P ace mdq plus capillary electrophoresis system

Glutamate content of CSF samples was measured using a capillary electrophoresis-laser induced fluorescence detection method developed in our laboratory [44 (link)] with some modifications. Neuropathic and sham-operated rats were sacrificed 14 days after pSNL operation. CSF samples were obtained by cisterna magna puncture and centrifuged at 2000× g, 4 °C for 10 min. The samples were then deproteinized by mixing with 2 volumes of cold acetonitrile and centrifuged at 20,000× g for 10 min at 4 °C. Supernatants were subjected to derivatization with NBD-F (1 mg/mL final concentration) in 20 mM borate buffer pH 8.5 for 20 min at 65 °C. One µM l-cysteic acid was used as an internal standard. Derivatized samples were analyzed by a P/ACE MDQ Plus capillary electrophoresis system coupled with laser induced fluorescence detector set to 488 and 520 nm excitation and emission wavelengths, respectively (SCIEX, Framingham, MA, USA). Separations were carried out in polyacrylamide coated fused silica capillaries (i.d.: 75 µm, effective/total length: 40/50 cm), using 50 mM HEPES buffer pH 7.0 containing 6 mM 6-monodeoxy-6-mono(3-hydroxy) propylamino-β-cyclodextrin at 15 °C by applying −27 kV constant voltage.

In our lab, a modified technique of capillary electrophoresis laser-induced fluorescence detection was established [83 (link)] for assessing the glutamate level in CSF samples. At 14 days after the pSNL, neuropathic and control rats were sacrificed with isoflurane. CSF samples were obtained by puncturing the cisterna magna, centrifuged at 2000× g and 4 °C for 10 min, and deproteinized by combining with two volumes of cold acetonitrile and centrifuging at 20,000× g for 10 min at 4 °C. Supernatants were derivatized using NBD-F (1 mg/mL final concentration) in 20 mM borate buffer pH 8.5 for 20 min at 65 °C. As an internal standard, 1 µM L-cysteic acid was utilized. A P/ACE MDQ Plus capillary electrophoresis system with a laser-induced fluorescence detector adjusted to 488 and 520 nm excitation and emission wavelengths, respectively (SCIEX, Framingham, MA, USA), was used to evaluate derivatized materials. Separations were performed in polyacrylamide-coated fused silica capillaries (i.d.: 75 µm, effective/total length: 40/50 cm) at 15 °C with a constant voltage of −27 kV while using a 50 mM HEPES buffer pH 7.0 containing 6 mM 6-monodeoxy-6-mono (3-hydroxy) propylamino-β-cyclodextrin.

Prior to euthanasia, the mice were acclimated to single capacity metabolic cages (Hatteras, Cary, NC, USA) overnight. The following day, urine collection tubes were replaced, and 24 h collection was initiated. This collection period concluded immediately prior to euthanasia. Samples were briefly centrifuged to remove debris and stored at −80 °C until analysis. Urine samples were analyzed via capillary electrophoresis for sodium, potassium, and chloride using a DxC 700 AU Chemistry Analyzer (Beckman Coulter, Brea, CA, USA). Urinary creatinine concentrations were calculated through direct potentiometry using a P/ACE MDQ Plus Capillary Electrophoresis System (Sciex, Redwood City, CA, USA). Urinary sodium–potassium ratios, total sodium excretion, and fractional excretion of sodium (FENa) were calculated using the values outlined above.