Rapid preconcentration for liquid chromatography-mass spectrometry assay of trace level neuropeptides

Abstract

Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MS(n)) has proven to be effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75 μm inner diameter (i.d.) capillary column packed with 10 μm reversed phase particles for increasing the throughput in CLC-MS(n) based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column has reduced analysis time from ∼30 min to 3.8 min for 5 μL sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A1-8 in 5 μL sample. The use of isotope-labeled internal standard lowered peptide signal variation to less than 5 %. This method was validated for in vivo detection of Leu and Met enkephalin with microdialysate collected from rat globus pallidus. The improvement in speed and stability makes CLC-MS(n) measurement of neuropeptides in vivo more practical.

Figure 1

Diagram and operation scheme of the dual valve dual pump LC-MSⁿ system. Valve positions shown for filling sample loop (A), sample loading onto column (B), rinsing column (C), and peptide elution and detection (D). Detailed description is given in text.

Figure 2

Influence of column I.D. and particle size on detection sensitivity. (A) Calibration curve showed column I.D. and particle size had little influence on sensitivity under the same volumetric elution flow rate. (B) Chromatogram of LE (5 µl 200 pM injected) showed no obvious peak broadening using column packed with 10 µm particle compared to column packed with 5 µm particle. All measurements were done with 3 replicates.

Figure 3

Influence of flow rate on detection sensitivity. (A) Peptide signal remained stable within the injection/rinsing flow rate range of 2.6 µl/min to 13.7 µl/min. (B) Calibration curve under different injection/rinsing flow rate (2.69 µl/min v.s. 13.44 µl/min) suggested no major sensitivity decrease when injecting peptide under high flow rate. (C). Increasing elution flow rate decreased peptide signal. Peptide standard: 200 pM LE, ME and DynA_1–8 dissolved in aCSF with 5% HAc. Injection volume: 5 µl. (D). Reconstructed ion chromatogram (RIC) of 5 µl 60 pM neuropeptide being injected onto the column. All measurements were done with 3 replicates.

Figure 4

Chromatogram of serial injections of 60 pM peptide standards in series. A wash injection with 20 µl 60:30:10 (v/v/v) isopropanol:acetonitrile:H₂O injected onto the column was carried out every 6 injections. The right image shows a zoomed-in view of one injection where 5 µl peptide standard was preconcentrated, rinsed, and separated for detection within 4 minutes.

Figure 5

In vivo measurement of LE and ME from rat brain dialysate. (A). Calibration curve of LE and ME from 5 pM to 3 nM (n=3) (B) LE’s peak area in 5 pM/50 pM standard and dialysate spiked with 5 pM/50 pM LE. (C). Serial injection of 9 basal dialysate sample in 37 minutes. Injection volume: 5 µl.

Figure 6

K⁺ stimulation profile of ME and LE. Potassium concentration: 75 mM. Stimulation time: 40 minutes. Number of replicates: 3. Error bar represents standard error of measurement (SEM).