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Exact quantification of the molar ratios of isotopologous mixed  O -methyl- O -methyl- d   3  -cellooligosaccharides (COS) comprising all combinations from fully methylated to fully deuteromethylated constituents within an individual degree of polymerization (DP) is the key step in the analysis of the substituent distribution over the polymer chains in methyl celluloses (MC). Deuteromethylation of MC is performed to level chemical differences, but due to a  m/z  range of 3 DP·ΔMe/Me- d   3  , bias during MS measurement cannot certainly be excluded. Therefore, ionization, ion transportation, and ion storage were studied with an electrospray ionization ion trap mass spectrometer (ESI-IT-MS) using binary equimolar mixtures of per- O -Me- and per- O -Me- d   3  -COS, defining the border cases of a particular Me/Me- d   3  -profile. Reference data of their molar ratio were determined after reductive amination with  m -amino benzoic acid by HPLC-UV. COS of DP2–6 were measured as their sodium adducts at  c  = 10 −6  M by syringe pump infusion. The impact of the RF voltage of the ion trap (TD), the octopole RF and DC voltages, and the  Cap Exit  potential on absolute and relative ion intensities were studied. Adapting the  Cap Exit  voltage was essential for correct quantification of DP2, while all COS of higher DP behaved insensitive with respect to bias. To check whether any bias occurs in the electrospray ionization process of the isotopologs, concentration-dependent measurements were performed with optimized instrumental settings for each DP. Intensity ratios IR =  I  (Me- d   3  )/ I  (Me) did not show any concentration-dependent trend and no selective ion suppression. Its decrease with DP observed under usually applied standard conditions ( smart mode ) is a consequence of discrimination according to  m / z  and can be overcome by appropriate instrumental settings of Oct 2 DC and TD. IR between 0.971 ± 0.008 and 1.040 ± 0.009 with no trend for DP (2-6) were obtained by averaging all measurements in the range 2 · 10 −7  to 2 · 10 −5  M total concentration. The DP-related optimized settings were applied to two MCs and compared with the results obtained under so far applied standard conditions. Graphical abstract

analytical and bioanalytical chemistry/analytical and bioanalytical chemistry

Impact of instrumental settings in electrospray ionization ion trap mass spectrometry on the analysis of multi-CH3-/CD3-isotopologs in cellulose ether analysis: a quantitative evaluation