Development of an electrospray ionization mass spectrometric method for the quantification of theophylline in horse serum.

This research discusses a new method developed for accurately determining the amount of the drug theophylline in horse serum through a process known as an electrospray ionization mass spectrometric method.

Development of the Method

• The scientists developed a fast and specific method to determine the amount of theophylline, a drug, in horse serum (a component of horse blood).
• There are three parts in this process: a protein precipitation extraction for sample preparation, liquid chromatography separation technique, and ionspray tandem mass spectrometry.
• Theophylline is taken out of the serum using a protein precipitation with acetonitrile, a colorless liquid. After this, the supernatants (the liquid that lies above a solid residue after precipitation, sedimentation, or centrifugation) are analyzed using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS), a detection method used for precise quantification and molecular identification of chemicals.

Chromatography Process

• Chromatography is performed using the specific analytical column. An isocratic mobile phase, which involves the use of both acetonitrile and 0.5% formic acid in water, is used. The mobile phase keeps a constant composition and helps to separate and propel the sample through the column. The flow rate is kept at 350 microL/min.

Calibration

• Calibration, a necessary process to ensure the results are reliable and accurate, was performed over a range of 0.1-20 ppm with a 1/concentration relationship. This means the measurements of theophylline are based on a linear scale.

Precision and Accuracy

• The intra-batch precision and accuracy of the process at LLOQ (Lower Limit of Quantification), medium, and high concentration levels were relatively good given the percentages provided. Similarly, the inter-batch precision and accuracy levels were also satisfactory.

Usage in Clinical Investigation

• This method for testing the level of theophylline in horse serum was considered successful and adheres to the generally accepted criteria used for bioanalytical assays.
• Due to its success, the method was successfully used during a clinical investigation.