Globally, cancer is a significant public health issue and a leading cause of death. ¹ Global Cancer Observatory (GLOBOCAN) estimates for 2020 indicate that 19.3 million cancer cases have occurred globally. India ranked third after China and the United States. According to GLOBOCAN’s prediction, the number of cancer cases in India is expected to rise to 2.09 million by 2040, representing a 57.5% increase from 2020. ² Hepatocellular carcinoma (HCC) is the most frequently occurring tumor among all primary liver cancers, accounting for 75–85% of cases. Patients with HCC are typically treated at an advanced stage, when a limited number of effective treatment options are available. Patients with advanced stages of HCC may benefit from systemic therapy. ^{3– 5} Despite improvements in early stage prognosis, the survival rate of patients with advanced illness remains poor.

Sorafenib and lenvatinib, tyrosine kinase inhibitors (TKIs), have been the first-line treatments for advanced HCC throughout the last decade, improving overall survival and quality of life, and have recently been replaced by immunotherapy. ^{6, 7} Sorafenib, a multiple-target tyrosine kinase inhibitor (TKI), inhibits angiogenesis by targeting proteins such as PDGFR-β, VEGFR-2, and c-KIT, and proliferation by targeting Raf-1, B-Raf, and kinase activity in the Ras/Raf/MEK/ERK signalling pathways, while extending the overall median survival of patients with advanced HCC. ⁸ According to the US Food and Drug Administration (FDA) recommendation, the approved dose of sorafenib is 400 mg (two tablets) twice daily without food. The dose may be reduced to 400 mg once daily depending on the adverse drug reactions experienced, such as hypertension, fatigue, weight loss, rash, hand-foot skin reaction, alopecia, and pruritus. ⁹ Lenvatinib, a multi-targeted receptor kinase inhibitor, inhibits the activity of FGFR 1, 2, 3, and 4; PDGFR α; VEGFR 1, 2, and 3; RET; and KIT. Lenvatinib binds to receptors and inhibits the PLCγ, Ras-Raf-ERK, and PI3K-AKT pathways, arresting tumor angiogenesis and cell death. ¹⁰ The median overall survival was 10.7 months in the sorafenib versus 12.8 months in lenvatinib group. ^{11, 12} Lenvatinib significantly surpassed sorafenib in terms of the ORR, PFS, and TTP. Its tolerance is adequate, and the most common adverse events (AEs) associated with therapy are hypertension, diarrhea, lack of appetite, and weight loss. ¹³ Poor adherence to oral chemotherapy is another major factor limiting long-term therapy. Inter-individual variability in pharmacokinetics is particularly pertinent in oncology, as anticancer agents are frequently administered at doses close to the maximally tolerable intensity, and most TKIs are considered to have a narrow therapeutic index. ¹⁴

Therefore, individualized dosing guided by pharmacokinetics could lead to less underdosing and reduced toxicity rates owing to overdosing. ¹⁵ Therapeutic Drug Monitoring (TDM) involves individualized drug dosage by maintaining plasma or drug concentrations within a therapeutic range or window. This clinical practice enables assessment of the efficacy and safety of medications for optimal patient benefits. ¹⁶ Determining sorafenib and lenvatinib plasma concentrations helps provide individualized treatment through dose adjustments to improve efficacy and avoid adverse effects. Generally, Asian patients do not tolerate the recommended dose in Western patients. ^{17– 19} According to FDA recommendations, sorafenib 400 mg (two tablets) twice daily and lenvatinib according to body weight (8 mg once daily if weight < 60 kg; 12 mg once daily if weight ≥ 12 kg), only a few patients can tolerate the total dose. Our preliminary data show that even at 25% or 50% of the recommended doses, therapeutic levels are achieved with a minimal adverse effects profile, and better patient adherence can increase quality of life.

Achieving maximum therapeutic efficacy with minimal iatrogenic toxicity is the primary objective of pharmacological treatment of patients with cancer. To achieve this goal, the dosage regimen should be adjusted for each patient. In regular clinical practice, although some individuals respond well to the standardized drug dosage regimen, others may experience lower efficacy and adverse effects. ²⁰ The association between dosage regimens and changes in drug and/or metabolite concentrations throughout the body over time has been studied using pharmacokinetics. ²¹ Dose adaptation based on therapeutic drug concentration, that is, therapeutic drug monitoring, can improve the treatment quality. Although TDM is routinely used for antibiotics and immunosuppressants, its role in the treatment of cancer is underutilized. ¹⁶ The criteria that need to be considered as candidates for TDM are tyrosine kinase inhibitors, which include large inter-individual PK variability, variable absorption rates, genetic variations in metabolizing enzymes, and interactions among co-medications. ^{22– 24} TKIs are mostly oral drugs that block the binding site of adenosine triphosphate (ATP) of tyrosine kinase receptors in malignant cells, thus preventing autophosphorylation of tyrosine residues and activating proteins involved in angiogenesis and tumor proliferation signalling pathways. ²⁵ TKIs are usually administered at fixed doses ²⁶ and orally. Steady-state methods provide a wide range of plasma concentrations with an inter-individual variability in trough concentration (C _trough) of up to23-fold. Sorafenib, an oral multi-kinase inhibitor with a recommended dosage of 400 mg twice daily, is a highly plasma-bound drug (99.5%). The liver metabolizes sorafenib via CYP3A4 enzyme and glucuronidation. High-fat meals reduce the bioavailability of sorafenib; therefore, they are either administered one hour before food or two hours after food intake. Close monitoring of patients receiving sorafenib therapy is required to determine toxicities. ^{8, 27, 28} According to previous studies and our pilot study, sorafenib concentrations ranged from 3.6 to 7 μg/mL. Improved overall survival was seen in patients with maximum sorafenib concentration C _max ≥4.78 μg/mL. ^{29– 31} The multi-targeted inhibitor lenvatinib was approved at 12 mg/day for patients with a body weight ≥ 60 kg and 8 mg/day for patients with a body weight < 60 kg. The drug is rapidly absorbed orally within 1-4 hours of administration (T _max). The enzymes CYP3A4 metabolize lenvatinib and are excreted in feces. Ninety-six to ninety-eight percent of the drug proteins are protein-bound. The incidence of gastrointestinal and skin toxicities is closely related to the concentration of lenvatinib. ^{32, 33} A study by Noda et al. ³⁴ demonstrated that lenvatinib’s target trough concentration in the treatment of HCC may range from 36.8 to 71.4 ng/mL in order to control disease and minimize grade 3 toxicity. Although the relationship between sorafenib and lenvatinib exposure and toxicity has been reported, data from the Indian population are lacking.

Here, we describe a sensitive, rapid, and simple LC-MS/MS method for the analysis of sorafenib and lenvatinib in human plasma using simple mobile phases (water and acetonitrile) with 0.1% formic acid, and extraction of analytes by simple protein precipitation with acetonitrile. The developed method reports a precise LC-MS/MS approach for the determination of sorafenib, sorafenib-n-oxide, and lenvatinib in hepatocellular carcinoma patients.

For three years, this prospective, interventional study was conducted in the Department of Medical Oncology, Amrita Institute of Medical Sciences, Kochi. The study protocol was approved by the Ethics Committee of Amrita School of Medicine (ECASM-AIMS-2021-293) on 15-06-2021 and was conducted as per principles of the Declaration of Helsinki and guidelines of the Indian Council of Medical Research.

Factors such as large inter-individual variability in drug absorption, genetic polymorphisms, drug interactions, and patient compliance with oral TKIs affect quality of life. Therefore, the implementation of therapeutic drug monitoring for TKIs can help reduce adverse events with maximum efficacy. The acceptance of TDM as a test in routine clinical practice can improve the survival of HCC patients with hepatocellular carcinoma. Therefore, this study has the potential to improve the clinical outcomes.