Normal phase SPE uses polar interactions as retention mechanism. These interactions include hydrogen bonding, dipole-dipole, induced dipole, π-π, etc. The interactions are between polar functional groups of analytes and polar sorbent chemistries.Reversed phase uses nonpolar, hydrophobic interactions as retention mechanism. These interactions include Van-der-Waals, London dispersion, π-π, etc. These interactions are between carbon rich, hydrophobic parts of both analytes and sorbent.When dealing with sample matrices that are only soluble in organic solvents, one would use a normal phase method. Examples of such matrices are oil, grease, or butter.Ideally, the sample is diluted with a nonpolar solvent (i.e. hexane) prior to sample loading. The sample can then be washed with hexane or 5% methylene chloride in hexane (analogous to washing with 5% MeOH in water in RP SPE methods to remove matrix interferences). Elution will be done with a polar solvent that can interrupt polar interactions between analytes and sorbent.Fractionation can be done by applying solvents in the order of increasing polarity or mixtures of solvents with different polarities.Below is a quick reference of solvents in ascending polarity, for Normal Phase SPE methods.<h3>Solvents in Polarity Order</h3><h5>From low to high&nbsp;</h5><ul><li>Hexane</li><li>Isooctane</li><li>Carbon tetrachloride</li><li>Toluene</li><li>Benzene</li><li>t-butyl methyl ether</li><li>Chloroform</li><li>Dichloromethane</li><li>Diethyl ether</li><li>Ethyl acetate</li><li>Tetrahydrofuran (THF)</li><li>Acetone</li><li>Acetonitrile</li><li>40% MeOH in acetonitrile</li><li>20% MeOH in diethyl ether</li><li>20% MeOH in dichloromethane</li><li>Isopropanol</li><li>Methanol</li><li>Water</li><li>Acetic acid</li></ul>

How is normal phase (NP) SPE different from reversed phase (RP) SPE? How do I decide which one to use?

When considering the mass of sorbent required to perform a solid phase extraction it is critical to consider the sample matrix (serum, plasma, urine, saliva, food, river water, soil etc). This information will allow you, with the use of a suitable look up table to determine the mass of SPE sorbent to use. The tables are produced to reflect the relative levels of interfering endogenous material present in the samples.&nbsp;The SPE device must have enough capacity to retain all analytes of interest together with any endogenous materials capable of retaining on the sorbent with the same type of interactions as the analyte. If there is insufficient sorbent, it is possible some analyte may pass unretained through the SPE device during sample loading, leading to lower recoveries and a loss of consistency of recovery between samples.<h3>Sample Volume</h3><h4>Determines Bed Mass/ Solvent Volumes</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_How_much_sample_can_I_load_onto_my_SPE_device_Is_it_linked_to_the_concentration_of_my_analyte_of_interest_12a9ab171c.jpg" alt="Phenomenex: How much sample can I load onto my SPE device? Is it linked to the concentration of my analyte of interest?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_How_much_sample_can_I_load_onto_my_SPE_device_Is_it_linked_to_the_concentration_of_my_analyte_of_interest_12a9ab171c.jpg 154w," sizes="100vw" width="983" height="992"><h3>Generic Loading Capacities:</h3><h4>Polymeric: 10 - 15% of Total Bed Mass | Silica: 5% of Total Bed Mass</h4>If we compare plasma and urine, we can load 500 µl of plasma onto a 60 mg polymeric sorbent such as <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?manufacturerItems=50&amp;search=Strata-X">Strata™-X</a>. Using the same 60 mg SPE tube 2 mL of urine could be loaded, indicating that urine contains significantly less endogenous material than plasma.For sample types not included in the table, the use of generic loading capacity, 5% of sorbent mass for silica materials, or 10-15% for polymeric materials are advised. The difference between silica and polymeric sorbent loading capacity is a function of available surface area, which is higher for polymeric materials and also ligand density – the entire surface of a polymeric material is essentially stationary phase, while on a silica material only the phase bonded to the silica takes part in the extraction mechanism for reversed phase or ion exchange extractions.

How much sample can I load onto my SPE device? Is it linked to the concentration of my analyte of interest?

<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

For most reversed-phase SPE applications using a silica-based SPE media (e.g. those using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1507">Strata™ C18-E</a>), cartridges (or wells in the case of 96-well plates) are initially conditioned using an organic solvent such as methanol, followed by rinsing with water or a buffer. If silica-based SPE media is allowed to dry out for extended periods of time during these conditioning steps, they may exhibit poor retention for target analytes, especially if the target compounds are polar in nature. It is therefore essential that SPE users pay close attention to their methods during this crucial conditioning step in order to obtain accurate and reproducible results.&nbsp;The figure below shows two <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1546">Strata C18-T </a>cartridges (1 gm/6 mL) that were loaded with 4 mL of a blue dye (CuCl2). The cartridge on the left (A) was first conditioned using 12 mL of methanol, then was dried for ten minutes under high vacuum (10” of Hg), followed by 12 mL of water. As you can see from the figure, the blue dye is found uniformly distributed throughout the sorbent bed. In contrast, the cartridge on the right hand side (B) was conditioned with methanol, followed immediately by water without any drying. Under these conditions, the blue dye is visible as a tight, focused band at the head of the sorbent bed. After elution, recovery from cartridge B was far greater than was the recovery from cartridge A (the dried cartridge). This difference in recovery can be attributed primarily to the poor retention of the dye compound due to drying of the sorbent bed.&nbsp;<h3>Tips to Prevent Accidental over-drying to prevent loss of Analyte</h3><ul><li>Don’t allow your bed to dry out at any step (unless it is specifically indicated in the method).</li><li>Use gravity flow as an alternative to vacuum to prevent over-drying.</li><li>Centrifuge at low velocities (e.g. 500 RPM) or use positive pressure.</li><li>Switch to an equivalent polymeric sorbent, such as Strata-X, which do not display shifts in retention and recovery due to periodic drying</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Over_Drying_of_Silica_Based_SPE_Cartridges_Can_Lead_to_Poor_Sample_Recovery_0839e0ef05.jpg" alt="Phenomenex: Over-Drying of Silica-Based SPE Cartridges Can Lead to Poor Sample Recovery: A. Silica C18 sorbent dried after conditioning with methanol; B. Silica C18 sorbent conditioned without drying." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Over_Drying_of_Silica_Based_SPE_Cartridges_Can_Lead_to_Poor_Sample_Recovery_0839e0ef05.jpg 245w," sizes="100vw" width="548" height="312">

Over-Drying of Silica-Based SPE Cartridges Can Lead to Poor Sample Recovery

When analysis of substances from serum or plasma takes place, it is likely that the samples will contain phospholipids, a major component of mammalian cell membranes. These phospholipids have a variety of structures and polarities, the more hydrophobic of those would gradually coat a reversed-phase HPLC column (such as a C18) over multiple injections. This process will result in a gradual increase in back pressure which is typically accompanied by a drop in separation efficiency. Selectivity changes may also occur.If UV detection is being utilized, this may be as far as the problem goes. If samples have simply been prepared by protein precipitation, which is quick and cheap to perform, shorter column lifetime may simply be accepted as an inevitable result of performing low-grade sample pretreatment. If MS detection is being used, ion suppression or ion enhancement may be seen. This is generally accepted to be caused by the co-elution of phospholipids with the compounds of interest. The presence of the phospholipids in the MS source may impact the ionization efficiency of the source at that point.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Why_Remove_Phospholipids_From_a_Sample_2_20c29e8927.jpg" alt="Phenomenex: Why Remove Phospholipids From a Sample? Figure 1: Assessment of column sensitivity following repeated injections of diclofenac in protein precipitated plasma vs using a sample preparation product" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Why_Remove_Phospholipids_From_a_Sample_2_20c29e8927.jpg 245w," sizes="100vw" width="682" height="408">As a result, the reliability of data produced may be affected by the presence of phospholipids because the amount of analyte could be underestimated (due to ion suppression) or overestimated (following ion enhancement). It is possible to check if this an issue in your separation by conducting the following experiment:<ul><li>Use a syringe pump to directly infuse a solution of your analyte of interest.</li><li>At the same time, prepare a blank serum/plasma sample using your usual sample preparation procedure. Inject this blank sample and run your usual HPLC method.</li><li>Monitor the ion current for your MS-MS transitions for the analyte of interest.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Why_Remove_Phospholipids_From_a_Sample_b2c759fc60.jpg" alt="Phenomenex: Why Remove Phospholipids From a Sample? Figure 2: Areas of ion suppression using protein precipitated plasm vs plasma extracted using a sample preparation product" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Why_Remove_Phospholipids_From_a_Sample_b2c759fc60.jpg 245w," sizes="100vw" width="694" height="332">Across the resulting chromatogram, areas of ion suppression or ion enhancement could be seen. If your analyte of interest elutes in one of these zones there is a possibility you may have problems with data integrity.The simplest solution to this issue is to use a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2182">sample preparation product</a> which allows for the simultaneous precipitation of plasma proteins whilst removing phospholipids from the resulting supernatant. Such products consist of tubes or 96-well plates that have membranes to remove precipitated proteins, and a sorbent to retain phospholipids. Samples are mixed with a precipitating solution, such as methanol or acetonitrile, before being pulled down through the filter plate and sorbent bed. The resulting samples are then free of both protein and phospholipids enabling reliable quantitation and improved column lifetime.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Why_Remove_Phospholipids_From_a_Sample_3_c2050c4e2e.jpg" alt="Phenomenex: Why Remove Phospholipids From a Sample? Figure 3: Phospholipid removal using Phree" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Why_Remove_Phospholipids_From_a_Sample_3_c2050c4e2e.jpg 245w," sizes="100vw" width="511" height="318">

Why Remove Phospholipids From a Sample?

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Why Remove Phospholipids From a Sample?

Over-Drying of Silica-Based SPE Cartridges Can Lead to Poor Sample Recovery

Sample Pre-treatment Procedures for Bioanalytical Samples

Considerations when working with solid samples and SPE

How much sample can I load onto my SPE device? Is it linked to the concentration of my analyte of interest?

How is normal phase (NP) SPE different from reversed phase (RP) SPE? How do I decide which one to use?

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