Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000, and QC coefficients from 0.53 to 3.2%. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. The best results were obtained for zinc sulphate in methanol, but only nine analytes were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120%, precision from 0.5 to 13%, and process efficiency from 65 to 108%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.

Department of Chemistry Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czech Republic

Department of Parasitology Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czech Republic

Prague Zoo Prague Czech Republic

Zobrazit více v PubMed

Douglas, Angela E. Multiorganismal Insects: Diversity and Function of Resident Microorganisms. Annu Rev Entomol. 2015;60: 17–34. doi: 10.1146/annurev-ento-010814-020822 PubMed DOI PMC

Nogge G. Significance of symbionts for the maintenance of an optimal nutritional state for successful reproduction in hematophagous arthropods. Parasitology. 1981;82: 101–104.

Puchta O. Experimentelle Untersuchungen über die Bedeutung der Symbiose der Kleiderlaus Pediculus Vestimenti Burm. Zeitschrift für Parasitenkd. 1955;17: 1–10. doi: 10.1007/BF00260226 PubMed DOI

Akman L, Yamashita A, Watanabe H, Oshima K, Shiba T, Hattori M, et al.. Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nat Genet. 2002;32: 402–407. doi: 10.1038/ng986 PubMed DOI

Říhová J, Novaková E, Husník F, Hypša V. Legionella becoming a mutualist: Adaptive processes shaping the genome of symbiont in the louse polyplax serrata. Genome Biol Evol. 2017;9: 2946–2957. doi: 10.1093/gbe/evx217 PubMed DOI PMC

Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, Clark JM, et al.. Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle. Proc Natl Acad Sci U S A. 2010;107: 12168–12173. doi: 10.1073/pnas.1003379107 PubMed DOI PMC

Nikoh N, Hosokawa T, Moriyama M, Oshima K, Hattori M, Fukatsu T. Evolutionary origin of insect-Wolbachia nutritional mutualism. Proc Natl Acad Sci U S A. 2014;111: 10257–10262. doi: 10.1073/pnas.1409284111 PubMed DOI PMC

Boyd BM, Allen JM, Nguyen NP, Vachaspati P, Quicksall ZS, Warnow T, et al.. Primates, Lice and Bacteria: Speciation and Genome Evolution in the Symbionts of Hominid Lice. Mol Biol Evol. 2017;34: 1743–1757. doi: 10.1093/molbev/msx117 PubMed DOI PMC

Iwakawa H, Fukui T, Fukuwatari T, Bamba S, Sasaki M, Tsujikawa T, et al.. Blood concentrations and renal clearance of water-soluble vitamins in outpatients with ulcerative colitis. Biomed Reports. 2019;10: 202–210. doi: 10.3892/br.2019.1191 PubMed DOI PMC

Zhang Y, Zhou WE, Yan JQ, Liu M, Zhou Y, Shen X, et al.. A review of the extraction and determination methods of thirteen essential vitamins to the human body: An update from 2010. Molecules. 2018;23: 1–25. doi: 10.3390/molecules23061484 PubMed DOI PMC

Erikson DW, Blue SW, Fecteau KM, Edelman AB, Jensen JT, Blithe DL. Simultaneous assay of segesterone acetate (Nestorone®), estradiol, progesterone, and estrone in human serum by LC–MS/MS. Contraception. 2020;102: 361–367. doi: 10.1016/j.contraception.2020.08.006 PubMed DOI PMC

Roelofsen-de Beer RJAC, van Zelst BD, Wardle R, Kooij PG, de Rijke YB. Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI–MS/MS. J Chromatogr B. 2017;1063: 67–73. doi: 10.1016/j.jchromb.2017.08.011 PubMed DOI

Fatima Z, Jin X, Zou Y, Kaw HY, Quinto M, Li D. Recent trends in analytical methods for water-soluble vitamins. J Chromatogr A. 2019;1606: 360245. doi: 10.1016/j.chroma.2019.05.025 PubMed DOI

Martin F, Giménez EC, Konings E. New methods for the analysis of water-soluble vitamins in infant formula and adult/pediatric nutritionals. J AOAC Int. 2016;99: 19–25. doi: 10.5740/jaoacint.15-0245 PubMed DOI

Bates CJ. Vitamin analysis. Ann Clin Biochem. 1997;34: 599–626. doi: 10.1177/000456329703400604 PubMed DOI

Redeuil, Karine M, Longet K, Bénet S, Munari C, Campos-Giménez E. Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry. J Chromatogr A. 2015;1422: 89–98. doi: 10.1016/j.chroma.2015.09.049 PubMed DOI

Shetty SA, Young MF, Taneja S, Rangiah K. Quantification of B-vitamins from different fresh milk samples using ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring methods. J Chromatogr A. 2020;1609: 460452. doi: 10.1016/j.chroma.2019.460452 PubMed DOI

Redeuil K, Benet S, Affolter M, Thakkar K S, Campos Gimenez E. A Novel Methodology for the Quantification of B-Vitamers in Breast Milk. J Anal Bioanal Tech. 2017;08. doi: 10.4172/2155-9872.1000352 DOI

Hampel D, York ER, Allen LH. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. J Chromatogr B Anal Technol Biomed Life Sci. 2012;903: 7–13. doi: 10.1016/j.jchromb.2012.06.024 PubMed DOI

Nguyen QH, Hoang AQ, Truong TMH, Dinh TD, Le TT, Luu THT, et al.. Development of Simple Analytical Method for B-Group Vitamins in Nutritional Products: Enzymatic Digestion and UPLC-MS/MS Quantification. J Anal Methods Chem. 2021;2021. doi: 10.1155/2021/5526882 PubMed DOI PMC

Jenčo J, Krčmová LK, Sobotka L, Bláha V, Solich P, Švec F. Development of novel liquid chromatography method for clinical monitoring of vitamin B1 metabolites and B6 status in the whole blood. Talanta. 2020;211: 120702. doi: 10.1016/j.talanta.2019.120702 PubMed DOI

Islam MA, Park E, Jeong B, Gwak YJ, Kim J, Hong WH, et al.. Validation of vitamin B5 (pantothenic acid) and B6 (pyridoxine, pyridoxal, and pyridoxamine) analyses in seafood. J Food Compos Anal. 2022;109: 104518. doi: 10.1016/j.jfca.2022.104518 DOI

El-Hawiet A, Elessawy FM, El Demellawy MA, El-Yazbi AF. Green fast and simple UPLC-ESI-MRM/MS method for determination of trace water-soluble vitamins in honey: Greenness assessment using GAPI and analytical eco-scale. Microchem J. 2022;181: 107625. doi: 10.1016/j.microc.2022.107625 DOI

Zhao L, Zhao X, Xu Y, Liu X, Zhang J, He Z. Simultaneous determination of 49 amino acids, B vitamins, flavonoids, and phenolic acids in commonly consumed vegetables by ultra-performance liquid chromatography–tandem mass spectrometry. Food Chem. 2021;344: 128712. doi: 10.1016/j.foodchem.2020.128712 PubMed DOI

Yang J, Wilson I, Rainville P. Evaluation of hybrid surface technology for the analysis of the B-group vitamins by LC-ESI-MS / MS. J Chromatogr B. 2022;1204: 123336. doi: 10.1016/j.jchromb.2022.123336 PubMed DOI

Robinson, Frank A. The vitamin B complex. J Am Pharm Assoc. 1951;40: 594–594. doi: 10.1002/jps.3030401125 DOI

Schrijver J, Speek AJ, Klosse JA, Van Rijn HJM, Schreurs WHP. A Reliable Semiautomated Method for the Determination of Total Thiamine in Whole Blood by the Thiochrome Method with High-Performance Liquid Chromatography. Ann Clin Biochem. 1982;19: 52–56. doi: 10.1177/000456328201900111 PubMed DOI

Talwar D, Davidson H, Cooney J, JO’Reilly DS. Vitamin B1 status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: Comparison with erythrocyte transketolase activation assay. Clin Chem. 2000;46: 704–710. doi: 10.1093/clinchem/46.5.704 PubMed DOI

Wang JL, Liu X, Schnute W. Simultaneous Analysis of Water-Soluble Vitamins in Vitamin-Enriched Beverages and Multivitamin Dietary Supplements by UHPLC-MS/MS. Appl Note 294. 2016. Available: https://tools.thermofisher.com/content/sfs/brochures/AN-294-LC-Vitamins-Beverages-Supplements-LPN3024-EN.pdf

Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R. Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 2003;785: 263–275. doi: 10.1016/s1570-0232(02)00914-5 PubMed DOI

Brusius M. Navigating the Vast Array of Sample Preparation Techniques for Biological Samples–Whole Blood. Chromatogr today. 2016; 40–47.

Xu, Raymond N, Rieser, Matthew J, El-Shourbagy, Tawakol A. Current Methods for the Quantitative Analysis of Pharmaceutical Compounds from Whole Blood Matrix Using Liquid Chromatography Mass Spectrometry. In: Tzanavaras, Paraskevas D, Zacharis, Constantinos K, editors. Reviews in Pharmaceutical & Biomedical Analysis. Soest: Bentham Science; 2010. pp. 52–62. doi: 10.2174/978160805190811001010052 DOI

Ocké MC, Schrijver J, Obermann-De Boer GL, Bloemberg BPM, Haenen GRMM, Kromhout D. Stability of blood (pro)vitamins during four years of storage at -20°C: Consequences for epidemiologic research. J Clin Epidemiol. 1995;48: 1077–1085. doi: 10.1016/0895-4356(94)00232-f PubMed DOI

Jansen EHJM, Beekhof PK, Cremers JWJM, Schenk E. Long-term (in)stability of folate and vitamin B12 in human serum. Clin Chem Lab Med. 2012;50: 1761–1763. doi: 10.1515/cclm-2012-0108 PubMed DOI

Lawrance P. Niacin (Vitamin B3)—A review of analytical methods for use in food. Gov Chem Program Rep. 2015.

Priemer DS, Gomaa S. Vitamin B2 (Riboflavin). In: Medscape [Internet]. Available: https://emedicine.medscape.com/article/2088605-overview

Talwar DK, Azharuddin MK, Williamson C, Teoh YP, McMillan DC, O’Reilly DSJ. Biological variation of vitamins in blood of healthy individuals. Clin Chem. 2005;51: 2145–2150. doi: 10.1373/clinchem.2005.056374 PubMed DOI

Mackey, Amy D, Davis, Steven R, Gregory, Jesse F. Vitamin B6. 10th ed. In: Shils, Maurice E, Shike M, Ross, Catharine A, Caballero B, Cousins, Robert J, editors. Modern Nutrition in Health and Disease. 10th ed. Baltimore: Lippincott Williams & Wilkins; 2005.

Biological, dietetic and pharmacological properties of vitamin B9

Biological Properties of Vitamins of the B-Complex, Part 1: Vitamins B1, B2, B3, and B5