Securityguard c18 pre column

An Agilent reversed-phase Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm particle size, 80 Å pore size) with a Phenomenex (Torrance, CA, USA) SecurityGuard C18 pre-column (4.0 mm × 3.0 mm) was used. LC parameters used were as follows: mobile phase A was 0.2% (v/v) formic acid in water, and mobile phase B was 0.2% (v/v) formic acid in acetonitrile. The gradient profile was: t = 0 min, 0% B; t = 0.5 min, 0% B; t = 5.5 min, 95% B; t=6.5 min, 95% B; t = 7.0 min, 0% B; and t = 9.5 min, 0% B. The column oven was set at 50 °C. The flow rate was 500 μL/min, and the sample injection volume was 10 μL.
For the analysis of organic acids, the mobile phases used were (A) 0.01% (v/v) formic acid in water, and (B) 0.01% (v/v) formic acid in methanol. The gradient profile was as follows: t = 0 min, 30% B; t = 2.0 min, 50% B; t = 12.5 min, 95% B; t = 12.51 min, 100% B; t = 13.5 min, 100% B; t = 13.6 min, 30% B and finally maintained at 30% B for 4.4 min. The column oven was set to 40 °C. The flow rate was 300 μL/min, and the sample injection volume was 10 μL.

For chromatography, an Agilent reversed-phase Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm particle size, 80 A pore size) with a Phenomenex (Torrance, CA, USA) Security Guard C18 pre-column (4.0 mm × 3.0 mm) was used for analyzing amino acids and biogenic amines. The parameters for LC–MS/MS analysis were as follows: Mobile phase A was 0.2% (v/v) formic acid in the water, and mobile phase B was 0.2% (v/v) formic acid in acetonitrile. The gradient parameters were t = 0 min, 0% B; t = 0.5 min, 0% B; t = 5.5 min, 95% B; t = 6.5 min, 95% B; t = 7.0 min, 0% B; and t = 9.5 min, 0% B. The chromatography column was set as 50 ºC. Ten microliters of samples was injected into the column with a flow rate of 300 µl/min.
For chromatography of organic acids, mobile phase A was 0.01% (v/v) formic acid in the water, and mobile phase B was 0.01% (v/v) formic acid in methanol. The gradient parameters were t = 0 min, 30% B; t = 2.0 min, 50% B; t = 12.5 min, 95% B; t = 12.5 min, 100% B; t = 13.5 min, 100% B; and t = 13.6 min, and finally 30% B for 4.4 min. The chromatography column was set as 40 ºC. Ten microliters of samples was injected into the column with a flow rate of 300 µl/min.

The LC–MS/MS system consisted of an API 3000 mass spectrometer (Sciex, Framingham, MA, USA) coupled to an UltiMate 3000 LC System (Dionex, Sunnyvale, CA, USA). Samples were separated using a ZORBAX Extend‐C18 column (Agilent, Santa Clara, CA, USA), preceded by a Securityguard C18 precolumn (Phenomenex, Utrecht, the Netherlands). Elution was done using a mixture of mobile phase A (0.1% formic acid in water (v/v)) and mobile phase B (methanol) in a 5 min gradient from 20% to 95% B, followed by 95% B that was maintained for 3 min and then re‐equilibrated at 20% B. Multiple reaction monitoring parameters were 490.2/383.1 (vemurafenib) and 496.2/389.1 (vemurafenib‐¹³C6). System control and data analysis were done using analyst^® 1.6.2 software (AB Sciex, Foster City, CA, USA).

Buparlisib was measured in samples from in vitro transport assays and in vivo pharmacokinetic studies using an LC-MS/MS system comprised of an UltiMate 3000 LC Systems (Dionex, Sunnyvale, CA) and an API 4000 mass spectrometer (Sciex, Framingham, MA). Samples were run through a Securityguard C18 pre-column (Phenomenex, Utrecht, The Netherlands) prior to separation on a ZORBAX Extend-C18 column (Agilent, Santa Clara, CA). Elution was done using a in a 5 minute gradient from 20% to 95% B (mobile phase A was 0.1% HCOOH in water (v/v) and mobile phase B was methanol). 95% B was maintained for 3 min followed by re-equilibration at 20% B. Multiple reaction monitoring was performed at 411.3/367.2 (buparlisib) and 418.2/138.4 (AZD8055). Analyst^® 1.6.2 software (AB Sciex; Foster City, CA) was used for system control and data analysis.

A targeted quantitative metabolomics assay called The Metabolomics Innovation Center (TMIC) Prime (TMIC PRIME^®) Assay was employed using a combination of direct injection (FIA) MS and reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was previously described by Zheng et al. [32 (link)]. Samples were derivatized prior to MS analysis. Amino acids, biogenic amines and derivatives, acylcarnitines, lipids, and glucose were derivatized with phenylisothiocyanate (PITC), while for organic acids, 3-nitrophenylhydrazine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and pyridine were added to achieve derivatization.
LC-MS/MS was used for the analysis of amino acids, biogenic amines and derivatives, and organic acids. An Agilent reversed-phase Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm particle size, 80 Å pore size) with a Phenomenex (Torrance, CA, USA) SecurityGuard C18 pre-column (4.0 mm × 3.0 mm) was used. The LC and MS parameters are described elsewhere [10 (link)]. Seven-point calibration curve was generated for each analyte.
The FIA-MS/MS method was employed for the analysis of lipids, acylcarnitines, and glucose; the LC autosampler was connected directly to the MS ion source by red PEEK tubing. Lipids, acylcarnitines, and glucose were analyzed semi-quantitatively.

An Agilent reversed-phase Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm particle size, 80 Å pore size), with a Phenomenex (Torrance, CA, USA) SecurityGuard C18 pre-column (4.0 mm × 3.0 mm), was used for the LC-MS/MS analysis of organic acids, amino acids, biogenic amines and derivatives.
The LC parameters used for the analysis of amino acids, biogenic amines and their derivatives were as follows: mobile phase A 0.2% (v/v) formic acid in water, and mobile phase B 0.2% (v/v) formic acid in acetonitrile. The gradient profile was as follows: t = 0 min, 0% B; t = 0.5 min, 0% B; t = 5.5 min, 95% B; t = 6.5 min, 95% B; t = 7.0 min, 0% B; and t = 9.5 min, 0% B. The column oven was set at 50°C. The flow rate was 500 μL/min, and the sample injection volume was 10 μL.
For the analysis of organic acids, the mobile phases used were A) 0.01% (v/v) formic acid in water, and B) 0.01% (v/v) formic acid in methanol. The gradient profile was as follows: t = 0 min, 30% B; t = 2.0 min, 50% B; t = 12.5 min, 95% B; t = 12.51 min, 100% B; t = 13.5 min, 100% B; t = 13.6 min, 30% B and finally maintained at 30% B for 4.4 min. The column oven was set to 40 °C. The flow rate was 300 μL/min, and the sample injection volume was 10 μL.