**1. Introduction**

The mass spectrometry (MS) is a technology enabling the measurement and analysis of multiple analytes with very high sensitivity and selectivity. The MS "use for purpose" includes the analysis of proteins and their modifications using either the top-down or the bottom-up approach, the analysis of multiple parameters for toxicology research, targeted analysis of active pharmaceutical compounds in patients' samples.

Recent years has brought a significant improvement of technology for ion-trap, the time-of-fligt, and triple quadrupole instruments resulting with a surge in applications for the medical, biological, and inorganic field. One of the greatest challenges for the MS was how to solve the problem of the insufficient ion transfer from the ionization source through different mass filters and to the detector. This challenge can be considered essentially solved due to changes in the design of ion-sources and subsequent mass filters such as ion funnels of different design, which enabled hugely improved ion focusing and ion transfer. However, there is still enough space to further improve the design of the interface to enable handling of larger ion currents generated by more powerful and more intense electrospray (ESI) ion sources. That would make bulky, expensive, and complex pumping systems obsolete. Actually, the greatest challenge of improving the MS is the ionization step, particularly when MS is used in combination with the liquid chromatography (LC). The combination of LC and MS (LC–MS) has advanced to the main workhorse in many laboratories, especially in biotechnology and medical laboratories. Since the low LC flow rate significantly increases the efficiency, the sensitivity, and the stability of the ESI, the use of separation columns with small inner diameters is recommended for the hyphenation of the two technologies. In cases with very low flow rates, e.g. 5–50 nl/min, a complete ionization of a substance can be achieved. On the other side, the use of such a low flow rates causes problems with flow's stability, reproducible flow gradient mixing, and stable ESI performance.

### **2. Clinical laboratory and mass spectrometry**

The laboratory medicine along with medical imaging procedures is one of the pillars of the modern diagnostics. The laboratory medicine has significantly benefitted from technical and technological development of analytical chemistry, miniaturization of instruments, and optimization of analytical methods.

Colorimeter and spectromphoter were the first modern analytical instruments to be used in a clinical laboratory. Since the time of their introduction, the art of

performing analyses and tests has significantly changed. In the 1950's additional developments were made and, in 1957 and 1959, respectively, the autoanalyzer, which is the precursor to the modern analytical systems, and the first RIA (immunoassay) for analysis of insulin were introduced. The introduction of RIA, which is still widely used, for insulin has significantly improved and changed the art of measuring a large of compounds.

The introduction of the mass spectrometry (MS) into the clinical laboratory had had the same revolutionary impact as the previously mentioned methods. A brief search for "mass spectrometry" and "clinical" from 1950 until 1970 results with only 9 publications! During the next 20 years, until 1990, the number of publications referring to the use of MS for different analyses in clinical laboratory jumped to 798! From the early 1990's until today, the steep rise of MS methods and approaches for analyses in clinical laboratory has steeply raised with more than 5000 publications from January 1st, 2020 – February 1st, 2021.

The significant rise in use of different MS approaches for clinical analyses correlates with improvements in ionization technologies, miniaturization of separation systems, notably of chromatographic systems (HPLC) [1], the significant and exciting improvements in sample preparation even of a single cell [2], and bioinformatic analysis. The mass spectrometry is applied for both "classical" clinical laboratory analyses and for analyzing samples for personalized and precision medicine. Undoubtly, the approaches and methods describe in current book are only a small part of possible applications.

In clinical laboratories, the analysis of clinical samples and monitoring levels of active compounds and their metabolites in e.g. patients' blood and urine samples are the main application fields. It is possible to perform specific detection of target analytes by applying MRM/SRM (multiple-reaction monitoring/selected-reaction monitoring) or SIM (single-ion monitoring) thus significantly enhancing the selectivity and sensitivity of the analytical method and provide targeted and highly specific analytical approach.
