, Volume 80, Issue 9, pp 1343–1352 | Cite as

Development of a Simple, Rapid, and Robust Isocratic Liquid Chromatographic Method for the Determination of Pyrimethamine and its Synthetic Impurities in Bulk Drugs and Pharmaceutical Formulations

  • Yonah H. Mwalwisi
  • Ludwig Höllein
  • Eliangiringa Kaale
  • Ulrike HolzgrabeEmail author


Pyrimethamine is an important antiparasitic drug in the treatment of malaria and toxoplasmosis and is often used in combination with either sulfadoxine, sulfalene, or sulfadiazine. Determining the content of pyrimethamine and investigating the related substances is currently possible applying either a compendial monograph utilizing thin layer chromatography as well as liquid chromatographic methods used by the respective manufacturers. To provide a simple method which is capable of determining the content of pyrimethamine and of resolving four of its potential synthetic impurities a very simple, cheap, precise, and accurate isocratic RP-HPLC method was developed. All analytes can be separated within a total runtime of 30 min and the method was linear within the concentration ranges of 0.12–0.740, 0.104–0.621, 0.120–0.710, 2.0–11.8, and 1.01–5.80 µg mL−1 for pyrimethamine, impurity A, impurity B, impurity C, and impurity D, respectively. These substances were separated by employing a Eurospher-II C18H column (250 × 4.6 mm, 5 µm particle size), a mobile phase being a mixture of a 0.05 M KH2PO4 buffer solution (pH 2.6) and methanol in the ratio 40:60 (v/v). The analysis was carried out at 30 °C, applying a flow rate of 1.2 mL min−1, and a detection wavelength of λ = 215 nm. The coefficients of determinations (R 2) for the five analytes were greater than 0.994 for pyrimethamine and all impurities. Results of recovery studies were within the range of 89.1–105.1% for all substances. In all tested genuine batches of pyrimethamine raw material impurities within the specified limits were present which is concurrent with results obtained from using the present manufacturer’s method.


Pyrimethamine RP-HPLC Quality control Bulk drug and tablet analysis Malaria Impurity profiling 


B. P.

British Pharmacopoeia


The European Directorate for the Quality of Medicines and HealthCare


High-performance thin layer chromatography


Catholic Academic Exchange Service

Ph. Eur.

European Pharmacopoeia

Ph. Int.

International Pharmacopoeia


Reversed-phase high-performance liquid chromatography


Tanzania Food and Drugs Authority


Thin layer chromatography


United States Pharmacopoeia



Thanks to the KAAD (Catholic Academic Exchange Services, Bonn, Germany) for awarding scholarship to Yonah Hebron Mwalwisi, to the European Directorate for the Quality of Medicines and HealthCare (EDQM) for the provision of samples and reference substances, the Tanzania Food and Drugs Authority (Dar es salaam, Tanzania) for facilitating the research stay of Yonah H. Mwalwisi at the Institute of Pharmacy and Food Chemistry at Würzburg, and Christine Erk (Institute for Pharmacy and Food Chemistry, Würzburg) for LC/MS support.

Author contributions

YHM, LH, and UH designed the study, YHM performed sample preparation and analysis, data collection, data analysis and interpretation. YHM and LH drafted the manuscript. UH and LH supervised the study and contributed in data interpretation. UH, LH, and EK reviewed of the manuscript. All authors have read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.


This work was funded by the Catholic Academic Exchange Services, Bonn, Germany (KAAD) by providing a scholarship to YHM.


  1. 1.
    Vardanyan RS, Hruby VJ (2006) Synthesis of essential drugs. Elsevier Science, AmsterdamGoogle Scholar
  2. 2.
    World Health Organization (2015) The International Pharmacopoeia, 5th edn. World Health Organization, GenevaGoogle Scholar
  3. 3.
    United States Pharmacopoeia 38 NF 33 (2015) United States Pharmacopoeial Convention, Rockville, MD, USAGoogle Scholar
  4. 4.
    British Pharmacopoeia (2015) British Pharmacopoeia Commission, London, UKGoogle Scholar
  5. 5.
    Raveendra V, Vemula B, Kumar SP, Singhvi I (2013) HPLC method development and validation for simultaneous estimation of sulphadoxine and pyrimethamine in tablet dosage form. Int J Pharm Sci 4:295–298Google Scholar
  6. 6.
    Wessels JC (2010) International Pharmacopoeia monographs: antimalarial dosage forms. Thesis submitted for the degree Doctor of Philosophy in Pharmaceutics, Potchefstroom, South AfricaGoogle Scholar
  7. 7.
    Mwalwisi YH, Tettey JN, Pournamdari M, Watson DG (2005) The chemical and pharmaceutical equivalence of sulphadoxine/pyrimethamine tablets sold on the Tanzanian market. J Clin Pharm Ther 30:575–581CrossRefGoogle Scholar
  8. 8.
    Amin NC, Blanchin MD, Ake M, Fabre H (2012) Capillary zone electrophoresis as a potential technique for the simultaneous determination of sulfadoxine and pyrimethamine in tablet formulations. J Pharm Biomed Anal 58:168–171CrossRefGoogle Scholar
  9. 9.
    Meena S, Sandhya SM (2013) Validated HPTLC method for simultaneous analysis of pyrimethamine and sulphadoxine in pharmaceutical dosage forms. J Chem 2013:1–6CrossRefGoogle Scholar
  10. 10.
    Hoellein L, Holzgrabe U (2014) Development of simplified HPLC methods for the detection of counterfeit antimalarials in resource-restraint environments. J Pharm Biomed Anal 98:434–445CrossRefGoogle Scholar
  11. 11.
    Hoellein L, Kaale EA, Mwalwisi YH, Schulze MH, Holzgrabe U (2016) Routine quality control of medicines in developing countries: analytical challenges, regulatory infrastructures and the prevalence of counterfeit medicines in Tanzania. TrAC 76:7660–7670Google Scholar
  12. 12.
    Khuluza FSK, Jähnke RWO, Heide L (2016) Use of thin-layer chromatography to detect counterfeit sulfadoxine/pyrimethamine tablets with the wrong active ingredient in Malawi. Malar J 15:2–7CrossRefGoogle Scholar
  13. 13.
    International Council on Harmonization (2006) Harmonized tripartite guideline impurities in new drug products Q3B(R2)Google Scholar
  14. 14.
    Mwalwisi YH, Hoellein L, Kaale E, Holzgrabe U (2016) Development of a simple, rapid, and robust liquid chromatographic method for the simultaneous determination of sulfalene, sulfadoxine, and pyrimethamine in tablets. J Pharm Biomed Anal 129:558–570CrossRefGoogle Scholar
  15. 15.
    Geetha G, Ganika Raju NK, Kumar BV, Gnana Raja M (2012) Analytical method validation: an updated review. Int J Adv Pharm Biol Chem 1:64–70Google Scholar
  16. 16.
    Chan CC, Lam H, Zheng XM (2010) Practical approaches to method validation and essential instrument qualification. Wiley, HobokenCrossRefGoogle Scholar
  17. 17.
    Wolcott RG, Dolan JW, Snyder LR, Bakalyar SR, Arnold MA, Nichols JA (2000) Control of column temperature in reversed-phase liquid chromatography. J Chromatogr A 869:211–230CrossRefGoogle Scholar
  18. 18.
    Böhme H, Hartke K, Bracher F (2015) Arzneibuch-Kommentar zum Europäischen Arzneibuch. Wissenschaftline Verlagsgesellschaft, StuttgartGoogle Scholar
  19. 19.
    Kleemann A, Engel J (1982) Pharmaceutical substances: synthesis, patents, application. Thieme, New YorkGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Yonah H. Mwalwisi
    • 1
    • 2
  • Ludwig Höllein
    • 1
  • Eliangiringa Kaale
    • 3
  • Ulrike Holzgrabe
    • 1
    Email author
  1. 1.Institute of Pharmacy and Food ChemistryUniversity of WürzburgWürzburgGermany
  2. 2.Tanzania Food and Drug AuthorityDar es SalaamTanzania
  3. 3.School of PharmacyMuhimbili University of Health and Allied SciencesDar es SalaamTanzania

Personalised recommendations