Not Every Matrix-Matched Calibration Is Made Equal – Case of Spinach

Matrix-matched calibrations are a popular option for complex matrices, such as food commodities. When it is employed, one can assume that the majority of matrix effects will be accounted for and there won’t be much difference between calibration standards and the tested samples. But are all matrix-matched calibrations equal?

In the case of QuEChERS extraction, there are three places where the standards can be spiked into the matrix (Figure 1), after complete extraction (option A), before cleanup (option B) and before the extraction (option C). In LC analysis, the majority of matrix effects come into play in the ion source, while in GC analysis, the majority of matrix effects happen in the inlet. To simplify the situation, I’m going to use data collected using GC-MS/MS.

Figure 1: Options for matrix-matched calibrations

 

Let’s look more closely at the extremes, options A and C.

Option A, spiking at the very end of the extraction and right before analysis, is particularly useful if you extract blanks ahead of time, the standards are expensive, or the standards are limited in amount. The amount of standard needed can be 10-15x lower than if we’d spike the commodity before extraction. The loss of analytes during extraction and cleanup steps is usually corrected by the use of internal standards.

In our study, we spiked the blank extract with standard to make the highest calibration point and used serial dilution with the blank extract to prepare the rest of the calibration solutions.

Option C, spiking into the commodity at the beginning of extraction, addresses the whole issue of extraction losses by having calibration solutions go through the entire process. However, this process consumes much more of the analyte standard due to the larger sample volumes involved.

In our study, we spiked the commodity with the standard to make the highest calibration point. After the full extraction with cleanup, we used serial dilution with the blank extract to prepare the rest of the calibration solutions.

So are there differences between Options A and C and what do they look like? Let’s look at the case of spinach!

Spinach is a highly pigmented commodity that makes the cleanup process difficult. The use of graphitized carbon black (GCB) does remove the chlorophyll, however, it can also take many planar pesticides with it. When calibration standards are made with cleaned up blanks (option A), a popular way to deal with the analyte loss is to use anthracene (or similar planar compound) as an internal standard. We’ve spiked spinach with 10 ppb of the GC Multiresidue Pesticide Kit (#32562) and anthracene (#33264) as an internal standard. Two calibration standards curves were prepared by preparing the highest calibration level in the matrix and further diluting with matrix blanks to obtain the calibration concentrations (Spinach A) and by extracting the commodity with the highest concentration level spiked. After cleanup further diluting with matrix blanks obtained the rest of the calibration concentrations (Spinach C).  We looked at the recoveries without any adjustments and corrected with anthracene for option A (Figure 2).

Figure 2: Pesticide residues recoveries in spinach grouped by percentage bracket

We can see that anthracene helps to shift from 86% of pesticide residues in the target recovery zone (70%-120%) to 93% and reduces the number of pesticide residues with recovery less than 20%. Option C even without any correction has 97% of pesticide residues in the target zone. Moreover, no pesticide residue has recovery below 50%. On the other hand, option C had 2 pesticides with a recovery above 120% compared to 1 when using option A. When we examine the pesticides that have recoveries below 70% (Figure 3), we can see that while anthracene works, it only works to an extent. Anthracene is only one internal standard, while the cleanup losses vary from 12% to 65%. So to help with the recoveries, more than one internal standard is needed.

Figure 3: Individual pesticide residues' recoveries in spinach

Processing your calibration standards along your samples can be beneficial for improving the quality of your data. Even when we use GC-MS/MS where the majority of matrix effects take place in the inlet, the method of preparation of samples can affect the results.

Extraction and cleanup products used: Q-sep AOAC 2007.01 QuEChERS Extraction Kit (#25852) and Q-sep QuEChERS dSPE Tubes (2 mL) with 150 mg MgSO₄, 50 mg PSA, 50 mg C18-EC, and 50 mg GCB (#26219)