Cto 20a oven

Mass spectrometry analysis was performed on a Shimadzu model CBM-20A chromatograph, equipped with Shimadzu LC-20AD pumps, a Shimadzu SPD-20A detector, Shimadzu CTO-20A oven, Shimadzu SIL 20AC autoinjector, and Bruker Daltonics maXis 3G mass spectrometer (Bruker Corp., Billerica, MA, USA), with a 4500-V capillary, 2-bar nebulizers, at 200°C, and a gas flow of 8 liter/min⁻¹ operating in MS and MS/MS data acquisition mode with the electron spray source in positive mode (ESI⁺). For the liquid chromatography, a reverse-phase C₁₈ (Phenomenex Luna) plus column (250 mm × 4.6 mm × 5 μm) was used, with a flow rate of 1.0 ml.min⁻¹. The elution system used a gradient system which consisted of a mixture of (i) acidified water (TFA 0.05%) and (ii) acetonitrile (ACN) for 10 to 70 min, reaching 80% ACN (48 ).
Nuclear magnetic resonance (NMR) was performed using a Bruker III 500-MHz spectrometer in deuterated water (D₂O), and the results were analyzed by unidimensional (1D) ¹H NMR and two-dimensional (2D) heteronuclear single quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) (48 ).

ORBI quantification in the solubility test was performed by a stability-indicating method with high performance liquid chromatography as described by Casedey and collaborators [48 (link)]. The chromatographic separation was achieved using a Prominence model Shimadzu™ liquid chromatograph (Kyoto, Japan) with a DGU-20A 3R degasser, SIL-20AC HT autosampler, CTO-20A oven, LC-20AD pump, and SPD-M20A diode array detector. The following chromatographic parameters were used. A Shim-pack CLC-ODS Shimadzu column (Kyoto, Japan) (dimensions 250 mm × 4.6 mm diameter and 5.0 µm particle size) at 25 °C was the stationary phase. The mobile phase used was a solution of 5% (v/v) acetic acid:methanol (80:20 v/v) at a flow rate of 0.7 mL·min⁻¹. UV detection was performed at a wavelength of 290 nm, and the injection volume of the sample in the chromatographic system was 20 μL.
A calibration curve was prepared using a stock solution in the mobile phase of ORBI Sigma standard (previously dried for 4 h at 105 °C in an oven) at 5 concentrations in triplicate (5, 10, 15, 20, and 30 µg·mL⁻¹). The results were analysed by linear regression of the peak area versus concentration. The regression equation for the calibration curve was y = 134.377x − 3751 with a correlation coefficient of (r) = 0.9999.

Briefly, a sample aliquot of 100 μl was protein-precipitated with 300 μl methanol. After centrifugation, 10 μl of the supernatant was injected into an AB SCIEX API-4000 tandem mass spectrometer (Foster City, CA, USA) equipped with electrospray ionization (ESI), LC-20AD pumps, a SIL-20A auto-sampler, and a CTO-20A oven (Shimadzu, Kyoto, Japan). Analyst 1.5.1 software was used for data acquisition and processing. Separations were conducted on a Luna C8 column (100 × 2.0 mm, 5.0 μm; Phenomenex Inc, USA), and the column temperature was maintained at 40 °C. A binary gradient consisting of solvent A (0.1% formic acid solution with 5 mM ammonium acetate) and solvent B (methanol) was employed for the elution of Cys A and FK506 (internal standard). The flow rate was kept at 0.45 ml·min⁻¹ by the following gradient program: 0–1.0 min, 75% B; 1.0–1.5 min, 75–99.5% B; 1.5–4.5 min, 99.5% B; 4.5–5.0 min, 99.5–75% B; 5.0–10.0 min, 75% B. The parameters of ESI source in positive mode were optimized as follows: curtain gas, 30 Arb; Gas 1, 65 Arb and Gas 2, 65 Arb; source temperature, 500 °C; ionization voltage, 5500 V. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode (m/z 1224.9 → 1112.8 for Cys A, m/z 821.4 → 768.3 for FK506).