(W1230-06-36) LC-MS/MS Method Development and Validation for Quantitative Analysis of CART Peptides in Cell Culture Supernatant and Human Plasma

Purpose: CART (cocaine- and amphetamine-regulated transcript) peptides have recently drawn significant interest from the scientific community because they have been shown to be involved in the regulation of food intake behavior which in turn influences the body weight and energy metabolism. These peptides are found in both the CNS and periphery and are believed to be involved in multiple physiological processes. The primary objective of this study was to develop and validate a robust LC-MS/MS method for the quantitative determination of CART-55 and CART-62 in human plasma or cell culture media. As part of this study, we evaluated the non-specific binding of these peptides and its impact on quantitation. The final method was used to quantitate concentrations of CART-55 and CART-62 in study samples.

Methods: A UHPLC system (Shimadzu Nexera X2 series) interfaced with a Sciex QTRAP 6500⁺ mass spectrometer was employed. Chromatographic separation was achieved with gradient elution using a Kinetex 2.6 µm, PS C18, 100A column (100 ´ 2.1 mm) at a flow rate of 0.3 mL/min. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Mass spectrometric parameters were optimized using neat standard solutions (400 ng/mL) in positive polarity mode. CART-55 and CART -62 molecular ions identified with M+7 and M+6 charged state, respectively as presented in Figure 1a, 1b and Table 1. Method validation studies were performed as per the U.S. Food and Drug Administration guidelines. Sample preparation involved protein precipitation. In brief, to 50 mL of study sample 10 µL of 1% ammonia solution was added and mixed briefly. Then 300 mL of precipitation solvent was added consisting of acetonitrile: water: formic acid (90:10:0.1, v/v/v) containing Biotin CART-55 (internal standard, IS, 10 ng/mL). Samples were mixed for 10 min at 2000 rpm (VWR shaker) and centrifuged for 5 min at 10 K rpm. The clear supernatant (340 µL) was removed and dried using refrigerated (-40 ^oC) concentrator (Labconco). The dried pellet was reconstituted in 60 µL of mobile phase A and B (1:1, v/v) and injected 30 µL on to the column. Analytical standards: CART 55-102 human (peptide purity ≥95; MW: 5245.2), CART 62-102 human, rat, mouse (peptide purity ≥95: MW: 4385.3) and Biotinyl CART 55-102 (purity ≥90: MW: 5485.5) analytical standards purchased from Phoenix Pharmaceuticals, INC. (Burlingame, CA). These analytical standards are highly water soluble. Primary and working stock solutions were prepared in water.

Results: The final method used a 7-minute chromatographic run with a Kinetex PS C18, 100´2.1mm column. The retention times of CART-55, CART-62, and IS were 2.72, 2.83, and 2.88 min respectively. Calibration curves were linear (with r value ³ 0.995) over ranges of 0.1 to 100 ng/mL and 0.5 to 100 ng/mL for CART-55 and CART-62 respectively. Intra-day and inter-day accuracy and precision values were within acceptable limits. Extraction recoveries for all analytes ranged from 77.7 -82.6 % for both the analytes and matrix effect was minimal in plasma. Stability of processed samples in the autosampler was maintained for at least 24 hr at 10 ^oC, and benchtop stability at room temperature was maintained for at least 7 hr. Both analytes were stable even after three cycles of freeze thaw, and remained stable for at least 43 days when stored at -80 ^oC. During method development it was determined that these peptides exhibit non-specific binding to tubes if they are stored in the absence of other protein such as BSA. The developed analytical method was successful in evaluating non-specific binding to the tubes and other regular containers at both high and low concentrations. Based on these observations, it is suggested to store standards of these peptides at higher concentrations ( >10µg/mL) or in the presence of other proteins such as BSA. At least 1% of protein content (e.g., BSA) in storage solutions reduces the non-specific binding significantly (Fig 2). Cell culture and human plasma samples were analyzed using this validated method.

Conclusion: A sensitive and reproducible bioanalytical method was developed for the simultaneous quantitation of CART-55 and CART-62 in cell culture and human plasma using LC-MS/MS. The developed method was validated per industry standard guidelines and met all acceptance criteria. The method was successfully used to analyze cell culture and human plasma samples.

Acknowledgements: This work is supported by a Cancer Prevention Research Institute of Texas Core Facilities Support Award (RP170003 to WCP).

Non-specific binding of peptides

CART-55 MS/MS spectral data

CART-62 MS/MS spectral data