Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC-MS/MS.

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.

Automated supported liquid extraction using 2D barcode processing for routine toxicokinetic portfolio support.

BACKGROUND: A new bioanalytical sample preparation approach has been developed to enhance the efficiency, reduce errors and improve the data quality supporting routine toxicokinetic (TK) study samples analysis, via the implementation of 2D barcode processing coupled with fully automated supported liquid extraction (SLE). RESULTS: A fully automated SLE was validated and used to determine TK drug concentrations of over 500 unknown samples via 2D barcode processing. Assay performance calculated from a total of 291 quality control samples over the period of validation through sample analysis demonstrated inter-day precision and accuracy within 10 and 7.3%, respectively. CONCLUSION: A new logistical approach implementing the use of 2D barcodes and automated SLE demonstrates the potential of a new methodology for the routine bioanalytical support of TK study sample analysis.

Simultaneous determination of plasma epinephrine and norepinephrine using an integrated strategy of a fully automated protein precipitation technique, reductive ethylation labeling and UPLC-MS/MS.

A novel, automated, simple, sensitive, specific, accurate, precise and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method has been developed for simultaneous determination of epinephrine (E) and norepinephrine (NE) in plasma by using the combination of a fully automated protein precipitation technique for plasma sample preparation, reductive ethylation labeling with UPLC-MS/MS. A simple protein precipitation procedure was used to clean up 50 microL calibration samples prepared in stripped human plasma and 50 microL quality control plasma samples containing 25 microL plasma and 25 microL stabilizing additives. The supernatants were subsequently dried down and then reconstituted with commercially available and cost-effective reductive ethylation labeling reagents, followed by UPLC-MS/MS analysis. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and quality control samples, shaking of 96-well plates, adding and transferring liquids. Processing time, which included the preparation of standards and quality control samples, protein precipitation and reductive ethylation labeling, is less than 2 h per 96-well plate. The chromatographic run time is 3.5 min per sample. The limits of detection of UPLC-MS/MS-based methods for E and NE, with/without reductive ethylation labeling, are 0.025/0.40 and 0.025/2.00 ng mL(-1), respectively. Reductive ethylation labeling of amino groups of E and NE affords 16 and 80 times increased detection sensitivity of corresponding native counterparts during the UPLC-MS/MS analysis. The linearity of this method was established from 0.05 to 25 ng mL(-1) for E and NE with accuracy and precision within 15% at all concentrations. The intra-run and inter-run assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QC samples prepared in commercially purchased plasma samples.

A novel simultaneous modeling approach to estimate linearity of pharmacokinetic parameters, including saturation of intestinal efflux, in the rat.

INTRODUCTION: Various independent methods exist for the estimation of linearity of pharmacokinetic parameters in vivo. A novel simultaneous modeling approach has been developed in the rat that in combination allows estimation of the rate and extent of duodenal absorption, hepatic first pass extraction and saturation of potential dose-dependent duodenal bioavailability (saturation of intestinal efflux). METHODS: Simultaneous modeling of plasma concentrations of a Pfizer compound in the rat were conducted using NONMEM after (1) accelerated intravenous, intraduodenal, and intraportal infusions over 5 h and (2) 5-min intravenous infusions of 0.28 and 1.4 mg doses. RESULTS: The data was best described by a two-compartment linear pharmacokinetic model with good agreement between observed and model predicted plasma concentrations following the various routes of administration. Clearance was estimated to be linear up to plasma concentrations of 1200 ng/ml. The estimated rate constants (+/-asymptotic errors) for intraduodenal absorption (KA), movement of drug from plasma to tissue (K23) and movement of drug from tissue to plasma (K32) were 0.645+/-0.107, 18.0+/-2.98, and 2.02+/-0.209 h(-1), respectively. The rate constants for drug elimination from the central compartment (K20) after 5-min intravenous infusion or accelerated infusion were 3.24+/-0.6 or 6.26+/-1.64 h(-1). The estimated maximal extent of first pass extraction was 17%. The model (including increasing duodenal bioavailability as the amount in the duodenum increases, from a minimum of 5% to a maximum estimated intraportal bioavailability value-83%) indicated a saturable intestinal efflux process. DISCUSSION: This novel study design and the proposed method for data analysis provides a robust and efficient means for assessing the linearity of multiple pharmacokinetic processes while accounting for the multi-compartmental distribution characteristics of the test compound.