what is the analytical tech? HPLC, GC, GCMS, LCMS, UV Vis, AAS................................?

LCMS-multiple reaction monitering

Hi Sivaleela, try to use Chemometrics, specially PLS1, together with full factorial experimental design ... it is strongly recommended to calibrate and predict the concentration of components in their mixtures ...

HI,

Send me the data and I will do it for you in a Excel sheet, with a several extra explanations that will allow you to do it yourself any time you want.

Miguel

Hi

I think you didn't understand my problem. Actually I want to run calibration curve for the mixture of standards. I have four standards, each standard have different concentration points, suppose for one compound 50 to 5000pg/uL, for the second 25 to 5000pg/uL, for the third 2000 to 5500pg/uL, for the fourth 100 to 5000pg/ul. I have to select eight concentration points per compound in between these ranges. I want to run only one calibration curve for the four compounds with eight concentration points.

for example the starting point is

50pg/uL for compound 1

25pg/uL for the compound 2

2000pg/uL for the compound 3

100pg/uL for the compound 4

In order to do above one I need to prepare all the above mentioned concentrations in one vial. so can you tell me whether I need to prepare individual stocks for the four and then mix into single vial or how to prepare?

Ok, I understand.

Both solutions would work. Maybe the most appropriated one, would be this in which the situation is simulated as in real terms. For example, in the following publication:"Hydrolysis optimization of mannan, curdlan and cell walls from Endomyces fibuliger grown in mussel processing wastewaters" We had the same problem. The hydrolytic compounds of cell walls were formed mainly by glucose and mannose, and we wanted to quantify them. So for the calibration curve in GC-MS we used a mixture of both compounds. We could use single calibration curves. However, we though that both compound (which are very similar) could interfere with each other.

Hope this helps.

M. A. Prieto

If you are using HPLC then for each standared solution you should have separate calibration plot.

Apologize me for my late ... you will find attached Excel file as a sample .. please place your spectral data of absorbance in columns as shown in sheet1, then place concentration data in sheet2 so that every mixture is place in row and is corresponding to every column of spectral data placed in sheet1 .. I mean row 1 (say concentrations 25, 100, 500) is corresponding to column 1, row 2 is corresponding to column 2 ... so on ... noting that the column values represent aborbances at different wavelengths with interval 1 nm or 2 nm .. while in the third sheet involves unknown spectra required to estimate the concentration of every component .. once I recieve your data I will manipulate and process using partial least squares method .. you will find the results at final at sheet 4 ... now, I am waiting you

no my concern is how to prepare single standard mixed solution for the mixture of four compounds, whether i have to prepare different stock solutions for each standard compound separately, and then by spiking each standard into one single flask to prepare mixed standard solution.

I think you got it

hi, if there's no interference among these componds, you can just prepare eight standard solutions with increasing concentration of each componds as you have mentioned above.

Good Luck!

As other mentioned if no interferences analyte each other then prepare standard covering all range required. But prepare blank and standards should be match of sample matrix

If you club all your calibration points you have total 16 points. Preparre 1 mg/mL stock of each solution. ( Compound 4 is 200 µl only. Dont worry, you will need to use just 10 µl from this stock.) .

Protocol:

In a vial containing 960 µL of diluent, spike 10 µl of each stock and mix well. You have a working stock of 100 µg/mL which has all 4 compounds.

Now step wise dilute this solution to get 250 ng/mL i.e 250 pg/µL. 10 times and 100 times dilution of this solution would give 2 spiking stocks of 25 pg/µL and 2.5 pg/µL. Prepare 2 mL of each of this solution.

Now you are ready to prepare final calibration curve by spiking different volumes of these solutions to get your calibration

Calibrants 1 to 8:

pg/µL spiking volume µL (2.5 µL spike soln)

25 10

50 20

100 40

250 100

500 200

750 300

1000 400

1500 600

Calibrants 9 to 16:

pg/µL spiking volume µL (25 µL spike soln)

2000 80

2500 100

3000 120

3500 140

4000 160

4500 180

5000 200

5500 220

Make up volume to 1 mL to get final concentration. Agian, first aliquote diluent in vial first and spike solution and mix.

I had actually listed excel columns. But the final posted data is jumbled. I guess, you will be able to make out.

Thank you so much I got it. I could able to understand what you want to convey.

I think you are close to the point. I want to make only 1 to 8 calibration points but not 1 to 16. The concentrations from 9 to 16 are also should be mixed with the 1 to 8 points. 2000 to 5500pg/uL is for the other compound.

Actually I want to analyse total four compounds together by using UPLC-MS/MS.

The concentration points (1 to 8) for the

compound-1: 25,50,100,250,500,1000,2500,5000pg/uL.

compound-2: 50,100,250,500,1000,1500,2500,5000pg/uL

compound-3: 100,250,500,750,1000,1500,2500,5000pg/uL

compound-4: 2000,2500,3000,3500,4000,4500,5000,5500pg/uL

Suppose if you consider first concentration point in that

the compound-1 should be with 25pg/uL

the compound-2 should be with 50pg/uL

the compound-3 should be with 100pg/uL

the compound-4 should be with 2000pg/uL

similarly the other concentration points also.

I think now you could be able to understand better.

Thanks

How does it matter, whether you run 8 points or 16 points? You have the points what you want in the calibration curve. After running all calibration points along with sample, you can choose different points for quantifying different compounds.

Hi All

Mr. Sivaleela Tallagadda you must differ between compound & Standard solution.

I understood what you want. you want to say :

comp. 1 2 3 4

Std. ________________pg/uL_______________

1 25 50 100 2000

2 50 100 250 2500

3 100 250 500 3000

4 250 500 750 3500

5 500 1000 1000 4000

6 1000 1500 1500 4500

7 2500 2500 2500 5000

8 5000 5000 5000 5500

here you had more than one concentration in one std. so you must prepare 4 stock solutions for 4 comp. one stock for one comp. for example stock for comp. 1 with concentration 10000 pg/uL

stock for comp. 2 with concentration 10000 pg/uL

stock for comp. 3with concentration 10000 pg/uL

stock for comp. 4with concentration 10000 pg/uL.

now use the dilution law C1*V1 = C2*V2 to know the volume which you must take from stock.

example to prepare std. 1 you must take from comp. 1 stock : 10000*V1(uL) = 25*1000(uL) => V1=2.5 uL

, from comp. 2 stock : 10000*V1(uL) = 50*1000(uL) => V1=5 uL

, from comp. 3 stock : 10000*V1(uL) = 100*1000(uL) => V1=10 uL

, from comp. 4 stock : 10000*V1(uL) = 2000*1000(uL) => V1=200 uL

and the volume of diluent is 1000-(2.5+5+10+200) = 782.5 uL

and so that for std. 2 , std. 3 and std. 4

that is the only way you must follow for this concentrations you need.

Thank you

Greetings Mr. Sivaleela Tallagadda,

The above suggestions given by fellow researchers are correct and applicable for your research :-) Since you are about to run these standards in UPLC-MSMS as a mixture of all of them, care should be taken so that they won't interact with each other. Besides, you have to make sure they have different retention time during elution. Good luck yeah.