Laboratory Testing Case Studies

Develop a Bioanalytical Method for a Synthetic Prostacyclin Analogue

Challenge: The advancement sought in this research conducted by Diteba was to develop and validate a specific, sensitive and reliable assay method that was capable of:

1. Exhibiting linear pharmacokinetics, i.e., proportional to dose administrated from approximately 10 pg/mL to 5 000 pg/mL;
2. Assessing clinical studies conducted for a population of patients with inoperable chronic pulmonary hypertension (which are usually dosed 45-125 ng/kg/min); and
3. Proving that the synthetic prostacyclin analogue plasma concentrations remain linearly proportional to doses up to 125 ng/kg/min.

This compound is a long-acting analogue of prostacycline and it is administered in low doses (1.25-15 ng/kg/min) by continuous intravenous infusion to patients with pulmonary hypertension.  The drug improves survival and the hemodynamics in these patients.  However, the treatment is limited by a very short half-life (1-2 minutes) and chemical instability requiring continuous intravenous infusion and refrigeration during administration.  Some research has been published with regards to biotransformation and pharmacokinetics for Beraprost, a synthetic analog of prostacyclin.

There were two major challenges in developing a suitable assay method for the drug quantification in plasma samples.  First, the low limit of quantification (LOQ = 10 pg/mL) and second, the very wide linearity range requirements made it difficult in selecting proper procedures for the preparation and  calibration and QC samples.  Furthermore, several of the endogenous homologues had been noted in human blank plasma and the likely interference with the detection of analytes.  In this situation it is difficult to find an appropriate sample of an authentic matrix with a sufficiently low analyte concentration to serve as a truly blank matrix.  Therefore alternative procedures had to be identified to prepare calibration or quality control (QC) samples.

Solution: The presence of an endogenous analyte also made the validation of chromatographic methods more challenging.  The validation was further complicated by the absence of official guidelines.  In order to ensure that the assay results have a comparable level of quality, in many cases the existing FDA method validation principles cannot be directly applied for samples with endogenous analytes.  Although several investigations describe some aspect of the kinetic profile of this compound, they did not lead to a better understanding of the pharmacokinetic characteristics.  A sensitive assay for this particular purpose is important in evaluating the pharmacokinetic parameters.  Interestingly, the analytical methods published for the compound have been very sparse even though the method has been used for some time.

As an essential part of method development protocol, Diteba evaluated and determined the proper procedure to prepare calibration and QC samples.  As mentioned above, several of the compound's endogenous homologues have been noted in human blank plasma and their interference with the detection of the analyte.  Among the approaches investigated to circumvent this problem, Diteba's scientists attempted the application of a method of standard addition, oxidation, and incubation of the samples in presence of oxygen and elevated temperature (37°C for 2 - 48 hours) to speed up depletion.  Diteba also tried enzymatic depletion of the endogenous analyte levels by spiking the analyte in artificial (surrogate) matrix or by stripping plasma with activated carbon.

Through the research conducted, it was determined a more selective approach to remove the endogenous analyte from blank plasma was required by using an affinity extraction.  The plasma samples were passed through a selected cartridge containing sorbent-bound antibodies directed specifically towards the analyte.  In principal, the only compound being removed is the analyte itself, along with potentially a small number of structurally-related compounds.

The spiked standard and QC samples were analyzed using tandem mass spectrometry (Waters UPLC/MS/MS TQD).  The mass transition (m/z) was monitored by negative ionization mode [M-H].  The reliability of the method was determined through duplicate analysis of standards which were prepared in and extracted using the aforementioned procedure at 11 concentrations (10.0, 20.0, 40.0, 80.0, 160, 320, 640, 1280, 2560, 4096, and 5120 pg/mL).  The mean percent accuracy value for the quality control samples of the method, run concurrently with the study samples, were 98.6%, 99.3% and 97.9% of theoretical values with a precision RSD of 4.56%, 6.33% and 4.12% at 30.0, 1920 and 3840 pg/mL respectively.

Result: Diteba developed and successfully verified an accurate, precise and specific LC/MS/MS assay method with a lower limit of quantification (LLOQ) of 10.0 pg/mL for a 0.5 mL aliquot of plasma.  The plasma samples were prepared with an optimized liquid-liquid extraction method.  The developed method clearly illustrated a linear relationship between the dose and the plasma concentrations, and it provides added assurance that, for the vast majority of patients, an increase in the infusion rate of drug yields a proportional increase in drug plasma concentrations.