Cover
Vol. 4 No. 1 (2026)

Published: June 1, 2026

Pages: 76-90

Review Article

Applications of HPLC in Pharmaceutical Analysis

Abstract

High-performance liquid chromatography (HPLC) is one of the most common analytical techniques used in pharmaceutical testing and in many other scientific fields. It is widely used because it can analyze different types of materials and give accurate and reliable results. HPLC is considered an important and trusted method in many laboratories. This narrative review gives a general explanation of HPLC, including its basic principles, simple instruments, and how the technique works with different samples. In pharmaceutical analysis, HPLC is mainly used in quality control laboratories. It is applied for drug assay, titration tests, impurity analysis, solubility studies, and stability testing. Many studies show that HPLC helps in ensuring the quality and safety of pharmaceutical products. In addition, HPLC is used in pharmacovigilance and toxicology to detect degradation products, identify counterfeit drugs, and find harmful or toxic compounds. These applications are important for protecting patient safety and supporting regulatory requirements. HPLC is also used in other fields such as environmental analysis, forensic science, and food analysis. In environmental studies, it helps detect pollutants and trace chemicals in water and soil samples. In forensic laboratories, HPLC is used to identify unknown substances in biological or chemical samples. In food analysis, it is applied to detect additives, contaminants, and residues. Although HPLC instruments have developed over time, proper method development, validation, and correct interpretation of results are still necessary to obtain reliable data for routine laboratory work and pharmaceutical regulatory use.

References

  1. Snyder LR, Dolan JW. Progress in high-performance liquid chromatography: patterns and future outlook. J Chromatogr A. 2021;1636:461774.
  2. Dong MW. New HPLC and UHPLC methods for analyzing drugs. LCGC North Am. 2022;40(2):18–26.
  3. Kazakevich Y, Lobrutto R. The growth of chromatographic procedures in labs that are controlled. TrAC Trends in Analytical Chemistry. 2020;130:115964.
  4. Vogt FG and Kord AS wrote a book on managing the life cycle of analytical methods in drug development. J. Pharm. Sci. 2021;110(2):421–430.
  5. Blessy M, Patel RD, Prajapati PN, Agrawal YK. Recent developments in the development of HPLC techniques that show stability. J Pharm Anal. 2020;10(2):111–122.
  6. Rozet E, et al. Quality by design used in analytical procedures. J Pharm Biomed Anal. 2020;178:112890.
  7. Reid GL and others Chromatography for checking the purity of drugs. Pharmaceutical Technology. 2021;45(5):30–39.
  8. Danezis GP, et al. HPLC-based techniques for ensuring food safety and detecting fraud. TrAC Trends Anal Chem. 2021;138:116224.
  9. Aronson JK. Data analysis in pharmacovigilance. Br J Clin Pharmacol. 2021;87(7):2771–27
  10. Richardson SD and Ternes TA. Analysis of emerging pollutants using LC–MS. Anal Chem. 2022;94(1):102–118.
  11. Pitt JJ. LC–MS in clinical and pharmacological analysis. Clin Biochem Rev. 2020;41(1):3–16.
  12. Peters FT and Remane D. LC–MS/MS for analytical toxicology. Analytical Bioanalytical Chemistry. 2021;413:25–37.
  13. Kümmerer K. Pharmaceuticals in the environment: analytical difficulties. Chemosphere. 2020;248:126013.
  14. Ternes TA and others The presence of drugs in water systems. Water Res. 2021;189:116631.
  15. Verlicchi P, et al. Pharmaceutical remnants in wastewater. Sci Total Environ. 740:140016.
  16. Maurer HH. New developments in clinical and forensic toxicology. Ther Drug Monit. 2021;43(1):6–15.
  17. Flanagan RJ. The function of chromatography in toxicological examination. Clin Biochem Rev. 2020;41(4):175–186.
  18. The World Health Organization. Pharmacovigilance: Making Sure Medicines Are Safe to Use. WHO in Geneva, 2020.
  19. Council for Harmonization in the World. ICH Q2(R2): Checking to see whether analytical procedures are correct. Geneva; 2022.
  20. The United States Pharmacopeia. USP <621> Chromatography. The USP Convention will be held in 2023.
  21. European Pharmacopoeia Commission. The European Pharmacopoeia. 11th ed. EDQM, Strasbourg, 2023.
  22. Massart DL, et al. Chemometrics in chromatographic evaluation. TrAC Trends in Analytical Chemistry. 2020; 130:115965.
  23. Brereton RG. Recognizing patterns in chromatographic data. Analytical Techniques. 2021;13:4203–4217.
  24. New UD. Improvements in the technology of HPLC columns. J Chromatogr A. 2020;1625:461281.
  25. Dong MW. Systems and uses of UHPLC. LCGC North Am. 2022;40(6):28–36.
  26. Anastas PT and Warner JC wrote "Green Analytical Chemistry Perspectives." Chemistry Green. 23:3405–3416 in 2021.
  27. Vogt FG. Chromatography and data integrity. J Pharm Innov. 2021;16:1–9.
  28. Borman P, et al. Transferring methods and making HPLC more reliable. J Pharm Biomed Anal. 2020;181:113101.
  29. Newton PN, et al. Low-quality drugs and analytical detection. Lancet. 2020;395:1985–1998.
  30. Petrovic M and others LC–MS for keeping an eye on the environment. A. Chromatogr. 1640:461950 in 2021.
  31. Nollet LML. HPLC for Food Analysis. 4th ed. CRC Press, Boca Raton, 2021.
  32. Andersen WC and Turnipseed SB. Residue analysis in food matrices. J Chromatogr A. 2022;1661:462739.
  33. Reid LM et al. Chromatography for checking the authenticity of food. Food Chem. 2021;356:129714.
  34. Danezis GP and others. Using LC methods to find food fraud. TrAC Trends in Analytical Chemistry. 2022;146:116493.
  35. Peters RJB and others Testing for drug residues in animals. Food Addit Contam. 2020; 37:1–14.
  36. Shah VP and others Revisiting the validity of bioanalytical methods. AAPS J. 2021;23:56.
  37. Jemal M. Bioanalysis using LC–MS/MS. Anal Chem. 2020; 92:705–725.
  38. Dressman JB and Krämer J. Testing for Dissolution in Pharmaceuticals. 3rd ed. CRC Press; 2021.
  39. Reid GL and others Management of the lifecycle of chromatographic techniques. Pharm Tech. 2022;46:24–33.
  40. Vogt FG and Kord AS talk about QbD ideas in analytical science. J Pharm Sci. 2021;110:195–204.
  41. Richardson SD. Advanced LC methods for analyzing water Anal Chem. 2021;93:124–141.
  42. Ternes TA, Joss A. Drugs in the environment. Environmental Science and Technology. 2020;54:889–904.
  43. Verlicchi P and Zambello E. Keeping an eye on hospital wastewater. Sci Total Environ. 755:142504 in 2021.
  44. Maurer HH and Meyer MR. LC–MS in forensic science. Drug Test Anal. 2020; 12:149–162.
  45. Deisingh AK. Finding fake pharmaceuticals. Anal Bioanal Chem. 2021;413:559–570.
  46. Peters FT, et al. Hyphenated methodologies in toxicology. Analytical Bioanalytical Chemistry. 2022;414:607–620.
  47. Brereton RG and Lloyd GR wrote "Chemometrics in Chromatography." Analyst. 2021;146:178–192.
  48. Kromidas S. Development of the HPLC Method. Wiley; 2021.
  49. Dong MW and Wysocki J. talk about modern gradient elution techniques. LCGC North America 2020;38:498–507.
  50. The World Health Organization. Making sure that drugs are safe and effective. WHO, Geneva; 2022.
  51. The European Medicines Agency. Guideline for impurities. EMA; 2020.
  52. FDA. Validation of analytical procedures and methods. FDA; 2022.
  53. Massart DL and Vandeginste BGM wrote a book called Chemometrics. Elsevier; 2020.
  54. Snyder LR, Kirkland JJ, Dolan JW. A Beginner's Guide to Modern Liquid Chromatography. 4th edition Wiley; 2021.
  55. Dolan JW. Comprehending retention and selectivity in HPLC. LCGC. 2020;38:56–64.
  56. Kazakevich Y. Tuning selectivity in RP-HPLC. J Chromatogr A. 2021; 1635: 461718.
  57. Hellinger R, and others HILIC uses in the analysis of drugs. J Pharm Biomed Anal. 2022;206:114355.
  58. Pitt JJ. Tandem mass spectrometry in clinical analysis. Clinical Biochemistry. 2021;90:1–10.
  59. Reid GL. What regulators anticipate from chromatographic data. Pharm. Regul. Aff. 2020;9:1000196.
  60. Flanagan RJ, et al. Difficulties in toxicological analysis. Clin Toxicol. 2021;59:481–493.
  61. Kümmerer K, et al. Environmental hazards associated with medications. Environ. 2022;161:107133.
  62. Ternes TA. What happens to drugs in water. Water Res. 2020;182:116040.
  63. Anastas PT. Green chemistry in analytical science. Green Chem. 2022;24:122–135.
  64. Danezis GP. Fingerprinting based on LC in food analysis. Control of Food. 2020;110:107007.
  65. Peters RJB. Strategies for analyzing food pollutants. TrAC Trends in Analytical Chemistry. 2021;136:116190.
  66. Maurer HH. Understanding chromatographic data in forensic science. Ther Drug Monit. 2022; 44:1–9.
  67. Newton PN. Keeping an eye on bad drugs. BMJ Global Health. 2020;5:e002505.
  68. Aronson JK. Safety signals and analytical proof for drugs. Drug Safety. 2021;44:35–44.
  69. Richardson SD. New problems in water analysis. Anal Chem. 2020;92:524–536.
  70. Verlicchi P. Pharmaceuticals in hospital wastewater. Sci. Total Environ. 2022;808:152138.
  71. Jemal M. Trends in bioanalytical LC–MS/MS. Biological analysis. 2021;13:1747–1760.
  72. Brereton RG. Recognizing patterns in LC data. Analytical Techniques. 2020;12:440–452.
  73. Nollet LML and Toldrá F. Food Analysis by HPLC. 2021 CRC Press.
  74. Peters FT. New things in forensic LC–MS. Drug Test Analysis. 2021;13:6–15.
  75. Kromidas S. Testing the strength of HPLC. J Chromatogr A. 2022;1670:462959.
  76. Dong MW. Managing the lifecycle of chromatographic techniques. LCGC North America 2023; 41:18–26.
  77. Vogt FG. Strategies for analytical control. Journal of Pharmaceutical Sciences 2022;111:143–152.
  78. Danezis GP. Trends in finding food fraud. TrAC Trends in Anal Chem. 2023;154:116641.
  79. Richardson SD. Improvements in environmental LC analysis. Anal Chem. 2023;95:102–118.
  80. Snyder LR and Dolan JW talk about what the future holds for liquid chromatography. J Chromatogr A. 2023;1700:463308.
  81. Saeed AM, Hamzah MJ, Ahmed NQ. Quantitative assay of aspirin and salicylic acid and heavy metals as impurities in Iraqi market aspirin tablets using different analytical methods. Int J Appl Pharm. 2018;10(5):167–172. doi:10.22159/ijap.2018v10i5.26820.
  82. Saeed AM, Hamzah MJ, Mohammed OJ. Validation of liquid chromatographic analytical method for determination of cephalexin and aspirin in pure and pharmaceutical preparations. Int J Pharm Res. 2020;12(Suppl 1):1625–1631.
  83. Merahge AH, Hamzah MJ, Al-Anbakey AM. Comparative analytical determination of isoniazid in pure and pharmaceutical preparations using spectrophotometric and RP-HPLC methods. Biochemical and Cellular Archives. 2019;19(2):3617–3622.
  84. Al-Kaffiji Hamzzah M.J, Al-Anbakey AMS. New chromogenic reagent for the spectrophotometric determination of chlorpromazine HCl in aqueous solutions and pharmaceutical formulations. Int J Pharm Pharm Sci. 2013;5(Suppl 3):606–611.
  85. Hamzah MJ, Alawad KM, Hameed TM, Hanna JS, Alanee RS, Najim HK, Ali RE, Qusay HL. Development of RP-HPLC method for the determination of isoniazid in pharmaceutical dosage forms. Bull Chem Soc Ethiop. 2026;40(1):1–9. doi:10.4314/bsce.v40i1.1
  86. Taqi RMM, Hammoudy SR, Hamzah MJ. Review of HPLC Methods for Determination of Azithromycin in Different Samples. Iraqi J Med Sci. 2022;20(1):77–82. doi:10.22578/IJMS.20.1.10