**1. Introduction**

The use of mass spectrometry in the clinical laboratory has become a standard for analysis of different substances such as antibiotics, for newborn screening, detection of immune-suppressive drugs, or the analysis of therapeutic antibodies used for the treatment of different diseases.

The focus of the use of mass spectrometry in clinical settings is the analysis of clinical samples and monitoring levels of active compounds and their metabolites in patients' blood and urine samples. The high sensitivity and specificity of the mass spectrometer and the possibility to perform specific detection of target analytes by applying MRM/SRM (multiple reaction monitoring/selected reaction monitoring) enable a targeted and highly specific analytical approach. The methods developed need a separation method in front of the MS and several companies such as Chromsystems (https://www.chromsystems.com/), ThermoFisher Scientific (https://www.thermofisher.com/at/en/home/clinical/diagnostic-testing/clinicalchemistry-drug-toxicology-testing/therapeutic-drug-monitoring.html) or BioRad (www.bio-rad.com), to name just a few, have developed fully verified and certified analytical systems. The interested reader is encouraged to search the internet for additional providers and systems.

Applying chromatography and mass spectrometry has its primary values in relatively fast detection and measuring of multiple analytes in a single sample with high sensitivity and high selectivity. In clinical routine, the key challenge for identifying

and analyzing active compounds is having the sensitivity of the analytical system needed and required to detect and quantify low-concentration analytes.

One of the challenges for using the MS in a clinical laboratory was the low ion yield, which significantly hampered the development of clinical applications. However, the development of new analytical systems, especially of new ion inlets and ion funnel designs with the most widely used electrospray ionization (ESI) sources has significantly improved ion focusing and ion transfer, which, finally, resulted in the overall increased sensitivity.

The quality of electrospray is highly dependable on separation conditions, i.e. mobile phase, presence or absence of salts, flow speed, column's inner diameter, etc. In proteomics, the use of columns with 50 μm or 75 μm ID is state-of-the-art. However, the columns operated at a low flow rate of several hundreds of nanoliters/ minute are still rare in clinical analysis although they can provide a significant increase in analysis's sensitivity.

However, currently, the use of nanoflow separation still cannot cope with the demand for high sample throughput in clinical applications. Currently, the closest compromise between sensitivity and throughput is the use of the microbore and capillary columns of 300 μm – 500 μm and 1 mm – 2 mm inner diameter.

A new and exciting application of mass spectrometry in the clinical environment is the use of "live-MS" during surgical operations. Further development of this approach will revolutionize the diagnostics and help surgeons in extracting e.g. tumors with higher accuracy and higher yield.
