Method Analyte and Internal Standards. Susan M. Deupree, Kelli ... Isotopically-labeled internal standards were used for all four analytes. Calibration standards ...
Susan M. Deupree, Kelli D. Goodman
Mitchell A. deLong
Tandem Labs—RTP
Aerie Pharmaceuticals, Inc.
LC/MS/MS Methodology for Quantification of AR-13323, AR-13324, AR-13503, and AR-13534 in Rabbit and Monkey Plasma
Purpose To develop and validate an analytical method to stabilize and extract two pairs of metabolically related enantiomers from rabbit and monkey plasma followed by separation and analysis using a chirally selective LC/MS/MS method.
Method Analyte and Internal Standards
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Method Summary The methods were developed to quantify four related compounds in rabbit and cynomolgus monkey plasma. The test article, AR-13324, is metabolized following ocular administration via esterases to the active metabolite AR-13503. Also of interest is the conversion of AR-13324 to its enantiomer, AR-13323, and its metabolite, AR-13534 (which is also the enantiomer of AR-13503) in vivo. Isotopically-labeled internal standards were used for all four analytes. Calibration standards and quality control samples were prepared in cynomolgous monkey or rabbit K2EDTA plasma containing an esterase inhibitor. Method analytes were extracted from biological matrix materials using protein precipitation. Chiral separation of the analytes was achieved using a Supelco® Astec® Chirobiotic V2 (4.6 x 150 mm) column operated at 40°C and an isocratic elution profile with a mobile phase composed of 65/35 (0.1% ammonium trifluoroacetate in methanol)/acetonitrile flowing at 1.5 ml/min. Peaks eluted between 2.1 and 9.0 minutes with a total run time of 12 minutes. A triple quadrupole mass spectrometer (AB Sciex® API 5000) was used for detection using electrospray ionization in positive ion mode with multiple reaction monitoring.
Method Development n
The four-analyte method required chiral separation of two pairs of enantiomers
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One pair of enantiomers metabolize into the other pair of enantiomers in rabbit/monkey plasma/ blood, requiring stabilization of biological samples
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The esterase activity was blunted by the addition of an inhibitor, bis(4-nitrophenyl) phosphate (BNPP), to blood at the time of collection
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The addition of formic acid to plasma samples prior to extraction improved both recovery and assay performance, presumably by disrupting protein-binding interactions
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Supelco® Chirobiotic V2 columns required equilibration to stabilize retention time when brought into service. A short equilibration sequence was also helpful following periods of idle instrumentation for the same reason.
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Carboxylesterase Action and Inhibition Carboxylesterases (CE) are ubiquitous enzymes that cleave a carboxylic acid ester into an acid and an alcohol. CE active sites may accommodate a variety of structures and no endogenous substrate has been identified. Therefore, they are thought to provide a protective function by scavenging exogenous molecules. Current and prospective therapeutic agents frequently possess carboxylic acid esters and are sensitive to cleavage by CE. In bioanalysis, it is important to recognize instances where method analyte concentrations might be impacted by enzymatic activity following collection and to take steps to preserve the sample. For the chiral analytical method presented here, it was of interest to determine the degree of inter-conversion in vivo of test article to its active metabolite and the metabolite of its enantiomer. Therefore, inhibiting CE activity ex vivo was critical. The compounds AR-13324 and AR-13323 rapidly degrade to AR-13503 and AR-13534, respectively, in rabbit plasma and blood. In monkey plasma and blood, only the R-enantiomer (AR-13323) was sensitive to enzymatic degradation. Processing the samples on ice did not sufficiently inhibit activity. The substrate bis(4-nitrophenyl) phosphate (BNPP) was found to be highly effective in stabilizing the compounds. The interior phosphate group of BNPP reacts to form a stable phosphate ester within the active site of the enzyme. Such irreversible inhibition is desirable ex vivo.
Since BNPP should be added to blood immediately upon collection, and as the volume of blood collected is difficult to control accurately, a range of additive concentrations was validated (1 - 3.2 mM). In practice, 20 uL of an 80 mM BNPP solution was added to vacutainers prior to collection. Theoretically, this allowed for a draw volume of 0.5 – 1.5 mL. However, since it was observed that stability was improved at 2 and 3.2 mM under some conditions (e.g., benchtop stability of AR-13323 in monkey plasma), a target volume of 0.5 – 0.8 mL was recommended to the collection facilities.
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Methodology Sample Preparation and Extraction n Thaw plasma samples on ice n
Add 0.0500 mL plasma aliquot to 0.0250 mL of 1% formic acid (aq) and mix
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Add 0.225 mL internal standard working solution (25.0 ng/mL each of the racemic isotopicallylabeled internal standards AR-13507 and AR-13508 in methanol with 0.1% formic acid)
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Mix thoroughly and centrifuge at 0-8 °C 3000 RCF for 10 min
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Using a Hamilton Star robotic liquid handling system, transfer 150 – 200 uL of supernatent to fresh plate
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Centrifuge at 0-8 °C 3000 RCF for 5 min
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Store supernatant at 0-8 °C until analysis
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Methodology (continued) Instrumental Conditions (LC)
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Methodology (continued) Instrumental Conditions (MS)
Results A linear range of 1.00-1000 ng/mL was validated and inter-run and intra-run precision and accuracy for four QC levels met GLP criteria. A range of inhibitor concentrations was validated, which stabilized all analytes over 6 freeze/thaw cycles and 24 and 6 hours of benchtop stability (on ice) for rabbit and monkey, respectively. The methods were demonstrated to be selective by the absence of matrix interferences at the retention times of interest. The ability to accurately and precisely quantify all analytes in 2% hemolyzed plasma was confirmed.
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Results (continued) Example Chromatogram: Matrix Control Blanks
Example Chromatograms – LLOQ
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Results (continued) Example Chromatograms – ULOQ
Standards Performance
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Results (continued) Linearity, Recovery, Matrix Factor Average correlation coefficients over three core validation runs as well as recovery and matrix factor are tabulated.
Quality Control Performance
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Results (continued) Stability Summary Stability experiments were conducted in plasma or blood with BNPP concentrations of 1, 2, and 3.2 mM with the exception of long-term storage stability, which was evaluated at 2 mM BNPP, correlating with the maximum collection volume recommended to the in-life facility. Samples were handled on ice.
*Benchtop stability was successfully demonstrated for 16 h in monkey plasma containing higher (2 and 3.2 mM) inhibitor concentrations. For this reason, the recommendation was made that study samples be collected at a minimum concentration of 2 mM BNPP.
Conclusion The validated chiral methods have been shown to be sensitive, selective, accurate, precise, and robust. The sample processing and extraction technique developed was shown to stabilize the analytes and prevent inter-conversion or loss between the time of collection and analysis. These methods were used in the GLP analysis of samples from pre-clinical studies.
References n
Cashman J, Perroti B, and Berkman C. Pharmacokinetics and molecular detoxification. Environ Health Perspect 1996; 104:23-40.
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Hatfield MJ and Potter PM. Carboxylesterase inhibitors. Expert Opin Ther Pat 2011; 21(8):1159-1171.
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