Development of a method for the direct analysis of peptide AM336 in monkey cerebrospinal fluid using liquid chromatography/electrospray ionization mass spectrometry with a mixed-function column.
A liquid chromatography/mass spectrometry (LC/MS) analytical procedure, using a single column for sample clean-up, enrichment and separation, has been developed for the determination of the peptide AM336 in monkey cerebrospinal fluid (CSF). CSF samples were injected and analyzed using a polymer-coated mixed-function high-performance liquid chromatography (HPLC) column with gradient elution and application of a timed valve-switching event. The mass spectrometer was operated in the positive electrospray ionization (ESI(+)) mode with single ion recording (SIR) at m/z 920. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9892. Assay precision and accuracy were evaluated by direct injection of AM336-fortified CSF samples at three concentration levels. Analyzed concentrations ranged from 99.93 to 113.1% of their respective theoretical concentrations with coefficients of variation below 9.0%. An evaluation of the signal-to-noise (S/N) ratio for a 200 ng/mL calibration standard, considered to be the lower limit of quantitation (LLOQ), resulted in an estimated limit of detection (LOD) of 31.2 ng/mL. Preliminary data suggest the possibility of using this method to analyze AM336 also in plasma samples, pending the successful outcome of additional investigations.
WIL Research Laboratories, Inc., Ashland, OH 44805, USA. wbu@wilresearch.com
Wei
W
Bu
Scott D
SD
Freer
Shelly M
SM
Hollar
Philip L
PL
Stetson
Robert A
RA
Boyd
John B
JB
Kurek
Daniel W
DW
Sved
D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D000882:Haplorhini / Q000134:cerebrospinal fluid*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D014688:Venoms / Q000134:cerebrospinal fluid*; D020960:omega-Conotoxins
C461486:AM336; D014688:Venoms; D020960:omega-Conotoxins