Chromatography Forum

hello every one Im seeking your help with my problem of,im trying to mesure a standard concentrations to make a calibration curve but, the drug and the IS  signals both are unstable in the plasma and liver samples and totally different from that of the water samples and much higher I USED two internal standard nilotinib 50nM and tolbutamide 100nM final conc. 

my sample preparation as follow :

plasma/water  7.5ul

st(0.1-3000) 15 ul

(nilotinib500nM+tolbutamid1000nM) 15ul

100%MeoH 112.5

liver 100-200 mg 

(nilotinib500nM+tolbutamid1000nM) 9ul+100% meOH 9000ul  

then added 4ul/mg of the mixture above to make  liver hemogenate  after that took 270 of the hemogenate with 30ul st solutions 

each of the plasma , water, liver samples where centrifuged with 15000rpm/10min/4c for tow times 

the took 100 ul into lcmsms well

mobile phase acetonitril and water

these are the response signal heights of nilotinib/dasatinib/tolbutamid

water0 796735 32 182684
0.1 781134 229 205622
0.3 672742 222 168218
1 684838 422 195346
3 711591 888 179249
10 770301 2947 192125
30 715084 7857 173846
100 728024 27931 183024
300 731337 57245 176130
1000 669012 221262 166505
3000 768826 692080 190855

plasma0 902755 99 209245
0.1 865941 147 184362
0.3 805266 524 183035
1 1334233 1514 229281
3 1153873 2679 187603
10 1037242 8367 196099
30 1089179 24129 226735
100 1199775 75324 183980
300 951273 198094 294584
1000 840437 574479 333974
3000 1123225 1318224 219058

liver0 505811 48 113874
0.1 88580 92 111167
0.3 101173 100 106828
1 105409 210 118305
3 157888 1081 119138
10 161908 2572 108209
30 179219 7988 104037
100 166083 28173 100921
300 149591 93436 141122
1000 190852 269647 100597
3000 169488 918190 101854
2017303_mix14 30723 9551 15235

I would say you still have lipides in your sample. I would look for an SPE method to get rid of the lipides. Another possibility is to freeze the sample for a while before centrifugation.