A new multi-target screening (MTS) procedure for drugs in blood and urine for toxicological analysis has been developed using a hybrid triple-quadrupole linear ion trap mass spectrometer (QTrap) for the fast detection and identification of 301 forensically important drugs, e.g. tranquilizers (benzodiazepines), hypnotics, drugs of abuse (opiates, cocaine, amphetamines, cannabinoids), antidepressants, neuroleptics, and some cardiac drugs, in one single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Samples were extracted either with liquid-liquid extraction or solid-phase extraction. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The advantage of this newly developed method is the possibility to detect and identify 301 drugs in one single LC/MS/MS run.

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An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions-in some cases solutions made of tablets-were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

Reproducibility of product ion spectra acquired using a liquid chromatography/triple-quadrupole mass spectrometry (LC/MS/MS) instrument over a 4-year period, and with three other LC/MS/MS instruments, one from the same manufacturer and two from a different manufacturer, was examined. The MS/MS spectra of 30 drug substances were generated in positive electrospray ionization mode at low, medium, and high collision energies (20, 35, and 50 eV). Purity and Fit score percentages against a 400-compound LC/MS/MS spectral library were calculated using an algorithm in which fragment intensity ratios and weighting factors were included. The long-term reproducibility study was conducted using a brand A instrument; after 4 years the reproducibility of the product ion spectra was still 94%, expressed as average Purity score. The inter-laboratory study involved two parts. Firstly, two LC/MS/MS spectral libraries, created independently in separate laboratories using brand A instruments, were compared with each other. The average Fit and Purity scores of spectra from one library against the other were better than 93 and 91%, respectively, when the same collision energies were used. Secondly, for the comparison of product ion spectra between brand A and brand B instruments, fragmentation conditions were first standardized for amitriptyline as the standard analyte. The average Fit scores of brand B spectra against the brand A spectral library varied between 79 and 85% at all three collision energies. These results indicate that, after standardizing the instrumental conditions, LC/MS/MS spectral libraries of drug substances are suitable for inter-laboratory use.