Hi. I am trying to develop a new analytical method by LC. Mi matrix is intraperitoneal fluid. I´ve read some articles and for the development of the method validation some authors use water as a matrix (they don´t use intraperitoneal fluid because i suposse matrix effect/recovery is not evident). I have calculated my recovery (Analyte in water VS Analyte in intraperitoneal fluid, both at the same concentration and same sample prep) and I have more analytical signal in intraperitoneal fluid samples because some of the volume is ocupped by proteins that i precipitate and this implies a lower volume and more concentrated sample.
Anybody can tell me why some authors can use water as a diluent for calibration/controls to determine my drug? I understand you can use water if you don´t have different signal response when you compare with biological matrix.
A method validation should take into account all matrix effects. Did the authors that used water for their validation have the same matrix as you? If not, they may not have observed any bias or matrix effect. If so, some matrices like cerebral spinal fluid, aqueous humor and even intraperitoneal fluid are difficult to obtain in sufficient quantity for a complete method validation. Keep in mind that the matrix used for a validation should represent a typical or average matrix. If the matrix is not available in quantity then options like water or better yet simulated body fluids (see link) need to be used. Did you look at the recovery in more than one sample of IP fluid, or from a pooled mixture of IP fluids from various sources? If so what were those recovery values?
http://www.dissolutiontech.com/DTresour/201108Articles/DT201108_A02.pdf
Hello Carlos, one of the approaches I have taken in the past when I have a unique matrix (like tumor or CSF) was to spike all the samples into plasma, and then run plasma standard curves and QCs. For example, take 5uL of your introperitoneal fluid and spike into 45uL of plasma. Then you can run your standards in plasma, and process them in the way that you would normally extract plasma (protein precipitation, SPE, LLE, etc.). That way the matrix for samples, standards and QCs is almost the same. You do have a 10 fold dilution to worry about, but if sensitivity is not an issue this is a common way to proceed.
Bruce. The authors have the same matrix like me, but they only say that they work with water for calibration curves and controls. The don´t talk about matrix effect or recovery. I only have two samples of intraperitoneal fluid of two patients with a low volume (I suposse is a difficult matrix to obtain). I´ll try to calculate recovery in both and check similarities. Thx for the file.
Chris. I can´t dilute my samples. It would be a problem for me to dilute. I noticed that I obtain the same response whit intraperitoneal fluid and Plasma so I am thinking about use this matrix instead water.
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