Mass Spectrometry Application Strategies of Dried Blood Spots Analysis

El análisis de manchas de sangre seca (DBS) es una tecnología de análisis bien conocida, cuyas primeras aplicaciones de rutina se remontan a la década de 1960. Los avances en los instrumentos de espectrometría de masas durante el siglo pasado, permitieron alcanzar la sensibilidad necesaria para abrir nuevos mercados. Sin embargo, algunas incertidumbres y la falta de comprensión de los métodos han impedido hasta el momento que la tecnología tenga una amplia aceptación en el mercado.En esta tesis doctoral se han desarrollado y validado diversas técnicas, condiciones y flujos de trabajo de análisis DBS, que demuestran la viabilidad y el potencial de aplicación de la tecnología DBS-LC-MS.Se han desarrollado métodos avanzados para su aplicación en el campo de la pediatría neonatal, en particular la prueba del talón en recién nacidos, donde se ha ampliado y estandarizado el panel de análisis.También se han investigado nuevos campos de aplicación, como la vigilancia de medicamentos terapéuticos y la toxicología forense. Se describe la aplicación de la farmacovigilancia remota de antirretrovirales en regiones de escasos recursos y se presentan nuevos enfoques analíticos para la vigilancia del abuso de alcohol. Finalmente, en esta tesis se ha introducido un método innovador con el que se pueden detectar más de 1.200 drogas ilícitas a partir de una sola gota de sangre.La investigación científica realizada se presenta en forma de compendio de publicaciones (6), que son incluidas en esta Memoria. Se adjuntan a modo de apéndice otros dos trabajos del candidato que no constan oficialmente en dicho compendio. Todos los trabajos constituyen una unidad temática coherente sobre la técnica de la DBS y su acoplamiento a la espectrometría de masas.This doctoral thesis is a compendium of dried blood spot (DBS) applications in the fields of newborn screening, forensic toxicology and therapeutic drug monitoring. DBS is a well-known analysis technology, which first routine applications date back to the 1960s. Advancements in mass spectrometry instruments during the last century, enabled to reach the required sensitivity to open up new markets. Some uncertainties and missing method understanding remain and this is holding back the technology from wide spread market acceptance. For the general scientific acceptance of this technology, several methods have been developed and validated within this work. Advanced methods for the field of newborn screening were developed, where the analysis panel has been extended and standardized. Goal of the first study was to transfer the amino acids and acyl carnitines analysis onto the automated DBS-MS 500 platform. Also, a steroid panel of 17OHP, cortisol and androstenedione was defined to exclude the 17OHP from the immune assay panel and to transfer this as well onto the fully automated DBS-LC-MS/MS. The conventional 17OHP enzyme-linked immunosorbent assay (ELISA) based on a manually punched DBS disc leads to a high percentage of false positive. First, cortisol increases when the babies are stressed, which also leads to elevated 17OHP concentrations and secondly, the ELISA has significant cross reaction potential with steroid sulfate which can be monitored with androstenedione. Both, the amino acid and acyl carnitine detection plus the integration of a steroid panel into the DBS-LC-MS/MS workflow was successfully achieved [1]. Newborn screening panels are not unified across borders and sometimes even differ within countries and laboratories. The implementation of the fully automated DBS platform could be a good starting point of standardization and unification of those programs. Here, a method based on an official newborn screening kit was introduced within this thesis. The analysis takes only 2 minutes per sample; however it is limited to amino acids and acyl carnitines only. The DBS extract is directly guided to the mass spectrometer without any column. This is a well-known procedure in newborn screening and allows speeding up the process to its optimum [2]. Therapeutic drug monitoring, especially in remote and rural areas is another upcoming application field of DBS. Several methods have been supported and co-authored, whereas the first method focuses of the three antiretroviral drugs nevirapine, efavirenz and lopinavir. The study highlights the transportation advantages of DBS, without any biohazard labeling neither requiring cooling chains. DBS were drawn in Tanzania, Africa, and sent to Switzerland where the samples went through different climate zones. Nonetheless, the samples showed very good results and stability of the monitored drugs [3]. We ran a follow up study on some of the samples approximately one year after the publication, where still all analytes could be recovered in the same concentration as published. Also the DBS sampling itself was investigated in a rural area of Tanzania [4] and the technique proved to be suitable. Another therapeutic drug, where a more efficient sampling process is required for mass drug administration (MDA) campaigns, is Ivermectin. This drug lowers the incidence of river blindness and lymphatic filariasis infections. Further, recent studies demonstrated that Ivermectin is also active against several other parasites and even against certain mosquitoes. As one of these mosquitoes is Anopheles gambiae, the major vector of malaria in Africa, Ivermectin could be mass administered to fight malaria. In other words, the drug makes the human blood lethal for the Anopheles gambiae and therefore reduces the number of vectors in an area. Still, more safety data is required from a larger population. Here, an according fully automated DBS method has been developed and validated. In addition, a comparison study to plasma samples, stability and hematocrit impact has been studied [5]. In forensic toxicology, it was shown that Dried Matrix Spot (DMS) is a suitable technology for large scale DBS applications. A fully automated method, where either 1200 drugs can be screened from a DBS and a quantitative follow up method focusing on the 28 most abundant drugs of abuse, was developed. This was documented within two publications, where the first publication was a proof of concept study to determine if the detection limits can be reached using the DBS-LC-MS/MS methodology [6]. The second, follow up publication was a specific method development and implementation. More than 1200 illicit drugs can be screened from a single DBS or Dried Urine Spots (DUS) within 20 minutes per sample using a Forensic Toxicology Database. The criteria of bio-analytical method validation guidelines were fulfilled, and the method was transferred into a routine laboratory successfully [7]. Furthermore, the discovery of phosphatidylethanol (PEth) as direct alcohol marker prolonged the window of detection for alcohol consumption to several weeks. PEth proved to be instable during storage of liquid blood samples. By using DBS sampling, this biomarker can be stabilized due to the inactivation of enzymatic activity. Also, for PEth, a fully automated DBS-LC-MS/MS was established for the determination of the two most abundant PEth homologs in a range from 20–1500 ng/mL. Automated DBS card handling and online solid phase extraction LC-MS/MS permits baseline separation and detection of PEth 16:0/18:1 and PEth 16:0/18:2 within 7 minutes per sample [8]. The methods from the various fields of application were presented at several conferences as oral presentations and posters, showing their interest from the scientific blood analysis community.<br /

Mass spectrometry application strategies of dried blood spots analysis

121 pags.-- Figuras,Tablas,Fotos.-- Tesis doctoral llevada a cabo para conseguir el grado de Doctor en la Universidad de Zaragoza.-- Lectura de la tesis: 19-09-2019.-- Sobresaliente "cum laude", con mención internacional.[EN] Dried blood spot analysis (DBS) is a well-known analysis technology, which first routine applications date back to the 1960s. Advancements in mass spectrometry instruments during the last century, enabled to reach the required sensitivity to open up new markets. Some uncertainties and missing method understanding remain and this is holding back the technology from wide spread market acceptance. In this doctoral thesis, various DBS analysis techniques, conditions and workflows have been developed and validated, demonstrating the feasibility and application of the DBS-LC-MS technology. Advanced methods for the field of newborn screening were developed, where the analysis panel has been extended and standardized. Also new fields of application, such as therapeutic drug monitoring and forensic toxicology were investigated. Drug monitoring of antiretroviral drugs in remote and resource poor regions are described and new approaches for alcohol abuse monitoring are presented. Finally, a breakthrough method, where over 1200 illicit drugs can be detected from one single blood droplet, was introduced within this thesis. The scientific research carried out is presented in the form of a compendium of publications (6), which are included in this report. Two other works of that do not appear officially in this compendium, are attached as an appendix. All the works constitute a coherent thematic unit on the technique of the DBS and its coupling to the mass spectrometry.[ES] El análisis de manchas de sangre seca (DBS) es una tecnología de análisis bien conocida, cuyas primeras aplicaciones de rutina se remontan a la década de 1960. Los avances en los instrumentos de espectrometría de masas durante el siglo pasado, permitieron alcanzar la sensibilidad necesaria para abrir nuevos mercados. Sin embargo, algunas incertidumbres y la falta de comprensión de los métodos han impedido hasta el momento que la tecnología tenga una amplia aceptación en el mercado. En esta tesis doctoral se han desarrollado y validado diversas técnicas, condiciones y flujos de trabajo de análisis DBS, que demuestran la viabilidad y el potencial de aplicación de la tecnología DBS-LC-MS. Se han desarrollado métodos avanzados para su aplicación en el campo de la pediatría neonatal, en particular la prueba del talón en recién nacidos, donde se ha ampliado y estandarizado el panel de análisis. También se han investigado nuevos campos de aplicación, como la vigilancia de medicamentos terapéuticos y la toxicología forense. Se describe la aplicación de la farmacovigilancia remota de antirretrovirales en regiones de escasos recursos y se presentan nuevos enfoques analíticos para la vigilancia del abuso de alcohol. Finalmente, en esta tesis se ha introducido un método innovador con el que se pueden detectar más de 1.200 drogas ilícitas a partir de una sola gota de sangre. La investigación científica realizada se presenta en forma de compendio de publicaciones (6), que son incluidas en esta Memoria. Se adjuntan a modo de apéndice otros dos trabajos del candidato que no constan oficialmente en dicho compendio. Todos los trabajos constituyen una unidad temática coherente sobre la técnica de la DBS y su acoplamiento a la espectrometría de masas.Peer reviewe

Validation of an automated extraction procedure for amino acids and acylcarnitines for use with tandem mass spectrometry for newborn screening

3 Tablas.- 1 Figura.- Datos suplementarios disponibles en la página web del editor.A certified reagent kit for newborn screening was transferred on a fully automated dried blood spot platform. The dried blood spot cards are directly eluted and the extract is online guided to tandem mass spectrometry instrument, where the amino acid and acyl carnitine panel is detected. The method takes 2 minutes per sample and requires no human interaction for up to 500 samples. The method is fully standardized through the automation and the usage of only certified consumables and reference material. The manual reagent kit was first modified to fit the automated platform, secondly validated and third, successfully transferred into a routine newborn screening laboratory.Peer reviewe

Automated drug screening of dried blood spots using online LC-MS/MS analysis

2 figuras, 2 tablasA fully automated method for the effective drug screening of large populations based on dried blood spot (DBS) technology is presented. DBSs were prepared, scanned, then spiked with deuterated standards, and directly extracted, before they were transferred online to an analytical liquid chromatography (LC) column and then to the electrospray ionization tandem mass spectrometry (ESI-MS/MS) system. The method was applied to DBS samples from two patients with back pain; codeine and oxycodone could be identified and quantified accurately below the level of misuse of 89.6 ng/mL and 39.6 ng/mL, respectively

Fully automated forensic screening of dried blood spots with MRM Spectrum Mode

Work presented at the 67th ASMS Conference on Mass Spectrometry and Allied Topics, June 2nd-6th, 2019, Atlanta, GA (USA).-- MP 099.Fully automated forensic screening of dried bloodspots was achieved by coupling a DBS-MS 500 system (CAMAG, Switzerland) to an LCMS system (Shimadzu, Japan). An increasing amount of prescription and illicit drugs drive clinical laboratories to more cost effective and faster screening methods in workplace drug testing, roadside testing, rehabilitation programs or post-mortem investigations without compromising established false positive or negative detection rates. Therefore, the increasing sample amount with a large panel of analytes needs to be handled in shorter analysis time and with simple sample preparation steps. In this study we present a fully automated dried bloodspot (DBS) extraction procedure coupled to an LC-MS/MS-system for forensic and toxicology screening of drugs and their metabolites. We use a new approach, the MRM Spectrum Mode, by measuring at least 5 MRM-transitions of each compound to generate a high confidence for the identification of the results.Peer reviewe

Fully automated forensic routine dried blood spot screening for workplace testing

3 Figuras, 3 Tablas.-- Sahar Y. Issa’s affiliation has been corrected to Faculty of Medicine, Alexandria University, Egypt.In this study, we describe the transfer of a new and fully automated workflow for the cost-effective drug screening of large populations based on the dried blood spot (DBS) technology. The method was installed at a routine poison control center and applied for DBS and dried urine spot (DUS) samples. A fast method focusing on the high-interest drugs and an extended screening method were developed on the automated platform. The dried cards were integrated into the automated workflow, in which the cards were checked in a camera recognition system, spiked with deuterated standards via an in-built spraying module and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. The target compounds were analyzed in positive multiple-reaction monitoring mode. Before each sample batch or analysis day, calibration samples were measured to balance inter-day variations and to avoid false negative samples. An internal standard was integrated prior the sample extraction to allow in process control. A total of 28 target compounds were analyzed and directly extracted within 5 min per sample. This fast screening method was then extended to 20 min, enabling the usage of a Forensic Toxicology Database to screen over 1,200 drugs. The method gives confident positive/negative results for all tested drugs at their individual cut-off concentration. Good precision (±15%, respectively ±20% at limit of quantification) and correlation within the calibration range from 5 to 1,000 ng/mL was obtained. The method was finally applied to real cases from the lab and cross-checked with the existing methodologies.Peer reviewe