Lc 4000 series

Measurement of gemcitabine in different samples was done by high performance liquid chromatography (HPLC) system (LC4000+ Series, Jasco, Tokyo, Japan). A HPLC system consists of LiChrospher 100 C18 column (4.6 × 250 mm, particle size 5 µm) with a 20 µL sample loop connected to UV-Visible detector (MD-4010, Jasco, Tokyo, Japan) and a software for data acquisition (ChromNAV 2.0, Jasco, Tokyo, Japan) was used. Elution of gemcitabine was achieved by a mobile phase consisting of acetonitrile and acetate buffer (97:3% v/v; pH 5). The column temperature was maintained at 25 °C, while the flow rate of mobile phase was 1 mL/min. Samples (20 µL) were injected and the detection was done at 280 nm [44 (link)]. Linear regression analysis indicates good linearity in the concentration of 25–800 ng/mL (r² = 0.995) and the retention time was estimated as 3.54 min. The LOQ (limit of quantitation) and LOD (limit of detection) of this method are 18.90 ng/mL and 6.23 ng/mL, respectively. The coefficient of variation and the accuracy range were 1.89%–8.25% and −2.67–9.05, respectively.

Dry cell biomass was obtained as follows: samples of 1.5–1.8 mL of well-mixed culture were added to previously weighed 2-mL tubes and centrifuged at 10,000 g for 15 min. The supernatant was stored for the analysis of extracellular metabolites, whereas the pellet was resuspended with 1 mL of distilled water and centrifuged again at 10,000 g for 15 min. The supernatant was discarded and tubes were dried at 37 °C until constant mass was reached (approximately 24 h). In the case of extracellular metabolites, glucose, ammonium, and glutamate were measured from the culture supernatant using an Analyzer Y15 (Biosystems, Barcelona, Spain) following the manufacturer’s instructions.
PHB was quantified using the crotonic acid method (Díaz-Barrera et al. 2016 (link)). Briefly, 3 mg of dry cell biomass were treated with 1 mL of ${\text{H}}_{\text{2}}{\text{SO}}_{\text{4}}$ and incubated at 90 °C and 700 rpm for 1 h. Samples were diluted 15 times, filtered through a 0.22 $\mu$ m PVDF filter, and assayed through a HPLC-UV system (LC-4000 series, Jasco, Japan) with an Aminex HPX-87 H ion-exclusion column.

The individual polyphenol and caffeine contents were determined by an ultrahigh-performance liquid chromatography (UHPLC) analysis according to Romano et al. [20 (link)] with some modifications. Five hundred milligrams of extract was weighed and dissolved in 0.5 mL of methanol. The mixture was vortexed for 30 s, sonicated for 20 min, and filtered with a 0.22-μm PES filter (Phenomenex, Torrance, CA, USA), then 10 μL was injected into the UHPLC system. A UHPLC system (Jasco LC-4000 Series, Tokyo, Japan) equipped with a PDA detector MD-4010, Column Oven CO-4061, UHPLC Semimicro Pump PU-4285, and a Spherisorb ODS2 (5 μm, 4.6 mm × 250 mm) C18 reversed-phase column (Waters, Arnhem, The Netherlands) was used. Caffeine detection was performed at 280 nm; chlorogenic acid, caffeic acid, and ferulic acid detection was performed at 330 nm; and 3,4 dihydroxy benzoic acid detection was performed at 260 nm using a photo diode array detector. The identification and quantification of the compounds was performed by comparing the retention times and areas between external standard peaks (dihydroxybenzoic acid, caffeic acid, chlorogenic acid, ferulic acid, and caffeine) and the peaks of the samples. The results are expressed as mg of individual compound/100 g of oil.