Uv 1280 spectrophotometer

Total myoglobin content was measured in non-aged steaks as not relevant changes were expected over ageing time. The method by Faustman and Philips [55 ], with minor modifications was followed. Previously thawed (4 °C, overnight) and minced (domestic grinder) 5 g of meat were homogenized in iced cold sodium phosphate buffer and set aside for 1 h (4 °C, darkness). Samples were then centrifuged (32,000× g) for 45 min and filtered through a Whatman filter paper (n °1, Whatman PLC, UK). Absorbance was read at 525 nm in a UV 1280 spectrophotometer (Shimadzu Corporation, Japan) and the total myoglobin concentration was estimated applying the extinction coefficient of 7.6 mM⁻¹ cm⁻¹ as proposed by Bowen [56 (link)].

Determination of the content of photosynthetic pigments was performed by the method of Arnon et al. (1956) [44 ] modified by Lichtenthaler and Wellburn (1983) [45 (link)] for chlorophyll “a”, “b” and total chlorophyll, and the method of Hager and Mayer-Berthenrath (1966) [46 (link)] for carotenoids. The material (15 fully developed, randomly selected leaves) was collected from each experimental variant, which were then divided into smaller fragments. From the prepared material, 0.03 g of fresh mass was obtained for the extraction of pigments. The samples were ground in a mortar with 10 cm³ of 80% acetone. The solutions were placed in test tubes and then transferred to a centrifuge to separate the liquid phase from the solid phase. Centrifugation was carried out for 10 min at 1500 rpm. The optical density of the samples was determined using a Shimadzu UV-1280 spectrophotometer (Japan). Determinations were made at wavelengths: 440, 645 and 663 nm. The content of dyes was calculated according to the formulas:


where Ek—extinction at a certain wavelength, V—amount of 80% acetone used for extraction and W—mass of fresh sample in grams, FM —fresh mass.

All the chemicals (cephalexin (Cayman Chemical, Tallinn, Estonia), sodium hydroxide (Chempur, Piekary Śląskie, Poland), hydrochloride acid (Chemical Company, Iași, Romania), sodium alginate (Carl Roth, Karlsruhe, Germany), calcium chloride (Chempur, Piekary Śląskie, Poland), sodium chloride (Chemical Company, Iași, Romania), ethanol (Chemical Company, Iași, Romania)) required in the experiments were of analytical purity and were used without further purification.
Yeast strain of Saccharomyces cerevisiae was kindly donated by Rompak Company (Pașcani, Romania).
A stock solution of cephalexin (Figure 1) with a concentration of 500 mg/L was prepared by dissolving the reagent in distilled water and kept at 4 °C in a closed vessel.
For the calibration curve, 0.02, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mL of CPX stock solution were placed in a series of volumetric flasks and the volumes were adjusted to 10 mL in order to obtain concentrations ranging between 1 mg/L and 30 mg/L. The samples absorbance was acquired at 260 nm with the help of a UV1280 spectrophotometer (Shimadzu, Tokyo, Japan). A calibration graph (absorbance vs. concentration) was plotted and a linear regression equation was recovered.
The other work solutions were obtained by appropriate dilutions. When necessary, NaOH (0.1 M) or HCl (0.1 M) were used to adjust the pH.

Ultraviolet and visible light (UV–Vis) absorption spectra of piplartine and its analogues were obtained with a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan). Purified piplartine and its analogues (1G, 1M, 6B and 14B) were solubilised in HPLC-grade acetonitrile and diluted to a concentration of 15 μg/mL. Because of its high absorbance and low solubility, the 19A analogue was further diluted to 7.5 μg/mL before measurement. Scans were performed using a quartz cuvette with a path length of 1 cm in the 200–450 nm range against a baseline recorded with acetonitrile at room temperature.

The plasma samples were depleted from albumin and immunoglobulin using ProteoPrep depletion columns (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Proteins from the urine samples were concentrated with Amicon Ultra-4 3 kDa centrifugal filter columns (Merck Millipore, County Cork, Ireland). The protein concentrations were determined by UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan) and samples with 4 μg of protein were taken for mass spectrometry. The proteins were reduced by dithiothreitol, alkylated by iodoacetamide, and digested by trypsin according to the manufacturer’s protocols (Thermo Scientific, Waltham, MA, USA). The samples were desalted by 10 μL C18 pipette tips from the same manufacturer in accordance with its protocol and dried before analysis.