DOI: 10.57755/f4tq-yk95
title: Chromatography analysis of MHV-1 infected cells

description: <p><span style="color:rgb( 0 , 0 , 0 );font-family:&#39;raleway&#39; , sans-serif;font-size:14px;text-align:justify;background-color:rgb( 251 , 251 , 251 )">The data include chromatography outcome data of analysis of 1mg of  cell culture sample infected or uninfected with MHV-1, murine coronavirus.  These are results of  one experiments and 2 technical replicates. </span></p>
description: <p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:700;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Chromatography analysis</span></p><p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:700;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Headspace solid-phase microextraction HS-SPME</span></p><p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:400;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Two 10 ml chromatography vials per infected and uninfected sample, 1ml each were subjected to chromatography analysis (two replicates per sample). Prior to analysis, 10 µg of 2-undecanone (≥99% purity; Sigma-Aldrich, Steinheim, Germany) was added as an internal standard to the frozen matrix. Volatile compounds were extracted using an SPME Arrow fiber (1.10 mm diameter, 20 mm length) coated with a DVB/CWR/PDMS phase (CTC Analytics AG, Zwingen, Switzerland). The fiber was conditioned at 250 °C for 2 minutes before each extraction, and reconditioned for 3 minutes following desorption. All incubations and extractions were performed using a</span><span style="font-family:&#39;calibri&#39; , sans-serif;background-color:transparent;font-weight:400;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt"> PAL autosampler</span><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:400;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt"> (CTC Analytics AG Zwingen Switzerland)</span><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:400;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt"> equipped with temperature-controlled modules. Samples were incubated at 70 °C for 15 minutes, followed by extraction at the same temperature for 30 minutes. Desorption was carried out in split 1 mode at 250 °C for 3 minutes. Helium of analytical grade purity (99.99997%; Air Products, Warsaw, Poland) was used as the carrier gas at a flow rate of 1 mL/min. Each sample was analysed in two technical replicates.</span></p><p><strong style="font-weight:normal"><br /></strong></p><p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:700;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Determination of VOCs</span></p><p><strong style="font-weight:normal"><br /></strong></p><p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:400;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Volatile organic compounds (VOCs) adsorbed on the SPME fiber were analysed using a Shimadzu GCMS-QP2020 system (Shimadzu Corporation, Kyoto, Japan) equipped with a single quadrupole mass spectrometer. Separation was achieved using a Zebron ZB-5 capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness; Phenomenex, Torrance, CA, USA). The oven temperature was initially held at 50 °C for 3 minutes, then ramped to 150 °C at a rate of 5 °C/min, followed by an increase to a final temperature of 250 °C at 15 °C/min, which was maintained for 5 minutes. The total chromatographic run time was 34.67 minutes. The mass spectrometer operated in electron ionization mode at 70 eV, with the interface and ion source temperatures set to 250 °C. Data acquisition was performed in full scan mode over a mass range of m/z 40–400, with spectra collected between 1 and 30 minutes of the chromatographic run.</span></p><p><strong style="font-weight:normal"><br /></strong></p><p style="line-height:1.2;margin-top:0pt;margin-bottom:0pt"><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;font-weight:700;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;white-space:pre-wrap;font-size:12pt">Identification of VOCs</span></p><p><br /><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;vertical-align:baseline;font-size:12pt">Identification of volatile organic compounds (VOCs) was carried out by comparing the experimentally obtained mass spectra and calculated linear retention indices (LRIs) with reference data. The LRIs were determined using a standard mixture of n-alkanes </span><span style="font-family:&#39;calibri&#39; , sans-serif;background-color:transparent;vertical-align:baseline;font-size:12pt">(C7–C</span><span style="font-family:&#39;calibri&#39; , sans-serif;background-color:transparent;vertical-align:baseline;font-size:12pt">30</span>; Sigma-Aldrich, Steinheim, Germany) run under identical chromatographic conditions. Mass spectral data were interpreted using automated and manual matching against multiple spectral libraries, including the NIST WebBook,<span style="font-family:&#39;calibri&#39; , sans-serif;background-color:transparent;vertical-align:baseline;font-size:12pt"> NIST23</span><span style="font-family:&#39;calibri&#39; , sans-serif;background-color:transparent;vertical-align:baseline;font-size:12pt"> d</span><span style="font-family:&#39;calibri&#39; , sans-serif;color:rgb( 0 , 0 , 0 );background-color:transparent;vertical-align:baseline;font-size:12pt">atabases. Where applicable, additional verification was performed through comparison with published literature data. Compounds were considered to be tentatively identified when both the mass spectrum match and LRI deviation fell within accepted confidence thresholds. This integrative approach enabled reliable annotation of VOCs present in the headspace of both virus-infected and uninfected cell cultures. </span></p>