Skip to main content
SpringerLink
Log in

Plasma kinetic study of folinic acid and 5-methyltetrahydrofolate in healthy volunteers and cancer patients by high-performance liquid chromatography

  • Original Articles
  • Folates, Antifolates, Folinic Acid, High Performance Liquid, Chromatography, Metabolism
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Summary

A reversed-phase HPLC method is described for the simultaneous determination of folinic acid, MTX, and their plasma metabolites 5-CH3−FH4 and 7-OH-MTX respectively. In addition, this technique allows the separation of FA another naturally occurring folate, and of AMT, used as internal standard.

Separation of these compounds was achieved on a Waters Spherical C18 column at a flow rate of 0.8 ml.min-1. Elution was carried out with 0.1 M sodium acetate buffer (pH 5.5) as solvent A and 7.5% acetonitrile 92.5% bidistilled water as solvent B. UV detection was performed at 280 nm. This method was applied in a pharmacokinetic study of folinic acid and its plasma metabolite 5-CH3−FH4 following two different protocols: (1) i. v. bolus injection of 50 mg calcium folinate in six healthy volunteers and (2) simultaneous i. v. bolus injections of 50 mg/m2 MTX and 50 mg/m2 folinic acid in four cancer patients. Mean apparent half-life values for folinic acid and its metabolite were 7.02±1.81 h and 3.90±0.86 respectively in the first protocol, 4.80±1.48 h and 4.74±1.47 h in the second protocol. MTX and 7-OH-MTX were also quantified in the second protocol and were found not to affect the pharmacokinetics of folinic acid and 5-CH3−FH4.

Since in vitro studies on metabolism of folinic acid might be of great interest in trying to assess the mechanism of action of the folates and the potential interaction of MTX and 7-OH-MTX in this mechanism via the metabolism, the chromatographic method we describe here has been adapted for the separation of all the potential intracellular monoglutamyl metabolites of folinic acid.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

FH2 :

dihydrofolate

FH4 :

tetrahydrofolate

5-CHO−FH4 :

5-formyltetrahydrofolate

10-CHO−FH4 :

10-formyltetrahydrofolate

5-CH3−FH5 :

5-methyltetrahydrofolate

5,10-CH=FH4 :

5,10-methenyltetrahydrofolate

MTX:

methotrexate

7-OH-MTX:

7-hydroxymethotrexate

AMT:

aminopterin

HPLC:

high-performance liquid chromatography

References

  1. Allegra CJ, Chabner BA, Drake JC, Lutz R, Rodbard D, Jolivet J (1985) Enhanced inhibition of thymidylate synthetase by Methotrexate polyglutamates. J Biol Chem 17: 260

    Google Scholar 

  2. Allen BA, Newman RA (1980) High performance liquid chromatographic separation of clinically important folic acid derivatives using ion-pair chromatography. J Chromatogr 190: 241–245

    Google Scholar 

  3. Baker H, Thomson AD, Feingol S, Frank O (1979) Role of the jejunum in the absorption of folic acid and its polyglutamates. Am J Clin Nutr 22 (2): 124–132

    Google Scholar 

  4. Bird OD, McGlohon VM, Vaitkus JW (1965) Naturally occurring folates in the blood and liver of the rat. Anal Biochem 12: 18–35

    Google Scholar 

  5. Birmingham BK, Greene DS (1983) Analysis of folinic acid in human serum using high-performance liquid chromatography with amperometic detection. J Pharm Sci 72: 1306–1309

    Google Scholar 

  6. Blakely RL (1969) In: Bicohemistry of folic acid and related pteridines. Wiley, New York, p 35–41

    Google Scholar 

  7. Bore P, Rahmani R, Cano JP, Just S, Barbet J (1984) Radioimmunoassay of 7-hydroxymethotrexate and methotrexate. Clin Chim Acta 141: 135–149

    Google Scholar 

  8. Branfman AR, McComish M (1978) Rapid separation of folic acid derivatives by paired-ion high pressure liquid chromatography. J Chromatogr 151: 87–89

    Google Scholar 

  9. Breithaupt H, Kuenzlen E (1982) Pharmacokinetics of methotrexate and 7-hydroxymethotrexate following infusions of high-dose methotrexate. Cancer Treat Rep 66: 1733–1741

    Google Scholar 

  10. Briggs DR, Jones GP, Sae-Eug P (1982) Isocratic separation of food folacin by high-performance liquid chromatography. J Chromatogr 246: 165–168

    Google Scholar 

  11. Cashmore AR, Dreyer RN, Horvath C, Knipe JO, Covard JK, Bertino JR (1980) Separation of pteroyl-oligo-γ-L-glutamates by high performance liquid chromatography. Methods Enzymol 66: 459–468

    Google Scholar 

  12. Chanarin I, Perry J (1970) Exchange between parenteral and tissue folate in man. Br J Haem 18: 691–698

    Google Scholar 

  13. Chapman SK, Greene BC, Streiff RR (1978) A study of serum folate by high performance ion-exchange and ion-pair partition chromatography. J Chromatogr 145: 302–306

    Google Scholar 

  14. Clifford CK, Clifford AJ (1977) High pressure liquid chromatographic analysis of food for folates. J. Assoc Offic Anal 60: 1248–1251

    Google Scholar 

  15. Day BP, Gregory III JF (1981) Determination of folacin derivatives in selected foods by high performance liquid chromatography. J Agric Food Chem 29: 374–378

    Google Scholar 

  16. Djerassi I (1967) Methotrexate infusions and intensive supportive care in the management of children with acute lymphocytic leukemia: follow up report. Cancer Res 27: 2561–2572

    Google Scholar 

  17. Dudman NPB, Slowiaczek Tatter Sall MHN (1982) Methotrexate rescue by 5-methyltetrahydrofolate or 5-formyltetrahydrofolate in lymphoblast cell lines. Cancer Res 42: 502–507

    Google Scholar 

  18. Fabre G, Cano JP, Catalin J, Just S (1983) In vitro approach to 7-hydroxymethotrexate interaction with methotrexate metabolism as tool of pharmacokinetic study. Int J Clin Pharm Res 6: 475–484

    Google Scholar 

  19. Fabre G, Cano JP, Iliadis A, Carcassonne Y, Favre R, Gilli R, Catalin J (1984) Assay of methotrexate and 7-hydroxymethotrexate by gradient-elution high-performance liquid chromatography and its application in a high-dose pharmacokinetic study. J Pharm Biomed Anal 2: 61–72

    Google Scholar 

  20. Fabre G, Fabre I, Matherly LH, Cano JP, Goldman ID (1984) Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro. J Biol Chem 259: 5066–5072

    Google Scholar 

  21. Fabre G, Matherly LH, Favre R, Catalin J, Cano JP (1983) In vitro formation of polyglutamyl derivatives of methotrexate and 7-hydroxymethotrexate human lymphoblastic leukemia cells. Cancer Res 43: 4648–4652

    Google Scholar 

  22. Goldin A, Vendetti JM, Kline I, Mantel N (1966) Eradication of leukaemic cells (L1210) by methotrexate and methotrexate plus citrovorum factor. Nature (Lond) 212: 1548–1550

    Google Scholar 

  23. Groff JP, Blakley RL (1978) Rescue of human lymphoid cells from the effects of methotrexate in vitro. Cancer Res 38: 3847–3853

    Google Scholar 

  24. Guilidori P, Galli-Kienle M, Stramentinoli G (1981) Liquid chromatographic monitoring of 5-methyltetrahydrofolate in plasma. Clin Chem 27: 2041–2043

    Google Scholar 

  25. Halpern RM, Halpern BC, Clark BR (1975) New approach to antifolate treatment of certain cancers as demonstrated in tissus culture. Proc Natl Acad Sci USA 72 (10): 4018–4022

    Google Scholar 

  26. Hoppner K, Lampi B (1983) Reversed phase high pressure liquid chromatography of folates in human whole blood. Nut Rep Intern 27: 911–919

    Google Scholar 

  27. Hoppner K, Lampi B (1982) The determination of folic acid (pteroyl monogintamic acid) in fortified products by reversed phase high pressure liquid chromatography. J Liq Chromatogr 5: 953–957

    Google Scholar 

  28. Horne DW, Briggs WT, Wagner C (1981) High pressure liquid chromatographic separation of the naturally occurring folic acid monoglutamate derivatives Anal Biochem 116: 393–397

    Google Scholar 

  29. Kamen BA, Cashmore AR, Dreyer RN, Moroson BA, Hsieh P, Bertino JR (1980) Effect of (3H)methotrexate impurities on apparent transport of methotrexate by a sensitive and resistant L1210 cell line. J Biol Chem 255: 3254–3257

    Google Scholar 

  30. Langone JJ, Levine L (1979) Immunoassay of leucovorin: use of 125I-labeled protein A to detect immunological binding. Anal Biochem 95: 472–478

    Google Scholar 

  31. Lankelma J, Van der Klein E, Jansen MJ Th (1980) Determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid by high performance liquid chromatography, using on column concentration and electrochemical detection. J Chromatogr 182: 35–39

    Google Scholar 

  32. Lankelma J, Van Der Klein E, Ramaekers F (1980) The role of 7-hydroxymethothotrexate during methotrexate anticancer therapy. Cancer Lett 9: 133–142

    Google Scholar 

  33. McMartin KE, Virayotha V, Tephly TR (1981) High pressure liquid chromatography separation and determination of rat liver folates. Arch Biochem Biophys 209: 127–136

    Google Scholar 

  34. May M, Bardos TJ, Berger FL, Lansord M, Rabel JA, Sutherland GL, Shive W (1951) Synthetic and degradative investigations on the structure of folinic acid. J Am Chem Soc 73: 3067–3075

    Google Scholar 

  35. Mehta BM, Hutchson D (1975) Assay for citrovorum factor (NSC-3590) in the presence of methotrexate (NSC-740). Cancer Chemother Rep 59: 935–937

    Google Scholar 

  36. Montgomery JA, Johnston TP, Thomas HJ, Piper JR, Temple C (1977) The use of microparticulate reversed-phase packing in high-pressure liquid chromatography of compounds of biological interest. Adv Chromatogr 15: 169–195

    Google Scholar 

  37. Nahas A, Nixon PF, Bertino JR (1972) Uptake and metabolism of N5-formyltetrahydrofolate by L1210 leukemia cells. Cancer Res 32: 1416–1421

    Google Scholar 

  38. Nixon PF, Bertino JR (1972) Effective absorption and utilization of oral formyltetrahydrofolate in man. New Engl J Med 286 (4): 175–180

    Google Scholar 

  39. Pinedo HM, Zaharko DS, Bull SM, Chabner BA (1976) The reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides. Cancer Res 36: 4418–4424

    Google Scholar 

  40. Reed LS, Archer MC (1976) Separation of folic acid derivatives by high performance liquid chromatography. J Chromatogr 121: 100–103

    Google Scholar 

  41. Reif VD, Reamer JT, Grady LT (1977) Chromatographic assays for folic acid. J Pharm Sci 66: 1112–1116

    Google Scholar 

  42. Reingold RN, Picciano MF (1980) Separation of folate derivatives by in situ paired-ion high pressure liquid chromatography. J Chromatogr 190: 237–240

    Google Scholar 

  43. Reingold RN, Picciano MF (1982) Two improved high-performance liquid chromatographic separations of biologically significant forms of folate. J Chromatogr 234: 171–179

    Google Scholar 

  44. Rothenberg SP, Da Costa M, Rosenberg Z (1979) A radiochemical assay for N5-formyltetrahydrofolic acid (citrovorum factor) in serum and urine. Anal Biochem 93: 483–488

    Google Scholar 

  45. Rustum YM, Campbell J, Zakrzewski S, Frank C, Arbuck S, Madajewicz S, Petrelli N, Mittelman A, Creaven P (1984) Pharmacokinetic and cellular determinants of 5-fluorouracil (FU) in combination with high-dose citrovorum factor (CF) in patients (pts) with advanced colorectal carcinoma (CRC). Proc Am Ass Cancer Res 25: 167

    Google Scholar 

  46. Sato JK, Moran RG (1984) Interaction of methotrexate and CF at folylpolyglutamate synthetase. Proc Am Assoc Cancer Res 25: 1233

    Google Scholar 

  47. Sirotnak FM, Chello PL, Moccio DM, Kisliuk RL, Combepine G, Gaumont Y, Montgomery J (1979) Stereospecifity at carbon 6 of formyl inhibitor tetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro. Biochem Pharmacol 28: 2993–2997

    Google Scholar 

  48. Stout RW, Cashmore AR, Coward JK, Horvath CG, Bertino JR (1976) Separation of substituted pteroyl monoglutamates and pteroyl oligo-γ-glutamates by high-pressure liquid chromatography. Anal Biochem 71: 119–124

    Google Scholar 

  49. Straw JA, Covey M, Szapary D (1981) Differences in the pharmacokinetics of the diastereoisomers of citrovorum factor in dogs. Cancer Res 41: 3936–3939

    Google Scholar 

  50. Straw JA, Szapary D, Wynn WT (1984) Pharmacokinetics of the diastereoisomers of leucovorin after intravenous and oral administration to normal subjects. Cancer Res 44: 3114–3119

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut I. Paoli-J. Calmettes, INSERM U 278, 232, boulevard de Sainte Marguerite, BP 156, F-13273, Marseille Cedex 9, France

    Brigitte Payet, Gérard Fabre, Nicole Tubiana & Jean-Paul Cano

  2. Faculté de Pharmacie, 27, boulevard Jean Moulin, F-13385, Marseille Cedex 5, France

    Brigitte Payet, Gérard Fabre, Nicole Tubiana & Jean-Paul Cano

Authors
  1. Brigitte Payet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gérard Fabre
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicole Tubiana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean-Paul Cano
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported by the “Fédérations Nationale et Départmentale des Centres de Lutte contre le Cancer”, by the “Ministère de la Recherche et de la Technologie” and by the “Association pour le Développement de la Recherche sur le Cancer”

Rights and permissions

Reprints and permissions

About this article

Cite this article

Payet, B., Fabre, G., Tubiana, N. et al. Plasma kinetic study of folinic acid and 5-methyltetrahydrofolate in healthy volunteers and cancer patients by high-performance liquid chromatography. Cancer Chemother. Pharmacol. 19, 319–325 (1987). https://doi.org/10.1007/BF00261481

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00261481

Keywords

Search

Navigation