quantitative determination of ergot alkaloids in maize...

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Ergocristine Analytical Standard: Romer Labs (biopure)

Ergocornine Analytical Standard, Romer Labs (biopure)

α-Ergocryptine Analytical Standard, Romer Labs (biopure)

Ergosine Analytical Standard, Romer Labs (biopure)

Ergotamine D-tartrate Analytical Standard, Sigma-Aldrich

Chemicals and Reagents

Individual stock standard solutions of each analyte were prepared in acetonitrile at a target concentration of 100 µg/mL.

Stock standards were combined to produce mixed working standard solutions in 50:50 acetonitrile:DI water (v/v) for use as instrument calibration standards and test portion fortification solutions.

Test MatricesThe method was validated using non-GM commercial varieties of dent corn grain, soybean seed and canola seed. A bulk sample of each matrix was ground and ho-mogenized using a centrifugal mill grinder to pass a 0.75 mm sieve. Ground sam-ples were stored in a freezer (ca. -20ºC) when not needed in the laboratory.

Test Portion Extraction• Weighed 2.5 g test portion of ground sample into a 50 mL plastic centrifuge tube.• Laboratory fortified test portions were spiked with a mixed working standard solution to achieve a

concentration of 5 ppb (LOQ) for each analyte.• Added 10 mL of 84:16 acetonitrile:ammonium carbonate (200 mg/L).• Extracted on a platform shaker at a speed of 350 rpm for 90 minutes then centrifuged at 3,000

rpm for 10 minutes.• Added 2 mL of the supernatant extract to a 15 mL plastic centrifuge tube containing 100 mg of

PSA (primary-secondary amine, Agilent Technologies) sorbent and vortex mixed. • Transferred 1.0 mL of the extract to a 15 mL glass culture tube and evaporated to dryness under a

stream of nitrogen. (N-Evap with heating block temperature of 60˚C.)• Added 1.0 mL of 50:50 acetonitrile:DI water (v/v) and vortex mixed. • Sonicated 5 minutes then filtered through a 0.2 µm syringe filter into an autosampler vial for

UHPLC-MS/MS analysis.

UHPLC-MS/MS Parameters• UHPLC System: Waters Acquity UPLC• MS Detector: Waters TQ Detector• UHPLC Column: BEH C18, 100 mm x 2.1 mm x 1.7 µm, Waters

Corporation• Column Temp.: 40˚C• Flow Rate: 0.5 mL/min.• Injection Volume: 3.0 µL• Expected Retention Times: See Figures 1a.-1e.• HPLC Gradient: Linear

Mobile Phase Gradient ProgramTime (min.) %A %B

0.00 80 200.10 80 205.00 65 355.01 0 1006.00 0 1006.01 80 207.50 80 20

A: 0.1% Formic Acid in DI Water.B: 0.1% Formic Acid in Acetonitrile

• MS Source: Electrospray• MS Polarity: Positive• MS Mode: Multiple Reaction Monitoring (MRM)

Figure 1a. Calibration Standard: 1 ng/mL Ergosine

Figure 1b. Calibration Standard: 1 ng/mL Ergotamine

Figure 1c. Calibration Standard: 1 ng/mL Ergocornine

Figure 1d. Calibration Standard: 1 ng/mL Ergocryptine

Figure 1e. Calibration Standard: 1 ng/mL Ergocristine

MRM Program for the Detection of Ergot Alkaloids

Analyte (RT Window)

Ion (m/z) Dwell Time (s) Cone Voltage Collision EnergyPrecursor ProductErgosine

(2.4-2.8 min.) 548 268 0.4 40 25 Ergotamine (2.8-3.5 min) 582 268 0.2 40 25Ergocornine (2.8-3.5 min.) 562 268 0.2 40 25Ergocryptine (3.5-3.9 min.) 576 268 0.4 40 25Ergocristine

(3.9-4.5 min.) 610 268 0.4 40 25

• Calibration: Linear, external standards• Calibration Range: 0.2-20 ng/mL• Weighting: 1/x• Minimum Correlation Coefficient (r): 0.990

Results/DiscussionMethod Validation: Spike and recovery was used to evaluate accuracy and precision. For each matrix, a single set of samples was prepared and analyzed by a single analyst. Each set contained a reagent blank, a single unfortified test portion and seven replicate fortifications at the target LOQ of 5 ppb. Analysis of reagent blanks and unfortified control test portions yielded no detectable ergot alkaloids.

Table 1. Spike and Recovery for Maize Grain

Analyte Mean (n=7) Recovery (%) Std. Dev. (%) RSD (%) Recovery Range

(%)Ergosine 81 9 11 63-89

Ergocornine 94 11 11 82-110Ergotamine 91 15 17 74-111Ergocryptine 93 7 8 84-101Ergocristine 89 7 8 78-101

Table 2. Spike and Recovery for Soybean Seed

Table 3. Spike and Recovery for Canola Seed

Analyte Mean Recovery (%) Std. Dev. (%) RSD (%) Recovery Range

(%)Ergosine 81 8 10 70-90

Ergocornine 81 14 17 60-101Ergotamine 75 11 14 63-94Ergocryptine 79 5 6 70-83Ergocristine 76 12 15 55-93

• Limit of Quantitation (LOQ): The method LOQ was established at 5 ppb for each analyte. The LOD was estimated to be 1.5 ppb, or about 30% of the LOQ.

• Linearity: The linear concentration range for each analyte was approximately 0.2-20 ng/mL. Minimum acceptable correlation coefficients were established at 0.990.

Figure 2a. Unfortified Canola Seed Extract, Ergosine

Figure 2b. Unfortified Canola Seed Extract, Ergotamine

Figure 2c. Unfortified Canola Seed Extract, Ergocornine

Figure 2d. Unfortified Canola Seed Extract, Ergocryptine

Figure 2e. Unfortified Canola Seed Extract, Ergocristine

Figure 3a. 5 ppb Fortified Canola Seed Extract, Ergosine

Figure 3b. 5 ppb Fortified Canola Seed Extract, Ergotamine

Figure 3c. 5 ppb Fortified Canola Seed Extract, Ergocornine

Figure 3d. 5 ppb Fortified Canola Seed Extract, Ergocryptine

Figure 3e. 5 ppb Fortified Canola Seed Extract, Ergocristine

Figure 4a. Ergosine Calibration Curve, 0.2-20 ng/mL

Figure 4b. Ergotamine Calibration Curve, 0.2-20 ng/mL

Figure 4c. Ergocornine Calibration Curve, 0.2-20 ng/mL

Figure 4d. Ergocryptine Calibration Curve, 0.2-20 ng/mL

Figure 4e. Ergocristine Calibration Curve, 0.2-20 ng/mL

ObjectiveThe objective of the research was to develop and validate an analytical method for the determination of the ergot alkaloids ergosine, ergocornine, ergotamine, ergocryptine and ergocristine in maize, soybean and canola seeds with a target limit of quantitation of 5 ppb for each analyte in each matrix. Analysis of these mycotoxins is required as part of a multi-toxin screen to support animal feeding trials conducted with laboratory diets formulated with genetically modified (GM) seed. The developed analytical procedure was based on a procedure described by Krsda et. al. for cereal grains and foodstuffs (1).

ConclusionA method for the extraction and UHPLC-MS/MS analysis of ergot alkaloids in maize grain, soybean seed and canola seed was developed and validated. The method demonstrated acceptable accuracy, precision, sensitivity and linear range. The validated method is now routinely employed to support toxin screens of laboratory animal diets formulated with GM seeds.Reference

(1) Krsda, R.; Stubbings, G. Macarthur, R. Simultaneous determination of the six major ergot alkaloids and their epimers in cereals and foodstuffs by LC-MS-MS. Anal. Bioanal Chem. 2008, 391: 563-576.

AbstractAn analytical method for the quantitative determination of the ergot alkaloids ergosine, ergocornine, ergotamine, ergocryptine and ergocristine in maize, soybean and canola seeds was developed and validated. Ergot alkaloids are secondary metabolites produced by the fungal genus Claviceps. They are responsible for ergotism in humans and other mammals that consume contaminated grains from outcrossing crops such as cereal grains. Although Claviceps species only infect specific cereal grain crops, cross contamination in non-outcrossing crops in grain handling facilities and equipment can be of concern. Therefore, screening of grains and seeds used to produce animal feeds may be necessary, especially feeds formulated for use in dietary toxicology studies. The validated method includes extraction of ground seed test portions using acetonitrile/ammonium carbonate solution followed by dispersive solid phase extraction (dSPE) cleanup. After evaporative solvent exchange, the sample solutions are filtered and analyzed by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS). The chromatographic separation was carried out on a C18 column utilizing an aqueous formic acid mobile phase gradient with acetonitrile. The ergot alkaloids are detected using electrospray ionization (ESI) in positive ion mode and quantified by multiple reaction monitoring (MRM). Assay precision and accuracy were determined on laboratory fortified seed test portions. The limit of quantitation (LOQ) was 5 µg/kg (ppb) and limit of detection (LOD) was 1.5 ppb.

Quantitative Determination of Ergot Alkaloids in Maize, Soybean and Canola Seeds by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry

Fred Claussen (claussen@eplbas.com)

Analyte Mean (n=7) Recovery (%) Std. Dev. (%) RSD (%) Recovery Range

(%)Ergosine 85 10 12 76-103

Ergocornine 98 14 15 76-117Ergotamine 74 5 7 68-82Ergocryptine 80 11 14 66-96Ergocristine 75 5 6 70-81