Db 5ms column

Approximately 20 mg of crude fat extract was mixed with 250 µL of internal standard (1.5 mg/mL of tetracosane dissolved in pyridine), followed by the addition of 250 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 50 µL of trimethylchlorosilane (TMSCl). Silylation was achieved by incubation at 70 °C for 30 min and lipid characterization was performed in a Gaseous Chromatographer coupled with a Mass Spectrometer (GC-MS), GCMS-QP2010 (Shimadzu (Kyoto, Japan)) equipped with an AOC-20i auto-injector and a DB-5 ms column (30 m × 0.25 mm diameter, 0.25 µm thickness). The elution program was the same as described elsewhere [60 (link)]. This started with 70 °C for 5 min followed by a temperature increase of 4 °C/min until 250 °C was achieved and a 2 °C/min increase until 300 °C, which was maintained for 5 min. The injection temperature was 320 °C and the split ratio was 100:0. Quantitative analysis was performed resorting to pure reference compounds (cholesterol, pentadecanol and palmitic acid), representative of the samples’ major lipophilic families.

The fermented and dried cocoa beans were milled (A11B, Ika) and subjected to simultaneous distillation/extraction for 2 hours using 4 mL of pentane as a solvent. The volatile concentrate obtained was stored at a temperature of 5 °C [52 (link),53 (link),54 (link)].
Aliquots (2 μL) of volatile concentrate was injected into a gas chromatograph coupled to mass spectrometry (GC-MS), in a Shimadzu QP-2010 Plus system, equipped with a DB-5MS column (30 m × 0.25 mm × 0.25 μm). The helium gas was used as the carrier gas with a flow rate of 1.2 mL/min. The temperature of the injector and the interface was 250 °C, and the oven temperature was adjusted to 60–250 °C, using a ramp of 3 °C/min. Electronic impact mass spectrometer at 70 eV and the ion source temperature at 220 °C were used.
Quantitative analysis of the chemical constituents was performed by peak-area normalization using a flame ionization detector (FID—Shimadzu, QP 2010 system) under the same conditions as GC-MS, except that hydrogen was used as a mobile phase. Chemical identification was carried out by comparing the mass spectra and retention indices (RI) with those of standard substances in the system libraries and with data from the literature [55 ,56 ,57 ]. The RIs were obtained using a homologous series of n-alkanes (C8–C24, Sigma-Aldrich Co, St. Louis, MO, USA).

To identify the plant secondary metabolites that may have contributed to the observed antileishmanial efficacy of ALE and ASE, GC-MS analysis was performed as described previously [16 (link)] using a Shimadzu QP2010 equipped with a DB-5MS column (30 m length, 0.25 mm i.d., film thickness 0.25 μm). The column temperature was increased gradually from 60 to 310°C at 5°C min⁻¹, and the injector and detector temperature was 260°C. Helium was employed as the carrier gas, set at a constant flow rate of 1.5 ml min⁻¹. The mass spectrums were produced in an electron impact ionization mode of 70 eV and the chemical constituents identified after correlation of the recorded mass spectra with the reference spectra of WILEY8.LIB and NIST08.LIB library, supplied with the software of the GC-MS system.

The EF and MB concentrations in the fumigation chambers were checked at 0.1, 1.0, 2.0, 4.0 h and 0.1, 1.0, and 2.0 h, respectively, to calculate the Ct product after fumigant injection into the chambers. This was conducted using a Shimadzu GC 17A gas chromatograph (Shimadzu, Kyoto, Japan) installed with a DB5-MS column (30 m × 0.25 mm i.d. × 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA) and a flame ionization detector (FID). Helium was used as a carrier gas at a flow rate of 1.5 mL/min. The oven, injector and detector temperature were maintained at 100, 250 and 280 °C, respectively. The EF and MB concentrations were calculated based on peak areas using external standards. The calibration curve standards were made by spiking a known volume of liquid EF into a 1 l Tedlar^® gas sampling bag (SKC Inc., Pittsburgh, PA, USA).
The Ct products were calculated based on the following equation as described in Ren et al. (2011) [32 (link)]: $$\mathrm{Ct}={{\displaystyle \sum }}^{\hspace{1em}}\frac{\left({\mathrm{C}}_{\mathrm{i}}+{ \mathrm{C}}_{\mathrm{i}+1}\right)\left({\mathrm{t}}_{\mathrm{i}+1}-{ \mathrm{t}}_{\mathrm{i}}\right)}{2},$$
where C = concentration of fumigant (mg/L), t = time of exposure (h), i = order of measurement, and Ct = concentration × time product (g h/m³).

The fatty acid profile was analyzed by GC-MS after their conversion to fatty acid methyl esters (FAME) following the methodology of O’Fallon et al. [37 (link)]. Samples were analyzed on a gas chromatograph mass spectrometer GCMS-QP2010 (Shimadzu, Kyoto, Japan) equipped with an AOC-20i auto-injector and a DB-5 ms column (30 m × 0.25 mm diameter, 0.25 µm thickness). The equipment operated under the following conditions: initial temperature, 70 °C for 5 min; temperature gradient, 4 °C min⁻¹; final temperature, 250 °C; temperature gradient, 2 °C min⁻¹; final temperature, 300 °C for 5 min; injection temperature, 320 °C; split ratio, 100:0. Identification of FAME was obtained by co-chromatography with authentic commercially available FAME standards (Supelco™ 37 Component FAME Mix, catalogue no. 47885-U, Supelco, Bellefonte, PA, USA). Total FAME content was quantified by comparison with a known amount of added nonadecanoic acid 19:0 (Thermo Fisher Scientific, Kandel, Germany) as internal standard. The internal standard (1000 mL, 2 mg mL⁻¹) was added prior to direct transmethylation to the ground seaweed powder.

To assess strain growth, the optical density at 600 nm (OD₆₀₀) was measured using a UV-2802PC; spectrophotometer (Unico, Shanghai, China). The concentrations of butyrate, butanol, and glucose in the fermentation samples were measured by HPLC using an Agilent 1260 system (Agilent Technologies, Santa Clara, CA, USA), equipped with an HPX-87H column (Bio-Rad Laboratories, Inc., Richmond, CA, USA) kept at 55 °C, with 5 mM H₂SO₄ at a flow rate of 0.5 mL/min as the mobile phase. The injection volume was 10 μL injection. For measurement of BB production, samples were taken from the solvent phase during fermentation, filtered, and immediately analyzed on a GCMS-QP2010 Ultra (Shimadzu, Japan) system equipped with a DB-5 ms column (30 m length, 0.25 mm inside diameter, 0.25 μm thickness, Agilent, USA). The flow rate of the helium carrier gas was 1 mL /min. The interface and ion source temperatures were set to 250 and 200 °C, respectively. The electron impact voltage was set to 70 eV. The m/z range was 35–500. The column temperature was initially set to 100 °C, after which it was increased to 250 °C at a rate of 20 °C/min, where it was held for 5 min.

The fatty acid profile was determined upon conversion of fatty acids to FAME, as previously described by Pinheiro et al. [17 (link)]. Briefly, to 300 mg of sample, 1 mL of the internal standard C19:0 (Thermo Fisher Scientific, Kandel, Germany) at 2 mg mL⁻¹ in methanol, 0.7 mL of KOH (10 N) in water and 5.3 mL of methanol were added. The tubes were incubated for 1.5 h at 55 °C. Afterwards, 0.58 mL of H₂SO₄ (24 N) was added and these were placed at the same temperature for another 1.5 h. Finally, 3 mL of hexane was added, followed by mixing and centrifugation. The hexane fraction was recovered and injected on a gas chromatograph mass spectrometer GCMS-QP2010 (Shimadzu, Kyoto, Japan) equipped with an AOC-20i auto-injector and a DB-5 ms column (30 m × 0.25 mm diameter, 0.25 µm thickness). The equipment operated under the following conditions: initial temperature, 70 °C for 5 min; temperature gradient, 4 °C min⁻¹; final temperature, 250 °C; temperature gradient, 2 °C min⁻¹; final temperature, 300 °C for 5 min; injection temperature, 320 °C; split ratio, 100:0. Identification of FAME was obtained by co-chromatography with authentic commercially available FAME standards. Total FAME content was quantified by comparison with the internal standard.