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A Highly Sensitive LC-MS/MS Method for the Quantification of Formoterol in Human plasma.

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Background:

Formoterol is a long-acting β2 agonist (LABA) used as a Bronchodilator in the management of asthma and chronic obstructive pulmonary disease. Recommended low doses are to be used as over dosing may lead to nausea, vomiting, headache, tremor, somnolence, palpitations, tachycardia, ventricular arrhythmias, metabolic acidosis, hypokalemia and hyperglycemia. Formoterol is used in powder form for inhalation. Due to this, a very low detection (sub pico gram level detection) in plasma is required for proper characterization of PK studies. Hence, a selective and sensitive method at a LLOQ of 0.4 pg/mL in human plasma has been developed.
 
Bioanalytical method:

The method for the quantification of Formoterol involves double extraction procedure i.e. protein precipitation followed by solid phase extraction to get rid of matrix effect. Formoterol-d6 has been used as internal standards (ISTD).  Chromatographic separation was achieved on Biphenyl 5µ, 100*4.6 mm column with a mobile phase consisting of 5 mM of ammonium formate buffer and methanol with gradient time program, with a total run-time of 9.0 minutes. Detection was carried out in positive electro-spray ionization mode using MRM transitions of 345.05→149.20 and 351.05→152.30 for Formoterol and Formoterol d6 respectively.
 
The method was validated in terms of linearity, stabilities and as per regulatory requirements. The validated method has a linearity range from 0.4 to 150 pg/mL for Formoterol. As Formoterol is unstable in blood, a stabilizer (buffer) has been used during the blood collection and plasma from the same has been used for the stability purpose and found to be stable in buffered plasma under the assay conditions. Excellent precision and accuracy were observed even at a LLOQ level i.e. 0.4pg/mL. Also, there was no matrix effect observed in normal human lots.
 
Conclusion:

The validated method is suitable for the analysis of samples from a single oral dose of 24 µg powder for inhalation dose under fasting condition to humans and will be applied to the analysis of study samples.
 
To conclude, a selective, simple, precise, accurate and highly sensitive LC-MS/MS method is now available for quantification of Formoterol in human plasma.

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