Passage
Electrospray ionization mass spectrometry (ESI-MS) employs a soft ionization technique that, unlike traditional ionization via electron bombardment, results in little to no fragmentation of target molecules. During electron bombardment, electrons collide with the analyte and cause the molecule to lose an electron whereas during ESI-MS, ions are produced by passing the analyte solution through an electrospray needle that has a potential difference applied with respect to a counterelectrode. This leads to the formation of charged droplets, which are repelled from the needle toward the counterelectrode and mass spectrometer, as shown in Figure 1.
Figure 1 ESI-MS ionization mechanismDuring the droplet's flight, the solvent is evaporated off analyte molecules. Ionization occurs through the protonation of basic sites on target molecules. Reaction 1 shows the number of protons n added to molecule M.M → (M + nH) ⁿ⁺Reaction 1Researchers used ESI-MS to quantify the formation of four pyridyloxobutyl-DNA (POB-DNA) adducts, shown in Figure 2. POB-DNA adduct formation involves highly mutagenic pyridyloxobutylating agents, which are formed from cytochrome P450-mediated hydroxylation of 4-(methylnitrosamino) -1-(3-pyridyl) -1-butanone (NNK) , an abundant N-nitrosamine carcinogen found in cigarette smoke. Adducts were produced in vitro by treating samples of calf thymus DNA with NNK. The DNA adducts were then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed via ESI-MS.
Figure 2 Molecular structure of POB-DNA adducts
Adapted from Lao Y, Villalta PW, Sturla SJ, Wang M, Hecht SS. Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem Res Toxicol. 2006.
-In a second experiment, the following DNA adducts are analyzed after separation by normal-phase HPLC. Which of the following is true regarding the separation of these adducts?

Question

Passage
Electrospray ionization mass spectrometry (ESI-MS) employs a soft ionization technique that, unlike traditional ionization via electron bombardment, results in little to no fragmentation of target molecules. During electron bombardment, electrons collide with the analyte and cause the molecule to lose an electron whereas during ESI-MS, ions are produced by passing the analyte solution through an electrospray needle that has a potential difference applied with respect to a counterelectrode. This leads to the formation of charged droplets, which are repelled from the needle toward the counterelectrode and mass spectrometer, as shown in Figure 1.

Figure 1 ESI-MS ionization mechanismDuring the droplet's flight, the solvent is evaporated off analyte molecules. Ionization occurs through the protonation of basic sites on target molecules. Reaction 1 shows the number of protons n added to molecule M.M → (M + nH) ⁿ⁺Reaction 1Researchers used ESI-MS to quantify the formation of four pyridyloxobutyl-DNA (POB-DNA) adducts, shown in Figure 2. POB-DNA adduct formation involves highly mutagenic pyridyloxobutylating agents, which are formed from cytochrome P450-mediated hydroxylation of 4-(methylnitrosamino) -1-(3-pyridyl) -1-butanone (NNK) , an abundant N-nitrosamine carcinogen found in cigarette smoke. Adducts were produced in vitro by treating samples of calf thymus DNA with NNK. The DNA adducts were then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed via ESI-MS.

Figure 2 Molecular structure of POB-DNA adducts
Adapted from Lao Y, Villalta PW, Sturla SJ, Wang M, Hecht SS. Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem Res Toxicol. 2006.
-In a second experiment, the following DNA adducts are analyzed after separation by normal-phase HPLC. Which of the following is true regarding the separation of these adducts?

Quizplus · Accepted Answer

11ecba2d_1280_ad33_a3bf_edea290969d3_MD0008_00 In contrast with RP-HPLC, normal-phase high-performance liquid chromatography (HPLC) and most other types of chromatography use a polar stationary phase and a nonpolar mobile phase. More polar molecules interact preferentially with the stationary phase (typically a silica gel that forms hydrogen bonds with polar solutes) and move more slowly through the HPLC column. Of the adducts shown, Adduct C is the most polar due to the presence of four -OH groups and two -NH groups. These groups allow Adduct C to form hydrogen bonds to the polar stationary phase with greater affinity than the other adducts. Therefore, Adduct C moves through the column most slowly and has the longest retention time and the latest elution. (Choice A) Compared to Adduct A, Adduct C has additional -OH groups. Accordingly, Adduct C is of greater polarity and has a longer retention time than Adduct A. (Choices B and D) Compared to the other adducts, Adduct B is largely nonpolar. Accordingly, Adduct B interacts least with the polar column and dissolves easily in the nonpolar solvent used in normal-phase HPLC. Therefore, Adduct B moves through the column fastest and elutes first. Educational objective: In normal-phase high-performance liquid chromatography, the stationary phase is polar whereas the mobile phase is nonpolar. Molecules of greater polarity move through the column more slowly (due to longer retention times) and elute later.

Passage Electrospray Ionization Mass Spectrometry (ESI-MS) Employs a Soft Ionization Technique