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Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

Fyrestam J, Bjurshammar N, Paulsson E, Johannsen A, Östman C - Anal Bioanal Chem (2015)

Bottom Line: In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry.Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae.Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science and Analytical Chemistry, Analytical and Toxicological Chemistry, 106 91, Stockholm, Sweden.

ABSTRACT
Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus

HPLC-MS/MS chromatogram of porphyrins extracted from P. gingivalis cultivated for 12 d.. Three porphyrins were detected in the sample: CPI (4 ng/g), CPIII (26 ng/g), and PPIX (214 ng/g)
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Fig5: HPLC-MS/MS chromatogram of porphyrins extracted from P. gingivalis cultivated for 12 d.. Three porphyrins were detected in the sample: CPI (4 ng/g), CPIII (26 ng/g), and PPIX (214 ng/g)

Mentions: There are large differences in the ESI+ ionization efficiency between the porphyrins. UPI contains eight carboxylic acid substituents, whereas PPIX contains only two. This could be a possible reason for the latter compound having a 40 times higher response than UPI in the Agilent/Sciex API 2000 LC/MS system. That the less polar PPIX has a higher response in ESI-MS compared with the more polar porphyrin UPI is most probably due to the various distributions of ions of different hydrophobicity in the droplets of the spray [25]. In the Perkin Elmer/Sciex API 2000 LC/MS system, the difference in response between the porphyrins was substantially lower. The retention times were different in the two systems, which meant that PPIX was ionized in different mobile-phase compositions in the two LC/MS systems (Figs. 5 and 6). Selection of mobile phases has shown to be important when ESI is used. Ionization efficiency is influenced by the solvent composition where high water content of the mobile phase tends to give decreased ionization efficiency. This is due to the high viscosity of water, leading to difficulties in forming a stable spray, whilst a high organic content results in a better aerosol formation. This effect can be seen when compounds of varying polarity are analyzed with gradient elution in RP-HPLC. Thus, there are no straight-forward conclusions that can be drawn regarding the differences in ESI ionization efficiency of the porphyrins observed in this study.


Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

Fyrestam J, Bjurshammar N, Paulsson E, Johannsen A, Östman C - Anal Bioanal Chem (2015)

HPLC-MS/MS chromatogram of porphyrins extracted from P. gingivalis cultivated for 12 d.. Three porphyrins were detected in the sample: CPI (4 ng/g), CPIII (26 ng/g), and PPIX (214 ng/g)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551553&req=5

Fig5: HPLC-MS/MS chromatogram of porphyrins extracted from P. gingivalis cultivated for 12 d.. Three porphyrins were detected in the sample: CPI (4 ng/g), CPIII (26 ng/g), and PPIX (214 ng/g)
Mentions: There are large differences in the ESI+ ionization efficiency between the porphyrins. UPI contains eight carboxylic acid substituents, whereas PPIX contains only two. This could be a possible reason for the latter compound having a 40 times higher response than UPI in the Agilent/Sciex API 2000 LC/MS system. That the less polar PPIX has a higher response in ESI-MS compared with the more polar porphyrin UPI is most probably due to the various distributions of ions of different hydrophobicity in the droplets of the spray [25]. In the Perkin Elmer/Sciex API 2000 LC/MS system, the difference in response between the porphyrins was substantially lower. The retention times were different in the two systems, which meant that PPIX was ionized in different mobile-phase compositions in the two LC/MS systems (Figs. 5 and 6). Selection of mobile phases has shown to be important when ESI is used. Ionization efficiency is influenced by the solvent composition where high water content of the mobile phase tends to give decreased ionization efficiency. This is due to the high viscosity of water, leading to difficulties in forming a stable spray, whilst a high organic content results in a better aerosol formation. This effect can be seen when compounds of varying polarity are analyzed with gradient elution in RP-HPLC. Thus, there are no straight-forward conclusions that can be drawn regarding the differences in ESI ionization efficiency of the porphyrins observed in this study.

Bottom Line: In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry.Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae.Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science and Analytical Chemistry, Analytical and Toxicological Chemistry, 106 91, Stockholm, Sweden.

ABSTRACT
Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus