ManNAc in Human Plasma
Method Development and Validation for the Quantitation of ManNAc in Human Plasma Using HILIC LC-MS/MS
Yifan Shi1; Meng Fang1; Michael Zhang1; Yinghe Li1; Amy Wang2; Ed Kerns2; Nuria CarrilloCarrasco2; Xin Xu2; Selwyn Yorke3; Bradley Gillespie4
1Alliance Pharma, Malvern, PA; 2TRND, NCATS, NIH, Rockville, MD; 3New Zealand Pharmaceuticals, Palmerston North, New Zealand; 4Clinical Research Directorate/Therapy for Rare and Neglected Diseases, Leidos Biomedical Research Inc.(formerly SAIC-Frederick, Inc.), Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702; 4Funded by the NCI Contract No. HHSN261200800001E.
The poster related to this Abstract will be presented at the American Society for Mass Spectrometry 2014 to be held in Baltimore, MD, June 2014.
GNE myopathy, previously known as hereditary inclusion body myopathy (HIBM) is an autosomal recessive, muscular disorder, characterized by progressive muscle weakness with onset in early adulthood. ManNAc (N-acetylmannosamine) is being investigated by NIH and New Zealand Pharmaceuticals, as this precursor of sialic acid, prevents the development of muscle disease in the mouse model of GNE myopathy. As an endogenous compound, calibration standards for analyzing ManNAc in human plasma need to be prepared in surrogate matrix. Alliance Pharma developed an LC-MS/MS method with a calibration range of 10 to 5000 ng/mL. The method has been validated thoroughly to support clinical trials of ManNAc in GNE myopathy patients.
As a highly hydrophilic compound, ManNAc is difficult to retain on reverse-phase columns. Thus, chromatography was developed using an amide column with acetic acid and trifluoroacetic acid in water and methanol as the mobile phases. The peak shape and sensitivity were satisfactory in HILIC mode. The instrument used was a Sciex API 4000 in the positive mode using an ESI source in conjunction with a Shimadzu 20 series HPLC
system. The samples were extracted by protein precipitation with acetonitrile in a phospholipid removal plate. Calibration standards, LLOQ and low QCs were prepared in surrogate matrix (bovine serum albumin), while medium, high and dilution QCs were made in pooled human plasma with pre-evaluated endogenous ManNAc level.
There were two major challenges in the method development. First, ManNAc has four hydroxyl groups and relatively low molecular weight (221 amu). Its chromatography in various reverse phase columns had poor peak shape and low sensitivity. This problem was addressed by using an amide column in HILIC mode with trifluoroacetic acid as mobile phase modifier. Second, the matrix background caused severe ion suppression which was
probably due to the phospholipids in acetonitrile crashed human plasma. The suppression was solved by utilizing a phospholipid removal plate in the extraction step. The method was evaluated by measuring QC samples at five concentrations over three days with an analytical range of 10 – 5000 ng/mL. The endogenous level of ManNAc in the human plasma pool used to prepare QCs (medium QCs and higher) was found to be 50.5 ng/mL. Fifty microliters of human plasma was extracted with ManNAc-d6 as the internal standard. The assay showed an excellent linearity (R2> 0.997). Accuracies ranged from 91.1% to 110.0%, and precision ranged from 2.3% to 6.7% in three runs. Short term stability of ManNAc in plasma was evaluated by testing 4 freeze-thaw cycles and 16 hours at room temperature. Reinjection stability of the sample extract was evaluated by making replicate injections from the same set of extracted samples immediately and approximately 72 hours after the samples were stored at 4 ºC. No matrix effect was observed by comparing the results from QCs prepared in matrix and prepared in neat solution. Long-term storage stability for 6 month at -70 ºC was established.
A sensitive and selective method was developed to quantify an endogenous compound, ManNAc, in human plasma using surrogate matrix.