A commercially available biliverdin sample was analyzed by means of HPLC/ESI–MS and NMR spectroscopy. It was been found that beside the main IXa 5Z,10Z,15Z isomer, the sample contains also the geometric isomer IXa 5Z,10Z,15E. It was also found the isomers behave differentially upon ‘‘in-source’’ fragmentation in negative ion mode (in contrast to the their behavior upon ‘‘in-source’’ fragmentation in positive ion mode and to their behavior upon MS/MS fragmentation in both modes): the relative abundances of deprotonated molecules and fragment ions are signiﬁcantly different for both isomers, which can be used as an analytical tool to differentiate between the isomers. Graphical abstract Keywords Biliverdin Geometric isomer Electrospray ionization Mass spectrometry NMR spectroscopy Introduction Biliverdin (BV) is a tetrapyrrolic pigment, a product of Electronic supplementary material The online version of this article heme catabolism. Usually the term ‘‘biliverdin’’ refers to (https://doi.org/10.1007/s00706-018-2161-7) contains supplementary the main isomer of biliverdin, namely to IXa isomer. material, which is available to authorized users. However, one should note that other positional isomers of ´ biliverdin have been identiﬁed (e.g. XIIIa or IXd)[1–11], & Rafał Franski firstname.lastname@example.org and also besides the ‘‘natural’’ (5Z,10Z,15Z) isomer the geometric isomers (e.g. (5Z,10Z,15E), at the exocyclic Faculty of Chemistry, Adam Mickiewicz University, double bonds are possible [12, 13]. Furthermore, the Umultowska 89B, 61-614 Poznan, Poland commercially available biliverdin may contain other posi- Adam Mickiewicz University, NanoBioMedical Centre, tional isomers as well . Therefore, we decided to check Umultowska 85, 61-614 Poznan, Poland using HPLC/ESI–MS if biliverdin obtained from a com- Adam Mickiewicz University, Centre for Advanced mercial source as hydrochloride contains other isomers. Technologies, Umultowska 89c, 61-614 Poznan, Poland 123 996 R. Fran´ski et al. The major ones were assigned to the Z,Z,Z-isomer of Results and discussion biliverdin, while the minor one to the Z,Z,E-isomer. The isomer ratio is about 5:1. The isomer structures were Figure 1 shows single ion chromatograms of ions ? - identiﬁed on the basis of a series of 1D selective NOE [BV ? H] and [BV-H] (m/z = 583 and 581) obtained experiments. For the Z,Z,Z-isomer the enhancement of upon HPLC/ESI–MS analyses. H-15 signal after irradiation of H-31 and H-30 was As clearly shown in Fig. 1, for both ions two chro- observed. For H-5 such an effect was recorded after irra- matographic peaks are obtained, thus the analyzed bili- diation of H-23 and H-21 or H-22 (vinyl group). In the verdin sample contains two isomers. Taking into account Z,Z,E-isomer the NOE effects between H-31 and H-30 as the height of the peaks in the positive ion mode, the ratio of well as H-30 and the vinyl group (H-32/33) were observed the main isomer to the minor isomer is about 5/1. A similar (Scheme 2). It is worth to add that an analogous geometric ratio was obtained by HPLC–UV/Vis analysis as shown in isomer has been described for biliverdin methyl ester the Supplementary Material. As described further a similar [12, 13]. ratio was also obtained by NMR spectroscopy. In the As mentioned earlier the isomers behave differently in negative ion mode the peak ratio is different (Fig. 1), it ESI/MS conditions in the negative ion mode (Fig. 1). indicates that the isomers may behave differentially upon Figure 4 shows the ESI mass spectra of both isomers in the ESI(-) conditions and this problem is discussed further in positive and negative ion mode at a cone voltage (CV) of the text. 50 V as representative examples. The spectra obtained at It can be taken for granted that the main isomer is IXa. other cone voltage values are shown in the Supplementary However, the question is what is the minor isomer. To Material. The cone voltage has the most profound effect on identify it by HPLC-ESI/MS we should have a respective the mass spectra obtained. Increase in this parameter leads isomer standards (to compare the retention times and ESI to the so-called ‘‘in-source’’ fragmentation/dissociation mass spectra). Fortunately, using NMR spectroscopy we (the pressure in this region is about 1 Pa), but a too low were able to identify the minor isomer also as IXa, how- cone voltage may cause a decrease in sensitivity (less ions ever, as Z,Z,E-isomer (obviously, the main isomer is reach the high vacuum region). The observed decomposi- Z,Z,Z), as discussed in detail below. The structures of both ? - tions of ions [BV?H] and [BV-H] in Fig. 4 are in good isomers (geometric isomers) are shown in Scheme 1. agreement with decomposition of this ions described The H NMR spectrum of biliverdin hydrochloride, elsewhere . recorded in acetonitrile/water solution consists of one set of As clearly seen in Fig. 4, the spectra of both isomers somewhat broadened signals, indicating a fast exchange in obtained in the positive ion mode are quite similar. There is the NMR time scale (Fig. 2). However, in methanol/water/ no difference in relative abundances of fragment ions. NaOD system (Fig. 3) two sets of signals were observed. ? - Fig. 1 Single ion chromatograms of ions [BV ? H] (top) and [BV-H] (bottom) 123 Identiﬁcation of a biliverdin geometric isomer by means of HPLC/ESI–MS and NMR spectroscopy… 997 Scheme 1 Fig. 2 H NMR spectrum of biliverdin sample in CD CN/D O (298 K) 3 2 There are only minor differences in relative abundances of there are differences in MS behavior of the isomers upon protonated dimers and sodium adducts. However, the ‘‘in-source’’ fragmentation in negative ion mode, however spectra of both isomers obtained in the negative ion mode there are no differences in MS behavior of the isomers are different. Beside the differences in relative abundances upon MS/MS fragmentation (in collision chamber). The of deprotonated dimers and sodium adducts, there is a fragmentation of ions upon MS/MS experiments occurs signiﬁcant difference in relative abundances of fragment later than that ‘‘in-source’’. Therefore, it is reasonable to ions (Fig. 4). It should be noted that in a few papers ESI/ conclude that before the isomer ions reach the collision MS has been successfully applied for biliverdin analysis chamber, they isomerize to the identical structure. The [16–21]. However, to the best of our knowledge, our fragmentation ‘‘in source’’ occurs almost immediately after ﬁnding is the ﬁrst one which demonstrates the different the transfer of the ions from solution to the gas phase, thus ESI/MS behavior of two biliverdin isomers. the fragmentation reﬂects the structural differences of the We also performed HPLC/ESI–MS/MS analysis of biliverdin isomers present in solution. commercial biliverdin sample. However, the MS/MS The key question is why the differences upon frag- spectra were very similar in both positive and negative ion mentation ‘‘in-source’’ of theisomers areinnegativeion mode. The results of HPLC/ESI–MS/MS analysis are mode and not in positive ion mode. It is reasonable that presented in the Supplementary Material. In other words, in positive ion mode, protonation occurs at nitrogen 123 998 R. Fran´ski et al. Fig. 3 H NMR spectrum of biliverdin sample in CD OD/D O/NaOD (298 K): full spectrum (top), selected regions (middle and bottom) 3 2 atom of C ring. Such protonated biliverdin molecules In negative ion mode, deprotonation of biliverdin may isomerize due to the resonance structures shown in molecule occurs at a carboxyl group. It is clear that the Scheme 3. isomerization of deprotonated biliverdin molecules is not 123 Identiﬁcation of a biliverdin geometric isomer by means of HPLC/ESI–MS and NMR spectroscopy… 999 Scheme 2 Scheme 3 Fig. 4 ESI mass spectra of bilverdin isomers obtained upon HPLC- ESI/MS analysis (CV = 50 V) 123 1000 R. Fran´ski et al. easy as the isomerization of protonated biliverdin However, for the main isomer decomposition of [BV-H] molecules. ion is amply compensated by the sensitivity increase (at To better understand the observed different behavior of higher cone voltage more ions reach the high vacuum the biliverdin isomers in the negative ion mode, we per- region). It is also worth adding that the decomposition of ions formed the breakdown plots of the respective ions, namely [BV-H] is compensated by the decomposition of ions - - - [2BV-H] , [BV-H] and the main fragment ion at m/ [2BV-H] . Taking into account the behavior of ions z = 285, against cone voltage. The breakdown plots are [2BV-H] (Fig. 5) it is clear that compensation of ion shown in Fig. 5. [BV-H] for the main isomer is more effective. As shown in Fig. 5 the gas phase stability of the We have performed the breakdown plots of respective [2BV-H] ion (deprotonated dimer) of main isomer is positive ions against cone voltage. As shown in the Sup- deﬁnitely higher than the gas phase stability of the plementary Material, the breakdown plots for both isomers [2BV-H] ion of the minor isomer (it is difﬁcult to are similar. rationalize why at a cone voltage of 70 V we deal with an increase in [2BV-H] ion abundances for both isomers). The gas phase stability of the [BV-H] ion of the main Conclusions isomer is also deﬁnitely higher than the gas phase stability of [BV-H] ion of the minor isomer. For both isomers Using HPLC-ESI/MS and NMR spectroscopy, the minor decomposition of ions [BV-H] begins from the cone isomer IXa 5Z,10Z,15E was found in a commercially voltage 40 V (fragment ion at m/z = 285 is formed, Fig. 5). available biliverdin sample (beside the main IXa - - Fig. 5 The breakdown plots of ions [2BV-H] , [BV-H] and main fragment ion at m/z = 285, against cone voltage (V). The abundances of ions correspond to the respective peak areas obtained upon HPLC-ESI/MS analysis 123 Identiﬁcation of a biliverdin geometric isomer by means of HPLC/ESI–MS and NMR spectroscopy… 1001 5Z,10Z,15Z isomer). The isomers behave differentially 2H, H-27), 2.35 (m, 4H, H25, 28), 2.19 (s, 3H, H-31), 2.14 upon ‘‘in-source’’ fragmentation in the negative ion mode (s, 3H, H-23), 2.08 (s, 3H, H-30), 1.86 (s, 3H, H-20) ppm. (in contrast to their behavior upon ‘‘in-source’’ fragmen- The HPLC-ESI/MS analyses were performed using a tation in the positive ion mode and to their behavior upon Waters model 2690 HPLC pump (Milford, MA, USA), a MS/MS fragmentation in both modes). It is difﬁcult to Waters/Micromass ZQ2000 mass spectrometer (single rationalize why this very geometric isomer is present in the quadrupole type instrument equipped with electrospray ion analyzed biliverdin sample. The geometric isomers are source, Z-spray, Manchester, UK). The software used was often formed as a result of exposure to light. However, our MassLynx V3.5 (Manchester, UK). Using an autosampler, sample had been stored in the dark and frozen. It should be the sample solutions were injected onto the XBridge C18 emphasized that our ﬁnding does not exclude such a column (3.5 lm, 100 9 2.1 mm i.d., Waters). The injec- commercial biliverdin sample from its use for scientiﬁc tion volume was 10 mm of biliverdin-containing solution (e.g. analytical) purposes. Quite the opposite, this sample at concentration 0.05 mg/cm . The solutions were analyzed may be useful for analysis of both isomers, at least for using linear gradient of CH CN–H O with a ﬂow rate of 3 2 semi-quantitative analysis. 0.3 cm /min. The gradient started from 0% CH CN—95% H O with 5% of a 10% solution of formic acid in water, reaching 95% CH CN after 10 min, and the latter con- Experimental centration was maintained for 10 min. The mass spectra were recorded in the m/z range Biliverdin (as hydrochloride) was obtained from Sigma- 200–1200, in positive and negative modes simultaneously Aldrich (Poznan ´ , Poland) and used without puriﬁcation. (during the HPLC/ESI–MS analyses the mass spectrometer H NMR spectra were recorded on Agilent DD2 800 was switched in the fast mode between the positive and spectrometer, operating at frequency 799.83 MHz. All negative ion modes). The electrospray source potentials spectra were measured at 298 K. The signal assignment has were: capillary 3 kV, lens 0.5 kV, extractor 4 V, and cone been made on the basis of 2D spectra (gCOSY, voltage 20–70 V (indicated in each mass spectrum shown). gHSQCAD, gHMBCAD) and 1D selective NOE mea- The source temperature was 120 C and the desolvation surements (mixing time 500 ms). Samples were prepared temperature 300 C. Nitrogen was used as the nebulizing by dissolution of 5 mg of biliverdin hydrochloride in and desolvation gas at the ﬂow rates of 100 and 300 dm /h, 3 2 2 0.7 cm of [ H] -methanol, containing 10% of [ H] -water respectively. 4 2 3 2 and 0.05% of NaOD or in 0.7 cm of [ H] -acetonitrile, Open Access This article is distributed under the terms of the Creative containing 10% of [ H] -water. Commons Attribution 4.0 International License (http://creative H NMR (CD CN/D O): d = 7.97 (bs, 1H, H-10), 6.69 3 2 commons.org/licenses/by/4.0/), which permits unrestricted use, dis- (bm, 1H, H-21), 6.51 (bs, 1H, H-15?), 6.49 (bs, 1H, H-5?), tribution, and reproduction in any medium, provided you give 6.17 (bm, 1H, H-32), 5.78 (bd, 1H, J = 11.4 Hz, H-22), appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were 5.64 (bd, 1H, J = 18.0 Hz, H-22), 5.41 (bs, 1H, H-33), made. 4.98 (bs, 1H, H-33), 3.23 (bs, 4H, H-24, H-27), 2.69 (bs, 4H, H-25, H-28), 2.28 (s, 3H, H-31?), 2.27 (s, 3H, H-23?), 2.10 (s, 3H, H-30), 1.48 (s, 3H, H-20) ppm. References H NMR data assigned to the 5Z,10Z,15Z-isomer (main) (CD OD/D O/NaOD): d = 7.02 (s, 1H, H-10), 6.73 (dd, 3 2 1. McDonagh AF, Lightner DA, Kar AK, Norona WS (2002) Bio- chem Biophys Res Commun 293:1077 1H, J = 11.7, 17.9 Hz, H-21), 6.52 (dd, 1H, J = 11.6, 2. Hirota K (1995) Biol Pharm Bull 18:48 17.6 Hz, H-32), 6.13 (s, 1H, H-15), 6.08 (s, 1H, H-5), 5.97 3. Suzuki Y, Sakagishi Y (1995) Anal Sci 11:699 (dd, 1H, J = 2.2, 17.6 Hz, H-33), 5.62 (dd, 1H, J = 1.6, 4. Heirwegh KPM, Blanckaert N, Van Hees G (1991) Anal Bio- 17.9 Hz, H-22), 5.57 (dd, 1H, J = 1.6, 11.7, H-22), 5.36 chem 195:273 (dd, 1H, J = 2.2, 11.6, H-33), 2.92 (m, 4H, H-24, 27), 2.35 5. Awruch J, Tomaro ML, Frydman RB, Frydman B (1984) Bio- chim Biophys Acta 787:146 (m, 4H, H25, 28), 2.18 (s, 3H, H-31), 2.13 (s, 3H, H-23), 6. Barrowman JA, Bonnett R, Bray PJ (1976) Biochim Biophys 2.11 (s, 3H, H-30), 1.84 (s, 3H, H-20) ppm. Acta 444:333 H NMR data assigned to the 5Z,10Z,15E -isomer (mi- 7. O’Carr P, Colleran E (1970) J Chromatogr 50:458 nor) (CD OD/D O/NaOD): d = 6.93 (s, 1H, H-10), 6.70 8. Heirwegh KPM, Fevery J, Blanckaert N (1989) J Chromatogr 3 2 496:1 (dd, 1H, J = 11.7, 17.9 Hz, H-21), 6.50 (dd, 1H, J = 11.6, 9. Schoch S, Lempert U, Wieschhoff E, Scheer H (1978) J Chro- 17.8 Hz, H-32), 6.13 (s, 1H, H-15), 6.03 (s, 1H, H-5), 5.87 matogr 157:357 (dd, 1H, J = 2.0, 17.8 Hz, H-33), 5.62 (dd, 1H, J = 1.6, 10. Hirota K, Yamamoto S, Itano HA (1985) Biochem J 229:477 17.9 Hz, H-22), 5.56 (dd, 1H, J = 1.6, 11.7, H-22), 5.37 11. Falk H (1989) The chemistry of linear oligopyrroles and bile pigments. Springer, Wien (dd, 1H, J = 2.0, 11.6, H-33), 2.92 (m, 2H, H-24), 2.89 (m, 123 1002 R. Fran´ski et al. 12. Holt RE, Farrens DL, Song P-S, Cotton TM (1989) J Am Chem 18. Abu-Bakar A, Arthur DM, Aganovic S, Ng JC, Lang MA (2011) Soc 111:9156 Toxicol Appl Pharm 257:14 13. Falk H, Grubmayr K, Haslinger E, Sehlederer T, Thirring K 19. Gorchein A, Lima CK, Cassey P (2009) Biomed Chromatogr (1978) Monatsh Chem 109:1451 23:602 14. Chen D, Brown JD, Kawasaki Y, Bommer J, Takemoto JY 20. De Matteis F, Lord GA, Lim CK, Pons N (2006) Rapid Commun (2012) BMC Biotechnol 12:1 Mass Spectrom 20:1209 15. Fran ´ ski R, Kozik T (2017) J Mass Spectrom 52:65 21. Niittynen M, Tuomisto JT, Auriola S, Pohjanvirta R, Syrjala P, 16. Stanojevic ´ JS, Zvezdanovic ´ JB, Markovic ´ DZ (2015) Pharmazie Simanainen U, Viluksela M, Tuomisto J (2002) Toxicol Sci 70:225 71:112 17. Weesepoel Y, Gruppen H, Vincken J-P (2015) Food Chem 173:624
Monatshefte für Chemie - Chemical Monthly – Springer Journals
Published: Feb 13, 2018
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