A Streamlined SALLE–LC–MS/MS Method for Rapid and Reliable Detection of Stimulants in Forensic Toxicology

A study conducted by the Georgia Bureau of Investigation Division of Forensic Sciences, (Decatur, Georgia) validated a salt-assisted liquid–liquid extraction (SALLE) with liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for detecting amphetamine-type stimulants (ATS) and cocaine metabolites in forensic toxicology. The method was tested on nine drugs, met all American Academy of Forensic Sciences (AAFS) 036 performance criteria, and demonstrated >80% recovery, minimal matrix effects (<20%), low LODs (5–25 µg/L), and sample stability for eight days. Applied to 150 real cases, results closely matched traditional GC-MS findings while reducing sample prep time by 67% and data-processing time by 80%, saving ~8 hours per batch. The Georgia Bureau of Investigation adopted the method, marking a major efficiency and reliability improvement in stimulant drug analysis. LCGC International spoke to Jon Stephenson of the Georgia Bureau of Investigation Division of Forensic Sciences about the study and the resulting paper (1) inspired by it.

What factors prompted the Georgia Bureau of Investigation to shift from traditional GC-MS methods to a SALLE-LC–MS/MS approach?

Legacy methods using GC/MS have served us well for many years, but they are labor-intensive, primarily due to the extensive sample preparation required. GC-MS also demands very clean samples to yield usable results. In contrast, LC-MS/MS is much more robust and tolerant of complex matrices, which allows us to significantly reduce sample preparation. That, along with its high sensitivity, makes LC-MS/MS a preferred option for our needs.

Can you explain why derivatization was necessary in the legacy GC-MS methods, and how the new method circumvents this step?

The previous procedures for analyzing ATS (amphetamine type stimulants) and cocaine (and their metabolites) were developed decades ago. While I can’t speak to all the original rationale, it was likely designed to maximize versatility across a limited number of instruments. Derivatization was used to overcome chromatographic challenges—particularly with amphetamines, which are difficult to analyze in their freebase form using a universal GC column. Adding an acetyl group improved volatility and retention time, resulting in better chromatographic separation and additional fragment ions for monitoring.

With LC-MS/MS, derivatization is no longer necessary. Our universal LC column provided excellent separation for all analytes, and we were able to identify two suitable transitions for MRM analysis without any analyte modification. This greatly simplified the workflow while maintaining analytical performance.

What are some of the key limitations of liquid–liquid extraction (LLE) and solid-phase extraction (SPE) in stimulant drug analysis?

Traditional LLE and SPE methods are often multi-step processes that include solvent evaporation, which can pose a problem with amphetamine-type stimulants (ATS) due to their volatility in freebase form. To prevent analyte loss during evaporation, samples must typically be pretreated with methanolic HCl to convert them into more stable salts. While this approach works, it introduces the potential for sample loss or contamination.

Both methods typically require larger sample volumes when used for GC-MS analysis and both techniques also usually require many additional cleanup steps, which increases time. LLE is typically a cost-effective option, where SPE has the added cost of the extraction columns.

How does SALLE improve upon traditional protein precipitation and LLE techniques in terms of matrix removal and analyte recovery?

In my experience, simpler preparation techniques generally yield more consistent results and are easier to manage over time. SALLE eliminates many of the transfer steps present in LLE, reducing variability from human handling and improving overall recovery consistency.

I’ve always appreciated protein precipitation for its simplicity and broad applicability across all drug classes acid, neutral, base, and amphoteric. However, it retains the aqueous portion of the blood, which can carry matrix effects. SALLE essentially enhances protein precipitation by fully removing both the solid and aqueous fractions of the blood. With just water and table salt, you gain an extra cleanup step without needing to evaporate and reconstitute the sample.

Why is solvent evaporation particularly problematic when analyzing amphetamine-type stimulants (ATS)?

Solvent evaporation can lead to significant analyte loss, particularly with volatile compounds like ATS in their freebase form. To mitigate this, labs often pretreat samples with methanolic HCl to convert the drugs into more stable salts. This helps prevent loss during the evaporation step, but it adds complexity to the workflow and still carries some risk.

What role does sample volatility play in method design, and how does the SALLE technique help address this issue?

For me, the volatility of drugs of interest is always something in the back of my mind when creating a new testing technique. I have seen complete loss of recovery for ATS in untreated samples during solvent evaporation. Since the SALLE technique described in this paper doesn’t use solvent evaporation, it avoids the issue completely.

How did the SALLE-LC–MS/MS method perform in terms of bias, precision, and recovery compared to AAFS 036 standards?

We validated the method against AAFS 036 standards, and it met all our predefined acceptance criteria across all tests. I was particularly impressed by the performance, even for analytes without dedicated deuterated internal standards.

Were there any specific challenges in achieving minimal ion suppression or enhancement, and how were they mitigated?

We really didn’t encounter any challenges with ion suppression or enhancements. I believe that since the sample preparation includes both the protein precipitation and the further separation of the aqueous blood fraction, we obtained better results in this test than we would have if we had just performed the protein precipitation alone.

How does the SALLE method maintain analyte integrity despite the lack of a solvent dry-down step?

I believe the SALLE method enhances analyte integrity. Over my 20-year career troubleshooting sample prep procedures, I’ve found that the simpler the process, the fewer things go wrong. SALLE’s simplicity—avoiding evaporation and minimizing handling—helps preserve analytes and improves overall method reliability.

What impact has this new method had on case throughput and overall laboratory efficiency?

The method has significantly improved our efficiency in confirming ATS and cocaine-related samples. Previously, our throughput was limited by solvent evaporator capacity—we couldn’t handle more than 50 samples per batch, and GC-MS runtimes were long. With SALLE and LC-MS/MS, we routinely process up to 100 samples per run, and the faster instrument runtimes have effectively cut our confirmation testing time in half.

How might the implementation of this SALLE method influence forensic laboratories beyond Georgia?

I hope this paper highlights the practical benefits of SALLE, especially its potential to improve laboratory efficiency. Many sample prep techniques exclude certain drug classes—acids, neutrals, or bases—but SALLE is broadly applicable. Since it doesn’t require proprietary reagents or equipment, there's no commercial push behind it. It’s just water, salt, and an organic solvent—but it works. In our laboratory, it’s now a go-to technique when updating legacy methods, and I hope it gains traction in toxicology as more labs recognize its value.

Do you foresee SALLE being adapted for other drug classes or biological matrices in the future? What are the next steps in further refining this method or expanding its validation scope?

Since SALLE doesn't rely on large pH shifts to extract drugs from the aqueous phase, I don't see it being limited by drug class. While we haven’t yet implemented it beyond ATS and cocaine confirmations, preliminary testing has shown good results across acidic, basic, neutral, and amphoteric compounds. We've also had success applying it to other biological matrices. As we phase out older methods, SALLE will be one of the first techniques we consider for new validations due to its many advantages.

References

1. Stephenson, J.; Austin, J.; Carney, B. et al. Quantitative Analysis of Stimulants in Whole Blood Using an Evaporation Free Precipitation Salt Assisted Liquid-Liquid Extraction (SALLE) Sample Preparation Approach. Forensic Toxicol. 2025, 43 (2), 377-384. DOI: 10.1007/s11419-025-00724-5