Biological sample pretreatment is the initial step in lipid analysis. Without pretreatment, most biological samples cannot undergo subsequent mass spectrometry analysis, regardless of the type of mass spectrometry detector used. The principles of sample pretreatment include removing unwanted substances to eliminate ion suppression, enhancing sensitivity, simplifying sample composition (such as reducing matrix background), enriching target analytes (such as preconcentrating samples), and ensuring the integrity of target analytes (such as avoiding analyte degradation).
Blood, urine, and tissue are commonly used samples for lipid analysis. However, these substances are more complex than other matrices due to their chemical composition, which includes proteins, various organic compounds, salts, etc., resembling the target analytes. Additionally, matrix effects are commonly encountered in quantitative analysis of biological samples, particularly in electrospray ionization mass spectrometry (ESI-MS), where signal suppression caused by unknown matrix interference is often observed.
Commonly used pretreatment methods in lipid analysis include protein precipitation, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Liquid chromatography-mass spectrometry (LC-MS) commonly employs LLE and SPE methods. SPE offers high recovery rates and good chromatographic performance but involves tedious and time-consuming lipid extraction steps. Strong acids or bases are required for eluting strongly polar analytes, making it unsuitable for detection systems like MS.
Currently, pretreatment techniques are rapidly advancing in two directions: automation of pretreatment devices and detection systems, and the development of more selective adsorbents such as immunoadsorbents, molecularly imprinted polymers, and restricted access materials (RAM).
The critical step in rapid analysis of biological samples is the pretreatment process. Before starting the analysis, experimenters need to determine the concentration range, quantity, matrix nature, volatility, reactivity, and lipophilicity of the analytes. Typically, the overall experimental strategy determines the pretreatment method of the samples. The effectiveness of sample pretreatment methods can be evaluated based on the reproducibility and recovery rate of the target analytes. Many details in mass spectrometry analysis are often crucial for quantitative analysis, such as inadequate sample mixing before pipetting, liquid residues in pipetting devices, and nonspecific binding on plastic products.
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Sample preparation in lipidomics, from lipid extraction, through fractionation to analysis (Domingues et al., 2018).
Dilution Method
The dilution method is the simplest and most cost-effective technique for biological sample pretreatment. It is also known as the "dilution and injection" method. This approach is advantageous due to its strong compatibility and high recovery rates of up to 100%. However, it lacks selectivity, leading to a significant decrease in response signal after sample dilution and noticeable matrix effects. The dilution method is primarily employed for simple matrices such as tears, urine, and cerebrospinal fluid, where matrix effects are minimal and sensitivity issues are absent. With the continuous advancement of high-sensitivity mass spectrometers and the application of stable isotope-labeled internal standards (SIL-IS), the dilution method has become a preferred choice for biological sample pretreatment due to its simplicity and ease of use. However, it is not suitable for analyzing samples containing complex matrices such as proteins and lipids.
Protein Precipitation Method (PPT)
The Protein Precipitation Method (PPT) is commonly used for samples like plasma or serum that contain soluble proteins. In this method, protein precipitants are added to the sample, causing proteins to precipitate out, effectively removing them. Various organic solvents like methanol, acetonitrile, or ethanol are typically used to denature the proteins. PPT offers several advantages: it works well for all polar lipid molecules, is fast, compatible with automation, and boasts high recovery rates, often close to 100%, retaining most small molecules. It's a simple, rapid, and convenient method, making it suitable for high-throughput applications, especially in lipidomics research.
However, PPT lacks selectivity and may precipitate all endogenous small molecules, potentially interfering with mass spectrometry ionization and reducing chromatographic column efficiency, leading to noticeable matrix effects and affecting data quality. Despite these limitations, PPT finds significant use in drug discovery due to its rapidity. It's commonly combined with LC-ESI-MS in drug development, particularly with the advent of stable isotope-labeled internal standards (SIL-IS), which help mitigate matrix effects by allowing for reduced sample injection volumes.
Studies evaluating different protein precipitation techniques have found that certain precipitants, like acetonitrile, trichloroacetic acid, and zinc sulfate, effectively remove proteins from plasma. Recent developments include high-throughput PPT filtration plates/tubes made from special materials, simplifying operation and increasing throughput. These plates retain phospholipids while removing proteins and phospholipids from serum or plasma. Additionally, disposable polytetrafluoroethylene tubes have been used for protein precipitation in trace compound analysis in blood samples, followed by direct analysis using paper spray ionization mass spectrometry, demonstrating high sensitivity and stability for various analytes.
Liquid-Liquid Extraction (LLE)
Liquid-Liquid Extraction (LLE) commonly utilizes organic solvents such as 1-chlorobutane, hexane, ethyl acetate, methyl tert-butyl ether (MTBE), or a mixture of two solvents. The choice of organic solvent depends on the polarity and solubility of the analyte, and adjusting the type of organic solvent or pH of the buffer solution can effectively optimize experimental conditions. Compared to methods like Protein Precipitation (PPT) or Solid-Phase Extraction (SPE), LLE offers stronger selectivity. Due to its initial selectivity, LLE allows for adjusting sample pretreatment conditions to balance recovery and selectivity (eliminating matrix background) as needed. When matrix effects are the primary concern, selectivity should be prioritized.
Using Stable Isotope-Labeled Internal Standards (SIL-IS) effectively corrects the entire extraction process, eliminating the need for 100% recovery rates or consistent recovery rates among different sample types. However, consistent recovery rates are generally required to ensure method stability. Attention should also be paid to both absolute and inter-sample recovery rates.
The advantages of LLE include:
- Suitability for non-polar and moderately polar analytes, which distribute well in the organic phase.
- The ability to increase sample volume to enhance analytical sensitivity. Increasing sample volume can amplify the signal, often surpassing the inhibitory effects of matrix effects.
- Predicting matrix background after extraction by determining the pH under extraction conditions, which aids in the development of other biological sample methods.
In the extraction of phospholipids from serum or plasma, Matrix Suppression is common in ESI-MS. Liu et al. demonstrated that establishing a database of phospholipid extraction under different conditions helps eliminate matrix suppression. Significant differences in the amount of extracted phospholipids and recovery rates of specific analytes were observed under different extraction conditions, particularly when the ratio of hexane to ethyl acetate varied.
Salt-assisted LLE and Carrier LLE are two commonly used derivative LLE methods. Salt-assisted LLE induces phase separation by adding salt (such as ammonium acetate, sodium chloride, potassium carbonate, or magnesium sulfate) to the water-soluble organic solvent-extracted target analyte solution, enhancing LLE efficiency. The advantages of salt-assisted LLE broaden the application range of LLE, from low to high lipophilicity analysis. However, a disadvantage is the higher matrix effect when extracting more endogenous substances. Carrier LLE utilizes high-surface-area inert solids to increase the contact area between the sample and organic solvent, mainly based on the distribution equilibrium of the target between the organic phase and adsorption on the solid-phase carrier. The advantages of Carrier LLE include high extraction efficiency, ease of automation, and resistance to emulsification.
Solid Phase Extraction (SPE)
Solid Phase Extraction (SPE) has been employed since the late 20th century for the extraction and enrichment of trace analytes from biological or environmental samples. SPE relies on various mechanisms of interaction between analytes and bonded solid-phase adsorbents. Analytes are adsorbed onto SPE cartridges, extraction plates, or columns, allowing for the removal of matrix during sample loading and washing, while retaining target analytes on the stationary phase for subsequent elution and analysis by LC-MS.
The development of SPE methods primarily considers factors such as the type of sorbent, bed volume, sample volume, loading, washing, and elution conditions. Consistency between SPE cartridges and variability between analytical batches can influence the performance of SPE methods. The advantages of SPE include its suitability for various types of substances, including small or large molecules, acids or bases, polar or non-polar compounds, and diverse sample matrices. SPE offers high recovery rates, good reproducibility, and strong selectivity, making it suitable for automation and widely applicable. However, SPE also has limitations such as lengthy development time, high cost, variability between different batches of SPE cartridges or extraction plates, and complexity of operation requiring skilled personnel.
In addition to traditional SPE, SPE combined with other separation mechanisms can form molecular exclusion material SPE, immunoadsorption SPE (IA-SPE), and molecularly imprinted polymer SPE (MIP-SPE). Molecular exclusion materials, first proposed in 1991, selectively allow only small molecules to enter their pores. Different molecular exclusion materials depend mainly on the surface functionalities, such as ion-exchange functional groups or hydrophobic functionalities. The advantages of molecular exclusion material SPE include direct injection of untreated plasma samples, combining the advantages of traditional SPE and molecular exclusion chromatography, making it easier to separate biological proteins from the matrix. IA-SPE involves the use of immunoadsorbents filled on SPE extraction plates or columns, containing specific antibodies against the target analyte, facilitating selective reversible antigen-antibody binding. This method is mainly used for extracting and enriching target analytes from complex matrices, offering high selectivity. However, the preparation of immunoadsorbents is challenging and costly, typically considered as a last resort. MIP-SPE operates similarly to the action of protein antibodies. MIP is a highly cross-linked polymer synthesized around template molecules, leaving behind cavities capable of specific binding with target analytes. MIP-SPE has been widely applied in various fields such as drug bioanalysis, food contaminant analysis, and environmental sample analysis, offering ease of synthesis, resistance to harsh conditions, and applicability for concentrating target analytes from large-volume samples in complex systems. Nevertheless, MIP-SPE also has limitations, including non-uniform binding sites and template molecule leakage.
In summary, IA-SPE and MIP-SPE both utilize fillers that interact specifically with the target analyte, offering high selectivity and strong enrichment capabilities compared to other methods but at a higher cost.
Reference
- Domingues, P., et al. "AACLifeSci Course Companion Manual Advanced Analytical Chemistry for Life Sciences; 2018."
