Determination of bradykinin and arg‐bradykinin in rat muscle tissue by microdialysis and capillary column‐switching liquid chromatography with mass spectrometric detection

Abstract

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.

Application of micro‐ and nano‐HPLC to the determination and characterization of bioactive and biomarker peptides

“…The concentration and mass LODs were l0.1 lg/L (l0.1 nM) and 50 pg, respectively. As expected, the LOD is lower than with UV detection (l1 nM) [7], despite the sample volume was 500 lL [8] versus 1000 lL [7]. The endogenous values obtained in rat dialysates for BD and Arg-BD were 0.1 and 0.4 lg/L, respectively, which are close to their LODs.…”

“…Saturation problems are especially important with biological samples, which usually have an abundant and complex background matrix of compounds. the analytes of interest such as microdialysis [8], affinity chromatography [9,10], or precipitation [11][12][13] can be used to circumvent saturation problems. The microdialysis technique allows the continuous sampling of substances from the interstitial space by inserting in the animal tissue a probe with a dialysis membrane, which is constantly perfused with a physiological solution.…”

“…With regard to MS detection, a micro-HPLC (150 mm6500 lm id, C 18 5 lm) -ESI-TOF MS system with a preconcentration precolumn (5 mm length61 mm id, C 18 5 lm) has been used for the quantitation of endogenous BD peptides in rat muscle tissue dialysates [8]. The BD peptides are released by muscle contractions and can increase blood flow, improving energy delivery to the working muscle.…”

“…The system was applied to the analysis of the human peptide hHEM-c 130-146, a bioactive peptide which exhibits a broad antimicrobial activity and corresponds to a C-terminal fragment of the c-hemoglobin. In the above commented micro-HPLC (150 mm6500 lm id, C 18 5 lm) -ESI-TOF MS method [8], (Tyr 8 )-BD was used as an internal standard to improve precision for the quantitation of BD peptides in rat muscle tissue dialysates. The within-day and between-day precisions were good enough (4.3-9.6% and 6.2-10.6%, respectively).…”

Fast and simple online sample preparation coupled with capillary LC‐MS/MS for determination of prostaglandins in cell culture supernatants

“…In cLC, large volumes can be injected using column-switching methods based on focusing on a trap column with subsequent transfer to an analytical column, thus improving the sensitivity [20,21]. Columnswitching systems can also be advantageously used for online sample preparation, which minimizes analysis time, sample loss, and errors due to several preparation steps [22,23].…”