Evaluation of the Puritox TC-M160 and Puritox MT-3000 Purification Columns for LCMSMS Utilizing AOAC Official and JAOAC Method Extractions
Julie L Brunkhorst, Heather McGhee, Kelley Renkemeyer, Jordon Bierbaum, Genna Owens, Nicole Laune, Kaitlyn Nobles, Jonathan Witte and Ronald Niemeijer
Trilogy Analytical Laboratory, Washington, MO 63090
The LC/MS/MS has the advantage over traditional analytical techniques by allowing for multi mycotoxin analyses. This technology does have inherent limitations due to matrix effects resulting in signal suppression or enhancement of the target analytes. The removal of the sample matrix utilizing a purification step prior to the analysis is one method that would help reduce this effect and allow for accurate quantification using an external calibration. An evaluation was performed on several matrices for the analysis of mycotoxins using the Puritox TC-M160 and Puritox MT-3000 Purification Columns LC/MS/MS. These purification columns are applicable for the most commonly found and regulated mycotoxins including Aflatoxin B1, B2, G1 and G2, Citrinin, Ochratoxin A, Zearalenone, Fumonisin B1, B2 and B3, Sterigmatocystin and various Type A and B Trichothecenes. This method uses an 84/16 Acetonitrile/water and 3/1 Methanol/water extractions to allow a rapid two step purification process prior to LC/MS/MS analysis. The removal of the sample matrix using these purification columns reduces the problem of signal suppression and enhancement often seen with LC/MS/MS analysis and also allows for the same extract to be used in the AOAC Official methods for confirmatory testing.
Various sample matrices including Cattle Feed, Corn, Corn Gluten Feed, Corn Gluten Meal, DDGS, Oat Hulls, Steepwater, etc. were analyzed by LCMSMS and HPLC for the detection and comparison of results for Aflatoxin, Deoxynivalenol, Fumonisin, Ochratoxin A and Zearalenone.
Samples were extracted with 84:16 Acetonitrile:water for Aflatoxin, Deoxynivalenol, Zearalenone and 3:1 Methanol:water for Fumonisin and Ochratoxin A.
These extracts were then purified with 2 different solid phase columns.
The 84:16 extracts were purified using the TC-M160 which is used for Aflatoxin, Type A and B Trichothecenes, Patulin, Zearalenone and Sterigmatocystin.
The 3:1 extracts were purified using the MT-3000 columns. These columns can be used for Aflatoxin, Beauvericin, Citrinin, Fumonisin, Ochratoxin A, Type A and B Trichothecenes, Sterigmatocystin and Zearalenone.
Both purification columns retain large molecules and contain several active sites that adsorb various interfering compounds such as fats, pigments, carbohydrates and proteins. Each column is operated using a one step process, the extract is pipetted into the top of the column packing and pushed through using a plunger leaving the purified extract in the collection tube.
Matrix calibration curves were used to calculate the individual mycotoxin concentrations that were analyzed by LCMSMS.
Aflatoxin by HPLC was performed using a modified AOAC 994.08
Deoxynivalenol by HPLC was performed using modified JAOAC Vol. 88, #4 2005
Fumonisin HPLC was performed using modified AOAC 2001.04
Ochratoxin A HPLC was performed using modified AOAC 2008.02
Zearalenone HPLC was performed using modified JAOAC Vol. 88, #6, 2005
Results listed below in the charts represent samples that were positive for each of the Mycotoxins.
Overall results compared well between methods utilizing the same extracts.
The clean up and recovery provided by the TCM-160 and MT-3000 columns are comparable to the accepted methods.