Limits...
Development of a Rapid LC-MS/MS Method for the Determination of Emerging Fusarium mycotoxins Enniatins and Beauvericin in Human Biological Fluids.

Belén Serrano A, Capriotti AL, Cavaliere C, Piovesana S, Samperi R, Ventura S, Laganà A - Toxins (Basel) (2015)

Bottom Line: The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%-103%) and the removal of the major interferences from the matrix.The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R² of 0.991-0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L(-1) and 40 ng·L(-1) in plasma and between 5 ng·L(-1) and 20 ng·L(-1) in urine).Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Toxicología, Departament de Medicina Preventiva I Salut Pública, Ciències de l'Alimentació, Toxicologia I Medicina Legal Facultat de Farmàcia, Universitat de València, València 46010, Spain. A.Belen.Serrano@uv.es.

ABSTRACT
A novel method for the simultaneous determination of enniatins A, A1, B and B1 and beauvericin, both in human urine and plasma samples, was developed and validated. The method consisted of a simple and easy pretreatment, specific for each matrix, followed by solid phase extraction (SPE) and detection by high performance liquid chromatography-tandem mass spectrometry with an electrospray ion source. The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%-103%) and the removal of the major interferences from the matrix. The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R² of 0.991-0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L(-1) and 40 ng·L(-1) in plasma and between 5 ng·L(-1) and 20 ng·L(-1) in urine). Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

No MeSH data available.


LC-SRM chromatograms of the single transitions of a urine sample extract fortified with the investigated analytes at 20 ng·L−1 (acquisition conditions are reported in the Experimental Section). For ENB1, this concentration level corresponds to its method detection limit.
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toxins-07-03554-f003: LC-SRM chromatograms of the single transitions of a urine sample extract fortified with the investigated analytes at 20 ng·L−1 (acquisition conditions are reported in the Experimental Section). For ENB1, this concentration level corresponds to its method detection limit.

Mentions: Therefore, for each analyte, the LODs and LOQs were extrapolated as reported in Section 3.5.3, considering the second most intense transition area and the sum of the transition areas, respectively. Then, standard solutions and samples fortified at the extrapolated level were prepared, processed and injected six times. Finally, the dataset was evaluated in terms of RSD of the areas and mean transitions ratio. An acceptable value of 20% of the RSD for quantification limits and 50% for the detection limits was arbitrarily set, whereas the acceptable differences established by 2002/657/EC [29] were considered for the ratio between the areas. When both conditions were respected, a more diluted sample was prepared, whereas a more concentrated sample was tested if not. To avoid repeating the operation too many times, concentration variations were 50%–100%. Results are shown in Table 4. Figure 2 and Figure 3 show, for all of the investigated analytes, the LC-SRM profile of each transition in plasma and urine samples, respectively, fortified at 20 ng·L−1. As can be seen, the experimental limits were of the same order of magnitude of the extrapolated ones. In addition, by adding to the matrix-matched calibration line for urine the concentration corresponding to experimental verified method detection limit (MDL), the R2 did not change significantly. These facts might be due to the restricted concentration range within which calibration lines were considered for expected concentration quantification (the range of linearity covers about three orders of magnitude). In some cases, MDLs and method quantification limits (MQLs) were very similar to each other or even the same: in these cases, for a concentration lower than that evaluated for MDL, they were not in compliance with the provision established by 2002/657/EC [29] regarding the mean value variation of the area ratio between the two transitions. Obviously, the areas of signals present in blanks were subtracted. This fact is important, because the distinction between quantifier and qualifier transition is no longer applicable. Looking at Figure 2 and Figure 3, it should appear clear why, although urine samples were concentrated ten times, whereas plasma samples were not concentrated, MQLs and MDLs are not very different. As can be seen, background signals are more intense for urine. Note that, for some analytes, 20 ng·L−1 is near or even under the MDL. This evaluation of MDLs and MQLs should be considered approximate as, although some biological variation was considered, the population was fairly homogeneous in terms of diet. Nevertheless, the limits reported here are five-times lower than those reported for plasma [17] and 500-times lower than those reported for ENA in urine [21].


Development of a Rapid LC-MS/MS Method for the Determination of Emerging Fusarium mycotoxins Enniatins and Beauvericin in Human Biological Fluids.

Belén Serrano A, Capriotti AL, Cavaliere C, Piovesana S, Samperi R, Ventura S, Laganà A - Toxins (Basel) (2015)

LC-SRM chromatograms of the single transitions of a urine sample extract fortified with the investigated analytes at 20 ng·L−1 (acquisition conditions are reported in the Experimental Section). For ENB1, this concentration level corresponds to its method detection limit.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591648&req=5

toxins-07-03554-f003: LC-SRM chromatograms of the single transitions of a urine sample extract fortified with the investigated analytes at 20 ng·L−1 (acquisition conditions are reported in the Experimental Section). For ENB1, this concentration level corresponds to its method detection limit.
Mentions: Therefore, for each analyte, the LODs and LOQs were extrapolated as reported in Section 3.5.3, considering the second most intense transition area and the sum of the transition areas, respectively. Then, standard solutions and samples fortified at the extrapolated level were prepared, processed and injected six times. Finally, the dataset was evaluated in terms of RSD of the areas and mean transitions ratio. An acceptable value of 20% of the RSD for quantification limits and 50% for the detection limits was arbitrarily set, whereas the acceptable differences established by 2002/657/EC [29] were considered for the ratio between the areas. When both conditions were respected, a more diluted sample was prepared, whereas a more concentrated sample was tested if not. To avoid repeating the operation too many times, concentration variations were 50%–100%. Results are shown in Table 4. Figure 2 and Figure 3 show, for all of the investigated analytes, the LC-SRM profile of each transition in plasma and urine samples, respectively, fortified at 20 ng·L−1. As can be seen, the experimental limits were of the same order of magnitude of the extrapolated ones. In addition, by adding to the matrix-matched calibration line for urine the concentration corresponding to experimental verified method detection limit (MDL), the R2 did not change significantly. These facts might be due to the restricted concentration range within which calibration lines were considered for expected concentration quantification (the range of linearity covers about three orders of magnitude). In some cases, MDLs and method quantification limits (MQLs) were very similar to each other or even the same: in these cases, for a concentration lower than that evaluated for MDL, they were not in compliance with the provision established by 2002/657/EC [29] regarding the mean value variation of the area ratio between the two transitions. Obviously, the areas of signals present in blanks were subtracted. This fact is important, because the distinction between quantifier and qualifier transition is no longer applicable. Looking at Figure 2 and Figure 3, it should appear clear why, although urine samples were concentrated ten times, whereas plasma samples were not concentrated, MQLs and MDLs are not very different. As can be seen, background signals are more intense for urine. Note that, for some analytes, 20 ng·L−1 is near or even under the MDL. This evaluation of MDLs and MQLs should be considered approximate as, although some biological variation was considered, the population was fairly homogeneous in terms of diet. Nevertheless, the limits reported here are five-times lower than those reported for plasma [17] and 500-times lower than those reported for ENA in urine [21].

Bottom Line: The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%-103%) and the removal of the major interferences from the matrix.The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R² of 0.991-0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L(-1) and 40 ng·L(-1) in plasma and between 5 ng·L(-1) and 20 ng·L(-1) in urine).Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Toxicología, Departament de Medicina Preventiva I Salut Pública, Ciències de l'Alimentació, Toxicologia I Medicina Legal Facultat de Farmàcia, Universitat de València, València 46010, Spain. A.Belen.Serrano@uv.es.

ABSTRACT
A novel method for the simultaneous determination of enniatins A, A1, B and B1 and beauvericin, both in human urine and plasma samples, was developed and validated. The method consisted of a simple and easy pretreatment, specific for each matrix, followed by solid phase extraction (SPE) and detection by high performance liquid chromatography-tandem mass spectrometry with an electrospray ion source. The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%-103%) and the removal of the major interferences from the matrix. The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R² of 0.991-0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L(-1) and 40 ng·L(-1) in plasma and between 5 ng·L(-1) and 20 ng·L(-1) in urine). Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

No MeSH data available.