RESUMEN
A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for a PEGylated adnectin therapeutic protein in cynomolgus monkey plasma. The validated method was performed using protein precipitation coupled with trypsin digestion, followed by LC-MS/MS detection of a surrogate peptide generated from the PEGylated adnectin protein. A tryptic peptide generated from a PEGylated adnectin protein analog was used as the internal standard to standardize the digestion, extraction, and quantitation processes. The protein precipitation extraction of the protein from cynomolgus plasma was performed using an acidic 2-propanol organic solution. Following the extraction, the supernatant was removed and a 45min trypsin digestion was performed at 60°C on the supernatant layer. The linear dynamic range of the assay was 50.0-25,000ng/mL. Chromatographic separation was performed with an Acquity BEH C18 (1.7µm particle size, 2.1mm×50mm) column using gradient elution. The assay proved to have robust accuracy, precision, and stability for the representative surrogate peptide of the PEGylated adnectin protein being evaluated. The validated method was implemented as a high throughput assay for a PEGylated adnectin protein using a similar PEGylated adnectin therapeutic protein as the internal standard that can be used for future monkey toxicokinetic (TK) studies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Fibronectinas/sangre , Macaca fascicularis/sangre , Polietilenglicoles/química , Espectrometría de Masas en Tándem/métodos , Tripsina/química , Animales , Precipitación Química , Fibronectinas/químicaRESUMEN
To support a first-in-human (FIH) clinical study in healthy volunteers, a human plasma assay, a 20-fold more sensitive method than the validated non-clinical LC-MS/MS assays, was requested. For the clinical assay, a LLOQ of 0.050 ng/mL for Compound A and 0.100 ng/mL for Compound B was desired to accurately determine the analyte concentrations in human plasma samples across all treatment groups. A design of experiment (DOE) investigation was performed in an effort to optimize the extraction procedure of the bioanalytical assay used to support the first in human study and future clinical studies. Three factors, extraction buffer pH (two pHs), volume ratio of organic solvent to plasma (two ratios), and extraction shake time (three times), were selected for the DOE. Both analytes were analyzed at a low concentration, 0.150 ng/mL, and a stable isotope label internal standard was used for each analyte. To estimate the recovery of each analyte from the extraction, the response ratio of each analyte over the respective internal standard was used, and to estimate matrix effects, the absolute response (peak area) of each analyte was used. The results of the DOE indicated that the three factors tested had a more significant effect on the extraction of the metabolite, Compound B, compared to that of the parent, Compound A. The extraction buffer pH had the greatest influence on Compound B and the volume of extraction solvent had an influence on both analytes. Unexpectedly, a longer extraction time caused an apparent decrease in the overall recovery for both analytes. This was presumably due to an increased extraction of interfering matrix components. Optimal conditions were achieved for the combined analysis of both compounds using the DOE approach.
Asunto(s)
Bioensayo/métodos , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión , Proyectos de Investigación , Espectrometría de Masas en Tándem , Bioensayo/normas , Tampones (Química) , Calibración , Cromatografía Líquida de Alta Presión/normas , Humanos , Concentración de Iones de Hidrógeno , Modelos Estadísticos , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Solventes/química , Espectrometría de Masas en Tándem/normas , Factores de TiempoRESUMEN
A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl(3):2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C(18) (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r(2)=0.9990) and hydrastine (r(2)=0.9983) over a range of 11.80 ng/mL to 17.64 µg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.