An analytical method for the quantitation (20-8,000 ppb) of Ergot Alkaloids in Wheat grain.
Michelle Mostrom, Kelly Benson, Brett Webb
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
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/
Before start
Steps
Standard Curve:
Prepare serial dilution
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.
Transfer 50µL of 500Parts per Billion (PPB) standard to poly vial for Standard 10
500 ppb stock standards in individual aliquots for one serial dilution
Make up from reference standards 500 ppb solution of 500 ppb of each ergot alkaloids.
Transfer 150 ul of 500ppb solution to amber vials and dry down under nitrogen.
Store these vials in freezer (-20C)
To use take out one vial and let warm to room temperature.
From your ISTD 100 PPB Dihyroergocristine in extracting solution make up 800 ul of 50 PPB Dihydroergocristine in extracting solution. (1:1 dilution)
Bring up vial in 150 ul of 50 PPB Dihydroergocistine in extracting solution. Mix well.
Use this to perform serial dilution of 10 standards. Use the same 50 PPB Dihydroergocristine in extracting solution to dilute with during serial dilution.
| A | B | C |
|---|---|---|
| Standard | PPB | 16x PPB |
| 10 | 500 | 8000 |
| 9 | 250 | 4000 |
| 8 | 125 | 2000 |
| 7 | 62.5 | 1000 |
| 6 | 31.2 | 500 |
| 5 | 15.6 | 250 |
| 4 | 7.8 | 125 |
| 3 | 3.9 | 62 |
| 2 | 2.0 | 32 |
| 1 | 1.0 | 16 |
The standard curve has the following ten Calibrators
PROCEDURE for sample preparation
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.
Add 40 mls of Extracting Solution. Cap and invert to mix.
Shake for 30 minutes on Horizontal Shaker at 150 +/- 10 Cycles per minute.
Let extract sit for at least 1 hour.
Filter entire extract thru Whatman 54 filter paper into a new labeled centrifuge tubes.
Transfer 4 mls of filtered extract to the glass tube provided with Mycosep kit.
Push the 4 mls thru Mycosep 150 Ergot Column.
Transfer 500ul of extract to a microcentrifuge tube.
Dilute with 500 ul of 100 PPB Dihydroergocristine in extracting solution
Final concentration of Dihydroergocristine is 50 PPB.
Centrifuge at 10,000 RPM for 5 mins
Transfer 100ul to Polypropylene vial for injection on QQQ.
Finale extract is a 16x dilution of sample.
Prepare 10-point Standard curve by serial dilution and run standard curve with samples daily.
LC-MS QQQ Method
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.
Samples are run with the LC by a method with the following parameters.
UHPLC Column : Agilent Zorbax Eclipse Plus C18 2.1 x 50 mm, 1.8 micron (P.N. 959757-902)
Guard Column: Zorbax Eclipse Plus C18, 2.1 x 5 mm, 1.8 micron, (P.N. 821725-901)
Mobile Phase: A: 3 mM ammonium carbonate in Water B: premixed Acetonitrile (90%) – Water (10%)
Auto Injector Parameters
| A | B |
|---|---|
| Injection Volume | 2 µl |
| Needle Wash | 5 seconds with Acetonitrile |
| Column Temp | 30.00 °C |
Binary Pump:
Flow: 0.200 mL/min
High Pressure Limit: 550 bar
Stop Time: 12.00 min
Post Time: 1.0 min
Gradient Program:
| A | B | C |
|---|---|---|
| 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% |
LC-MS QQQ
| A | B |
|---|---|
| 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 |
MRM transitions
| A | B | C | D | E | F | G | H | I | J |
|---|---|---|---|---|---|---|---|---|---|
| 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 |
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.
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| # | 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 |
DATA ANAYSIS
Anaylze data in mass hunter software.Note ISTD is run as analyte and not used in quantitation.
Analyze Batch.
The calibration curve is linear, ignore origin and weighed 1/x for all compounds.
Navigate thru Batch table to see compounds, curves and results.
Check integration of peaks and verify if compounds are correctly identified.
Check integration and response of the ISTD in each sample and standards.Verify that each sample has been injected.
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.
CALCULATIONS
Final extract is .0625 equivalence so the value from the curve needs to be multiplied by 16.
The standard curve is set to detect the following range: 16 ppb to 8000ppb.The detection limit is the lowest standard run.
LLOQ (Lower limit of Quantification): 20 ppb
ULOQ (Upper limit of Quantification): 8000 ppb
