Also check https://www.mdpi.com/2072-6651/11/5/297/pdf

Kindly check https://core.ac.uk/download/pdf/12042031.pdf

Also check https://www.mdpi.com/2072-6651/11/5/297/pdf

Dear Dr. Abdellaoui,

starting with 5 gr of sample, and considering that fumonisins does not stick to any of the surfaces, you have 5 grams in 25 ml of MeOH, so you have 0.2 gram per ml. Then you take 10 ml out of 25 (corresponding to 2 grams of sample). Then, you mix 10 ml of this solution with 40 ml PBS (so you have 0.04 gram per ml). You take 10 ml of this mix, corresponding to 0.4 gram. Once dried and redissolved in 400 microliters you have 0.4 gram in 400 microliters, that is i mg per microliter. You take 30 microliters, add 30 microliters of OPA, so you have 0.5 mg per microliter. If you inject 30 microliters of this solution you inject the corresponding to 15 mg of sample.

Filtered extracts before or after purification (2μ l) and stan-dards were spotted on thin-layer chromatographic plates(Merck n°5553, VWR, Fontenay sous bois, France).Separation was carried out in 1-butanol-acetic acid-water(20+10+10, v/v/v). After a final drying step, plates weresprayed with a mixture containing methanol, p-anisalde-hyde, acetic acid and sulfuric acid (85-0.5-10.5 vol) and hea-ted 10 min at 110°C.Quantification was performed by using a spectrophotoden-sitometer (Shimadzu CS930, Shimadzu Corp., Kyoto, Japan)set at 600 nm. The system was coupled to a personal compu-ter data processing system. Linear response was obtainedover a range of 50-800 ng FB1. For each plate, 9 extractswere spotted, after dilution if necessary, along with 4 diffe-rent standard amounts of FB1 (50, 100, 200 and 400 ng). Thedetection limits in culture material were 30-50 μ g/g of initialdry corn when crude extracts were spotted, and 3-5 μ g/g ifextracts were purified. (PDF) Production and purification of fumonisins from a highly toxigenic Fusarium verticilloides strain. Available from: https://www.researchgate.net/publication/44104396_Production_and_purification_of_fumonisins_froma_highly_toxigenic_Fusarium_verticilloides_strain [accessed Dec 02 2020].

Alessandra Ricelli thank you Dr Alessendra for your answer. Just I am curious, because while making standard we used the same method such adding 30 ul of OPA to 30 ul of standard, then injected 30 ul of mix. so is it possible omit the last part of dilution through use of OPA for sample? also based on calculation of sample concentration using standard curve which its unit is expressed by ppm, should we use that value and multiplied by the 1000 mg and divided by b15 mg to get the amount per gram of sample? sorry for my questions.

Najib Abdellaoui

let me try to answer with a different approach (do correct me if I am wrong)

Suppose you spike 5 mg of fumonisin in this 5 g of sample (maybe grain like corn, which is certified free of fumonisin) as part of a recovery study.

1.Ground this 5 g of sample with 25 mL of methanol: ACN: water (25:25:50). (you still have 5 mg of fumonisin here)

2. Shake for 20 min and centrifuge for 10 min at 2500 xg. (assuming no losses, you still have 5 mg in the supernatant)

3. Filter supernatant through filter paper. (assuming no losses, you still have 5 mg)

4.Take 10 mL of extract, mix with 40 mL of PBS and filter through microfiber filter. (10 mL out of 25 mL, means you now have 2 mg in the 10 mL)

5.Pass 10 mL of diluted extract through Vicam column (all fumonisin will be trapped here), wash column using 10 mL of PBS, and elute fumonisin by passing 1.5 mL of HPLC grade methanol. collect the elute in vial. (assuming no losses, you still have 2 mg)

6.Dry down methanol elute. (assuming no losses, you still have 2 mg)

7.Redissolves elute in 400 uL of ACN: water (50:50). (you still have 2 mg)

8.Mix 30 uL of well-mixed redissolved elute with 30 uL OPA reagent. (taking 30 uL out of 400 uL means you have (30/400)* 2 mg = 0.15 mg here in the 30 uL)

9.Inject 30 uL. (now you are injecting half of the 60 uL or 0.075 mg)

How to calculate the dilution factor for this protocol to get amount of mycotoxin per g of sample?

Answer: The dilution factor is 5 mg/0.075 mg=66.67 times.

Whatever the results from new sample of 5 g following the above protocol, you need to times them by 66.67 to get the amount of fumonisin in 5 g of sample (if per g divide by 5)

Note: in recovery studies your percentage recovery limit range is usually from 96 to 104%.

M. Y. Shukor thanks a lot for your detailed explanation, I have few questions if possible:

-while making the standard, we used same amount of OPA 30 ul plus 30 ul of different concentration. so while calculating the dilution factor, is it ok to omit the last step while injecting?

-while making the standard curve, is it ok to use ppm as unit or should use concentration as unit ug/mL?.

Najib Abdellaoui Suppose you prepare a series of standard concentration of fumonisin from 1 to 5 mg/L in a final volume of 30 uL. Then you add 30 uL of OPA for each concentration and only inject 30 uL. Since the sample and the standards are treated the same manner, you can ignore the the contribution of OPA in your calculation if you work with concentration. The dilution factor I used above deals with thinking in the amount basis. If you wish to work with concentration, you have to rework from the beginning but you have to be careful. The HPLC standard curve can both be presented with either amount or concentration as the x-axis unit. I prefer to work with amount and converts it to concentration later.

In terms of ppm (mg/L) as the concentration of choice, this is equivalent to the concentration unit ug/mL.

M. Y. Shukor thanks a lot for your answer, just to be clear when you use the term amount are you referring to ppm unit? for standard curve, is it better to use ppm as unit?

Najib Abdellaoui ppm is concentration not amount. ppm is mg per L or ug per mL. For standard curve you can use ppm or amount in the form of ug Fumonisin. The trick is to know your calculations, which can be a headache but must be mastered.

M. Y. Shukor thanks a lot for your help and detailed explanation. as you mentioned, I got confused for which unit to use amount or concentration ppm or ug/ml.

again thanks a lot.

M. Y. Shukor in step 4, we mix 10 ml of extract with 40 ml of PBS, then took 10 ml of it and pass it through column. so the amount should be 0.4 mg instead of 2 mg. is it correct?.

Najib Abdellaoui Yes. Since the dilution factor here is 1/(10/(10+40))=5 and 0.4 is a fifth of 2.0 mg. My suggestion is to pass all of the 50 ml total mixture into the column provided that the column can adsorb all of the fumonisin. This will definitely increase the detection limit of the system.

M. Y. Shukor we followed the protocol for the extraction of fumonisin. so I will follow your suggestion to get a more accurate results. thank you for your time and help.