(M1330-02-08) Chromatographic Method for Determination of 6-mercaptopurine in Skin Permeation Assays

Purpose: 6-mercaptopurine monohydrate (6-MP) is widely used in leukemia chemotherapy, but limited information is available about its cutaneous absorption. Yet, the possibility of cutaneous absorption of 6-MP raises concerns about the safety of healthcare professionals and caregivers who come into contact with the drug during the administration and dispensing of its dosage forms [1]. Routine handling may result in the involuntary absorption of the drug, potentially leading to serious side effects [2]. This study aimed to validate an analytical method using high-performance liquid chromatography (HPLC) for quantifying 6-MP in the skin, allowing the assessment of its cutaneous permeation. Moreover, the utility of the developed method has been put to the test in a permeation assay to assess for the first time the potential risk of the drug cutaneous exposure.

Methods: For chromatographic determination of 6-MP, a C18 reversed-phase column was used as the stationary phase. The mobile phase was composed of acetonitrile and water in a 95:5 ratio (v/v), eluted isocratically at a 1.0 mL/min flow rate. The injection volume was 20 µL, and the column oven was maintained at 25°C. Detection was performed at 327 nm. The method was validated to quantify 6-MP in the different layers of the skin for permeation assay purposes, following the guidelines of the International Conference on Harmonization (ICH). The porcine skin layers extracts used for method validation were obtained by the tape stripping technique with adhesive tape, allowing to obtain three distinct extracts: stratum corneum (SC), hair follicles (HF), and remaining skin (RS) (Figure 1) [3]. To evaluate the developed method, a skin permeation assay was performed using porcine ear skin to assess the potential for contamination of the chemotherapeutic 6-MP while handling its tablet. The tests were conducted using a modified Saarbruecken permeation model [4].

Results: The validation of the method demonstrated high selectivity against skin interferents (Figure 2). No appreciable peaks related to the interferents were observed. Moreover, the presence of skin interferents in the analytical sample did not modify the chromatographic performance parameters of the analyte, evidencing that the components of the skin matrix did not compromise the quantification. The method showed linearity (R=0.999), precision, and sensitivity, with detection and quantification limits of 0.14 μg/mL and 0.42 μg/mL, respectively. The developed extraction procedure allowed the recovery of the drug from the stratum corneum, hair follicle, and remaining skin in a reproducible manner, minimizing the impact of enzymatic metabolism in these matrices. Using the developed method, a preliminary skin permeation test on porcine skin showed that a relevant amount of drug permeated the RS even in a solid state and for a short period (30 min). In fact, the dose permeated in this layer of the epidermis represents approximately 2% of the total dose in a tablet, which gives an indication of the high permeability of this molecule and the potential risk of adverse effects. Although the quantified 6-MP is in an inactive form, its retention in the skin raises concerns regarding occupational and unintentional exposure, especially due to its high toxicity.

Conclusion: Thus, the method described constitutes an essential tool in assessing the risk of topical contamination of 6-MP and can be used in long-term studies of skin permeation of this drug, contributing to the safety of health professionals, caregivers, and patients. The results here reinforce the need to adopt strict preventive measures, such as personal protective equipment and hygiene protocols, to minimize risks to patients, caregivers, and healthcare professionals.

References: [1] Mulla H, Buck H, Price L, Parry A, Bell G, Skinner R. Acceptability of a new oral suspension formulation of mercaptopurine in children with acute lymphoblastic leukaemia. Journal of Oncology Pharmacy Practice. 2015;22(3):387-395. doi:10.1177/1078155215577808
[2] Connor TH, McDiarmid MA. Preventing Occupational Exposures to Antineoplastic Drugs in Health Care Settings. CA Cancer J Clin. 2006;56(6):354-365. doi: 10.3322/canjclin.56.6.354
[3] Angelo T, Pires FQ, Gelfuso GM, da Silva JKR, Gratieri T, Cunha-Filho MSS. Development and validation of a selective HPLC-UV method for thymol determination in skin permeation experiments. Journal of Chromatography B. 2016;1022:81-86. doi: 10.1016/j.jchromb.2016.04.011
[4] Lima SGB, Pinho LAG, Sa-Barreto LL, Gelfuso GM, Gratieri T, Cunha-Filho M. Granules of finasteride and cyclodextrin obtained by hot-melt extrusion to target the hair follicles. Powder Technol. 2021;391:311-320. doi: 10.1016/j.powtec.2021.06.027

Acknowledgements: The authors declare that they have no conflict of interest. The authors acknowledge the University of Brasilia (UnB/Brazil), Studies and Projects Funding Agency (FINEP/Brazil), and Federal District Research Support Foundation (FAP-DF/Brazil).


Figure 1. Scheme of the tape stripping technique with adhesive tape, allowing the obtaining of porcine layers skin separation, i.e., stratum corneum, hair follicles, and remaining skin.


Figure 2. Chromatograms of 6-MP in acetonitrile in nominal concentration of 5 µg/mL containing different interferents from the skin layers (stratum corneum, hair follicle, and remaining skin).


Figure 3. Amount of 6-MP quantified in the different layers of the skin (stratum corneum - SC, hair follicle - HF, and remaining skin - RS) after the skin permeation test.