High-Speed Sensitive Analysis of Sunitinib and Related Metabolites Through UPLC-MS/MS Featuring CORTECS™ Premier C18 Columns With Maxpeak™ High Performance Surfaces (HPS) Technology

Tyrosine Kinase Inhibitors (TKIs) are a class of small molecule drugs used to alter cell growth pathways for the treatment of cancer. Specifically, Sunitinib, a TKI on the market, is available to treat a variety of cancers including pancreatic cancer. Here we create a method to support the therapeutic drug monitoring of Sunitinib and its active metabolite. We accomplish this by utilizing the new CORTECS Premier C₁₈ Columns with MaxPeak™ (HPS) Technology combined with MS detection on the Waters™ Xevo™ TQ Absolute System. This method provides sensitive, linear, and reproducible results for the analysis of Sunitinib and its active metabolite. 

Benefits

• CORTECS Premier C₁₈ Columns with Max Peak offered 5x more height and area signals when compared to traditional stainless-steel systems and columns
• Chromatographic separation is achieved in under two minutes
• Dynamic sensitive linearity was accomplished across the therapeutic dosing range

Tyrosine kinase inhibitors (TKIs) are class of small molecule pharmaceuticals that alter biological signaling pathways to modify cell growth. TKIs are applied as cancer treatments, to help prevent the growth and spread of tumors. Sunitinib is one of the many TKIs available for cancer treatment on the market.¹ Sunitinib is approved for variety of cancers, such as pancreatic cancer, in which it has increased the overall survival of patients.² As drugs are absorbed by the body, metabolites of the parent drug may form. In the case of Sunitinib, the main active metabolite is N-desethyl sunitinib and has been reported to cause toxicity in higher concentrations.³ Therefore, methods supporting the therapeutic drug monitoring (TDM) of Sunitinib, and its metabolites must be reliable and sensitive to ensure safe and effective cancer treatments.

Given the sensitivity needed for TDM of Sunitinib, we applied the new CORTECS Premier C₁₈ Columns featuring new MaxPeak HPS Technology. The MaxPeak HPS Technology has been shown to mitigate some undesirable metal and analyte interactions leading to lower responses in chromatography.^4,5 Further, MaxPeak HPS Technology has been tested on TKIs and provided positive changes in the analysis of these compounds.⁶

Here we develop a method to separate and quantify Sunitinib and its main active metabolite in under two minutes. This method covers therapeutic ranges that are documented in literature, making it applicable to the clinical sector of Sunitinib analysis. Further, we showcase the benefits CORTECS Premier C₁₈ Columns featuring MaxPeak HPS Technology has on the chromatography for this method. 

Stock and standard preparations were designed based on a meta-analysis conducted for bioanalytical assays for TKIs.⁷

Stock Standard Preparation

Sunitinib was purchased from Selleck Chem (Houston, TX). N-Desethyl Sunitinib Hydrochloride was purchased from Sigma Aldrich (Milwaukee, WI). Sunitinib-d10 and N-Desthyl Sunitinib-d5 from Toronto Research Chemicals (Toronto, ON). Stocks were individually prepared at 10 µg/mL in 4 mL amber vials diluted in 100% Dimethyl-Sulfoxide (DMSO). Any salt factors were taken into consideration during preparation. Stocks were stored at 2 °C–8 °C. Given the physical properties of DMSO, stocks solidified in cold storage. Vials were wrapped in foil and thawed slowing slowly in an incubator set at 35 °C for approximately 1 hour. Foil and dark preparation conditions were used due to the observed light sensitivity of Sunitinib.⁸ Thawed stocks were allowed to equilibrate to ambient room temperature prior to standard preparation. 

System Suitability Standards Preparation

Stocks were diluted using 50% Methanol in Deionized Water (diluent). Deuterated standards are individually spiked into plasma at 1% of the total volume at a 1 ng/mL concentration. The use of deuterated standards acted as an internal standard or IS for quantitative purposes for each analyte. A mixed standard containing Sunitinib and N-Desethyl Sunitinib in an equal concentration was prepared using concentrated intermediate stocks, that were then spiked into IS containing plasma at 4% of the total volume. Spiked plasma calibration standards underwent protein precipitation. Reactions took place in 1.5 mL centrifuge tubes using 100% Acetonitrile at 3x the volume of plasma. Precipitation reactions were vortexed for approximately 5 seconds. Then, centrifugated at 15,000 rpm and 4 °C for 5 minutes. Finally, 100 µL of the supernatant was combined with 100 µL of diluent in a low volume amber HPLC vial.

Linearity and Limit of Quantitation Standards Preparation

Stocks were diluted using 50% Methanol in Deionized Water (diluent). Deuterated standards are were individually spiked into plasma at 1% of the total volume at a 1 ng/mL concentration. The use of deuterated standards acted as an internal standard or IS for quantitative purposes for each analyte. Individual calibration curve points were prepared using concentrated intermediate stocks, that were then spiked into IS containing plasma at 4% of the total volume. Spiked plasma calibration standards underwent the same protein precipitation as described above.

Method of Separation Results

This method was reproducible in the retention and separation of Sunitinib, N-Desethyl Sunitinib, and related deuterated standards After 10 injections. The %RSD for area and retention time for IS corrected analytes were < 5% (Table 2 and Table 3). Below, an overlay chromatogram of the 10 injections provides a clear picture of the method’s performance (Figures 1a and 1b). 

This method was compared on a stainless-steel system to highlight the improvements of CORTECS Premier C₁₈ Columns with MaxPeak HPS Technology has on TKIs analysis. 10 injections of the system suitability standard were injected onto each of the instrument configurations. Below, results for both systems are compared (Tables 4 and 5, Figures 2a and 2b). 

The MaxPeak Premier HPS Ssystem set-up offered 5x the height and area response when compared to the Traditional Stainless-Steel system set-up. This will offer advantages for TKIs testing as these increases will lead to more sensitive detection of analytes. 

Linearity Results

Linearity was performed on SUN and NDSUN to demonstrate the quantitative suitability for this method. The linear range was established by referencing TDM studies done on Sunitinib. The therapeutic plasma concentration range for Sunitinib was suggested by The British Journal of Clinical Pharmacology to be from “37.5–60 ng/mL for continuous dosing and 50–80 ng/mL for intermittent dosing”.⁹ Therefore, we covered a plasma concentration range from 0.1 ng/mL to 100 ng/mL for both SUN and NDSUN. These ranges achieved lower quantitation limits compared to previous SUN TDM methods in literature.^10,11 The curves for SUN and NDSUN are below (Figures 3a and 3b).

Limit of Quantitation Results

The lowest curve point for both SUN and NDSUN was injected 6 times to establish reproducibility of the lower level quantitative limits (LLOQs). According to the Food and Drug Administration, current bioanalytical assay validation guidelines suggest that the accuracy and precision for the LLOQ should be with in +/- 20% RSD.¹² Tables 6 and 7, and figures 4a and 4b provide a clear picture of the LLOQ results for this method. 

Here, the use of CORTECS Premier C₁₈ Columns and Systems with MaxPeak HPS Technology improved the analysis of TKIs when compared to a traditional stainless-steel chromatography set up. Heights and areas of SUN and NDSUN increased 5-fold using MaxPeak HPS Technology. Further, this method enables for TDM across a dynamic linear range while providing reproducible results in under 2 minutes. 

1. Kim A, Balis FM, Widemann BC. Sorafenib and Sunitinib. The Oncologist [Internet]. 2009 Aug 1 [cited 2022 Oct 24];14(8):800–5. Available from: https://academic.oup.com/oncolo/article/14/8/800/6398036.
2. Faivre S, Niccoli P, Castellano D, Valle JW, Hammel P, Raoul J-L ., et al. Sunitinib in Pancreatic Neuroendocrine Tumors: Updated Progression-Free Survival and Final Overall Survival From a Phase Iii Randomized Study. Annals of Oncology [Internet]. 2017 Feb 1 [cited 2020 Apr 28];28(2):339–43. Available from: https://www.annalsofoncology.org/article/S0923-7534(19)32208-2/fulltext.
3. Takasaki S, Kikuchi M, Kawasaki Y, Ito A, Arai Y, Yamaguchi H, et al. Severe Toxicity Induced by Accumulation of Active Sunitinib Metabolite in a Japanese Patient With Renal Cell Carcinoma: A Case Report. Journal of Medical Case Reports. 2017 Feb 1;11(1).
4. Shah D, Smith K, Yang J, Hancock P. Analysis of Fourteen Organic Acids in Various Beverages Using the ACQUITY UPLC H-Class PLUS and ACQUITY QDa Mass Detector. Waters Application Note: 720007289, 2021.
5. Waters Corporation. CORTECS Columns Applications Notebook, 720004739, 2014.
6. Layton C, Rainville P. Advantages of Using MaxPeak™ HPS™ Technology for the Analysis of Targeted Cancer Growth Inhibitor Therapies. Waters Application note, 720007565, 2022.
7. Retmana IA, Beijnen JH, Sparidans RW. Chromatographic Bioanalytical Assays for Targeted Covalent Kinase Inhibitors and Their Metabolites. Journal of Chromatography B [Internet]. 2021 Jan 1 [cited 2022 Oct 24];1162:122466. Available from: https://www.sciencedirect.com/science/article/pii/S1570023220313428?via%3Dihub.
8. Jolibois J, Schmitt A, Royer B. A Simple and Fast LC-MS/MS Method for the Routine Measurement of Cabozantinib, Olaparib, Palbociclib, Pazopanib, Sorafenib, Sunitinib and Its Main Active Metabolite in Human Plasma. Journal of Chromatography B. 2019 Nov;1132:121844.
9. Westerdijk K, Krens SD, Graaf WTA, Mulder SF, Herpen CML, Smilde T, et al. The Relationship Between Sunitinib Exposure and Both Efficacy and Toxicity in Real‐World Patients With Renal Cell Carcinoma and Gastrointestinal Stromal Tumour. British Journal of Clinical Pharmacology. 2020 May 26;87(2):326–35.
10. Zhang M, Liu X, Chen Z, Jiang S, Wang L, Tao M, et al. Method Development and Validation for Simultaneous Determination of Six Tyrosine Kinase Inhibitors and Two Active Metabolites in Human Plasma/Serum Using UPLC–MS/MS for Therapeutic Drug Monitoring. Journal of Pharmaceutical and Biomedical Analysis. 2022 Mar;211:114562.
11. de Bruijn P, Sleijfer S, Lam M-H, Mathijssen RHJ, Wiemer EAC, Loos WJ. Bioanalytical Method for the Quantification of Sunitinib and Its N-Desethyl Metabolite SU12662 in Human Plasma by Ultra Performance Liquid Chromatography/Tandem Triple-Quadrupole Mass Spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2010 Mar;51(4):934–41.
12. Bioanalytical Method Validation Guidance for Industry [Internet]. FDA. U.S. Food and Drug Administration; 2018 May [cited 2022 Dec 23]. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry.