Beckman system gold hplc

Free amino acid content was determined in lyophilized samples of mature grapes. Extraction was performed by adding 25 mL of Milli-Q H₂O to 1 g of grape berry powder, and the quantification of natural free amino acids (excluding tryptophan) was assessed in a Biochrom 30 Amino Acid Analyzer with a weak acidic cation exchange resin acting as the stationary phase (200 × 4.6 mm column) and a number of weak acidic Li-citrate buffers acting as the mobile phase. Stepwise pH, temperature and salt concentration gradients were applied. Detection after post column derivatization with Ninhydrin (135 °C ) at 570 or 440 nm was performed (Ansynth Service B.V., Roosendaal, The Netherlands). For tryptophan quantification, the sample solutions were diluted in Milli-Q H₂O (1:10), and analysis was performed using a Beckman System Gold HPLC (Beckman, Brea, CA, USA) equipped with an Allsphere C8 column, 250 × 4.6 mm, stationary phase, (P.J. Cobert Associates, Inc. St. Louis, MO, USA) and using a phosphate buffer/MeOH gradient (mobile phase). Detection was performed by fluorometry, with the emission wavelength set at 340 nm and the excitation wavelength set at 280 nm (Ansynth Service B.V., Roosendaal, The Netherlands).

Liposomal formulations were characterized in citrate buffer (10 mM citrate buffer, 145 mM NaCl, pH 6.0) according to their mean hydrodynamic diameter, polydispersity index in a Zetasizer Nano S (Malvern Instruments, Malvern, UK), and surface charge in a Zetasizer Nano Z (Malvern Instruments, Malvern, UK). The absence of quercetin crystals was evaluated by microscopy over time, and the lipid quantification was performed by the Rouser method [17 (link)].
Drug quantification was performed by high-performance liquid chromatography (HPLC) in a Purospher^® STAR RP-18 endcapped (5 µm) HPLC column, 4.6 × 250 mm (Merck, Darmstadt, Germany), using a Beckman System Gold HPLC (Beckman Coulter, Carlsbad, CA, USA). Liposomes containing drug were disrupted with methanol, with the concomitant solubilization of quercetin, and filtered through a PTFE membrane with 0.2 µm pore diameter before quantification at 360 nm with a mobile phase consisting of methanol/water acidified with 1 mM trifluoroacetic acid (70:30, v/v).
Nanoformulations were evaluated in terms of loading capacity, defined as the final drug-to-lipid ratio (µg quercetin/µmol lipid)–([Drug]/[Lipid])f–, and the incorporation efficiency (I.E.) that was determined according to the following equation: $$\mathrm{I}.\mathrm{E}.=\frac{\left(\frac{\left[\mathrm{Drug}\right]}{\left[\mathrm{Lipid}\right]}\right)\mathrm{f}}{\left(\frac{\left[\mathrm{Drug}\right]}{\left[\mathrm{Lipid}\right]}\right)\mathrm{i}}\times 100$$

Diclofenac in plasma samples, were analysed by using a validated HPLC method as previously described (Naidoo et al., 2007 (link)). In brief, plasma samples (200 µl) were mixed with diethyl ether (400 µl), 0.3 M potassium dihydrogen phosphate (400 µl) and vortexed. The organic layer was separated in an ice bath (methanol/solid carbon dioxide), evaporated to dryness and dissolved in 400 µl mobile phase. Samples were analysed on a Beckman System Gold HPLC (Beckman Instruments, Fullerton, CA, USA). Separation was achieved with a C18 column (250 mm × 4.6 mm × 5 µm; Thermo Scientific, Runcorn, UK). The mobile phase consisted of 0.05 M sodium dihydrogen phosphate (pH  = 4.85 to 4.89):CH₃CN, 42.5:57.5. One hundred (100) µl of the reconstituted samples was injected onto the column at 1 ml/min in an isocratic run. Detection of diclofenac was carried out at 275 nm. The total runtime per sample was 8 min with retention times of 4.9 min. Control values (100–0.39 µg/ml) showed a mean accuracy of 99%. When sufficient time points were available, samples were analysed by non-compartmental pharmacokinetic analysis (Thermo Fisher Kinetica 5.1, USA) using the standard settings.

Samples were analyzed on a Beckman System Gold HPLC (model 126, Beckman Coulter Life Sciences, Brea, CA, USA) with a TDA detector (Model 302, Viscotek, Malvern Instruments Ltd., Worcestershire, UK), using a Zorbax 300SB-C3 column (4.6 × 150 mm; 5 μm) and a Zorbax 300SB-C3 Guard 5 μm pre-column (Agilent, Santa Clara, CA, USA). Mobile phase used for gradient elution: A = 0.1% (v/v) TFA in water and B = 0.1% (v/v) TFA in ACN. The elution program comprised a flow rate of 1.5 mL/min with linear gradient from 5% B to 59% B over a period of 2 min, a flow rate of 1.5 mL/min at 59% B for 3 min, a flow rate of 1.5 mL/min with linear gradient from 59% B to 100% B over a period of 2 min and a flow rate of 1.5 mL/min at 100% B for 5 min. The column temperature was controlled at 40 °C. Detection was performed at UV 219 nm. Sample volume injected was 10 μl. OspA desorption from aluminum gel was performed by adding 150 mg of tri-sodium citrate per 1 ml of vaccine and incubation overnight at 5 °C under agitation. Aluminum gel was removed by centrifugation 10 min at 2190 g and supernatant was concentrated using ultrafiltration spinners 10 k MWCO according supplier recommendations (ThermoFischer, Rockford, IL, USA).