Bioanalytical Method Validation. Applicazione di un metodo di validazione per l’analisi di droghe d’abuso su matrice urinaria
Author(s)
Giovagnoli, Maria
Date Issued
May 16, 2014
Type
Doctoral Thesis
Abstract
The present work arose from experiences and studies carried out during a PhD course at the UOC Toxicology Clinical Analysis Laboratory of the Hospital of Belcolle, ASL Viterbo.
In particular, we developed the validation method applied in the research of many analytes in the urine matrix, indicating the presence of even the lowest traces of these, in the screening of addiction substances.
The first part of the paper describes the daily activities of a forensic toxicology laboratory diagnostics in the field of forensic medicine, current Regulations and the Guidelines and Standards for ISO certification. Essentially it follows the process undergone by the sample organic: from its choice for the collection and to the chain of custody, that guarantee of the genuineness of the procedures from all points of view. In reference to the real Chemical and Toxicological Analysis I will refer to the choice of the type of analysis, distinguishing those of the first screening ( immunochemical methods ) from those of acknowledgment (gas chromatography and liquid chromatography coupled with mass spectrometry) which specifically concern my work. Having therefore fixed the reference standard and tested the sensitivity, the cut-off is detected, i.e. the process of exclusion of the samples that do not contain substances or their metabolites or which have a concentration below the threshold concentration. The comparison with the reference standard in the dyadic relationship with the quality controls contribute to the validation of analytical data that is submitted to Reporting and transmission of the data. The core of my thesis is devoted mostly to the coupling or ifenation of gas chromatography and / or high- performance liquid chromatography with mass spectrometry (GC / MS - HPLC/MS), which has significantly extended the applications of both techniques. The high sensitivity also allows the identification and dosage of traces of compounds not otherwise identifiable in very complex mixtures.
This limit of detection (identification) is then the lowest concentration that allows to assess the presence or absence of an analyte qualitatively and is indicated with the term LOD (Limit of Detection) and must be determined in each laboratory in reference to the instrumentation which is being used. The limit of quantification (LOQ - Limit of Quantitation ) is the lowest concentration of analyte that can be calculated with predetermined precision and accuracy, generally considering the signal attributed to the analyte wich is about 10 times higher than the standard deviation of the blank.
However, the laboratory does not only provides an analytical result, but the output of the data is conditioned by a series of chemical- toxicological assessments that can be performed only by those with experience and the necessary know how. What is required to Forensic Toxicologist is in fact the interpretation of the analytical data obtained; the interpretation regards the meaning of the positivity or negativity in relation to a series of parameters such as the type of biological sample examined and the analytical techniques used. Because of the changes in the field of drug abuse, control programs and collaboration with other laboratories are needed in order to continuously update and optimize the analytical procedures.
The mass spectrometer enhances the detection method although showing a destructive process . It can be associated with the liquid chromatograph for the detection of addiction substances . The operating principle is simple enough : the sample is introduced into a chamber where it is ionized and fragmented. These electrically charged fragments are made to migrate , and their rate of division will be based on the ratio molecular weight / charge . The detectors for signal transduction will do the rest obtaining spectrograms useful for conclusions. In the second part of the PhD, it has been emphasized the technical aspect of the equipment supplied. The sample is introduced in such a way that inside the chamber the vacuum is ensured. This is fragmented and ionized by the ionizer. In our case it is an electron-impact ionisation (ESI) : an incandescent filament of tungsten, produces electrons, travelling towards an anode situated opposite. This acceleration makes the electrons travelling at a very high energy (70 eV ) . Since to ionize a molecule an energy of 13-14 eV is required , the analyte is not only ionized but also fragmented.
The fragments eventually reach the analyzer . Actually we will examine all the methods of ionization and all type of analyzers common in laboratory practice investigations.
In other words, there are different types of analyzers, but in our case for the ifenation a quadrupole analyzer is used and a deeper analysis has been devoted to it, with respect to structure and function. This consists of four molybdenum metal bars that surround the path taken by the ions in the path between the ionizer and the detector. During this journey , the electromagnetic potential of the bars is kept oscillating , or if in the moment T0 two bars are positive and two are negative , the positive T1 instantly becomes negative and vice versa. Acting on the frequency of these oscillations , the fragments will move with a sinusoidal path toward the detector. So these fragments will move at a speed according to the charge and molecular weight. The signal from the photomultiplier ( the detector ) , is processed by a computer which will be able to use the signal to track the mass spectrum.
At our Toxicology laboratory HPLC-ESI-MS/MS tests are performed using a UltiMate 3000 UHPLC system of DIONEX with remote control ( via management software instrument or its override) , equipped with auto sampler thermostatically controlled and a module for the ' housing of the analytical column also with thermostat. The UltiMate 3000 HPLC system offers UHPLC compatibility across all modules , ensuring maximum performance for all users and all laboratories . The HPLC system was interfaced to a triple quadrupole mass spectrometer API 3200 Q TRAP, equipped with ESI ion source.
The mass spectrometer is equipped with a pump for infusion added to conduct experiments under FIA ( Flow Injection Analysis) . The system API 3200 Small Benchtop available, increases the power and performance of the Mass Spectrometry by the presence of the triple quadrupole . The operator can choose a higher resolution depending on the issues to be solved (eg . Fixing the resolution individually loaded 0.5-0,6 atomic mass unit, or amu ( atomic mass unit ) tuning with half the molecular weight; this can help in the search for double charge ions . The low resolution especially in Q3, often improves sensitivity in the MRM ( multiple
reaction monitoring ) mode used for quantitative analysis. The above mentioned MRM is the application of SRM
( Selected Reaction Monitoring ) to the ions produced by one or more precursor ions.
In the system 3000 of DIONEX UHPLC ultimate separation was achieved using a column chromatographic analytical Phenomenex 2.6U Kinetex XB- C18 100 A ( 100 x 2.10 mm ), preceded by a precolumn Phenomenex STRATA -X CW 25 microns on line 20 Extraction, 2 mm. Starting from the chemical standards and the deuterated analogues ( internal standard ) at different concentrations of the analytes, analyzed solutions in mobile phase are prepared. Both in Loading (load guard), and in the chromatography.
We will start by adding to 1 ml of urine, 30 L of methanol solutions previously described, the analytes, and 30 L of the solutions of internal standards.
By the hydrolysis reaction it has been possible to detach the analyte from the compound The system in use MQ4 can determine the various elements up to concentrations of less than one part per billion or PPB ( below one part for billion).
So the path of the Drug Screening Validation. was applied to the following analytes:
Amphetamines methamphetamine MDA MDMA EDDP morphine benzoylecgonine codeine Cocaine methadone
THC COOH
LSD Norbuprenorphine The whole procedure on-line SPE UHPLC-MS/MS is applied to samples of each standard in previously hydrolyzed urine samples and to mix samples containing the analytes considered at different concentrations . The solid phase extraction (SPE) is an extraction for adsorption, a physical process between a solid phase and a liquid, wherein the solid phase has a greater affinity for the compound to be isolated, as regards the solvent in which the same compound is dissolved. The adsorbent C18 bound to non polar groups allows good absorption and good chromatographic separation by HPLC.
In our laboratory at the end of the path through API3200QT , the ions are reported as Extracted ion
chromatogram ( XIC ) using MRM (Multiple Reaction Monitoring ) , i.e. the application of the SRM (Selected Reaction Monitoring ) ions produced by one or more precursor ions .
In a research analysis of data standard samples are used to build charts " dose / response " that represent the calibration curves or straight lines.
The results obtained have allowed us to determine the calibration curve: 10, 30 75, 150, 300 ng/ml, show good linearity (LOL). The Limit of Detection (LOD): 5 ng/ml. The limit of quantification (LOQ): 10 ng/ml. In conclusion, the innovative contribution in the field of Forensic Toxicology, allowed to obtain a calibration curve, suitable for the rapid execution of the confirm tests for clinical and forensic purposes.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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