Interference from lipids is a common problem for laboratories measuring trace residues in fatty foods or complex biological matrices: their presence in a sample can cause significant matrix interferences, resulting in ion suppression, and therefore can negatively impact sample analysis. Lipids can build up in the analytical column and instrument, which decreases column lifetime and increases the frequency of instrument maintenance.
Traditional methods for lipid removal often sacrifice analyte recovery due to nondiscriminant removal of sample matrix. Advances in sample preparation technologies make it possible to remove lipids without compromising analyte recovery while improving data quality for more reliable analysis.
Effective matrix removal
Bond Elut Enhanced Matrix Removal—Lipid (EMR—Lipid) from Agilent Technologies (Wilmington, Del.) selectively removes lipids in challenging, high-fat sample matrices without hindering analyte recovery. This innovative sorbent technology delivers cleaner matrix background profiles, improves data quality and reduces the need for sample-related troubleshooting. Cleaner samples mean better data quality and reliability, less instrument downtime and increased productivity.
In addition to enhancing accuracy, reproducibility, precision and trace-level quantitation in complex sample matrices, the sorbent reduces the amount of matrix entering the MS. This is especially valuable for labs running routine high-matrix samples, in particular, in the analysis of multiclass and multiresidue analytes in high lipid samples.
This article compares traditional and modern methods for lipid removal, and provides examples using QuEChERS and solvent extraction followed by dispersive solid-phase extraction (dSPE) workflows.
Pesticide analysis for LC/MS/MS
Avocado is a high-fat matrix. The high lipid content can hurt method performance. With EMR–Lipid, fats were effectively removed, allowing for the analysis of 44 pesticides by LC/MS, as shown in Figure 1. Of the 44 pesticides, 95% had a recovery of 70–120%, except for 2,4-D acid and cyprodinil. The latter gave recoveries just below 70% with good relative standard deviation (RSD). Method reproducibility was less than 10% RSD (n = 6) for 91% of the pesticides at 5 ng/g, 100% at 50 ng/g and 98% at 200 ng/g. All other RSD values were well under 20% using the sorbent.
Figure 1 – Quantitation results for 44 representative pesticides in avocado using the Agilent Bond Elut EMR—Lipid workflow. Accuracy and precision data were calculated based on 18 replicates at three different concentrations (Agilent ZORBAX RRHD Eclipse Plus C18 column, 1290 Infinity LC, 6490 Triple Quadrupole LC/MS).Pesticide analysis for GC/MS
The EMR sorbent is equally effective prior to GC/MS analysis, as was found when using QuEChERS AOAC extraction followed by EMR—Lipid cleanup.
Figure 2 depicts overlaid GC/MS full-scan chromatograms for an avocado matrix blank and the chromatographic profiles obtained with EMR—Lipid, C18/PSA and zirconia cleanup. The chromatogram from the sample without dSPE cleanup (black trace) shows a high abundance of matrix interferences, which will impede the analysis of target analytes. Chromatograms from extracts treated with C18/PSA (blue) and zirconia sorbent (green) show 36 and 55% matrix removal, respectively. The significant amount of matrix remaining in the samples will still create interferences, making target analyte analysis difficult. Near-baseline removal of these interferences, corresponding to 95% matrix removal, is achieved with the EMR—Lipid dSPE trace (red), resulting in a cleaner sample profile for easier data analysis.
Figure 2 – GC/MS full-scan chromatogram overlay of avocado matrix blanks from a QuEChERS AOAC extraction followed by dSPE using Agilent Bond Elut EMR—Lipid (red), zirconia (green), PSA/C18 (blue) or no cleanup (black) (Agilent J&W DB-5ms Ultra Inert GC column, 7890A GC, 7000C Triple Quadrupole GC/MS).EMR—Lipid is useful for the analysis of pesticides in fatty matrices, such as avocado, since less matrix in the sample has a positive effect on instrument performance. Time spent troubleshooting variability in results, reanalyzing samples and analyzing data is reduced. Cleanup is done using a simple dSPE technique in the QuEChERS workflow.
Analyzing veterinary drugs in bovine liver
Multiclass veterinary drugs typically contain a wide variety of compounds, including many polar and labile types. Matrices for veterinary drug applications are usually of animal origin, e.g., meat, liver or kidney, and often contain fat, protein and other complex components that can cause matrix interferences. An optimized EMR—Lipid method was compared with a traditional QuEChERS method that uses C18 dSPE and zirconia sorbent cleanup. Figure 3 shows the evaluation for selected veterinary drugs extracted from a typically fatty matrix, bovine liver. The optimized EMR—Lipid protocol provided significant improvements in recovery and precision of the problematic analytes, especially compared to zirconia sorbent. Most analytes (93%) had 70–120% recoveries when using EMR—Lipid, in comparison to the QuEChERS and zirconia protocols. Zirconia gave low recoveries for the fluoroquinolone and tetracycline classes.
Figure 3 – Selected analyte recoveries of veterinary drugs in bovine liver to compare the Agilent Bond Elut EMR—Lipid protocol with traditional protocols (Agilent Poroshell 120 EC-C18 column, 1290 Infinity LC, 6490 Triple Quadrupole LC/MS).Compared to other traditional cleanup techniques, the EMR—Lipid workflow saved time and effort while maintaining high recovery and precision. The methodology is extremely simple and drops into existing workflows to meet performance demands.
Analysis of PAHs by GC/MS
The following highlights the analysis of polycyclic aromatic hydrocarbons (PAHs) from salmon by GC/MS. Salmon was chosen as a representative sample due to its high fat content relative to other seafood. The optimized procedure streamlines the workflow and takes advantage of the EMR—Lipid dSPE cleanup step.
Absolute recovery was 62–98% without the use of internal standards (Table 1). Two compounds—indo[1,2,3-cd]pyrene and benzo[g,h,i]pyrene— gave recoveries slightly below 70%. The PAH absolute recoveries decreased with increasing molecular weight due to reduced solubility in ACN. However, most recoveries were high and easily corrected using the internal standards. Internal standard absolute recoveries were also high. Despite the solubility limitation of ACN, this method provided good-to-excellent recoveries and highly reproducible results in the high-fat salmon sample.
Table 1 – Accuracy, absolute recoveries, RSDs of PAHs in salmon (n = 6)*
As is typical with protocols using enhanced matrix removal, the high efficiency of the sorbent permits a larger sample size, which improves overall method sensitivity. For conventional EMR—Lipid protocols, water is added to activate the sorbent before dSPE. In this salmon protocol, the supernatant from extraction was transferred directly to the EMR—Lipid tube without the addition of water. The water present in the sample provided adequate sample preparation for GC/MS SIM (selected ion monitoring) analysis. Immediate mixing at each transfer step is essential to suspend the solids and ensure maximum interaction with sorbent and avoid clumping. Recoveries can be effectively corrected with the internal standards to give high accuracy and precision.
Conclusion
Lipid removal is important for laboratories using LC/MS/MS, GC/MS/MS and GC/MS to analyze food samples with high fat content. The methods described are simple to align with existing QuEChERS and solvent extraction workflows. The advanced sorbent technology in Enhanced Matrix Removal—Lipid provides high precision, reduces matrix effects, improves data integrity and defensibility and increases productivity.
Additional reading
- Zhao, L. and Lucas, D. Multiresidue Analysis of Pesticides in Avocado with Agilent Bond Elut EMR–Lipid by LC/MS/MS. Agilent Publication, p/n 5991-6098EN.
- Zhao, L. and Lucas, D. Multiresidue Analysis of Pesticides in Avocado with Agilent Bond Elut EMR–Lipid by GC/MS/MS. Agilent Publication, p/n 5991-6097EN.
- Zhao, L. and Lucas, D. Multiresidue Analysis of Veterinary Drugs in Bovine Liver by LC/MS/MS. Agilent Publication, p/n 5991-6096EN.
- Zhao, L. and Lucas, D. PAH Analysis in Salmon with Enhanced Matrix Removal. Agilent Publication, p/n 5991-6088EN.
April DeAtley is sample preparation product manager, and Limian Zhao and Derick Lucas are R&D applications scientists, Agilent Technologies, 2850 Centerville Rd., Wilmington, Del. 19808, U.S.A.; tel.: 800-227-9770; e-mail: [email protected]; www.agilent.com