By Rashmi on Thursday, May 6, 2004 - 11:24 am:
Hi
I am trying to repoduce a method reported in a paper regarding extraction of nicotine from serum samples. The method is:serum sample (1ml) was alkalinized by a 50 microliter of 10 M NaOH. After addition of internal standard, the mixture was extracted with 4 ml of dichlormethance by shaking for 10 mins. After centrifugation at 1000 g for 10 min, 25 microliter conc HCl was dded to organic fraction. The organic fraction was evaporated at 40 degrees in nitrogen stream and the residue was redissolved in 100 micoliters of mobile phase and then 80 microliters portion of the sampple was injected in HPLC.
The way i did it was i spiked the blank serum sample (200 microliter) with a known quantity of nicotine. To this i added 10 microliters of 10 M NaOH and extracted with 4 ml dichloromethane by shaking for 10 min. After centrifugation at 1500 rpm for 10 min, i added 25 microliters of conc HCL, and evaporated the organic fraction under nitogen stream at 40 degrees. The residue was redissolved in 100 microliters mobile phase and then 50 micoliter portion of the sample was injected in HPLC.
However i had the following problems,
1)When i was evaporating the organic fraction under nitrogen stream, a white colored residue was formed at the bottom. Is this because of NaOH added earlier or HCL or is it contamination from serum. How do i remedy this.
2) Also when i injected extracted sample into HPLC i got a double peak at the retention time of the drug. When i inject the nicotine sample dissolved in water, i dont get a double peak.
What do i do?
By Anonymous on Thursday, May 6, 2004 - 07:52 pm:
What are your HPLC conditions?
By rashmi on Friday, May 7, 2004 - 06:39 am:
These are the chromatographic conditions
Column : Xterra RP 18 , 50 * 4.6 mm , 30.5 micron
Mobile phase: Sodium peclorate buffer containing ammonium hydroxide (85 %) + Water (15%)
Flow rate: 1ml/min
Wavelenght: 261 nm
Integration mode: Area
Retention time for nicotine: 3.6 mins
By rashmi on Friday, May 7, 2004 - 11:41 am:
Am sorry the column with particle size of 3.5 micron not 30.5 micron
By Uwe Neue on Friday, May 7, 2004 - 02:36 pm:
I do not know, why the HCl is added in the procedure. The very acidic residue is likely to overload the pH control in your mobile phase. What is the ammonia concentration?
By Alex on Thursday, May 13, 2004 - 12:11 am:
Just a thought to the acid addition: upon acid addition most likely nicotine hydrochloride is formed, additional HCL should evaporate theoretically. The hydrochloride is definitely less volatile then the nicotine itself.
If performing then analysis by myself I wouldn't add acid, If a had to write a student or fool-proof protocol I would consider this option.