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How do I construct a calibration curve for plasma spiked drug samples?
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Kevin Thorpe
How do I construct a calibration curve for plasma spiked drug samples?
plasma error percentage can be easily determined by dividing area of drug in plasma over area of drug in solvent without plasma multiplying by 100, so high percentage include high extraction efficiency of drugs in that plasma applying concentration of drug and drug area.
plasma error percentage can be easily determined by dividing area of drug in plasma over area of drug in solvent without plasma multiplying by 100, so high percentage include high extraction efficiency of drugs in that plasma applying concentration of drug and drug area.
From my experience, the calibration curve should be prepared by spiking an appropriate amount of the drug solutions into the blank plasma to obtain desired final concentrations. Then, the spiked plasma smaples for the calibration curve will be extracted/processed as same as study samples. I think there is no gold standard for liquid-liquid extraction. You just have to try several extraction procedures until you obtain the extraction method that give a high recovery and reproducibility. Evaporation of the extraction solvent is carried out if the extraction solvent is not compatible with the separation/detection method e.g. HPLC. Moreover, evaporation allow you to concentrate your sample. You can evaporate all extraction solvent and reconstitute your samples with a suitable amount and type of reconstitution solvent.
From my experience, the calibration curve should be prepared by spiking an appropriate amount of the drug solutions into the blank plasma to obtain desired final concentrations. Then, the spiked plasma smaples for the calibration curve will be extracted/processed as same as study samples. I think there is no gold standard for liquid-liquid extraction. You just have to try several extraction procedures until you obtain the extraction method that give a high recovery and reproducibility. Evaporation of the extraction solvent is carried out if the extraction solvent is not compatible with the separation/detection method e.g. HPLC. Moreover, evaporation allow you to concentrate your sample. You can evaporate all extraction solvent and reconstitute your samples with a suitable amount and type of reconstitution solvent.
In my opinion, it's quite strange to prepare a calibration curve like that. The matrix/preparation of calibration standards should be the same as your study samples. I'm not sure what is your expected ULOQ (or the highest concentration in the calibration curve) but the concentration at 1 mg/mL is too high for me. If I were you, I would prepare a stock meloxicam solution at 1 mg/mL in an organic solvent like acetonitrile or methanol. Then, I will dilute the stock solution about 10-20 times with the same organic solvent or 50% organic solvent in water to obtain an intermediate stock meloxicam solution. From the intermediate stock meloxicam solution, I will serially dilute to obtain spiking solutions which are going to be spiked to blank plasma. The total spiking volume is less than 5% of the final volume. At this point, I can adjust the concentrations of spiking solutions that give the desired final concentrations of meloxicam in plasma. My calibration curve will be constructed using spiked plasma. So, my calibration standards are processed as same as my study sample.
In my opinion, it's quite strange to prepare a calibration curve like that. The matrix/preparation of calibration standards should be the same as your study samples. I'm not sure what is your expected ULOQ (or the highest concentration in the calibration curve) but the concentration at 1 mg/mL is too high for me. If I were you, I would prepare a stock meloxicam solution at 1 mg/mL in an organic solvent like acetonitrile or methanol. Then, I will dilute the stock solution about 10-20 times with the same organic solvent or 50% organic solvent in water to obtain an intermediate stock meloxicam solution. From the intermediate stock meloxicam solution, I will serially dilute to obtain spiking solutions which are going to be spiked to blank plasma. The total spiking volume is less than 5% of the final volume. At this point, I can adjust the concentrations of spiking solutions that give the desired final concentrations of meloxicam in plasma. My calibration curve will be constructed using spiked plasma. So, my calibration standards are processed as same as my study sample.
Thanks for the response. If my standard drug solutions are prepared in the mobile phase and my plasma spiked samples are prepared in a different way (e.g. addition of organic solvent + centrifugation + evaporation + addition of mobile phase), then would this be a problem in the study design? Because I have found significant differences in the peak area using two different drug standard procedures at the same concentration. When I employed the evaporation step in the preparation of drug standards, the peak area was greater, whilst the other step gave me a lower PA value (and could not calculate extraction efficiency since the PA was lower than that of the plasma spiked sample). How do we determine the volume of reconstitution solvent? Will that affect the concentration of the sample?
Thanks for the response. If my standard drug solutions are prepared in the mobile phase and my plasma spiked samples are prepared in a different way (e.g. addition of organic solvent + centrifugation + evaporation + addition of mobile phase), then would this be a problem in the study design? Because I have found significant differences in the peak area using two different drug standard procedures at the same concentration. When I employed the evaporation step in the preparation of drug standards, the peak area was greater, whilst the other step gave me a lower PA value (and could not calculate extraction efficiency since the PA was lower than that of the plasma spiked sample). How do we determine the volume of reconstitution solvent? Will that affect the concentration of the sample?
Q. Dear, Sir, I have to construct a Meloxicam calibration curve for Rat plasma spiked drug samples in 100ml solution for getting 1000 µg/ml first stock solution from that I will prepare different serial dilution, for getting 1000 µg/ml plasma spiked drug samples first stock solution, I am adding 100mg drug into 99ml solution and then adding 1ml of extracted plasma sample, am I going to correct way? please answer me.
Q. Dear, Sir, I have to construct a Meloxicam calibration curve for Rat plasma spiked drug samples in 100ml solution for getting 1000 µg/ml first stock solution from that I will prepare different serial dilution, for getting 1000 µg/ml plasma spiked drug samples first stock solution, I am adding 100mg drug into 99ml solution and then adding 1ml of extracted plasma sample, am I going to correct way? please answer me.
plasma error percentage can be easily determined by dividing area of drug in plasma over area of drug in solvent without plasma multiplying by 100, so high percentage include high extraction efficiency of drugs in that plasma applying concentration of drug and drug area.
plasma error percentage can be easily determined by dividing area of drug in plasma over area of drug in solvent without plasma multiplying by 100, so high percentage include high extraction efficiency of drugs in that plasma applying concentration of drug and drug area.
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From my experience, the calibration curve should be prepared by spiking an appropriate amount of the drug solutions into the blank plasma to obtain desired final concentrations. Then, the spiked plasma smaples for the calibration curve will be extracted/processed as same as study samples. I think there is no gold standard for liquid-liquid extraction. You just have to try several extraction procedures until you obtain the extraction method that give a high recovery and reproducibility. Evaporation of the extraction solvent is carried out if the extraction solvent is not compatible with the separation/detection method e.g. HPLC. Moreover, evaporation allow you to concentrate your sample. You can evaporate all extraction solvent and reconstitute your samples with a suitable amount and type of reconstitution solvent.
From my experience, the calibration curve should be prepared by spiking an appropriate amount of the drug solutions into the blank plasma to obtain desired final concentrations. Then, the spiked plasma smaples for the calibration curve will be extracted/processed as same as study samples. I think there is no gold standard for liquid-liquid extraction. You just have to try several extraction procedures until you obtain the extraction method that give a high recovery and reproducibility. Evaporation of the extraction solvent is carried out if the extraction solvent is not compatible with the separation/detection method e.g. HPLC. Moreover, evaporation allow you to concentrate your sample. You can evaporate all extraction solvent and reconstitute your samples with a suitable amount and type of reconstitution solvent.
More
VOTE
In my opinion, it's quite strange to prepare a calibration curve like that. The matrix/preparation of calibration standards should be the same as your study samples. I'm not sure what is your expected ULOQ (or the highest concentration in the calibration curve) but the concentration at 1 mg/mL is too high for me. If I were you, I would prepare a stock meloxicam solution at 1 mg/mL in an organic solvent like acetonitrile or methanol. Then, I will dilute the stock solution about 10-20 times with the same organic solvent or 50% organic solvent in water to obtain an intermediate stock meloxicam solution. From the intermediate stock meloxicam solution, I will serially dilute to obtain spiking solutions which are going to be spiked to blank plasma. The total spiking volume is less than 5% of the final volume. At this point, I can adjust the concentrations of spiking solutions that give the desired final concentrations of meloxicam in plasma. My calibration curve will be constructed using spiked plasma. So, my calibration standards are processed as same as my study sample.
In my opinion, it's quite strange to prepare a calibration curve like that. The matrix/preparation of calibration standards should be the same as your study samples. I'm not sure what is your expected ULOQ (or the highest concentration in the calibration curve) but the concentration at 1 mg/mL is too high for me. If I were you, I would prepare a stock meloxicam solution at 1 mg/mL in an organic solvent like acetonitrile or methanol. Then, I will dilute the stock solution about 10-20 times with the same organic solvent or 50% organic solvent in water to obtain an intermediate stock meloxicam solution. From the intermediate stock meloxicam solution, I will serially dilute to obtain spiking solutions which are going to be spiked to blank plasma. The total spiking volume is less than 5% of the final volume. At this point, I can adjust the concentrations of spiking solutions that give the desired final concentrations of meloxicam in plasma. My calibration curve will be constructed using spiked plasma. So, my calibration standards are processed as same as my study sample.
More
VOTE
Thanks for the response. If my standard drug solutions are prepared in the mobile phase and my plasma spiked samples are prepared in a different way (e.g. addition of organic solvent + centrifugation + evaporation + addition of mobile phase), then would this be a problem in the study design? Because I have found significant differences in the peak area using two different drug standard procedures at the same concentration. When I employed the evaporation step in the preparation of drug standards, the peak area was greater, whilst the other step gave me a lower PA value (and could not calculate extraction efficiency since the PA was lower than that of the plasma spiked sample).
How do we determine the volume of reconstitution solvent? Will that affect the concentration of the sample?
Thanks for the response. If my standard drug solutions are prepared in the mobile phase and my plasma spiked samples are prepared in a different way (e.g. addition of organic solvent + centrifugation + evaporation + addition of mobile phase), then would this be a problem in the study design? Because I have found significant differences in the peak area using two different drug standard procedures at the same concentration. When I employed the evaporation step in the preparation of drug standards, the peak area was greater, whilst the other step gave me a lower PA value (and could not calculate extraction efficiency since the PA was lower than that of the plasma spiked sample).
How do we determine the volume of reconstitution solvent? Will that affect the concentration of the sample?
More
VOTE
Q. Dear, Sir, I have to construct a Meloxicam calibration curve for Rat plasma spiked drug samples in 100ml solution for getting 1000 µg/ml first stock solution from that I will prepare different serial dilution, for getting 1000 µg/ml plasma spiked drug samples first stock solution, I am adding 100mg drug into 99ml solution and then adding 1ml of extracted plasma sample, am I going to correct way? please answer me.
Q. Dear, Sir, I have to construct a Meloxicam calibration curve for Rat plasma spiked drug samples in 100ml solution for getting 1000 µg/ml first stock solution from that I will prepare different serial dilution, for getting 1000 µg/ml plasma spiked drug samples first stock solution, I am adding 100mg drug into 99ml solution and then adding 1ml of extracted plasma sample, am I going to correct way? please answer me.
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