Development, validation and method stability study of a LC-MS method to quantify leuprolide (Hormone analog) in human...

Area of Science:

• Analytical Chemistry
• Pharmacokinetics
• Biotechnology

Background:

• Leuprolide, a Gonadotropin Releasing Hormone (GnRH) analog, requires precise quantification in biological matrices.
• Existing methods for leuprolide measurement may lack the sensitivity, speed, or simplicity needed for certain applications, particularly in clinical settings.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS) assay for quantifying leuprolide in human plasma.
• To assess the stability of leuprolide under various conditions using the developed assay.

Main Methods:

• Human plasma samples were spiked with leuprolide and a stable isotope internal standard.
• Leuprolide was extracted using C18 solid-phase microextraction (MEPS) and analyzed via LC-MS/MS.
• Chromatographic separation was performed on a Waters Atlantis HILIC C18 column with a mobile phase of acetate buffer and acetonitrile.
• Detection utilized electrospray ionization in positive mode with multiple reaction monitoring (MRM).

Main Results:

• The assay demonstrated a linear dynamic range of 0.0500-40 ng/ml with a lower limit of quantification (LLOQ) of 0.0500 ng/ml.
• Full analytical validation confirmed specificity, precision, accuracy, matrix effect, and stability.
• The method achieved acceptable precision and accuracy across the standard curve range.

Conclusions:

• The developed LC-MS/MS assay is a simple, rapid, precise, and accurate method for quantifying leuprolide in human plasma.
• This assay provides valuable data on solution stability and is a suitable alternative for leuprolide quantification in human clinical trials.