Agilent ion trap xct plus mass spectrometer

Hormones were analysed as described previously (Albacete et al., 2008 (link)). Briefly, 0.5g of each sample were homogenized, in triplicate, with liquid nitrogen, and extracted with 2.5ml of methanol:H₂O (80:20). Samples were centrifuged and the precipitates were re-extracted with another 2.5ml of the methanol:H₂O mixture. The two supernatants were mixed and passed through a SepPak Plus C18 cartridge (SepPak Plus, Waters, Millford, MA, USA). Samples were evaporated and the residues were dissolved in a methanol:H₂O (20:80) mixture and filtrated. 8 µl of sample were injected in an Agilent 1100 Series HPLC (AgilentTechnologies, Santa Clara, CA, USA), equipped with a micro-wellplate autosampler and a capillary pump, connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA,USA) using an electrospray interface.

The expression of ZP proteins was studied using proteomic analysis in Mastomys coucha, Mus mattheyi, and Mus pahari ZP. Ovaries were trimmed using small scissors and dissected to remove fat and connective tissue. The solubilized ZP was obtained according to the protocol previously described by our group (Izquierdo-Rico et al., 2009 (link); Jiménez-Movilla et al., 2009 (link)). Solubilized ZP was also obtained from oocytes, for which oocyte ZP was solubilized at 65°C in PBS buffer for 30 min. The analysis was carried out on an HPLC-MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA) equipped with a μ-well-plate autosampler and a capillary pump, and connected to an Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an electrospray (ESI) interface.

Protein, creatinine, ferritin and CRP in urine were measured by previously described methods [9 (link), 13 (link)].
Automated assays using commercial kits were used for IgG and GGT (Beckman Coulter, Inc., Brea, CA, USA) and RBP and NAG (Dyazime, Poway, CA, USA) measurements. Analyses were performed in an automatic analyzer (Olympus AU400, Hamburg, Germany). All four assays when validated in urine showed inter and intra-assay imprecision lower than 15% and linearity under dilution resulted in linear regression equations with correlation coefficients (R²) higher than 0.98 in all cases.
SDMA was measured by liquid chromatography-mass spectrometry (LC-MS) following a previously described method [14 ]. The LC-MS system consisted of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped connected to an Agilent Ion Trap XCT Plus Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an electrospray interface (ESI). This assay showed an inter and intra-assay imprecision lower than 4%, high linearity with serial dilutions (R² > 0.98) and recoveries after spiking different concentrations of SDMA to a canine serum were 96–103%.