VAMS™ and AEDs: an exciting new LC-MS/MS method
A microsampling pioneer in Italy has recently developed, validated, and submitted for publication a new LC-MS/MS method for measuring concentrations of a range of antiepileptic drugs (AEDs) using Volumetric Absorptive Microsampling (VAMS™) technology. Dr. Ugo de Grazia and his collaborators at Fondazione IRCCS Istituto Neurologico Carlo Besta Neurological Hospital in Milano have established a useful example for facilitating easier antiepileptic drug monitoring and for dealing extraction difficulties of small drugs on different physical-chemical properties.
During their work, Dr. de Grazia and his team had to overcome significant challenges around extraction. “In the very first experiments, recovery and reproducibility were very low or very variable,” says Dr. de Grazia. “Then we had the idea that saved our lives and definitively changed our landscape. We found that by rehydrating blood on well desiccated tips for few seconds, we achieved the requirements needed for developing a robust and reliable method as requested by international FDA or EMA guidelines on biochemical method development.”
This experimentation was worth it, Dr. de Grazia says, because of the advantages of making VAMS™ work in his environment. “In our institution we hospitalize about 6,000 patients a year, of which about 1,300, or 22%, are children. Many of these children - but also adults - are affected by different forms of epilepsy and require TDM. When we became aware of this novel device for microsampling, we immediately thought it could be very useful for children because it’s less painful. Moreover, this kind of therapy is long lasting and requires continuous monitoring. Our patients come from all over Italy, traveling sometimes long distances, or very frequently, or both. So, we thought that if we have a reliable tool for microsampling, we can try to reduce patient’s effort in reaching our specialized laboratory to have their TDM, thus improving the patient’s quality of life.”
De Grazia also highlights the advantages of VAMS™ in the context of clinical laboratory workflow. “In planning our study, we also considered lab benefits, such as reduced costs with regards to dried blood microsampling - e.g., no cold chain or biohazard shipping – along with less need for a phlebotomist if samples are collected at home; no need to undertake processing of blood samples to prepare plasma; and finally assay compatibility with in-house routine chromatographic techniques.”
Dr. De Grazia is performing his current work in collaboration with Prof. Philip Patsalos and his team at National Epilepsy Research Centre in the UK, a globally known force in the world of research into therapeutic drug monitoring for antiepileptics. Dr. de Grazia credits Neoteryx with helping to establish and solidify this partnership. “Dr. James Rudge, technical director at Neoteryx, introduced me to Prof. Patsalos, as we share professional interest in microsampling and expertise in TDM of AEDs, and we both are members of International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). At the time, we had the first results on Mitra sampling applied to AED TDM, which we were going to show at the Neoteryx corporate workshop in MSACL EU congress in Salsburg (Austria) in 2017. Prof. Patsalos was impressed by those results and we exchanged some mails and calls to start a collaboration about this topic.”
Aside from demonstrating the possibility of using VAMS™ and Mitra devices to submit biological samples for the therapeutic drug monitoring of antiepileptic drugs, Dr. de Grazia’s research has created some other important discoveries. One of the most exciting, says Dr. de Grazia, is an ability “to highlight the different behavior between the drugs that are able to permeate the membrane of red blood cells and accumulate within, like zonisamide, and those that do not, like perampanel. This property was very exciting because it reveals a biological phenomenon and may be useful in all coming studies on small molecules - e.g., new drugs - to assay if they permeate, or don’t permeate, red blood cells. We can change our idea of total concentration of molecule since, until today, we referred to plasmatic concentration - drug bound fraction plus free fraction - not considering the reservoir of a drug within red cells, which may contribute to plasmatic drug equilibrium.”
Based on his challenges involving extraction, Dr. de Grazia makes some specific suggestions for working with VAMS™ and Mitra, which are likely to prove useful for other scientists and researchers sure to follow in his stead.
“Mitra is a powerful and versatile device that opens a new era in microsampling, but you may need some time to learn to handle and become confident with it,” says de Grazia. He outlines some essential first steps:
- Be sure tips are well dried before starting the extraction procedure to avoid carrying too much hemoglobin in samples.
- Rehydrate tips in water before extraction.
“Perhaps these are not universal laws,” Dr. de Grazia says, “but we found them mandatory in our study.”
He suggests that, after these preliminary steps, you should consider “if the molecule you are interested in is water soluble or not.”
- If it’s soluble in water, proceed thought the method recovering water used for rehydration - now including the drug - adding a precipitation step to be sure you don’t have any hemoglobin in your sample.
- If it’s not water-soluble, you may consider including one more step of extraction by the mean of a mixture water: organic - methanol, acetonitrile, or what is better suitable for your molecule - and then proceed further by precipitation. Depending on the nature of studied drug, consider testing different combinations of the mixture water: organic solvent.
“A comprehensive tutorial about Mitra sampling and derived analytical methods explaining and discussing all stages of VAMS™ procedures on different matrix has been recently published,” Dr. de Grazia says in conclusion. “VAMS™ applications and automation are discussed, too.”