Development and validation of a sensitive ELISA for quantitation of Grastim (rhG-CSF) in rat plasma: Application to a pharmacokinetic study.

Grastim is bacterially produced recombinant counterpart of human granulocyte colony stimulating factor (G-CSF). It has biological activity similar to that of endogenous G-CSF. In the present work a sensitive, accurate, precise and enzyme-linked immunosorbent assay (ELISA) for the quantitation of G-CSF in rat plasma was developed and validated. The ELISA method employed a technique in which anti-human-G-CSF was adsorbed onto 96-well maxisorp plates and used to capture the G-CSF in rat plasma samples. The captured G-CSF was then detected using streptavidin-HRP amplification system. Absolute recovery was >90% from rat plasma. The validation includes assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLOQ), storage stability (30 days) in rat plasma and assay specificity. The standard curve for G-CSF was linear (R2 > 0.996) in the concentration range 4.88-625 pg/mL. The LLOQ was established at 4.88 pg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 15, 250 and 500 pg/mL, were in the range 3.00-8.66% relative standard deviation (RSD) and 1.03-4.69% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.28-110.79% of the nominal values. The assay shows dilutional linearity and specificity. Stability of G-CSF was established for 30 days at -80 degrees C and through three freeze-thaw cycles. The validated assay was successfully employed for the assessment of pharmacokinetic disposition of G-CSF in rats.

Development and validation of a chiral liquid chromatographic method, based on Chiralpak to quantify enantiomers of (+/-)-DRF 2725 in rat plasma: lack of inversion of ragaglitazar (S(-)-DRF 2725) to its antipode in plasma.

A selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method for the separation of individual enantiomers of DRF 2725 [R(+)-DRF 2725 and S(-)-DRF 2725 or ragaglitazar] was obtained on a chiral HPLC column (Chiralpak). During method optimization, the separation of enantiomers of DRF 2725 was investigated to determine whether mobile phase composition, flow-rate and column temperature could be varied to yield the base line separation of the enantiomers. Following liquid-liquid extraction, separation of enantiomers of DRF 2725 and internal standard (I.S., desmethyl diazepam) was achieved using an amylose based chiral column (Chiralpak AD) with the mobile phase, n-hexane-propanol-ethanol-trifluoro acetic acid (TFA) in the ratio of 89.5:4:6:0.5 (v/v). Baseline separation of DRF 2725 enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Nominal retention times of R(+)-DRF 2725, S(-)-DRF 2725 and I.S. were 15.8, 17.7 and 22.4 min, respectively. The standard curves for DRF 2725 enantiomers were linear (R(2) > 0.999) in the concentration range 0.3-50 microg/ml for each enantiomer. Absolute recovery, when compared to neat standards, was 70-85% for DRF 2725 enantiomers and 96% for I.S. from rat plasma. The lower limit of quantification (LLOQ) for each enantiomers of DRF 2725 was 0.3 microg/ml. The inter-day precisions were in the range of 1.71-4.60% and 3.77-5.91% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. The intra-day precisions were in the range of 1.06-11.5% and 0.58-12.7% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 83.4-113% and 83.3-113% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Both enantiomers and I.S. were stable in the battery of stability studies viz., bench-top (up to 6 h), auto-sampler (up to 12 h) and freeze/thaw cycles (n = 3). Stability of DRF 2725 enantiomers was established for 15 days at -20 degrees C. The application of the assay to a pharmacokinetic study of ragaglitazar [S(-)-DRF 2725] in rats is described. It was unequivocally demonstrated that ragaglitazar does not undergo chiral inversion to its antipode in vivo in rat plasma.