance to water and oil has led to widespread use in diverse applications, including water- and stain-repellent coatings for textiles, paints, personal care items, firefighting foams, fluoropolymer-based lubricants and additives, as well as grease- and water-resistant food packaging. Due to their extensive use, PFAS have been found in various environmental and biological samples, such as <strong>food, drinking water, wastewater, and human blood</strong>.</p></div><div><div><div><div><h3><strong>Why PFAS Testing Is Crucial?</strong></h3></div></div></div></div><div><p>Like many other chemicals, PFAS can cause<strong> </strong>various <strong>harmful health effects</strong>, depending on factors such as the level, duration, and route of exposure, as well as individual characteristics like age, sex, ethnicity, overall health, and genetic background. PFAS exposure has been linked to various health effects in both sexes, with some impacts supported by high certainty evidence—such as <strong>thyroid disease, liver damage, kidney cancer, and testicular cancer</strong>.</p><p>In females, PFAS may also affect pregnancy outcomes and fetal development, including increased miscarriage risk, reduced birth weight, and delayed mammary gland development. Because of their toxic nature, these compounds have been classified as “persistent organic pollutants” by the<strong> Stockholm Convention</strong> (including PFOS, PFOA, PFHxS, and their salts) and by the <strong>European Commission</strong> (including PFOA, PFOS, PFNA, and PFHxA). Their durability, chemical stability, harmful effects, and tendency to build up in living organisms make them a serious risk to both environmental and human health; hence, PFAS testing is crucial for monitoring and mitigating these threats.</p></div><div><div><div><div><h3><strong>PFAS Testing Techniques</strong></h3></div></div></div></div><div><p>PFAS testing employs advanced techniques to detect, identify, and quantify both known and emerging compounds across diverse environmental matrices.</p><h5><strong>Liquid Chromatography-Mass Spectrometry (LC-MS/MS)</strong></h5><p>LC-MS is a <strong>commonly used PFAS testing method</strong>. Samples are first processed using suitable preparation techniques, such as <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>solid phase extraction </strong></a>(SPE). The resulting extracted samples are then examined using LC/MS/MS with calibration performed via standards. Target analytes are identified with help of isotopically labeled surrogate standards. . Additionally, product ion ratios are compared with those from standards to support identification. Once confirmed, the concentration of each identified target compound is determined based on the response of its main product ion using external standard calibration.</p></div><div><div><div><div><h4><strong>Gas Chromatography-Mass Spectrometry (GC-MS)</strong></h4></div></div></div></div><div><p>Non targeted analysis of PFAS has largely been dominated by LC-MS methods, although GC-MS can access a complementary chemical space, especially for volatile and semivolatile PFAS. Studies show that LC‑MS covers most ionic PFAS; GC‑MS captures neutral/volatile precursors less amenable to LC, highlighting the need for GC-based approaches. GC is particularly valuable for identifying novel PFAS and transformation products from sources like manufacturing waste, incineration, wastewater, and consumer products.</p></div><div><div><div><div><h4><strong>Total Organic Fluorine (TOF)</strong></h4></div></div></div></div><div><p>Total Organic Fluorine (TOF) analysis is an indirect method used to estimate the overall burden of per- and polyfluoroalkyl substances (PFAS) and other organofluorine compounds in a sample. Instead of targeting individual PFAS molecules, TOF methods quantify the sum of organically bound fluorine.</p><p>Because this process measures the organic fluorine present within a sample, it provides a helpful bulk measurement of fluorine from all PFAS present, including those which may not be covered by targeted LC-MS/MS methods.</p></div><div><div><div><div><h4><strong>High Resolution Mass Spectrometry (HRMS)</strong></h4></div></div></div></div><div><p>HRMS is increasingly used to detect emerging PFAS, with two main strategies applied for identification: suspect screening (SS) and non-target screening (NTS). SS is a more direct method, particularly effective when tandem mass spectrometry (MS/MS) reference spectra are available. In contrast, NTS is critical for uncovering new PFAS compounds in the absence of a suspect list, using fragment-based or homologue-based techniques.</p><p>HRMS contributes significantly by offering precise mass measurements, which are essential for identifying unknown substances. Since PFAS often form homologous series, patterns in accurate mass, retention time, and common fragments (such as CF₂, C₂F₄, and CF₂O) help in their recognition.</p></div><div><div><div><div><h3><strong>Choosing the Right Testing Method</strong></h3></div></div></div></div><div><p>C-MS/MS is the primary technique used for targeted PFAS analysis due to its high sensitivity and accuracy. It is validated by the U.S. EPA through established methods such as 1633, 533, and 537.1 for detecting PFAS in various environmental and drinking water samples. For laboratories seeking reliable and compliant workflows, choosing a comprehensive <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/industries/pfas">P<strong>FAS analysis solution</strong></a> that aligns with these regulatory methods is essential for accurate detection and quantification.</p><ul><li><strong>EPA Method 1633</strong> is designed for use under the Clean Water Act (CWA) to measure the levels of 40 different PFAS in various sample types including water, soil, biosolids, sediment, and biological tissue, using <strong>LC-MS/MS</strong>. Weak anion exchange solid phase extraction (WAX-SPE) is employed for extracting various sample matrix types, and PFAS compounds are quantified through calibration with isotopically labeled internal standards.<br>&nbsp;</li><li><strong>EPA Method 533</strong> utilizes weak anion exchange solid phase extraction (WAX-SPE) followed by LC-MS/MS to detect 25 specific PFAS in drinking water, including short chain PFAs. This method employs tandem mass spectrometry and isotope dilution to improve analytical selectivity.<br>&nbsp;</li><li><strong>Method 537.1 </strong>involves extraction via SDBL reversed phase SPE combined with LC-MS/MS for detection of 18 specific PFAs compounds, including the novel GenX compound. This method involves <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/documents/pfas-in-drinking-water">PFAS detection in drinking water</a> samples.</li></ul></div>

Guide to PFAS Testing Methods

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