The Sample Preparation Blog

Solid phase extraction methods usually require a large amount of elution solvent to pull everything off of the sorbent bed. However, evaporating and reconstituting those samples can take a lot of time. So is it possible to do an elution that doesn’t require evaporation and reconstitution? We were able to develop a method that resulted in recovery of all compounds, all without evaporating our samples.

We started by using the method that we developed when determining our optimal sample size for a 10 mg sorbent bed. We analyzed our 100 compound drugs of abuse panel in urine and used a 50 uL sample volume. We decided to try different volumes of 2 separate elution solvents: 98:2 methanol/ammonium hydroxide and 2.5% formic acid in water. We wanted to make sure to elute all of our compounds of interest without putting a too basic or too aqueous solution onto our mass spec. We tried the following combinations:

	98:2 MeOH/NH4OH	2.5% formic acid (aq)
Combination 1	1 x 150 µL	1 x 100 µL
Combination 2	2 x 75 µL	2 x 75 µL
Combination 3	2 x 50 µL	2 x 50 µL
Combination 4	1 x 100 µL	1 x 100 µL

Of all of the elution combinations, Combination 1 resulted in the highest recoveries for most of our analytes of interest. The below chart compares our microelution protocol with a “normal SPE protocol” that included evaporation and reconstitution.

There were a few compound classes that had decreased recoveries, including tricyclic antidepressants (nortriptyline), selective serotonin reuptake inhibitors (duloxetine), and some opioids (EDDP). While the recoveries were decreased for these compounds, we did still achieve recovery of all analytes in our panel. We did not see an increase in matrix effects when using our microelution protocol, which means our samples were fairly clean!

While a 10 mg sorbent bed mass isn’t usually used with a microelution with no evaporation, it is possible to do it! Depending on the compounds being analyzed, recoveries can be as good as recoveries seen when using a “normal” SPE protocol.

Have you ever encountered problems loading your samples onto an SPE or SLE plate?  Aside from sample viscosity or cartridge blockages, a good troubleshooting step to start with is ensuring that you are achieving complete frit coverage on the load.  Recently, the Biotage Analytical Applications Team was tasked with generating a workflow for some basic compounds in both urine and blood matrices.  We decided to try a variety of different techniques to develop a comparative workflow to accommodate routine, clinical, and forensic applications.  As the target compounds do not require hydrolysis or unbinding from proteins, we ultimately decided on a very simple 1:1 dilution with a buffer for sample pre-treatment, and we applied this to each extraction media type we investigated.  This resulted in a total loading volume of 200 µL.  Among the techniques tested was, you guessed it, ISOLUTE SLE+, where we encountered some interesting challenges!

Continue reading There and Square Again: A Frit Coverage Tale

We’ve all been there. We prepare a batch of samples to analyze, prime our systems, warm up the detector, and within 2-3 injections we receive an error message stating that an integral component has failed for one reason or another.  While basic troubleshooting can resolve many issues commonly encountered with mass analyzers (specifically, LC-MS/MS systems), more serious problems can require a visit from your local service engineer, ultimately resulting in a period of instrument downtime.  This can be a dreadful experience, especially for a routine production laboratory.  However, as the old adage goes, “an ounce of prevention is worth a pound of cure,” the same can be said for LC-MS/MS operation.

Continue reading Why Adequate Sample Preparation Is Not The ONLY Thing to Consider for Successful, Robust Analysis

In this blog post, we will discuss how to determine the optimal sample volume for a sorbent bed mass.  We have developed an adequate solid phase extraction method for our laboratory. However, we’re starting to notice some oddities when we extract and analyze our samples. Could we be losing compounds from our sample?

Continue reading Determining the Optimal Sample Size for your Sorbent Bed Mass

This is a question that I am often asked by very smart people that just haven’t had time to learn the process. An optimized sample preparation method is critical for an accurate, specific, robust clinical LC-MS/MS assay.  Once the LC and MS/MS conditions for the compounds of interest are optimized, and method parameters like desired LOQ/LOD, analytical measurement range and specimen volume used for analysis have been established, sample prep method development can begin.  A good sample prep method can improve accuracy and precision, provide longer LC column lifetime, and keep your LC-MS/MS system clean.

Continue reading How do I Develop a Sample Prep Method?

You’ve just decided to develop a solid phase extraction (SPE) method for a new assay in your laboratory. However, there are so many decisions to be made! Which company do I choose? Which product do I use? Polymer based sorbent or silica based? Cation or anion exchange? While this seems incredibly difficult, it doesn’t have to be that hard! Today, we’re going to walk through this process of figuring out how to develop a starting point for our SPE method.

Continue reading How do I come up with a “starting point” for my SPE method?

LC/MS/MS seems to be the new gold standard in sample analysis nowadays. However, some labs (like forensic labs) are a little slower to get LC/MS/MS instruments and are still analyzing samples using GC/MS. The majority of published articles in peer-reviewed journals are for LC/MS/MS so those working in laboratories with GC/MS instrumentation may not know how to change the sample preparation to better fit GC/MS.

First and foremost, GC/MS methods tend to need a higher sample volume than LC/MS/MS methods. GC/MS is a less sensitive instrument, which means more sample is needed to achieve lower limits of quantitation (LOQs). Many LC/MS/MS methods only require 50-200 uL of sample. GC/MS methods usually require 1-3 mL of sample.

Because of the higher sample volume needed for GC/MS analysis, higher mass sorbent beds in the extraction cartridge are usually needed as well. The larger sorbent bed allows for a higher amount of the compounds of interest to bind to the sorbent bed. Because of the larger sorbent bed, higher volumes of wash solvent and elution solvent are needed as well.

GC/MS also can’t be used for super volatile compounds because of the temperature of the injection port. For many methods, the injection port can be anywhere from 150⁰C to 300⁰C. Many compounds can’t hold up to these temperatures. Because of this, derivatization becomes necessary for many compounds when analyzing by GC/MS. Very simply, derivatization involves adding a structure onto your compound of interest that acts as a stabilizer. This adds time to your sample preparation as an incubation period is necessary in order to make sure that the compounds in your sample are fully derivatized.

When reconstituting samples for LC/MS/MS analysis after the extraction and dry down are completed, the mobile phase starting conditions are usually used as a reconstitution solvent. For example, if the LC gradient in a method starts as 90% aqueous mobile phase A and 10% organic mobile phase B, the reconstitution solvent would be that same 90:10 mix. In GC/MS analysis, the reconstitution solvent needs to be 100% organic, usually ethyl acetate or acetonitrile. The reconstitution volume is usually much lower in GC/MS than in LC/MS/MS. Since GC/MS is a less sensitive instrument, the less solvent used to reconstitute the sample, the more concentrated the compounds are as they are injected into the GC/MS.

These are some of the biggest differences between sample preparation methods for GC/MS and LC/MS/MS analysis. As can be seen above, the differences between methods are minimal, which makes it a fairly simple process to develop a new method for one or the other instrument type. If any questions come up during this process, reach out and ask us!

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

Last time we wrapped up tuning our analytes to the mass spectrometers optics. However, let’s take a break from that and have a look at one of the more irritating issues in LC/MS method development: signal loss. I’m not talking about signal attenuation, which can be much more convoluted to diagnose; but rather a complete loss of signal – nothing, not even a blip on your TIC. So where do we begin?

Continue reading Troubleshooting Loss of Signal: Where did my peaks go?

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.

Continue reading When should I choose SPE instead of SLE?