Reducing adsorption to improve recovery and in vivo detection of neuropeptides by microdialysis with LC-MS

Abstract

Neuropeptides are an important class of neurochemicals; however, measuring their concentration in vivo by using microdialysis sampling is challenging due to their low concentration and the small samples generated. Capillary liquid chromatography with mass spectrometry (cLC-MS) can yield attomole limits of detection (LOD); however, low recovery and loss of sample to adsorptive surfaces can still hinder detection of neuropeptides. We have evaluated recovery during sampling and transfer to the cLC column for a selection of 10 neuropeptides. Adding acetonitrile to sample eliminated carryover and improved LOD by 1.4- to 60-fold. The amount of acetonitrile required was found to have an optimal value that correlated with peptide molecular weight and retention time on a reversed phase LC column. Treating AN69 dialysis membrane, which bears negative charge due to incorporated sulfonate groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold. The effect appeared to be due to reducing electrostatic interaction between peptides and the microdialysis probe because modification increased recovery only for peptides that carried net positive charge. The combined effects improved LOD of the entire method by 1.3- to 800-fold for the different peptides. We conclude that peptides with both charged and hydrophobic regions require combined strategies to prevent adsorption and yield the best possible detection. The method was demonstrated by determining orexin A, orexin B, and a novel isoform of rat β-endorphin in the arcuate nucleus. Dialysate concentrations were below 10 pM for these peptides. A standard addition study on dialysates revealed that while some peptides can be accurately quantified, some are affected by the matrix.

Figure 1

Plumbing diagram for cLC system used in this work. The autosampler loads sample. A selection valve is used to switch between a high pressure pump for rapid sample loading and column rinsing and a gradient LC pump for elution. Electrospray voltage applied at a tee at column inlet. The flow path of sample is highlighted in red to illustrate the surfaces that contact peptide during loading and injection. Sample needle, sample loop and connection tubing between the injection valve and column were all made from fused silica capillaries.

Figure 2

Optimal acetonitrile concentration for each individual peptide and its correlation to peptide properties. (A, B, C and D) Peptide signal, measured as peak area from reconstructed ion chromatograms (RICs) normalized to the highest for each peptide (n = 3), as a function of acetonitrile volumetric percentage in sample. 8 μL of peptides were injected at 100 pM. Balance of sample solution was Ringer’s solution and FA was added to a final concentration of 0.5%. Peptides are plotted in groups by optimal acetonitrile concentration for clarity. (E) Correlation of peptide molecular weight with optimal acetonitrile percentage of peptides. R2 = 0.77 for a linear fit. (F) Correlation of peptide retention time on a reverse phase column with the optimal acetonitrile percentage of peptides. R2 = 0.56 for a linear fit. To correlate retention time with optimal acetonitrile percentage, acetonitrile with 0.1% FA was used as MPB and a shallower gradient than the detection gradient was used to better separate peptides: 0–1 min: 2%–20% MPB, 1–9 min: 20–30% MPB, 9–10 min: 30–90% MPB, 10–12 min: 90% MPB, 12–12.1 min: 90–0% MPB, 12.1–15 min: 0% MPB. Data is average from 9 replicate injections on three cLC columns. Error bars = ± 1 standard deviation (SD) for all graphs.

Figure 3

Representative calibration curve of three peptides analyzed in vivo. Standards were prepared with 0 or 25 % acetonitrile added with balance of solvent being Ringer’s solution (0.5% FA). Each data point is the average of 3 replicate injections and error bar represents ± 1 SD.

Figure 4

In vitro relative recovery of peptides from probes constructed from AN69 membrane (n = 4), AN69 membrane treated with PEI (n = 4), and CMA 12 probe that uses PAES membrane (n = 3). PEI modification significantly increased recovery for orexin B, mouse β-endorphin, rat β-endorphin, Gal, Sub P, Deacetylated α-MSH and OFQ (* paired t-test, p < 0.05). CMA probe was only tested for orexins, mouse β-endorphin, α-MSHs and DynA1-17. Error bar represents standard error of mean (SEM).

Figure 5

(A) Overlaid RIC for orexin A, β-endorphin and orexin B from in vivo dialysate collected using either PEI modified AN69 membrane or unmodified AN69 membrane. two rats were used for each type of probe as pilot study. (B) Comparison between standard addition curve and standard calibration curve using dialysate collected by PEI modified probes. Standard addition curve points are average of three rats on three different days and error bar is + 1 SEM. 60 μL dialysate was collected from each rat and spiked with proper volume of acetonitrile and FA to produce 25% acetonitrile and 0.5% FA in final sample, aliquoted to 6 vials and spiked with peptide standard to a final concentration of 0, 0.5, 1, 2, 5, 10 pM of all three peptides. The peptide signal from spiked dialysate was normalized to the peptide signal from highest standard (10 pM) on that day.

Figure 6

(A) Response to K+ stimulation for orexin A, β-endorphin and orexin B from in vivo dialysate collected using PEI modified probe. Points are mean for 6 animals. All data are normalized to the average of the first 4 fractions, which are considered 100% of baseline. * indicates statistically different from baseline by t test with p < 0.05. Dialysate were collected into autosampler vials spiked with proper volume of acetonitrile and FA to produce final concentration of 25% acetonitrile and 0.5% FA. (B) In vitro probe response to concentration change for same peptides. Probe was sequentially placed in stirred vials containing 1 nM, 6 nM and 0 nM peptide standard. Data points are mean for experiments with 4 different probes. All error bars are ± 1 SEM. Bars indicating stimulation and concentration application are corrected for dead volume of the system.