External (absolute) response type

<p>Hello</p><p></p><p>Instrument is Acuity UPLC/TQD.  I am developing a Methotrexate method.  This is an antineoplastic drug and I need to be able to detect and quantitate down to 0.02 µmole/L.  In determining the sensitivity of the method I developed thus far I tried making a calibration curve using external (absolute) response type.  This is the first time I ever done that.  Usually I use an internal standard.  Anyway my results were quite good giving me correlation coefficient r of 0.999492.  I was quite surprised by this. </p><p></p><p>Question is how safe it would be not to use an internal standard.  Deuterated Methotrexate is expensive and hard to use (insoluble).  I could try some other very polar material but why bother if my curve is more than acceptable.  The extraction is a protein precipitation so I do worry about ion suppression which a deuterated internal standard could compensate for.</p><p></p><p>Anyway I am seeking advice in this matter.</p><p></p><p>TIA</p><p></p><p>Mike</p>