G1329a autosampler

The chromatographic conditions used for the analysis were based on compendial methods for the API [8 ]. HPLC-DAD coupled with tandem mass spectrometry (MS/MS) was conducted with Agilent instrumentation using G1311A pump, G1329A autosampler, G1330A autosampler thermostat, G1316A column compartment, G1315B diode array detector, G6340 mass spectrometer modules (Agilent Technologies, Santa Clara, California, USA). Instrument software comprised Agilent ChemStation B.01.03 with 6300 Series Ion Trap LC/MS software Version 6.1. The following chromatographic conditions were used: 0.9 mL/min flow rate; Agilent column ZORBAX SB-C18 3.0 mm x 250 mm, 5 μm; 60 °C column temperature; 10 μL injection volume (MPA powder samples); 20 μL injection volume (finished product samples). The diode array detector (DAD) was set at 254 nm, where all spectra were stored from 190 nm to 400 nm. The mass spectrometer used the following parameters: capillary - 3500 V; Nebulizer 50.0 psi; Dry Gas 12.0 L/min; Dry Temp 350 °C; alternating polarity; Scan: 50–500 m/z; ESI Source.

The Merck Hitachi Lachrome Elite HPLC system (Japan) with an L-7100 pump, an L-7200 autosampler, L-7350 column oven, L-7450A DAD detector, and Ezochrom Elite software was employed for the selectivity test and Agilent Technologies 1200 series HPLC system with a G1310A isocratic pump, G1314A UV detector, and G1329A autosampler was used for all other tests. The LiChrospher 100 RP18 column (5 μm, 125 mm length, 4.0 mm inner diameter) was utilized in this study and it is from Merck (Darmstadt, Germany). The C18 column with 5 μm, 125 mm length, 4.0 mm inner diameter was kept at room temperature. A mixture of acetonitrile and water (60:40, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The UV detector was regulated for 236 nm and the injection volume w 10 μL.

An Agilent 6490 triple quadrupole LC-MS system (Agilent Corporation, MA, USA) equipped with G1311 A quaternary pump, G1322 A vacuum degasser, G1329 A autosampler, and G1316 A thermostat was used for the UPLC-QqQ MS/MS analysis. The mobile phase consisted of acetonitrile containing 0.05% formic acid (solvent A) and water containing 20 mmol ammonium acetate (solvent B). The stepwise linear gradient was optimized as follows: 0–20 min, linear from 95% to 70% A; 20–21 min, linear from 70% to 50% A; 21–24 min, held at 50% A; 24–25 min, linear from 50% to 95% A; and 25–30 min, held at 95% A for equilibration of the column. The flow rate was 0.3 mL/min. The injection volume was 3 μL. The separation was achieved at 25°C using an optimized Waters ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm).
The analytes were determined by monitoring the precursor-product transition in the MRM mode using ion polarity switching mode. To ensure the desired abundance of each compound, the CE values and other parameters were optimized and were as follows: cycle time, 300 ms; gas temp, 200°C; gas flow, 14 L/min; nebulizer, 20 psi; sheath gas flow, 11 L/min; capillary voltage, 3 kV; nozzle voltage, 1.5 kV; and Delta EMV(+), 200 V. The optimized mass transition ion pairs (m/z) and CE values for neurotransmitters are shown in Table 1. The MRM chromatograms of 8 neurotransmitters are shown in Figure 1.

An Agilent 1200 Series HPLC instrument equipped with a G1311A quaternary pump, a G1322A degasser, a G1329A autosampler, and a G1314B ultraviolet-visible detector from Agilent Technologies (Palo Alto, CA, USA) was employed. Instrument was controlled with the Agilent ChemStation software. Chromatographic separation was performed in reversed-phase mode by using a Kinetex C₁₈ (100 x 4.6 mm i.d., 2.6 µm particle size) column from Phenomenex (Torrance, CA, USA) at room temperature. Gradient elution employing 0.1% (v/v) formic acid aqueous solution (solvent A) and methanol (solvent B) as mobile phase components was applied following the next elution program: 0–2 min, isocratic step at 5% B; 2–4 min linear gradient from 5 to 25% B; 4–12 min, at 25% B; 12–14 min, from 25 to 45% B; 14–16 min, at 45% B; 16–18 min, from 45 to 95% B; 18–20 min, at 95% B; 20-21 min, back to initial conditions at 5% B; and from 21–30 min, at 5% B for column re-equilibration. The mobile phase flow rate was 1 mL/min and the injection volume was 20 µL. Direct UV-absorption detection at 280 nm was employed.

A high-performance liquid chromatographic method coupled with MS/MS detection (HPLC- MS/MS) was developed at the ERDC laboratories for the determination of VRD in human plasma. The method was validated according to the “FDA Bio-analytical Method Validation Guidelines 2003”. Assay linearity was verified for VRD at concentration range of 3–350 ng/mL with regression coefficients (R2) of 0.996. The described method is proven to be sensitive, accurate and reproducible within the lower limits of quantification of 3 ng /mL for VRD.
The HPLC-MS/MS system consisted of HPLC, Agilent series 1200 (Agilent Technologies Deutschland GmbH, Waldbronn, Germany), used with a mass spectrometer detector, Agilent 6420 with Triple Quad G1311A quaternary pump, G1329A auto sampler, G1322A vacuum degasser and mass hunter software. Chromatography was performed (75% acetonitrile: 25% buffer (ammonium formate 20 mM + 0.2% (v/v) formic acid in water) as mobile phase at a flow rate of 0.35 ml/min and a reversed phase column Intersil ODS -3 (4.6 mm × 50 cm, dp 5 µm Sigma–Aldrich, USA) temporized at 25 ◦C. Sildenafil was used as an internal standard (IS). Full details of the analysis procedure will be published in a separate article.

The procedure for the extraction of soluble polysaccharides from grape skins was carried out as described by Gil-Cortiella and Peña-Neira [80 (link)]. The quantification of the polysaccharides fractions was carried out using dextrans to assess the molecular weight of polymeric fractions and pectins as the external standard to quantify the polysaccharides using previously described developed and validated methodologies [79 (link),80 (link)].
The analysis of soluble polysaccharides was carried out by using a high-performance size exclusion chromatography–refractive index detection (HPSEC-RID) system (Agilent 1260 Infinity Series liquid chromatograph; Santa Clara, CA, USA) consisting of a G1329A autosampler, a G1362A refractive index detector, a G1315D diode array detector, a G1311B quaternary pump, a G1316A column oven. The chromatograph was connected to an Agilent Chem Station data processing station (version B.04.03).