HPLC A Praactical User''''S Guide Part 9 docx

METHODS DEVELOPMENT 155

Figure 12.4 Systematic methods development: samples. (a) Heat plasma blank; (b) plasma

blank spiked with standards; (c) 80% SFE window; (d) heated plasma/standards; (e) SFE win￾dowed/plasma standards; (f) SFE windowed patient sample.

cartridge. You elute the cartridge with 2 mL each of 60%, 80%, and 100%

MeOH, add 1 mL of IS to each cut, dilute 100×, and shoot each sample into

the HPLC system. The 60% wash is contaminated with B and a trace of A.

You repeat, moving the window frame to the left by eluting with 55%, 80%,

and 100% MeOH. All your peaks are in the 80% window (Fig. 12.4c); none

show up in either the 55% or 100% washes. Quantitization against the inter￾nal standard’s peak height shows no loss of peaks B or D on the SPE cartridge.

You remove the other half of the pooled blood sample from the refrigera￾tor. You mix 4 mL of blood with 1 mL each of the four standards in acetoni￾trile, sonicate, heat in boiling water, and centrifuge.You place 2 mL of the super

and 1 mL of IS 100× in a 100-mL volumetric flask, dilute with mobile phase,

and inject into the HPLC. (Note: You are looking for loss of standards by

adherence to precipitated protein.) Peaks A, C, and D are present; the last two

standard peaks quantitate correctly (Fig. 12.4d). You take 2 mL of the remain￾ing plasma plus standards supernatant, dilute it 5-fold with water, and place it

on an activated SFE cartridge column. You elute with 55%, 80%, and 100%

MeOH in water containing 1% acetic acid. The 80% fraction is mixed with IS,

diluted and run. It shows a much narrower polar peak, compound B as a shoul￾der on the polar peak from the plasma peak, resolved peaks A, C, D, and IS,

and a small amount of the latter running nonpolar peaks (Fig. 12.4e). All four

standards give correct peak height response factor to the IS peak.We are ready

to accept patient standards.

Two more comments are necessary. The internal standard is added to

correct for injection variations. The way it was used in the last step, it was also

checking for standard recovery from the protein precipitation step. It is mildly

dangerous to use the same internal standard for two purposes. If the quanti￾tization was not correct, it would have been necessary to repeat both the injec￾tions and the precipitation with another internal standard to find the problem.

Also, you must check for possible interfering drugs (ones co-eluting with our

standards) that might be given to patients taking these target drugs. I would

use the plasma blank spiked with standards and IS to look for these interfer￾ences by changes in response factors of the standards. This study can be post￾poned for our work right now.

To run a patient sample, you will need to go through exactly the same

deproteination, SFE cartridge extraction, IS addition, mobile phases dilution,

and injection steps (Fig. 12.4f). From the peak heights relative to the IS height,

we can now quantitate the amount of each drug in the patient’s blood. To

insure linearity, you may need to dilute our windowed plasma blank and spike

it with different levels of each standard and plot calibration curves for each

compound, but basically, our methods development is done.

12.3 GRADIENT DEVELOPMENT

It is sometimes not possible to develop an isocratic separation for complex

mixtures of compounds. Binary gradient methods development starts with a

156 SAMPLE PREPARATION AND METHODS DEVELOPMENT