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Rationale: Precise assessment of renal glomerular filtration rate (GFR) is essential for the early detection of chronic kidney disease. AcSDKP-NH(2), an analogue of the endogenous tetrapeptide AcSDKP, is not degraded in vivo and is freely filtered by the kidney and eliminated in urine; for that reason this analogue is an ideal candidate marker for the assessment of GRF after administration to humans. Proof-of-concept demonstration and lack of toxicity in animals have allowed an ongoing clinical study in which AcSDKP-NH(2) was administered intravenously at a dose of 100 µg and compared with currently available GFR markers. The use of the AcSDKP analogue in clinical practice requires that this novel marker be associated with an analytical method that combines specificity, robustness and high accuracy. We have developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay and compared it with an existing enzyme immunoassay (EIA) for AcSDKP-NH(2)., Methods: Human urine and plasma samples from the clinical study were analyzed by EIA and LC/MS/MS. Before LC/MS/MS assessment, AcSDKP-NH(2) was extracted using mixed-mode cation-exchange solid-phase extraction cartridges. Chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC), before analysis with an electrospray ionization triple quadrupole mass spectrometer., Results: Mass spectrometry, through the use of an internal standard, tailored sample preparation and chromatographic separation, has better intra- and inter-assay precision (accuracies between 95 and 101% with CVs <8% for LC/MS/MS vs. accuracies between 90 and 115% with CVs <18% for EIA) and allows greater steadiness in intra-subject concentrations during the infusion (4.4% for LC/MS/MS vs. 8.6% for EIA). Moreover, the LC/MS/MS assay circumvents matrix effects observed in certain instances for the EIA and which may reduce its accuracy., Conclusions: Although the EIA can provide sufficient information in most subjects, the LC/MS/MS assay associated with this new marker should be the reference method., (Copyright © 2011 John Wiley & Sons, Ltd.)

We have investigated the potential and robustness of the off-line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI-MS), for further applications in the screening of single-nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion-exchange solid-phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of ∼70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI-MS analysis of a model 114-bp PCR product performed on the LTQ-Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 ± 7.11 ppm can be routinely obtained using an external calibration. The PCR/ESI-MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ-Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A ↔ G) switch, i.e. a 16 Da difference, in binary mixtures of ∼ 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI-MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T ↔ A) switch (9 Da mass difference) was successfully identified in a 114-bp PCR product., (Copyright © 2010 John Wiley & Sons, Ltd.)

Apelin peptides are of great interest owing to their involvement in physiological and pathological processes and they have been proposed as novel biomarkers for heart failure. The plasma concentrations of bioactive peptides of 12 (apelin-12), 13 (apelin-13) and pyroglutamyl apelin-13 (apelin-p13), 17 (apelin-17) and 36 (apelin-36) amino acids are reported to range from 20 to 4000 pg/mL in healthy subjects. As standard immunoassays cannot specifically quantify each apelin peptide, we have developed a sensitive and targeted multiplexed liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for each plasma apelin fragment. The approach was based on a cation-exchange extraction step of apelin forms present in human plasma. Apelin-12, -13, -p13, -17 and -36 were quantified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Stable isotope-labeled internal standards were used for quantification. Following assay validation, apelin peptide stability in plasma was investigated. Ten plasma samples from healthy donors were analyzed both with a standard immunoassay and with our LC/MS/MS method. The immunoassay results for the ten healthy donors showed immunoreactive plasma apelin concentrations ranging from 208 to 466 pg/mL. The lower limits of detection of our LC/MS/MS assay ranged from 10 to 50 pg/mL for apelin-12, -13, -p13, -17, and -36. Surprisingly, none of the five expected circulating forms of apelin was detected. These results question the nature and/or the concentration of circulating apelin peptides as well as the specificity of the immunoassays that have hitherto been used for clinical applications., (Copyright © 2010 John Wiley & Sons, Ltd.)

In order to increase the sensitivity of liquid chromatography/mass spectrometry (LC/MS) assays of recombinant proteins for pharmacokinetics studies, we have developed an immuno-mass spectrometry assay for EPI-hNE4, a 6237 Da protein currently developed for respiratory distress syndromes. After immunocapture of the analyte in human plasma with magnetic beads coated with anti-EPI-hNE4 antibodies, the intact protein was eluted and separated in reversed-phase LC and then analysed by tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode. The problem of analytical interference due to endogenous binding antibodies was addressed by successive steps of acidification and neutralisation before immunocapture. Furthermore, potential variations in the recovery of analyte during sample extraction were compensated for by addition of an internal standard recognised by the antibodies. The precision of the assay remained therefore below 15%. A significant increase in assay sensitivity was achieved since the extraction step allowed sample concentration and removal of matrix components interfering with the electrospray ionisation process. Using 0.4 mL of plasma, a limit of quantification at 0.5 ng/mL (80 pM) was reached, which represents a 10-fold improvement in sensitivity over our previous work using sample precipitation. This technique was able to monitor EPI-hNE4 kinetics in the plasma of human subjects for 36 h after an intravenous administration of 0.125 mg/kg., (Copyright 2007 John Wiley & Sons, Ltd.)

This work reports the use of reverse-phase liquid chromatography coupled to electrospray ion trap (QIT) mass spectrometry for the analysis of the metabolome in rat urine. An injection of 20 microL of urine into the chromatographic system is followed by a slow gradient elution and mass spectrometric detection in the scanning mode from m/z 100-1000 in both positive and negative modes. Over a time scale of 90 min, 30 and 20 resolved peaks were observed in the positive and the negative modes, respectively, corresponding to the presence of a few hundred m/z ratios. By using a QIT analyzer, data-dependent tandem mass spectrometry of selected m/z ratios identified several compounds in rat urine and characterized various chemical families, including carboxylic acids, amines, sulfated compounds, glucuronides and glycosides, by the observation of characteristic fragment ions or neutral losses. The method has been applied to the investigation of the chronic toxicity of heavy metals in rat urine. A few tens of m/z ratios, differing in intensity more than threefold from control values, were observed in both positive and negative modes. The time variations for some selected ions suggest that LC/ESI-MS could allow selective characterization of biomarkers in response to specific toxic compounds., (Copyright 2003 John Wiley & Sons, Ltd.)