Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE-enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2-2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.

Rationale The choice of mobile phase components and optimal ion source, mainly electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), is a crucial part in liquid chromatography/mass spectrometry (LC/MS) method development to achieve higher sensitivity and lower detection limits. In this study we demonstrate how to rigorously solve these questions by using ionization efficiency scales. Methods Four ionization efficiency scales are used: recorded with both APCI and ESI sources and using both methanol‐ and acetonitrile‐containing mobile phases. Each scale contains altogether more than 50 compounds. In addition, measurements with a chromatographic column were also performed. Results We observed a correlation between calibration graph slopes under LC conditions and logIE values in ESI (but not APCI) thereby validating the use of logIE values for choosing the ion source. Most of the studied compounds preferred ESI as an ion source and methanol as mobile organic phase. APCI remains the ion source of choice for polycyclic aromatic hydrocarbons. For APCI, both acetonitrile and methanol provide similar ionization efficiencies with few exceptions. Conclusions Overall the results of this work give a concise guideline for practitioners in choosing an ion source for LC/MS analysis on the basis of the chemical nature of the analytes.

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We constructed for the determination of lactose a bienzymatic biosensing system based on a fibre-optical oxygen sensor and two enzymes -β-galactosidase (β-gal, from Aspergillus oryzae, Sigma Aldrich, EC 3.2.1.23) and glucose oxidase (GOD, from A. niger, Sigma Aldrich, EC 1.1.3.4) and analysed how the calculation of biosensor output signal parameters, used for the calibration of lactose biosensors, is influenced by the data collection period during the transient phase of the signal rising in case no preliminary incubation period with β-gal was applied. The calculation of reaction steady state and kinetic parameters from the biosensor signal revealed that longer data collection periods resulted in more accurate biosensor calibration curves with bigger slopes, while in case of slower reactions the calculated reaction parameters had their maximal values already if data were collected for 600 seconds. For reactions where enzyme concentrations were higher (0.027-0.071 IU/mL β-gal and 2.03-5.33 IU/mL GOD), the steady state signal was not achieved even within 1 hour from the initiation of the reaction and the calculated reaction parameters continued to change. Although the sensor signal was decreasing continuously, the reaction parameters calculated from the transient phase data were suitable for biosensor calibration if the data of at least 500 seconds were taken into consideration.