help on accurately preparing 1ug/ml drug in oil mixture

[darasc0211]

Hi,

I am working in an analytical lab and need to prepare a 1ug/ml formulation (1mg drug in 1 Liter of 0.1% BHT in Oleic acid). In order to precisely reach this super low concentration, I believe I need to weight the 1 L 0.1% BHT in Oleic acid due to the viscosity, how do I accuratelly weigh it? Or please provide other good ideas on accurately prepare this formulation?

Thanks a lot.

[gstaepels]

Hi,

I would go for a series of dilutions.
Example: 100 mg drug (weighted accurately and record the weight) dissolved in 100 ml in a volumetric flask (=1000 mg/l). 5 ml of this solution is pipetted in a volumetric flask and diluted to 100 ml (= 50 mg/l). 5 ml of this solution is pipetted in a 250 ml volumetric flask and diluted (= 1 mg/l).
Your weighting, pipetting and filling the volumetric flask are very important. You can calculate the accurate concentration, if you take the initial weigth of your sample into account.

[darasc0211]

Hi,

Thanks for your reply.

But the thing is the solution is oil based so it's not accurate to use pipet. And it will stick on the glassware therefore won't be completely transferred by pouring or pipetting. Any other suggestions.

Thank.

[Peter Apps]

Weighing the oil is sound practise, but weighing 1 mg of drig is likely to be challenging unless you have a six- figure balance, so you do need to do serial dilutions. The dilution scheme will depend on what the smallest mass is that you can weigh with sufficient accuracy.

Peter

[darasc0211]

Thanks for both of your responses. Because I have two doses (300ug/ml and 1ug/ml, both use 0.1% BHT in oil as vehicle), I am thinking of diluting from 300ug/ml, but every step has to be weighed instead of measuring. Her is my plan.

1. 300ug/ml high dose formulation preperation:
weigh 3mg API into a container
add 0.89g/ml(oil density) x10ml=8.9g oil (equals 10ml oil), so API conc=3/10ml=300ug/ml
Add 10mg BHT, so BHT con=10mg/10ml=1mg/ml=0.1%
This is 300ug/ml drug in 0.1%BHT in oil formulation

2. prepare 1ug/ml low dose formulation:
Assuming high dose density is close to oil density 0.89g/ml, so weigh 0.89g (equal to 1ml) of 300ug/ml formulation,
add 299mlx.89g/ml=266.11g of oil, API concentraion =1mlx300ug/ml / (299ml+1ml)=1ug/ml
then add 300mg BHT. BHT conc=300mg/300ml=1mg/ml=0.1%
(this is 1ug/ml drug in 0.1%BHT in oil formulation)

The calculation is based on assumption that solution density is not significantly affected by adding BHT. But I am worried after adding BHT, there will be some kind of affect to my final concentration?

I think I can apply a correction factor to the API weight by calculating the actual formulation concentration after HPLC test.

But is there a better way to accurately do this?

Thanks a lot.

[HW Mueller]

Neither of your two proposals correspond to the methods to express concentrations which chemists have been using for ages. There is the Molarity (mol, etc., in 1L solution, not 1l solvent) and Molality (mol, gram in 1kg solvent). If you think you can not use volumetric instruments then use Molality.

[Peter Apps]

You can eliminate all uncertainties about densities by doing everything on a mass basis, and expressing "concentrations" as mass fractions (i.e. mg/Kg etc).

What balance are you proposing to use ?

Peter

[darasc0211]

I will use a micro balance to weight the API, (up to 0.001mg accuracy), and a large scale balance for the excipients. That's all we have in our lab.

Thanks.

[darasc0211]

You can eliminate all uncertainties about densities by doing everything on a mass basis, and expressing "concentrations" as mass fractions (i.e. mg/Kg etc).

What balance are you proposing to use ?

Peter

Peter:

Could you give an example as of how to prepare the 1ug/ml from 300ug/ml via expressing "concentrations" as mass fractions (i.e. mg/Kg etc)? I am still confused

Thanks a lot.

[Peter Apps]

A balance reading to micrograms is the right tool for this job. If you have a certified mass piece of 3 mg check weigh it immediatley before and after you weigh your API.

Mass fractions are simple: if you weigh 3 mg of API and dissolve it in 8.9 g of oil (with BHT at 0.1%), as you propose, you have a mass fraction of 3mg/8.9g = 0.3371mg/g. If you take 1g of that solution and add clean oil (with BHT at 0.1%) to a total weight of 300 g you have diluted by the same factor as you propose, and your final mass fraction is 1.1236 ug/g, or 1.0000 ug/ml, which is what you wanted.

Note here that there are no assumptions about densities of oil plus BHT or oil plus API.

When you prepare your samples (which presumably are also in oil) do that by weight as well, run your calibrations as mass fractions (not concentrations), and you will get the results for the samples as mass fractions. If you want them as concentrations (i.e. mass of API per volume of sample) then correct for the densitiy of the samples (which is the density of the oil).

Throught the process, rather than wasting time getting to exact target weights (which is equivalent to making a volume to the mark) get close to it and record the actual weight (which has no equivalent with volumetric glassware), and then use that actual value in all the calculations. With a spreadsheet this is fast, easy and accurate.

Peter

[darasc0211]

Hi, Peter:

Thanks for the detailed explaination and this is very helpful. I added my understandings to your suggestions in Blue. Could you give me a yes/no confirmation to each item and also answer to my questions. I only have 20mg of drug to play with for the whole development so I have to make sure I don't waste any drug. I am very appreciated to your kind help.

A balance reading to micrograms is the right tool for this job. If you have a certified mass piece of 3 mg check weigh it immediatley before and after you weigh your API. (We have a balance ranging up to 31 mg, and read up to 3 decimal points. I think that should do the job you described below)

Mass fractions are simple: if you weigh 3 mg of API and dissolve it in 8.9 g of oil (with BHT at 0.1%). (Here it's still assumed that the density of 0.1% BHT in oil is the same as the density of the oil (0.89g/ml), and that's why you dissolve in 8.9g. Here is what I am thinking: prepare 0.1% BHT in oil, weight some amount of 0.1% BHT in oil in a volumetric flask and calculate the density. Because volumetric flask has a precise volume, the density should be precise. Based on this density I can decide the weight of the oil(with BHT at 0.1%). What do you think?

as you propose, you have a mass fraction of 3mg/8.9g = 0.3371mg/g. If you take 1g of that solution and add clean oil (with BHT at 0.1%) to a total weight of 300 g you have diluted by the same factor as you propose, and your final mass fraction is 1.1236 ug/g, or 1.0000 ug/ml, which is what you wanted. (This is a smart way! From here on the density doesn't matter any more. This should be my bulk blend which will be used to fill the capsules)

Note here that there are no assumptions about densities of oil plus BHT or oil plus API. (density still needs to be decided at the first step but later one it's not necessary).

When you prepare your samples (which presumably are also in oil) do that by weight as well,

( Please let me know which one do you mean by "prepare your samples" and if my approach below is appropriate:
1. If you are talking about how to fill a capsule, I agree I should do by weight as stated above: the capsule I'll use can hold 1ml solution, so I'll weigh 1g of solution which should contain 1ug or 300ug of API depending on which bulk solution I will use, and inject to each capsule and seal.
2. If you are talking about extracting sample into diluent and get ready for the HPLC analysis, my plan is to drop each capsule into a volumetric flask. From the weight of solution weighed into each capsule and the API(mg)/total solution(g) ratio of the bulk solution, I can calculate the API amount in Volumetric flask. I also know the final volume because I am using a volumetric flask. So I can get the theoretical concentration and don't need to calibrate as mass fraction, is that correct?)

run your calibrations as mass fractions (not concentrations) and you will get the results for the samples as mass fractions. If you want them as concentrations (i.e. mass of API per volume of sample) then correct for the densitiy of the samples (which is the density of the oil).
(I don't think I need mass fractions, See same reason in 2 above)

Throught the process, rather than wasting time getting to exact target weights (which is equivalent to making a volume to the mark) get close to it and record the actual weight (which has no equivalent with volumetric glassware), and then use that actual value in all the calculations. With a spreadsheet this is fast, easy and accurate.

Peter

[Peter Apps]

Hi, Peter:

Thanks for the detailed explaination and this is very helpful. I added my understandings to your suggestions in Blue. Could you give me a yes/no confirmation to each item and also answer to my questions. I only have 20mg of drug to play with for the whole development so I have to make sure I don't waste any drug. I am very appreciated to your kind help.

A balance reading to micrograms is the right tool for this job. If you have a certified mass piece of 3 mg check weigh it immediatley before and after you weigh your API. We have a balance ranging up to 31 mg, and read up to 3 decimal points. I think that should do the job you described below)[/color]3 decimals of mg is ug which is plenty of resolution for what you need

Mass fractions are simple: if you weigh 3 mg of API and dissolve it in 8.9 g of oil (with BHT at 0.1%). (Here it's still assumed that the density of 0.1% BHT in oil is the same as the density of the oil (0.89g/ml), and that's why you dissolve in 8.9g. Here is what I am thinking: prepare 0.1% BHT in oil, weight some amount of 0.1% BHT in oil in a volumetric flask and calculate the density. Because volumetric flask has a precise volume, the density should be precise. Based on this density I can decide the weight of the oil(with BHT at 0.1%). What do you think? If you are going to put oil+BHT into a volumetric flask to determine density then you can just as well do this step volumetrically; weigh 3 mg of API into the volume of oil+BHT. It might have been clearer if I had suggested using 10g of oil+BHT and adding 3.371 mg of API - if you want to work to a mass per volume concentration by working gravimetrically you have to know the density of the matrix before you start

as you propose, you have a mass fraction of 3mg/8.9g = 0.3371mg/g. If you take 1g of that solution and add clean oil (with BHT at 0.1%) to a total weight of 300 g you have diluted by the same factor as you propose, and your final mass fraction is 1.1236 ug/g, or 1.0000 ug/ml, which is what you wanted. (This is a smart way! From here on the density doesn't matter any more. This should be my bulk blend which will be used to fill the capsules)

Note here that there are no assumptions about densities of oil plus BHT or oil plus API. (density still needs to be decided at the first step but later one it's not necessary). you need to knw the density before you start

When you prepare your samples (which presumably are also in oil) do that by weight as well,

( Please let me know which one do you mean by "prepare your samples" and if my approach below is appropriate:
1. If you are talking about how to fill a capsule, I agree I should do by weight as stated above: the capsule I'll use can hold 1ml solution, so I'll weigh 1g of solution which should contain 1ug or 300ug of API depending on which bulk solution I will use, and inject to each capsule and seal.This is not what I meant by sample prep. It will not work as stated, 1 g of oil is 1.12 ml which is too much for the capsule
2. If you are talking about extracting sample into diluent and get ready for the HPLC analysis, my plan is to drop each capsule into a volumetric flask. From the weight of solution weighed into each capsule and the API(mg)/total solution(g) ratio of the bulk solution, I can calculate the API amount in Volumetric flask. I also know the final volume because I am using a volumetric flask. So I can get the theoretical concentration and don't need to calibrate as mass fraction, is that correct?)Since you know the amount of API in the capsule, and therefore in the solution, why do you need to measure it by HPLC ?

run your calibrations as mass fractions (not concentrations) and you will get the results for the samples as mass fractions. If you want them as concentrations (i.e. mass of API per volume of sample) then correct for the density of the samples (which is the density of the oil).
(I don't think I need mass fractions, See same reason in 2 above) I do not see a calibration (in the sense of plotting detector response vs amount of API) anywhere in your plan

Throughout the process, rather than wasting time getting to exact target weights (which is equivalent to making a volume to the mark) get close to it and record the actual weight (which has no equivalent with volumetric glassware), and then use that actual value in all the calculations. With a spreadsheet this is fast, easy and accurate.

Peter

[darasc0211]

Hi, Peter:

I have some more thoughts below. Please comment. Thanks a lot.

Hi, Peter:

Thanks for the detailed explaination and this is very helpful. I added my understandings to your suggestions in Blue. Could you give me a yes/no confirmation to each item and also answer to my questions. I only have 20mg of drug to play with for the whole development so I have to make sure I don't waste any drug. I am very appreciated to your kind help.

A balance reading to micrograms is the right tool for this job. If you have a certified mass piece of 3 mg check weigh it immediatley before and after you weigh your API. We have a balance ranging up to 31 mg, and read up to 3 decimal points. I think that should do the job you described below)[/color]3 decimals of mg is ug which is plenty of resolution for what you need

Mass fractions are simple: if you weigh 3 mg of API and dissolve it in 8.9 g of oil (with BHT at 0.1%). (Here it's still assumed that the density of 0.1% BHT in oil is the same as the density of the oil (0.89g/ml), and that's why you dissolve in 8.9g. Here is what I am thinking: prepare 0.1% BHT in oil, weight some amount of 0.1% BHT in oil in a volumetric flask and calculate the density. Because volumetric flask has a precise volume, the density should be precise. Based on this density I can decide the weight of the oil(with BHT at 0.1%). What do you think? If you are going to put oil+BHT into a volumetric flask to determine density then you can just as well do this step volumetrically; weigh 3 mg of API into the volume of oil+BHT. It might have been clearer if I had suggested using 10g of oil+BHT and adding 3.371 mg of API - if you want to work to a mass per volume concentration by working gravimetrically you have to know the density of the matrix before you start. One more question here: If I want to start with mass fraction instead of mass per volume, theoretically I should weigh 3 mg of API and dissolve it in 8.91 g of oil (with BHT at 0.1%) instead of 8.9g. Because 8.9g is the weight of oil only (0.89g/mlx10ml), and the weight of BHT is 0.01g which is 0.1% of oil (i.e. 1mg/ml), therefore the total should be 8.91g. Although in reality this may not matter because 8.91 and 8.90 is not much different, but am I thinking right? Please comment. as you propose, you have a mass fraction of 3mg/8.9g = 0.3371mg/g. I think here I should use a mass fraction of 3mg/(8.91+0.003)g=3mg/8.913g=0.3366mg/g, because 8.913g is actually the weight of all the components in the solution. Comments? If you take 1g of that solution and add clean oil (with BHT at 0.1%) to a total weight of 300 g you have diluted by the same factor as you propose, and your final mass fraction is 1.1236 ug/g, or 1.0000 ug/ml, which is what you wanted. (This is a smart way! From here on the density doesn't matter any more. This should be my bulk blend which will be used to fill the capsules)

Note here that there are no assumptions about densities of oil plus BHT or oil plus API. (density still needs to be decided at the first step but later one it's not necessary). you need to knw the density before you start

When you prepare your samples (which presumably are also in oil) do that by weight as well,

( Please let me know which one do you mean by "prepare your samples" and if my approach below is appropriate:
1. If you are talking about how to fill a capsule, I agree I should do by weight as stated above: the capsule I'll use can hold 1ml solution, so I'll weigh 1g of solution which should contain 1ug or 300ug of API depending on which bulk solution I will use, and inject to each capsule and seal.This is not what I meant by sample prep. It will not work as stated, 1 g of oil is 1.12 ml which is too much for the capsule
2. If you are talking about extracting sample into diluent and get ready for the HPLC analysis, my plan is to drop each capsule into a volumetric flask. From the weight of solution weighed into each capsule and the API(mg)/total solution(g) ratio of the bulk solution, I can calculate the API amount in Volumetric flask. I also know the final volume because I am using a volumetric flask. So I can get the theoretical concentration and don't need to calibrate as mass fraction, is that correct?)Since you know the amount of API in the capsule, and therefore in the solution, why do you need to measure it by HPLC ?My client requested an HPLC analysis on Assay and impurities on these two formulations.
run your calibrations as mass fractions (not concentrations) and you will get the results for the samples as mass fractions. If you want them as concentrations (i.e. mass of API per volume of sample) then correct for the density of the samples (which is the density of the oil).
(I don't think I need mass fractions, See same reason in 2 above) I do not see a calibration (in the sense of plotting detector response vs amount of API) anywhere in your plan

Throughout the process, rather than wasting time getting to exact target weights (which is equivalent to making a volume to the mark) get close to it and record the actual weight (which has no equivalent with volumetric glassware), and then use that actual value in all the calculations. With a spreadsheet this is fast, easy and accurate.
The target dose is 1ug API in each capsule, so I think I have to get to the exact target weight.

Peter

[Peter Apps]

So, to summarize so far:

Your client wants you to put exactly 1ug of API into each capsule, then take it out again and assay it for API and impurities. And the customer is always right !

You can break this down into two parts: part 1 putting the API and carrier into the capsule, part 2 assaying API and imputities.

The API that goes into the capsule has to be dissolved in oil+BHT. As long as there does not have to be exactly 1 ml of oil in the capsule you can make gravimetric dilutions similar to the scheme we have already sorted out, recording actual weights at each stage, calculate the mass fraction of API in carrier, and then weigh an appropriate amount of API in carrier into each capsule to give you 1 ug of API inthe capsule. That is just simple arithmatic.

If you want to assay the API in each capsule you have to calibrate the analysis, which is not part of your current plan.

What concentrations of impurities do you want to detect ? Say 1% for argument's sake, you then have to detect 1% of 1ug of impurity = 10 ng of impurity in each sample. Can you do that ? And of course that analysis has to be calibrated as well.

Peter

[kwfx]

Hi, Peter:

Now the client agreed to increase the dose to 3ug/ml so I though I could directly weigh and dilute. Here is what I did:

1. prepare a solution containing 8.9g OA and 10mg BHT, 2. decide the density of the solution using volumetric flask, 3. weight 0.3mg API and add API along with weighing boat in the container, 4. add density x 100 ml of the solution prepared in step 1 to the API.

But this doesn't give me a very good result. I have 109%, 85%, 102% so they are not consistent. Maybe determine density with volumetric flask was not accurate enough??

Next I will completely ignore density determination and follow what we've discussed by diluting from a higher concention solution by w/w. i.e.:
1. weigh 3mg and add a solution containing 8.9g OA and 10mg BHT in solution, w/w=3mg/(8.9g OA+0.01g BHT+0.003gAPI) =3/8.913=0.3366mg/g=336.6ug/g (Could you check this calculation for me? I mentioned in my previous email but you didn't address it. This will directly affect my calculation on HPLC assay% result. Thanks).
2. then weigh 1g of this solution and add up to total of 100g.--this should be 3ug/ml formulation, so w/w=336.6/100=3.36ug/g.
3. weigh 1g of 3.36ug/g into a capsule, I think I can accurately weigh 1g so my final API in capsule shouldn't differ too much.
4. Add 1 capsule to volumetric flask and extract with 0.2% TFA and Ethanol and analyze by HPLC. I am also in the process of developing a stability indicating method.

Could you confirm with my calculation again.

I appreciate your help.