Neutral loss analysis of amino acids by desorption electrospray ionization using an unmodified tandem quadrupole mass spectrometer

Abstract

A new method to analyze free amino acids using desorption electrospray ionization (DESI) has been implemented. The method is based on the neutral loss mode determination of underivatized amino acids using a tandem quadrupole mass spectrometer equipped with an unmodified atmospheric interface. Qualitative and quantitative optimization of DESI parameters, including ESI voltage, solvent flow rate, angle of collection and incidence, gas flow and temperatures, was performed for amino acids detection. The parameters for DESI analysis were evaluated using a mixture of valine, leucine, methionine, phenylalanine and tyrosine standards. A few microliters of this mixture were deposited on a slide, dried and analyzed at a flow rate of 2 microL/min. The optimal ionization response was obtained using laboratory glass slides and an equivalent solution of water/methanol doped with 2% of formic acid. The method specificity was evaluated by comparing product ion spectra and neutral loss analysis of amino acids obtained either by DESI or by electrospray ionization flow injection analysis (ESI-FIA). To evaluate the quantitative response on amino acids analyzed by DESI, calibration curves were performed on amino acid standard solutions spiked with a fixed amount of labelled amino acids. The method was also employed to analyze free amino acids from blood spots, after a rapid solvent extraction without other sample pretreatment, from positive and negative subjects. The method enables one to analyze biological samples and to discriminate healthy subjects from patients affected by inherited metabolic diseases. The intrinsic high-throughput analysis of DESI represents an opportunity, because of its potential application in clinical chemistry, for the expanded screening of some inborn errors of metabolism.

What can we learn from ambient ionization techniques?

“…The samples usually maintain their original chemical/physical/biological states without external interference before ionization [1,23]. Biological [24 -27], pharmaceutical [4, 28 -30], environmental [31][32][33][34], food [35][36][37][38], and forensic samples [39 -42] have been successfully examined by ambient mass spectrometry; typical examples of such analyses have been summarized in recent reviews [1,8,43,44]. Although the chemical composition and origins of samples can change dramatically in different studies, solid surfaces, solutions, and liquid mixtures are usually the states to be characterized by ambient mass spectrometry.…”

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

“…Specific OXC ion chromatogram, at (m/z) 253 > 180, shows one peak at RRT of 1.40, due to the elution of OXC, and another peak at RRT of 0.97 min, due to elution of DHD (named DHD 2) fragmented on cone and generating a main fragment at (m/z) 253. In fact, in an our previous paper [22] we have demonstrated that there is an interference in the OXC channel due to the ion source fragmentation of DHD at a cone voltage of 25 V. Despite MRM guarantees high specificity in drug assay, it is known that mass selective detection may produce compromised results due to interferences [17]. To avoid the fragmentation of DHD in source it is possible to use a cone voltage of 5 V obtaining just the (m/z) 271 molecular ion [22], but during the chromatographic run time the 4 MRM channels were acquired continuously and even if DHD voltage was set at 5 V, the voltages of other channels (OXC, MHD and CYE) were set at 25 V. So, in these conditions, during the assay there is also the generation of the DHD fragment of (m/z) 253 when the MS method operates with a cone voltage of 25 V. To avoid this interference we have increased the chromatographic resolution to separate DHD from OXC, completely.…”

“…In fact, in an our previous paper [22] we have demonstrated that there is an interference in the OXC channel due to the ion source fragmentation of DHD at a cone voltage of 25 V. Despite MRM guarantees high specificity in drug assay, it is known that mass selective detection may produce compromised results due to interferences [17]. To avoid the fragmentation of DHD in source it is possible to use a cone voltage of 5 V obtaining just the (m/z) 271 molecular ion [22], but during the chromatographic run time the 4 MRM channels were acquired continuously and even if DHD voltage was set at 5 V, the voltages of other channels (OXC, MHD and CYE) were set at 25 V. So, in these conditions, during the assay there is also the generation of the DHD fragment of (m/z) 253 when the MS method operates with a cone voltage of 25 V. To avoid this interference we have increased the chromatographic resolution to separate DHD from OXC, completely. Data comparison about the determination of DHD, following both reactions at (m/z) 271 > 180 (DHD 1) and (m/z) 253 > 180 (DHD 2), showed a good quantitative response for DHD 1 and DHD 2 with better results for the latter; thus we have used the (m/z) 253 > 180 MRM reaction for DHD determination in patients samples.…”

Biomarker Characterization by MALDI–TOF/MS

“…Electrospray-assisted laser desorption/ionization mass spectrometry was used to characterize glycated hemoglobin (HbA1c) in whole blood for rapid diagnosis of diabetes [88]. Desorption electrospray ionization mass spectrometry, direct analysis in real-time mass spectrometry, and paper spray ionization mass spectrometry were respectively used to analyze dried blood spots and urine to diagnose inborn metabolite disorders or monitor drug levels [89][90][91][92][93][94]. In addition to analysis of biological fluids, AMS is an efficient technique for directly characterizing biomolecules in tissues for disease diagnosis or classification [95][96][97][98][99][100].…”