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.
Bu
Wei
W
WIL Research Laboratories, Inc., Ashland, OH 44805, USA. wbu@wilresearch.com
Freer
Scott D
SD
Hollar
Shelly M
SM
Stetson
Philip L
PL
Boyd
Robert A
RA
Kurek
John B
JB
Sved
Daniel W
DW
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