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Method Development and Validation for the Quantitation of HMF and HMFA in Human Plasma Using LC-MS/MS

Jun 1, 2015 11:43:00 AM / by Dr. Feng Li

5-Hydroxymethylfurfural (HMF) is a water-soluble heterocyclic organic compound derived from sugars. HMF binds with high affinity to intracellular sickle hemoglobin (HbS). In vivo studies using transgenic sickle mice showed that orally administered HMF inhibits the formation of sickled cells in the blood. NIH and its collaborators conducted investigations into the possibility of HMF as a treatment of Sickle cell disease (SCD) which is characterized by an abnormality in the oxygen-carrying haemoglobin molecule in red blood cells. A method for the quantitation of HMF and one of its major metabolites, 5-hydroxymethyl-2-furoic acid (HMFA), has been developed and validated in commercially available human plasma by Alliance Pharma.

LCMSMS method development HMF.png


The standards and QCs were spiked with stable-isotope labeled HMF/HMFA as internal standards and extracted by protein precipitation with 0.1% formic acid in acetonitrile in a phospholipid removal plate. The eluent was evaporated to dryness and the residue was reconstituted with acetonitrile:water (10:90). The analysis was conducted utilizing a Schimadzu Prominence 20AC HPLC system coupled with SRM detection in ESI positive mode for HMF and in ESI negative mode for HMFA on a Sciex API 4000 Q Trap mass spectrometer. Chromatographic separation was achieved using an Atlantis T3 column with 0.02% acetic acid in water and 4 mM ammonium formate in methanol as the mobile phases.

LC-MS Conditions

Chromatographic Conditions
HPLC: Shimadzu LC-20AC
Column:Waters, Atlantis T3, 100x2.1mm, 3 μm
Column Temperature: 40oC
Mobile phase A: 0.02% acetic acid in water
Mobile phase B: 4 mM ammonium Formate in methanol
Flow rate: 0.6 mL/min

MS/MS Detection

Mass spectrometer: Sciex API 4000 Q Trap
Source temperature: 550oC
Ion transition monitored:
HMF: ESI positive mode
m/z 126.9 → 53.1
HMFA: ESI negative mode
m/z 141.0 → 69.2

Precision and Accuracy of Spiked QCs for HMF

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (5 ng/mL) in one validation run, and at low (15 ng/mL), medium (150 ng/mL) and high concentrations (1500 ng/mL) over three validation runs.

Precision and Accuracy of Spiked QCs for HMFA

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (0.1 μg/mL) in one validation run, and at low (0.3 μg/mL), medium (6 μg/mL) and high concentrations (75 μg/mL) over three validation runs.


  • A selective and sensitive HPLC-MS/MS method for the quantification of HMF and HMFA in commercially available human plasma was developed. 
  • Great retention and selection of highly hydrophilic compounds were achieved using carefully selected HPLC column and optimized mobile phases.
  • Phospholipid removal plate was used to decrease the ion suppression resulted from the phospholipids in the protein precipitation extract.
  • The method was validated as linear, accurate, precise and reproducible.



Meng Fang, Yifan Shi, Yinghe Li, Michael Zhang, Bradley Gillespie, Warren Stern, Amy Wang, Nora Yang, and Xin Xu
Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355; Leidos Biomedical Research Inc., Frederick, MD 21701; AesRx, LLC, Newton, MA 02466; TRND, National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Dr., Rockville, MD 20850

Topics: lc-ms/ms

Dr. Feng Li

Written by Dr. Feng Li

Feng (Frank) Li, Ph.D., is President of Alliance Pharma; he obtained his Ph.D. degree in Bioanalytical Chemistry jointly from Concordia University and the National Institute of Scientific Research (Canadian Doping Control Center) in Montreal, Canada. Subsequently, Dr. Li did his post-doctoral fellowship at the Biomedical Mass Spectrometry Facility at the Mayo Clinic in Rochester, Minnesota. Furthermore, Dr. Li has an M.Sc. degree in Natural Product Chemistry and a B.S. in Pharmacy. He has held leadership roles in the Department of Drug Discovery Metabolism at Phoenix International Life Sciences, Inc., a major CRO at the time in Montreal, Canada, which was later acquired by MDS Pharma Services; in the Drug Analysis group in the Department of Drug Metabolism and Pharmacokinetics (DMPK) at GlaxoSmithKline; and in the Drug Metabolism group in the Department of Drug Safety and Disposition at Cephalon, Inc. Dr. Li has extensive DMPK experience in discovery and developmental phases of drug development. With more than 20 years in the pharmaceutical biotechnology, and CRO industry, he is well versed in bioanalytical techniques for both qualitative (drug metabolite identification) and quantitative (PK/TK) drug analysis and has published numerous articles in the area of drug metabolite identification and quantitation.