Lisa Delahaye (Presenter)
Ghent University
Bio: Lisa Delahaye studied Pharmaceutical Sciences at Ghent University. In 2016 she obtained her degree as Master in Drug Development. In January 2017 she started a PhD at the Laboratory of Toxicology at the Faculty of Pharmaceutical sciences at Ghent University. Her PhD focuses on the quantitative determination of protein adducts in dried blood spots and other microsamples. Currently she is working on albumin adducts, but also other protein adducts, such as hemoglobin adducts, will be included during the course of her PhD.
Authorship: Lisa Delahaye (1); Christophe P. Stove (1)
(1) Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University
| Short Abstract The risk assessment in case of a paracetamol intoxication can be improved by monitoring the NAPQI-albumin adduct, compared to the current guidelines. The potential value of this biomarker has been pointed out by several studies, using a 1 µM cut-off value for treatment. It can also be used to tailor paracetamol therapy in critically ill children and patients with liver disease, who have an increased risk for paracetamol-induced liver damage. Therefore, we aim to develop a method for the combined determination of paracetamol and the NAPQI-albumin adduct in 10 µL VAMS (volumetric absorptive microsamples) via LC-MS/MS. |
Long Abstract
Introduction
Paracetamol is one of the most used analgesics worldwide. When used in the proper pharmaceutical dose it has a very good safety profile. However, paracetamol intoxications, which can result in hepatotoxicity, are frequently seen. N-acetyl-para-benzoquinone imine (NAPQI), a reactive hepatic paracetamol metabolite, is the cause of this hepatotoxicity, as it forms adducts with cysteine residues of nearby proteins. The current assessment of the need for treatment with N-acetylcysteine, mainly based on the Rumack-Matthew nomogram, has several drawbacks, and thus a more sensitive and specific clinical biomarker for paracetamol-induced hepatotoxicity is desirable. Several reports have pointed out the potential clinical value of the NAPQI-albumin derived APAP-Cys to assess paracetamol-induced liver damage, and a cut-off value of 1 µM has been proposed. This biomarker can also be used to monitor the in vivo exposure to NAPQI in critically ill children, who often need a high dose of paracetamol, and liver disease patients, who have an increased risk of paracetamol intoxications. Therefore the combined determination of paracetamol and NAPQI-albumin, allowing to combine two risk assessment procedures, would be an added value in the clinical field.
The proposed analyses include minimally invasive sampling techniques (volumetric absorptive microsampling or VAMS), which can be performed by minimally trained, non-medical staff. Also, these samples are known to improve the stability of many analytes, and to facilitate storage and transportation issues. VAMS is a minimally invasive sampling technique, in which a fixed amount of capillary blood (10 µl) from a finger prick is collected. This is a relatively new alternative for regular blood sampling, with several important benefits, e.g. small sample volume; increased analyte stability, fewer difficulties with respect to sample handling, storage and transport, and less (not) influenced by the hematocrit effect compared to dried blood spots.
Methods
A VAMS-based method for the combined determination of paracetamol and APAP-Cys (derived from NAPQI-albumin after protein digestion) is being developed. Given the different requirements in sensitivity, a different instrument set-up is used for both analytes. This is done by two subsequent steps: (1) extraction with a methanolic solution to obtain paracetamol and (2) direct digestion (i.e. without prior extraction) with an aqueous pronase solution to determine APAP-Cys.
For paracetamol, UHPLC (Waters) is combined with MS/MS (AB SCIEX API 4000), while for APAP-Cys HPLC (Shimadzu) is combined with MS/MS (AB SCIEX 5500 QTrap). Optimization of the LC-MS/MS methods included the comparison of different mobile phases in combination with different (U)HPLC-columns, the testing of different gradient elution programs and the adaptation of source- and compound dependent parameters. The sample preparation procedure needed to be adjusted and optimized in order to be able to quantitate both compounds from one VAMS.
Results
For the quantitation of paracetamol, an ACQUITY UPLC® HSS T3 column (2.1 mm x 100 mm x1.8 µm) was chosen as it gave the best results in terms of peak shape. Mobile phases consisting of 95/5 water/MeOH (0.01% FA) (A) and 95/5 MeOH/water (0.01% FA) (B) at a flow rate of 0.55 mL/min gave sufficient sensitivity and a good peak shape. The calibration curve ranged from 100 ng/mL (set as LLOQ) to 100 µg/mL. For the LC-MS/MS method to determine APAP-Cys, a Chromolith® Performance RP-18e 100 mm x 4.6mm column equipped with a guard cartridge was chosen as it gave the best results in terms of peak shape. A mobile phase consisting of water (0.1% FA) (A) and MeOH (0.1% FA) (B) at a flow rate of 1.2 mL/min turned out to be the best option.
For the sample preparation procedure, a methanolic extraction (80/20 MeOH/water (0.01% FA)) is used to retrieve paracetamol from the VAMS. 300 µL is added to the VAMS and placed in the Thermoshaker for 10 minutes (1000 rpm; 22 °C). Subsequently the sample is centrifuged (10000 G, 10 min) and 250 µL of the supernatant is diluted with 750 µL water (0.01% FA) and transferred to the LC-MS/MS.
In a second step, the VAMS is washed with a methanolic solution (80/20 MeOH/water), and a second time with water to eliminate APAP-Cys not derived from NAPQI-albumin. The pronase solution is added to the VAMS and incubated for 24h at 37°C. The digest is filtered with a 3 kDa ultrafilter and transferred to the LC-MS/MS.
Preliminary experiments show that this sample preparation procedure should be able to meet the requirement of determination of both compounds from one VAMS, but the extraction procedure needs further optimization to be able to obtain a quantitative result for APAP-Cys.
Conclusion
The combined determination of both paracetamol and NAPQI-albumin, together with a minimally invasive sampling technique can be an added value in the risk assessment procedure of paracetamol overdoses.
References & Acknowledgements:
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