Home Chromatographic methods development for clinical practice: requirements and limitations
Article
Licensed
Unlicensed Requires Authentication

Chromatographic methods development for clinical practice: requirements and limitations

  • Lenka Kujovská Krčmová ORCID logo EMAIL logo , Bohuslav Melichar and František Švec ORCID logo
Published/Copyright: July 1, 2020
Become an author with De Gruyter Brill
Submit Manuscript Author Information
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 58 Issue 11

Abstract

Development of a chromatographic method in bioanalysis is a challenging and complex procedure with many pitfalls and often unexpected reversals that can require several months to accomplish. Even an experienced analytical team must contend many limitations mainly in connection with the strict requirements imposed on current clinical research. These restrictions typically persist throughout the whole development process, from clinical trial assignment, across optimization of extraction of biological materials and chromatographic separation, to validation and data interpretation. This paper describes questions and their possible answers raised during the pre-analytical phase such as use of modern sample preparation techniques in clinical methods, application of internal standards, as well as selection of stationary phases and detection techniques in the analytical phase. Validation problems and interpretation of results are demonstrated with three typical examples of characteristics to be considered, i.e. recovery, matrix effect, and limit of detection vs. lower limit of quantification.

Keywords: clinical research; clinical study; laboratory medicine; liquid chromatography; validation
Corresponding author: Assoc. Prof. Lenka Kujovská Krčmová, PhD, The Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; The Department of Clinical Biochemistry and Diagnostics, University Hospital, Sokolská 581, 500 05 Hradec Králové, Czech Republic, Phone: +420 49583337, Fax: +420 495834841, E-mail:

Funding source: Charles University, Faculty of Pharmacy in Hradec Králové

Funding source: University Hospital in Hradec Králové

Funding source: Ministry of Health of the Czech Republic

Funding source: Ministry of Health of the Czech Republic

Funding source: Ministry of Health of the Czech Republic

Funding source: PERSONMED – Center for Development of Personalized Medicine in Age-Related Diseases

Funding source: STARSS project co-funded by ERDF

Acknowledgments

The authors gratefully acknowledge Filip Hátle for technical support. Thanks are also due for the financial support by Charles University, Faculty of Pharmacy in Hradec Králové (Project SVV 260 458), project MH CZ – DRO (University Hospital in Hradec Králové, 00179906), the Ministry of Health of the Czech Republic, grants numbers 18-03-00130, 17-28882A, and 17-29241A, PERSONMED – Center for Development of Personalized Medicine in Age-Related Diseases, Reg Nr. CZ.02.1.01/0.0/0.0/17_048/0007441 co-financed by the ERDF state budget of the Czech Republic. FS gratefully acknowledges the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF.

  1. Research funding: Thanks are due for the financial support by Charles University, Faculty of Pharmacy in Hradec Králové (Project SVV 260 458), project MH CZ – DRO (University Hospital in Hradec Králové, 00179906), the Ministry of Health of the Czech Republic, grant numbers 18-03-00130, 17-28882A, and 17-29241A, PERSONMED – Center for Development of Personalized Medicine in Age-Related Diseases, Reg Nr. CZ.02.1.01/0.0/0.0/17_048/0007441 co-financed by the ERDF state budget of the Czech Republic. FS gratefully acknowledges the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

References

1. World Health Organization. Clinical trials. Available from: https://www.who.int/topics/clinical_trials/en/ [Accessed 20 Sep 2019].Search in Google Scholar

2. Hammerling, JA. A review of medical errors in laboratory diagnostics and where we are today. Lab Med 2011;43:41–4. https://doi.org/10.1309/LM6ER9WJR1IHQAUY.Search in Google Scholar

3. Racek, J. Klinická biochemie, 2nd ed. Prague: Galén; 2006:23 p.Search in Google Scholar

4. Adaway, JE, Keevil, BG, Owen, LJ. Liquid chromatography tandem mass spectrometry in the clinical laboratory. Ann Clin Biochem 2014;52:18–38. https://doi.org/10.1177/0004563214557678.Search in Google Scholar PubMed

5. Hooshfar, S, Bastlett, MG. Hazards in chromatographic bioanalysis method development and applications. Biomed Chromatogr 2017;31:e3859. https://doi.org/10.1002/bmc.3859.Search in Google Scholar PubMed

6. Khamis, MM, Adamko, DJ, El-Aneed, A Strategies and challenges in method development and validation for the absolute quantification of endogenous biomarker metabolites using liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev 2019;39:1–22. https://doi.org/10.1002/mas.21607.Search in Google Scholar PubMed

7. Patil, R, Bhaskar, R, Ola, M, Pingale, D, Chalikwar, S. Bioanalytical method development and method validation in human plasma by using LC MS/MS. Indo Am J Pharm 2019;06:5176–83.Search in Google Scholar

8. Kucerova, K, Kujovska Krcmova, L, Mikanova, Z, Matysova, L, Solich, P. Could urinary retinol be used as a new biomarker of kidney damage?. Trac-Trends Anal Chem 2017;95:57–61. https://doi.org/10.1016/j.trac.2017.07.025.Search in Google Scholar

9. Namera, A, Saito, T. Recent advances in unique sample preparation techniques for bioanalysis. Bioanalysis 2013;5:915–32. https://doi.org/10.4155/bio.13.52.Search in Google Scholar PubMed

10. Kabir, A, Locatelli, M, Ulusoy, HI. Recent trends in microextraction techniques employed in analytical and bioanalytical sample preparation. Separations 2017;4:1–15. https://doi.org/10.3390/separations4040036.Search in Google Scholar

11. Abdel-Rehim, M. Microextraction by packed sorbent in bioanalysis. Bioanalysis 2009;1:687–91. https://doi.org/10.4155/bio.09.61.Search in Google Scholar PubMed

12. Ahmadi, M, Elmongy, H, Madrakian, T, Abdel-Rehim, M. Nanomaterials as sorbents for sample preparation in bioanalysis: a review. Anal Chim Acta 2017;958:1–21. https://doi.org/10.1016/j.aca.2016.11.062.Search in Google Scholar PubMed

13. Zuloaga, O, Olivares, M, Navarro, P, Vallejo, A, Prieto, A. Dispersive liquid–liquid microextraction: trends in the analysis of biological samples. Bioanalysis 2015;7:2211–25. https://doi.org/10.4155/bio.15.141.Search in Google Scholar PubMed

14. Tang, YQ, Weng, N. Salting-out assisted liquid–liquid extraction for bioanalysis. Bioanalysis 2013;5:1583–98. https://doi.org/10.4155/bio.13.117.Search in Google Scholar PubMed

15. Ebrahomzadeh, H, Molaei, K, Asgharinezhad, AA, Shekari, N, Dehghani, Z. Molecularly imprinted nano particles combined with miniaturized homogenous liquid–liquid extraction for the selective extraction of loratadine in plasma and urine samples followed by high performance liquid chromatography-photo diode array detection. Anal Chim Acta 2013;767:155–62. https://doi.org/10.1016/j.aca.2013.01.013.Search in Google Scholar PubMed

16. Hamidi, S, Alipour-Ghorbani, N, Hamidi, A. Solid phase microextraction techniques in determination of biomarkers. Crit Rev Anal Chem 2018;48:239–51. https://doi.org/10.1080/10408347.2017.1396885.Search in Google Scholar PubMed

17. Filipou, O, Bitas, D, Samanidou, V. Green approaches in sample preparation of bioanalytical samples prior to chromatographic analysis. J Chromatogr B 2017;1043:44–62. https://doi.org/10.1016/j.jchromb.2016.08.040.Search in Google Scholar PubMed

18. Gjelstad, A, Andresen, AT, Dahlgren, A, Gundersen, TE, Pederson-Bjergaard, S. High-throughput liquid–liquid extraction in 96-well format: parallel artificial liquid membrane extraction. LC GC Eur 2017;30:10–7.Search in Google Scholar

19. Pan, J, Jiang, X, Chen, Y. Automatic supported liquid extraction (SLE) coupled with HILIC-MS/MS: an application to method development and validation of erlotinib in human plasma. Pharmaceutics 2010;2:105–18. https://doi.org/10.3390/pharmaceutics2020105.Search in Google Scholar PubMed PubMed Central

20. Bojko, B, Cudjoe, E, Pawliszyn, J, Wasowicz, M. Solid-phase microextraction. How far are we from clinical practice?. Trac-Trends Anal Chem 2011;30:1505–12. https://doi.org/10.1016/j.trac.2011.07.008.Search in Google Scholar

21. Srinivasan, B, Tung, S. Development and applications of portable biosensors. J Lab Autom 2015;20:365–89. https://doi.org/10.1177/2211068215581349.Search in Google Scholar PubMed

22. Filipiak, W, Bojko, B. SPME in clinical, pharmaceutical and biotechnological research – How far are we from daily practice?. Trac-Trends Anal Chem 2019;115:203–13. https://doi.org/10.1016/j.trac.2019.02.029.Search in Google Scholar

23. Kataoka, H, Saito, K. Recent advances in SPME techniques in biomedical analysis. J Pharmaceut Biomed 2011;54:926–50. https://doi.org/10.1016/j.jpba.2010.12.010.Search in Google Scholar PubMed

24. Roszkowska, A, Miekus, N, Baczek, T. Application of solid phase microextraction in current biomedical research. J Sep Sci 2019;42:285–302. https://doi.org/10.1002/jssc.201800785.Search in Google Scholar PubMed

25. Couchman, L. Turbulent flow chromatography in bioanalysis: a review. Biomed Chromatogr 2012;26:892–905. https://doi.org/10.1002/bmc.2769.Search in Google Scholar PubMed

26. Chromatography Today. Turbulent flow chromatography: an evolving solution for bioanalysis. Available from: https://www.chromatographytoday.com/article/bioanalytical/40/christophe_chassaing_sarah_robinson/turbulent_flow_chromatography_an_evolving_solution_for_bioanalysis/498 [Accessed: 14 Apr 2020].Search in Google Scholar

27. Michopoulos, F, Edge, AM, Theodoridis, G, Wilson, ID. Application of turbulent flow chromatography to the metabolomics analysis of human plasma: comparison with protein precipitation. J Sep Sci 2010;33:1472–79. https://doi.org/10.1002/jssc.200900789.Search in Google Scholar PubMed

28. Sahena, F, Zaidul, ISM, Jinap, S, Karim, AA, Abbas, KA, Norualini, NAN, et al. Application of supercritical CO2 in lipid extraction – a review. J Food Eng 2009;95:240–53. https://doi.org/10.1016/j.jfoodeng.2009.06.026.Search in Google Scholar

29. Rios, A, Zougagh, M, de Andres, F. Bioanalytical applications using supercritical fluid techniques. Bioanalysis 2010;2:9–25. https://doi.org/10.4155/bio.09.167.Search in Google Scholar PubMed

30. Adaway, JE, Keevil, BG, Owen, LJ. Liquid chromatography tandem mass spectrometry in the clinical laboratory. Ann Clin Biochem 2015;52:18–38. https://doi.org/10.1177/0004563214557678.Search in Google Scholar PubMed

31. Honour, JW. Development validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem 2011;48:97–111. https://doi.org/10.1258/acb.2010.010176.Search in Google Scholar PubMed

32. European Medicines Agency. Guideline on bioanalytical method validation. Available from: https://www.ema.europa.eu/ema/index.jsp?curl=pages/includes/document/documentdetail.jsp?webContentId=WC500109686%26mid=WC0b01ac058009a3dc [Accessed: 14 Apr 2020].Search in Google Scholar

33. U.S. Food and Drug Administration. Analytical procedures and methods validation for drugs and biologics. Available from: https://www.fda.gov/files/drugs/published/Analytical-Procedures-and-Methods-Validation-for-Drugs-and-Biologics.pdf [Accessed: 14 Apr 2020].Search in Google Scholar

34. Krcmova, L, Solichova, D, Plisek, J, Kasparova, M, Sobotka, L, Solich, P. Miniaturisation of solid phase extraction method for determination of retinol, alpha- and gamma-tocopherol in human serum using new technologies. Int J Environ Anal Chem 2010;90:106–14. https://doi.org/10.1080/03067310903267299.Search in Google Scholar
Received: 2020-04-16
Accepted: 2020-05-25
Published Online: 2020-07-01
Published in Print: 2020-10-25

© 2020 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Biotin interference in cardiac troponin immunoassay – where the wild things are?
  4. Review
  5. Laboratory-related issues in the measurement of cardiac troponins with highly sensitive assays
  6. Mini Review
  7. Chromatographic methods development for clinical practice: requirements and limitations
  8. Opinion Paper
  9. Harmonising EQA schemes the next frontier: challenging the status quo
  10. Genetics and Molecular Diagnostics
  11. Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients
  12. A novel mitochondrial m.14430A>G (MT-ND6, p.W82R) variant causes complex I deficiency and mitochondrial Leigh syndrome
  13. Obesity status modifies the association between rs7556897T>C in the intergenic region SLC19A3-CCL20 and blood pressure in French children
  14. General Clinical Chemistry and Laboratory Medicine
  15. Influence of reagent lots and multiple measuring systems on estimating the coefficient of variation from quality control data; implications for uncertainty estimation and interpretation of QC results
  16. Electrophoretic α1-globulin for screening of α1-antitrypsin deficient variants
  17. A continued method performance monitoring approach for the determination of pediatric renin samples – application within a European clinical trial
  18. Pilot study for cystic fibrosis neonatal screening: the Cuban experience
  19. Validation of the analytical performance of the NOVEOS™ System, a system which improves upon the third-generation in vitro allergy testing technology
  20. IgE cross-reactivity measurement of cashew nut, hazelnut and peanut using a novel IMMULITE inhibition method
  21. Sexual dimorphism in the cerebrospinal fluid total protein content
  22. Current state of the morphological assessment of urinary erythrocytes in The Netherlands: a nation-wide questionnaire
  23. Reference Values and Biological Variations
  24. Within-subject and between-subject biological variation of first morning void urine amino acids in 12 healthy subjects
  25. Proenkephalin as a new biomarker for pediatric acute kidney injury – reference values and performance in children under one year of age
  26. Hematology and Coagulation
  27. Quality performance for indirect Xa inhibitor monitoring in patients using international external quality data
  28. Cardiovascular Diseases
  29. Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay
  30. Short- and long-term biological variation of cardiac troponin I in healthy individuals, and patients with end-stage renal failure requiring haemodialysis or cardiomyopathy
  31. Infectious Diseases
  32. Monocyte distribution width (MDW) as a screening tool for sepsis in the Emergency Department
  33. Performance of a Toxo IgM prototype assay for the diagnosis of maternal and congenital Toxoplasma infections
  34. Letters to the Editors
  35. Evaluation of an ELISA for SARS-CoV-2 antibody testing: clinical performances and correlation with plaque reduction neutralization titer
  36. Preliminary evaluation of Roche Cobas Elecsys Anti-SARS-CoV-2 chemiluminescence immunoassay
  37. Hypoalbuminemia and elevated D-dimer in COVID-19 patients: a call for result harmonization
  38. Total pathway to method validation
  39. Derivation of performance specifications for uncertainty of serum C-reactive protein measurement according to the Milan model 3 (state of the art)
  40. FGF23 measurement in burosumab-treated patients: an emerging treatment may induce a new analytical interference
  41. Use of a modified IDS-ISYS intact PTH assay for intraoperative PTH measurements
  42. Agreement of dried blood spot lyso-Gb3 concentrations obtained from different laboratories in patients with Fabry disease
  43. Influence of delayed separation of plasma from whole blood and centrifugation protocol on Zn plasma concentration
  44. A survey of order of draw on inpatient wards and adherence to EFLM-COLABIOCLI recommendations
  45. Successful implementations of automated minimum re-test intervals to overcome ferritin over-requesting in a Spanish hospital laboratory
  46. Remarkable pseudoleucocytosis induced by mild cryoglobulinemia
  47. Massive hemolysis due to Clostridium perfringens: a laboratory’s perspective
https://doi.org/10.1515/cclm-2020-0517
Keywords for this article
clinical research; clinical study; laboratory medicine; liquid chromatography; validation

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Biotin interference in cardiac troponin immunoassay – where the wild things are?
  4. Review
  5. Laboratory-related issues in the measurement of cardiac troponins with highly sensitive assays
  6. Mini Review
  7. Chromatographic methods development for clinical practice: requirements and limitations
  8. Opinion Paper
  9. Harmonising EQA schemes the next frontier: challenging the status quo
  10. Genetics and Molecular Diagnostics
  11. Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients
  12. A novel mitochondrial m.14430A>G (MT-ND6, p.W82R) variant causes complex I deficiency and mitochondrial Leigh syndrome
  13. Obesity status modifies the association between rs7556897T>C in the intergenic region SLC19A3-CCL20 and blood pressure in French children
  14. General Clinical Chemistry and Laboratory Medicine
  15. Influence of reagent lots and multiple measuring systems on estimating the coefficient of variation from quality control data; implications for uncertainty estimation and interpretation of QC results
  16. Electrophoretic α1-globulin for screening of α1-antitrypsin deficient variants
  17. A continued method performance monitoring approach for the determination of pediatric renin samples – application within a European clinical trial
  18. Pilot study for cystic fibrosis neonatal screening: the Cuban experience
  19. Validation of the analytical performance of the NOVEOS™ System, a system which improves upon the third-generation in vitro allergy testing technology
  20. IgE cross-reactivity measurement of cashew nut, hazelnut and peanut using a novel IMMULITE inhibition method
  21. Sexual dimorphism in the cerebrospinal fluid total protein content
  22. Current state of the morphological assessment of urinary erythrocytes in The Netherlands: a nation-wide questionnaire
  23. Reference Values and Biological Variations
  24. Within-subject and between-subject biological variation of first morning void urine amino acids in 12 healthy subjects
  25. Proenkephalin as a new biomarker for pediatric acute kidney injury – reference values and performance in children under one year of age
  26. Hematology and Coagulation
  27. Quality performance for indirect Xa inhibitor monitoring in patients using international external quality data
  28. Cardiovascular Diseases
  29. Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay
  30. Short- and long-term biological variation of cardiac troponin I in healthy individuals, and patients with end-stage renal failure requiring haemodialysis or cardiomyopathy
  31. Infectious Diseases
  32. Monocyte distribution width (MDW) as a screening tool for sepsis in the Emergency Department
  33. Performance of a Toxo IgM prototype assay for the diagnosis of maternal and congenital Toxoplasma infections
  34. Letters to the Editors
  35. Evaluation of an ELISA for SARS-CoV-2 antibody testing: clinical performances and correlation with plaque reduction neutralization titer
  36. Preliminary evaluation of Roche Cobas Elecsys Anti-SARS-CoV-2 chemiluminescence immunoassay
  37. Hypoalbuminemia and elevated D-dimer in COVID-19 patients: a call for result harmonization
  38. Total pathway to method validation
  39. Derivation of performance specifications for uncertainty of serum C-reactive protein measurement according to the Milan model 3 (state of the art)
  40. FGF23 measurement in burosumab-treated patients: an emerging treatment may induce a new analytical interference
  41. Use of a modified IDS-ISYS intact PTH assay for intraoperative PTH measurements
  42. Agreement of dried blood spot lyso-Gb3 concentrations obtained from different laboratories in patients with Fabry disease
  43. Influence of delayed separation of plasma from whole blood and centrifugation protocol on Zn plasma concentration
  44. A survey of order of draw on inpatient wards and adherence to EFLM-COLABIOCLI recommendations
  45. Successful implementations of automated minimum re-test intervals to overcome ferritin over-requesting in a Spanish hospital laboratory
  46. Remarkable pseudoleucocytosis induced by mild cryoglobulinemia
  47. Massive hemolysis due to Clostridium perfringens: a laboratory’s perspective
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 11.9.2025 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2020-0517/html
Scroll to top button