TITLE: An analytical method for the quantitation (20-8,000 ppb) of Ergot Alkaloids in Wheat grain.
AUTHORS: Michelle Mostrom, Kelly Benson, Brett Webb

[DESCRIPTION]
Ten ergot alkaloids are quantified in wheat and rye grains at concentrations ranging 20-8,000 ppb using HPLC-MS/MS. Briefly, 5 grams of ground sample is mixed with 40 mL of an extraction solution (ACN-water; 84/16, v/v + 200 mg/L Ammonium Carbonate, pH 8.5) was added to each tube, agitated for 30 min on a horizontal shaker at 150 cycles per min, let to sit for 1h to ensure adequate extraction of analytes and filtered through a Whatman 54 filter paper into a new tube. An aliquot of 4mL was pushed through Mycosep 150 Ergot column. An aliquot of 500 mL was mixed with 500 mL of  dihydroergocristine at 100 ng/mL in the extraction buffer (internal standard) and centrifuged at 10 000 RPM for 5 min to remove any possible particulate matter. An aliquot of 100 mL was transferred into autosampler vial and injected (2 mL) into Agilent 6460C UHPLC–MS/MS equipped with guarded Agilent Zorbax Eclipse Plus C18 column (2.1 50mm, 1.8 micron).

Validation data (in-house and via collaborative studies such as Blinded Method Test) are available in the following publication: https://pubmed.ncbi.nlm.nih.gov/33394021/

[STEPS]
SECTION: Standard Curve:
1. Prepare serial dilution
SECTION: Standard Curve:
1.2. Transfer 50 µL of 500 Parts per Billion (PPB) standard to poly vial for Standard 10  

SECTION: Standard Curve:
1.1. Make up a 500 PPB solution of 500 PPB each of Ergocornine, Ergocorninine, Ergocristine, Ergocristinine, Ergocryptine, Ergocryptinine, Ergosine, Ergosinine, Ergotamine, and Ergotaminine from reference standards.

SECTION: Standard Curve:
2. 500 ppb stock standards in individual aliquots for one serial dilution
SECTION: Standard Curve:
2.1. Make up from reference standards 500 ppb solution of 500 ppb of each ergot alkaloids.
SECTION: Standard Curve:
2.2. Transfer 150 ul of 500ppb solution to amber vials and dry down under nitrogen.
SECTION: Standard Curve:
2.3. Store these vials in freezer (-20C)
SECTION: Standard Curve:
2.4. To use take out one vial and let warm to room temperature. 
SECTION: Standard Curve:
2.6. Bring up vial in 150 ul of 50 PPB Dihydroergocistine in extracting solution. Mix well.
SECTION: Standard Curve:
2.5. From your ISTD 100 PPB Dihyroergocristine in extracting solution make up 800 ul of 50 PPB Dihydroergocristine in extracting solution. (1:1 dilution)
SECTION: Standard Curve:
2.7. Use this to perform serial dilution of 10 standards. Use the same 50 PPB Dihydroergocristine in extracting solution to dilute with during serial dilution.
SECTION: Standard Curve:
2.8. 
 

SECTION: PROCEDURE for sample preparation
3. Weigh out 5 grams of dried and ground sample into Centrifuge tube:
Wheat ground on Perten Mill or Glen Mill particle size within 100-355 um.
Wheat sample moisture is typical of wheat in long-term storage conditions or < 14 to 15% moisture. If upon physical evaluation it appears/feels wet samples are dried overnight in 60C oven.
SECTION: PROCEDURE for sample preparation
5. Shake for 30 minutes on Horizontal Shaker at 150 +/- 10 Cycles per minute.
SECTION: PROCEDURE for sample preparation
7. Filter entire extract thru Whatman 54 filter paper into a new labeled centrifuge tubes.
SECTION: PROCEDURE for sample preparation
8. Transfer 4 mls of filtered extract to the glass tube provided with Mycosep kit.
SECTION: PROCEDURE for sample preparation
9. Push the 4 mls  thru Mycosep 150 Ergot Column.
SECTION: PROCEDURE for sample preparation
10. Transfer 500ul of extract to a microcentrifuge tube.
SECTION: PROCEDURE for sample preparation
11. Dilute with 500 ul of 100 PPB Dihydroergocristine in extracting solution
SECTION: PROCEDURE for sample preparation
12. Final concentration of Dihydroergocristine is 50 PPB.
SECTION: PROCEDURE for sample preparation
13. Centrifuge at 10,000 RPM for 5 mins
SECTION: PROCEDURE for sample preparation
14. Transfer 100ul to Polypropylene vial for injection on QQQ.
SECTION: PROCEDURE for sample preparation
15. Finale extract is a 16x dilution of sample.
SECTION: PROCEDURE for sample preparation
16. Prepare 10-point Standard curve by serial dilution and run standard curve with samples daily.
SECTION: PROCEDURE for sample preparation
4. Add 40 mls of Extracting Solution. Cap and invert to mix.
SECTION: PROCEDURE for sample preparation
6. Let extract sit for at least 1 hour.
SECTION: LC-MS QQQ Method
17. Analysis is carried out on an Agilent 6460C Triple Quad LC/MS.It is carried out with positive electrospray ionization in Dynamic MRM mode using three major transitions per target compound.
SECTION: LC-MS QQQ Method
18. Samples are run with the LC by a method with the following parameters.

SECTION: LC-MS QQQ Method
18.1. UHPLC Column: Agilent Zorbax Eclipse Plus C18 2.1 x 50 mm, 1.8 micron (P.N. 959757-902)


SECTION: LC-MS QQQ Method
18.5. Binary Pump:
    Flow: 0.200 mL/min
    High Pressure Limit: 550 bar
    Stop Time: 12.00 min
    Post Time: 1.0 min

 Gradient Program:

   Time    A%    B%    0.70 min    95%    5%    1.3 min    50%    50%    8.0 min    10%    90%    10.0 min    10%    90%    12.0 min    95%    5%  
 
SECTION: LC-MS QQQ Method
18.4. 
Auto Injector Parameters
 
AB AB  Injection Volume    2 µl    Needle Wash    5 seconds with Acetonitrile    Column Temp    30.00 °C     
 
SECTION: LC-MS QQQ Method
18.3. Mobile Phase: A: 3 mM ammonium carbonate in Water B: premixed Acetonitrile (90%) – Water (10%)
SECTION: LC-MS QQQ Method
18.2. Guard Column: Zorbax Eclipse Plus C18, 2.1 x 5 mm, 1.8 micron, (P.N. 821725-901)
SECTION: LC-MS QQQ Method
19. LC-MS QQQ
 
 
       Agilent 6460C Triple Quadrupole Parameters                Ionization mode         Positive ESI with Agilent   Jet Stream                Scan type         Dynamic MRM                      Gas temperature         200 °C                Gas Flow         8 L/min                Nebulizer pressure         45 psi                Sheath gas temperature         400 °C                     Sheath gas flow         12 L/min                     Capillary voltage         3000 V                     Nozzle voltage         500 V                     Delta EMV         500                     Cycle Time         500 ms               
 
 
SECTION: LC-MS QQQ Method
20. MRM transitions
 
       Compound   Name         RT         Prec   Ion         Prod   Ion         Frag (V)         CE (V)         Cell Acc (V)         Ret window         Polarity             ION            1. Ergosine         5.4         548.0         530.2         150         15         4         1         Positive         Qual            1. Ergosine         5.4         548.0         223.1         150         35         6         1         Positive         Quant            1. Ergosine         5.4         548.0         208.0         150         50         4         1         Positive         Qual            2. Ergotamine         5.7         582.1         277.1         140         25         6         1         Positive         Qual            2. Ergotamine         5.7         582.1         223.1         140         35         6         1         Positive         Quant            2. Ergotamine         5.7         582.1         208.0         140         45         4         1         Positive         Qual            3. Ergocornine         6.4         562.1         305.1         140         25         6         1         Positive         Qual            3. Ergocornine         6.4         562.1         277.1         140         30         4         1         Positive         Qual            3. Ergocornine         6.4         562.1         223.1         140         35         6         1         Positive         Quant            4. Ergocryptine         6.9         576.0         305.1         120         25         6         1         Positive         Qual            4. Ergocryptine         6.9         576.0         291.1         120         25         4         1         Positive         Qual            4. Ergocryptine         6.9         576.0         223.1         120         35         6         1         Positive         Quant            5. Ergocristine         7.1         610.0         305.0         140         25         4         1         Positive         Qual            5. Ergocristine         7.1         610.0         268.2         140         25         7         1         Positive         Qual            5. Ergocristine         7.1         610.0         223.1         140         40         6         1         Positive         Quant            6. Ergosinine         7.5         548.2         530.1         141         14         4         1         Positive         Qual            6. Ergosinine         7.5         548.2         223.1         141         34         5         1         Positive         Quant            6. Ergosinine         7.5         548.2         208.0         141         50         4         1         Positive         Qual            7. Ergotaminine         7.9         582.2         277.1         144         26         5         1         Positive         Qual            7. Ergotaminine         7.9         582.2         223.1         144         34         5         1         Positive         Quant            7. Ergotaminine         7.9         582.2         208.1         144         50         5         1         Positive         Qual            8. Ergocorninine         8.3         562.2         305.1         140         30         5         1         Positive         Qual            8. Ergocorninine         8.3         562.2         277.1         140         30         4         1         Positive         Qual            8. Ergocorninine         8.3         562.2         223.1         140         38         5         1         Positive         Quant            9. Ergocryptinine         8.9         576.2         305.1         138         30         5         1         Positive         Qual            9. Ergocryptinine         8.9         576.2         291.1         138         30         4         1         Positive         Qual            9. Ergocryptinine         8.9         576.2         223.1         138         38         5         1         Positive         Quant            10. Ergocristinine         9.1         610.2         305.1         139         30         5         1         Positive         Qual            10. Ergocristinine         9.1         610.2         268.0         139         24         7         1         Positive         Qual            10. Ergocristinine         9.1         610.2         223.1         139         38         5         1         Positive         Quant            11. Dihydroergocristine         6.6         612.8         350.1         178         26         4         1         Positive         Quant            11. Dihydroergocristine         6.6         612.8         270.1         178         34         4         1         Positive         Qual            11. Dihydroergocristine         6.6         612.8         253.1         178         38         4         1         Positive         Qual      
 
SECTION: LC-MS QQQ Method
21. QQQ Time
To minimize the matrix going into the spray chamber, the HPLC flow should be diverted into waste from 10 minutes to the end of the analysis.
 
     #      Start time      Scan type      Div Valve      Delta EMV (+)      Delta EMV (-)      Stored        1      0      Dynamic MRM      To MS      500      0      x        2      10      Dynamic MRM      To Waste      0      0      x   
 
SECTION: DATA ANAYSIS
22. Anaylze data in mass hunter software.Note ISTD is run as analyte and not used in quantitation.
SECTION: DATA ANAYSIS
23. Analyze Batch.

SECTION: DATA ANAYSIS
24. The calibration curve is linear, ignore origin and weighed 1/x for all compounds.
SECTION: DATA ANAYSIS
25. Navigate thru Batch table to see compounds, curves and results.
SECTION: DATA ANAYSIS
26. Check integration of peaks and verify if compounds are correctly identified.
SECTION: DATA ANAYSIS
27. Check integration and response of the ISTD in each sample and standards.Verify that each sample has been injected.
SECTION: DATA ANAYSIS
28. Total Ergots is set up as a MRM compound in Mass Hunter Quant software Edit and is set up to add up all of the Ergots.It is shown in the quant results table and reported out as Total Ergot.
SECTION: CALCULATIONS
29. Final extract is .0625 equivalence so the value from the curve needs to be multiplied by 16. 

SECTION: CALCULATIONS
30. The standard curve is set to detect the following range: 16 ppb to 8000ppb.The detection limit is the lowest standard run.
SECTION: CALCULATIONS
31. LLOQ (Lower limit of Quantification): 20 ppb
SECTION: CALCULATIONS
32. ULOQ (Upper limit of Quantification): 8000 ppb