Metabolomics Sample Preparation in Toxicology and Clinical Testing

The metabolome is a highly dynamic collection of molecules, with metabolite concentrations undergoing rapid fluctuation in response to stimuli [1]. Proper sample handling during collection, processing, and storage is essential to maintain metabolite integrity. Inadequate procedures can lead to metabolite degradation or changes that distort the true metabolic state, resulting in inaccurate data [1].

Key considerations in metabolite extraction include:

- Sample type: Different biological matrices (tissues, blood, cultured cells, urine, etc) require tailored approaches. 

- Timing of collection: Metabolite levels are significantly influenced by a range of variables relating to time of day (circadian rhythm, nutritional status, physical activity) and should be collected under similar conditions [1]. 

- Metabolism quenching: Rapidly halting enzymatic activity preserves the metabolite profile from the time of collection [1]. 

- Solvent selection: Solvent mixture is selected based on the chemical properties of the target metabolites and to maximize metabolite recovery [2]. 

- Efficient drying: Solvent removal should occur with minimal thermal degradation of metabolites [3]. 

Nitrogen evaporators have several distinct advantages over other concentration methods:

Prevention of Thermal Degradation

Many metabolites are heat-sensitive and can degrade when exposed to high temperatures [3]. Nitrogen evaporation allows for solvent removal at room temperature or slightly elevated temperatures, preserving the chemical integrity of metabolites.

Efficient Solvent Removal

The nitrogen evaporator uses a stream of nitrogen gas to gently blow over the sample surface, accelerating solvent evaporation without excessive heat application. This process is effective at removing common solvents such as methanol, acetonitrile, dichloromethane, and hexanes.

Sample Concentration

Detecting low-abundance metabolites often requires concentrating the samples. Nitrogen evaporators excel at this task by reducing total sample volume, making samples more amenable to analytical instrumentation such as gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS).

Speed and Throughput

Nitrogen evaporators can provide faster drying compared to traditional evaporation methods such as lyophilization or rotary evaporation. The MULTIVAP system is also beneficial as it can process multiple samples simultaneously with uniform evaporation rates across the sample set.

Medical Lab Evaporates Methanol Quickly

Researchers at the University of Pennsylvania transitioned from a centrifuge-style unit to a MULTIVAP evaporator for removing excess methanol in their metabolite analysis workflow.

They reported significantly faster sample preparation due to two key advantages:

1. Higher temperature range: While the centrifuge system was limited to 15-35°C, the MULTIVAP high-temperature dry evaporator can operate from 30°C up to 120°C, accounting for solvents with a higher boiling point. 

2. Controlled nitrogen flow: The ability to increase nitrogen flow on demand contributed to much faster solvent removal, attributed to the adjustable flow meter. 

Biology Lab Concentrates Metabolites Without Thermal Degradation

The European Molecular Biology Laboratory (EMBL) utilizes Organomation's MICROVAP small vial evaporator in their protocol for extracting polar metabolites from cell cultures. Using a standardized protocol ensures reliable extraction and recovery of metabolites, providing a robust foundation for accurate metabolomics analysis. The main principles outlined in this protocol can be readily adapted to accommodate the specific requirements of different sample types and studies.

Tumor Analysis Aided by Dichloromethane Evaporation Ahead of GC-MS

At the University of Chicago, the Muir Laboratory uses its MULTIVAP nitrogen evaporator to streamline sample preparation ahead of GC-MS analysis, in their research with pancreatic cancer. Their team studies tumor microenvironments and how this can affect tumor growth by measuring metabolites and amino acids.

With the MULTIVAP nitrogen evaporator, the lab is able to process batches of 40+ samples, drying off excess methanol, dichloromethane, and hexanes. This has improved the lab's workflow by significantly reducing dry-down time and accelerating overall sample preparation. In this research, the drying step is critical prior to derivatization, which modifies analytes to enhance their separation during gas chromatography. Another benefit is the system's flexibility, which allows researchers to turn off nitrogen flow to unused rows when processing smaller batches, conserving gas.

UHPLC Sample Prep Expedited by Evaporation to Dryness

At the Hospital for Sick Children in Toronto, researchers utilize Organomation's MICROVAP sample concentrator to prepare human and mouse biological samples for chromatographic analysis. Their work focuses on creatine deficiency syndromes, which can alter gene expression, negatively affecting metabolism and both neurological and muscular systems.

The lab processes various sample types including urine, plasma, CSF, and tissue homogenates. Their protocol includes:

- Protein precipitation with TCA and methanol
- Derivatization
- Evaporation to dryness at 60 °C for 30 minutes using the MICROVAP
- Resuspension in methanol

The researcher noted that their MICROVAP has been used daily for five years, demonstrating its reliability and durability in a high-demand research environment.

Plate Dryer Supports Urine Analysis in Toxicology Lab

Physicians Toxicology, a clinical laboratory specializing in drug screening through urine analysis, incorporates well plate evaporators into its workflow for processing drug screening panels. These panels typically target the detection of opioids, amphetamines, benzodiazepines, and cannabinoids. MICROVAP systems enable efficient sample preparation, supporting downstream analysis with advanced techniques such as LC-MS/MS for confirmatory testing. Their consistent, reliable use over five years highlights their durability and strong performance in a high-throughput clinical setting.

Saliva Samples Prepared for LC-MS/MS via Multiple Well Plate Evaporator

A clinical toxicology laboratory in California has integrated Organomation's triple 96-well MICROVAP evaporators into their workflow for urine and oral fluid drug testing. This facility offers diagnostic services that support healthcare providers in making informed prescribing decisions, addressing the challenge that nearly 1 in 5 patients do not respond to their prescribed medications. Their comprehensive process includes DNA extraction, specialized liquid handling systems, a tracking system, and LC-MS/MS analysis — with MICROVAP evaporators playing a crucial role in sample concentration.

The triple-plate MICROVAP design integrates three 96-needle manifolds and three heat blocks on a single platform, enabling the simultaneous evaporation of up to three 96-well microplates, potentially processing as many as 288 samples at once. This high-throughput capability, along with uniform heating across platforms, supports the laboratory's commitment to rapid turnaround times with results typically available within 24 hours. The systems operate up to 12 hours daily, five days a week, demonstrating exceptional durability.

Metabolomics sample preparation is a crucial step that directly impacts data quality and the biological interpretation of results. Organomation’s nitrogen evaporation systems offer valuable support in this process, especially in applications such as toxicology and clinical testing.

Test Tube Evaporators

• The N-EVAP series is particularly valuable for metabolomics sample preparation as it addresses several critical challenges:

- Metabolite Preservation: The gentle nitrogen blowdown technique helps preserve heat-sensitive metabolites that can degrade when exposed to high temperatures [3].  

- Solvent Removal Flexibility: Compatible with a range of tube sizes, enabling researchers to process varying sample volumes tailored to specific metabolite concentration needs. 

- Controlled Evaporation: Individual gas flow control at each sample position ensures consistent sample preparation, which is essential for obtaining reliable data. 

- Temperature Control: Water bath models are available with a wide temperature range enabling optimization for different solvents used in metabolite extraction protocols. 

 • The MULTIVAP system addresses high-throughput needs in metabolomics research: 

- Batch Capability: Processes up to 100 samples simultaneously, essential for large-scale studies. 

- Standardization: Provides consistent evaporation conditions across samples, reducing sample variability which can skew the biological interpretation. 

- Metabolite Integrity: Gentle nitrogen dry down protects delicate metabolites from degradation, particularly important for unstable compounds in toxicology testing [1]. 

- Efficiency: Significantly reduces sample preparation time compared to traditional methods, allowing for faster results and turnaround time in clinical settings. 

96 Well Plate Evaporators

• The MICROVAP Microplate Evaporator single-plate system supports modern metabolomics workflows by effectively addressing the following needs:

- Microplate Compatibility: Directly supports the 96-well format widely used in high-throughput studies. 

- Precise Temperature Control: Digital temperature control up to 130°C (±2°C accuracy) which enables optimization for different metabolite classes and extraction solvents. 

- Versatility: Accommodates both standard microplates and deep well plates, for various sample volume requirements. 

- Standardized Processing: Ensures uniform evaporation across all wells, critical for comparative studies. 

• The MICROVAP Triple Plate system addresses advanced needs in high-volume clinical and toxicology laboratories:

- Maximum Throughput: Simultaneous processing of three 96-well plates significantly increases laboratory capacity for large-scale studies. 

- Workflow Integration: Can seamlessly integrate with automated liquid handling systems used in modern metabolomics workflows. 

- Space Efficiency: The compact design maximizes laboratory space while maintaining high sample processing efficiency. 

- Consistent Results: Uniform heating across all three platforms ensures reproducible sample preparation, critical for reliable data collection. 

Both types of evaporators contribute to effective metabolomics sample preparation, highlighting the value of Organomation’s robust, well-engineered systems for metabolic sample analysis.

Citations: 

1. González-Domínguez, R., González-Domínguez, Á., Sayago, A., & Fernández-Recamales, Á. (2020). Recommendations and Best Practices for Standardizing the Pre-Analytical Processing of Blood and Urine Samples in Metabolomics. Metabolites, 10(6), 229. https://doi.org/10.3390/metabo10060229
2. Smith, L., Villaret-Cazadamont, J., Claus, S. P., Canlet, C., Guillou, H., Cabaton, N. J., & Ellero-Simatos, S. (2020). Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions. Metabolites, 10(3), 104. https://doi.org/10.3390/metabo10030104
3. Fang, M., Ivanisevic, J., Benton, H. P., Johnson, C. H., Patti, G. J., Hoang, L. T., Uritboonthai, W., Kurczy, M. E., & Siuzdak, G. (2015). Thermal Degradation of Small Molecules: A Global Metabolomic Investigation. Analytical chemistry, 87(21), 10935–10941. https://doi.org/10.1021/acs.analchem.5b03003