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Hi! Chromatography experts, I am currently adapting and doing some modifications in a published method in determining glyburide in human plasma.
The published method HPLC conditions are the ff: column: Kingsorb C8;15cm x 4.6 mm i.d;3µm with a C8 precolumn insert C8 (Phenomenex); mobile phase: 45% buffer solution (0.05M NH4H2PO4):40% acetonitrile:15%methanol adjusted to pH 5.7;flow rate 1.0mL/min;excitation wavelength 235nm;emission wavelength 354nm Fluorescence detector. The sample preparation was direct precipitation using acetonitrile and 5% CuSO4 solution in water; injection volume is 25µL. Ketoconazole was used as internal standard. The retention time for ketoconazole and glyburide were 6.7 and 9.6 minutes. The LOQ is 5ng/mL.
In our laboratory, we modify some HPLC conditions, we used a different brand of C8 column (Phenomenex) with a C8 guard column; same mobile phase but our pH is 3.1;same wavelength settings of Fluorescence detector. We used a different Internal standard, glipizide since it was available in the lab and it is a structure analogue of glyburide. The pH was set to 3.1 so glipizide will not be eluted that very early and be able to separate from an endogenous interference.The flow rate is 1.5mL/min;the retention time for glipizide is 4.4mininutes while for glyburide is 11.5minutes. Using the same sample preparation we still can’t obtain the LOQ 5ng/mL. I set it higher to 10ng/mL but still we can’t attained it. The signal to noise ratio is only 2. I try sample preparation from other published method, liquid-liquid extraction using DCM:Hexane(1:1) with 1:6 ratio to plasma volume but there are many interfering peak to glyburide and LOQ of 10ng/mL was not obtained.I try using the previous method again which is direct precipitation with acetonitrile 1:1 ratio and increase the volume of 5% CuSO4 in water ( from 50µL to 150µL);glyburide is quite diluted under this condition perhaps that's why I'm not able to attain a S/N ratio = 10.I was thinking of concentrating the supernatant. The supernatant (500µL) is a mixture of acetonitrile and water, If I evaporate this with nitrogen gas under 60 degrees celcius , perhaps I could concentrate glyburide but I worry that glyburide might be degraded under this temp. condition.
I am exhausted modifying the method, please enlighten me if I am making a sensible modifications.
Kindly suggest other sample preparation modification I could do to obtained an LOQ 10ng/mL of glyburide in human plasma.
Thank's in advance!
The published method HPLC conditions are the ff: column: Kingsorb C8;15cm x 4.6 mm i.d;3µm with a C8 precolumn insert C8 (Phenomenex); mobile phase: 45% buffer solution (0.05M NH4H2PO4):40% acetonitrile:15%methanol adjusted to pH 5.7;flow rate 1.0mL/min;excitation wavelength 235nm;emission wavelength 354nm Fluorescence detector. The sample preparation was direct precipitation using acetonitrile and 5% CuSO4 solution in water; injection volume is 25µL. Ketoconazole was used as internal standard. The retention time for ketoconazole and glyburide were 6.7 and 9.6 minutes. The LOQ is 5ng/mL.
In our laboratory, we modify some HPLC conditions, we used a different brand of C8 column (Phenomenex) with a C8 guard column; same mobile phase but our pH is 3.1;same wavelength settings of Fluorescence detector. We used a different Internal standard, glipizide since it was available in the lab and it is a structure analogue of glyburide. The pH was set to 3.1 so glipizide will not be eluted that very early and be able to separate from an endogenous interference.The flow rate is 1.5mL/min;the retention time for glipizide is 4.4mininutes while for glyburide is 11.5minutes. Using the same sample preparation we still can’t obtain the LOQ 5ng/mL. I set it higher to 10ng/mL but still we can’t attained it. The signal to noise ratio is only 2. I try sample preparation from other published method, liquid-liquid extraction using DCM:Hexane(1:1) with 1:6 ratio to plasma volume but there are many interfering peak to glyburide and LOQ of 10ng/mL was not obtained.I try using the previous method again which is direct precipitation with acetonitrile 1:1 ratio and increase the volume of 5% CuSO4 in water ( from 50µL to 150µL);glyburide is quite diluted under this condition perhaps that's why I'm not able to attain a S/N ratio = 10.I was thinking of concentrating the supernatant. The supernatant (500µL) is a mixture of acetonitrile and water, If I evaporate this with nitrogen gas under 60 degrees celcius , perhaps I could concentrate glyburide but I worry that glyburide might be degraded under this temp. condition.
I am exhausted modifying the method, please enlighten me if I am making a sensible modifications.
Kindly suggest other sample preparation modification I could do to obtained an LOQ 10ng/mL of glyburide in human plasma.
Thank's in advance!
