Zb 1701 column

Manufactured by Phenomenex

Sourced in New Zealand

The ZB-1701 column is a gas chromatography (GC) column designed for the separation and analysis of a wide range of organic compounds. It features a stationary phase consisting of a 100% dimethylpolysiloxane coating, which provides high thermal stability and inertness. The column dimensions are 30 m length, 0.25 mm internal diameter, and 0.25 μm film thickness.

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ZB-1701 GC capillary column (14 citations) ZB-1701 (5 citations)

12 protocols using zb 1701 column

GC-MS Analysis of Organic Compounds

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We analysed the samples on a GC-MS system—a GC7890B coupled to an MSD5977A operated at 70 eV electron ionization (Agilent Technologies)—equipped with a ZB-1701 column, 30 m × 0.25 mm × 0.15 μm with 5 m guard column (Phenomenex), at a constant flow rate of 1.0 mL/min of helium. Based on a published protocol^{39 (link)}, samples (1 µL) were injected under pulsed splitless mode (for trace analyses) with the inlet at 290 °C (180 kPa for 1 min, 50 mL/min purge flow after 1 min). The GC oven temperature was initially held at 45 °C for 2 min, after which it was raised to 180 °C at 9 °C/min and held for 5 min. Subsequently, the temperature was raised to 220 °C at 40 °C/min and held for 5 min. The temperature was raised again to 240 °C at 40 °C/min and held for 11.5 min. Finally, the temperature was raised to 280 °C at 40 °C/min and held for 10 min. The interface was kept at 250 °C and the quadrupole at 130 °C. The detector operated in scan mode, started after 5.5 min with a mass range between 38–550 atomic mass units at 2.9 scans/s. Negative controls were injected at the beginning of each run and QCs were run every seventh sample.

Chia A.R., de Seymour J.V., Wong G., Sulek K., Han T.L., McKenzie E.J., Aris I.M., Godfrey K.M., Yap F., Tan K.H., Shek L.P., Lee Y.S., Kramer M.S., Karnani N., Chong M.F, & Baker P.N. (2020). Maternal plasma metabolic markers of neonatal adiposity and associated maternal characteristics: The GUSTO study. Scientific Reports, 10, 9422.

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GC-MS Analysis of Derivatized Compounds

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Derivatized samples were analyzed with an Agilent 7890B GC system coupled to a 7000 Triple Quadrupole GC-MS system. The column was Phenomenex ZB-1701 column (30 m x 0.25 mm x 0.25 μm), with the following oven program: initial temperature 50°C/ hold 2 min; increment at 10°C/min to 140°C/ hold 0 min; increment at 20°C/min to 182°C/ hold 1 min; and increment at 50°C/min to 280°C/ hold 0 min. Run time 16.1 min. Samples (1 μL) were injected using split mode (20:1, 28 mL/min split flow). The column gas flow was held at 1.4 mL/min of He. The temperature of the inlet was 280°C, the interface temperature 230°C, and the quadrupole temperature 150°C. The column was equilibrated for 2 min before each analysis. The mass spectrometer was operated in single-ion monitoring (SIM) mode between 9.9 and 14.0 min. A segment for each compound was defined: benzyl-formate at 9.9 min, fragments m/z 136 and 137; benzyl-acetate at 11.0 min, fragments m/z 150, 151, 152 and 153; benzyl-propionate at 12.2, fragments m/z 164, 165, 166 and 167; and benzyl-butyrate at 13 min, fragments m/z 178,179,180, and 182. All segments at 2.5 cycles/s. Agilent MassHunter Workstation Software (version B.07.01 SP1) was employed for automated data processing, using peak area for absolute concentrations calculations.

Katz S., Grajeda-Iglesias C., Agranovich B., Ghrayeb A., Abramovich I., Hilau S., Gottlieb E, & Hershberg R. (2023). Metabolic adaptation to consume butyrate under prolonged resource exhaustion. PLOS Genetics, 19(6), e1010812.

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Biochemical Indicators of SLOS Severity

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The primary biochemical indicator of disease severity was the (7-DHC+8-DHC)/Cholesterol ratio (14 (link)). However, because this represents the first investigation of the association between biochemical variables and sleep in SLOS, we also independently examined associations between 7-DHC, 8-DHC, and total plasma cholesterol. Plasma sterol concentrations were measured by capillary-column gas chromatography on a Perkin Elmer gas chromatograph (model AutoSystemXL) with a CP-Wax57 column (25M, 0.32 mm ID; 0.25 μm film; Chrompack Co., Rariton, NJ) or Agilent gas chromatograph (model 6890N) with a ZB1701 column (30M; 0.25 mm ID; 0.25 μm film; Phenomenex). Internal standards (5α-cholestane or epicoprostanol) and authentic cholesterol standards were used for calibration (18 (link)).
Mevalonic acid concentration was determined in 24-hr urine samples using a radioenzymatic isotope dilution method reported previously and expressed in μmol/day (29 (link)). Plasma concentrations of 24(S)-hydroxycholesterol (24S) were determined by liquid chromatograph-mass spectrometry (LC-MS) as previously described (30 (link)). The within- and between-run precision (coefficients of variation) for 24S measurements was less than 8.5% across the range measured with a lower limit of detection of 38 ng/ml.

Freeman K.A., Olufs E., Tudor M., Roullet J.B, & Steiner R.D. (2016). A Pilot Study of the Association of Markers of Cholesterol Synthesis with Disturbed Sleep in Smith Lemli-Opitz Syndrome. Journal of developmental and behavioral pediatrics : JDBP, 37(5), 424-430.

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Acetate Derivatization and Quantification

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Acetate was derivatized as before (Tumanov et al., 2016 (link)). Briefly, 50 μL 1-propanol was added to 200 μL medium in a 2-mL microfuge tube. 40 μL 1 mM ²H₃-acetate (Sigma) was added, and the tube was placed on ice in the fume hood, followed by 50 μL pyridine and 5 min of incubation on ice. Next, 100 μL 1 M NaOH was added, followed by 30 μL methyl chloroformate (MCF), vortexing for 20 s, and, finally, addition of 300 μL of tert-butyl methyl ether (MTBE). Samples were vortexed for 2 min and centrifuged at 10,000 × g for 5 min. The top layer was transferred to GC-MS vials and analyzed with an Agilent 7890B GC and an Agilent 7000 Triple Quadrupole GC-MS system with a Phenomenex ZB-1701 column (30 m × 0.25 mm × 0.25 μm). Data were extracted with Agilent Mass Hunter B.06.00 software, from MS intensities of mass-to-charge ratio (m/z) 61 and 63 ions, corresponding to ¹²C- and ¹³C-acetate, respectively, which were quantified with ²H₃-acetate (m/z of 64).

Bulusu V., Tumanov S., Michalopoulou E., van den Broek N.J., MacKay G., Nixon C., Dhayade S., Schug Z.T., Vande Voorde J., Blyth K., Gottlieb E., Vazquez A, & Kamphorst J.J. (2017). Acetate Recapturing by Nuclear Acetyl-CoA Synthetase 2 Prevents Loss of Histone Acetylation during Oxygen and Serum Limitation. Cell Reports, 18(3), 647-658.

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GC-MS Quantification of Acetate Isotopomers

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The acetate samples were analyzed with an Agilent 7890B GC system coupled to a 7000 Triple Quadrupole GC-MS system. The column was Phenomenex ZB-1701 column (30 m × 0.25 mm × 0.25 μm), with an oven program as described in Table 1. Samples (2 μL) were injected using split mode (0.5 bar, 25 mL/min split flow). The column gas flow was held at 1.0 mL of He per min. The temperature of the inlet was 280 °C, the interface temperature 230 °C, and the quadrupole temperature 200 °C. The column was equilibrated for 2 min before each analysis. The mass spectrometer was operated in scan mode between 2.2 and 2.7 min with a mass range of 30–150 AMU at 1.47 scans/s. Agilent Mass Hunter B.06.00 software together with R-based MetabQ software were employed for automated data processing using peak heights of m/z 61, 63, and 64 ions used to quantify ¹²C, U-¹³C, and ²H₃-acetate, respectively (the peak shapes were consistently highly symmetric, and using either peak area or peak heights gave equivalent results) [30 (link)]. Peak heights of ¹²C and U-¹³C acetate were compared to the ²H₃-acetate peak height, and absolute concentrations were obtained from a calibration curve.Table 1

GC temperature program for acetate analysis

Start temperature (°C)	Ramp (°C/min)	End temperature (°C)	Hold time (min)
45	–	45	0.8
45	25	60	0
60	50	190	0

Tumanov S., Bulusu V., Gottlieb E, & Kamphorst J.J. (2016). A rapid method for quantifying free and bound acetate based on alkylation and GC-MS analysis. Cancer & Metabolism, 4(1), 17.

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Serum Metabolite Quantification Protocol

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Serum separated by centrifugation (7000 × g, 3 min) using Beckton Dickson vials was stored at −80 °C until analysis. Samples were prepared as previously described by Kelley with slight modifications [17 (link)]. Coprostan-3-ol (20 μg, Sigma) added to each 50 μl sample was the surrogate internal standard. Saponified, extracted and dried samples were derivatized with bis-trimethylsilytrifluoroacetamide plus 1 % trimethylchlorosilane (ThermoFisher) for 1 h (60 °C) and injected onto a Trace 1310GC TSQ 8000 Evo Mass Spectrometer (Thermo Scientific) using a ZB-1701 column (Phenomenex). Retention times were confirmed using standards (Sigma), and the National Institutes of Standards and Technology mass spectral library (data version 14).

London E., Tatsi C., Soldin S.J., Wassif C.A., Backlund P., Ng D., Biesecker L.G, & Stratakis C.A. (2020). Acute Statin Administration Reduces Levels of Steroid Hormone Precursors. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme, 52(10), 742-746.

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Quantifying Plasma 5α-Cholestanol by GC-MS

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GC-MS measurement of elevated plasma 5α-cholestanol has been described [7 (link),9 ]. In brief, internal standard (epicoprostanol) was added to plasma samples or calibrants generated using 5α-cholestanol. Sterols were saponified by the addition of ethanol/KOH and the aqueous phase was extracted with hexane. Dried sterols were derivatized with BSTFA and the trimethylsilyl ether derivative of 5α-cholestanol was measured using GC (splitless injection) performed with a ZB1701 column (30m, 0.25mmID, 0.25μm film thickness, Phenomenex, Torrance, CA) coupled to a mass spectrometer (Agilent GC 6890N and MS 5975; Santa Clara, CA). Mass spectra were collected in selected ion mode (with m/z= 355 and 370 ions monitored for epicoprostanol and m/z= 306 and 305 ions for 5α-cholestanol).

DeBarber A.E., Luo J., Giugliani R., Souza C.F., Chiang J.(., Merkens L.S., Pappu A.S, & Steiner R.D. (2014). A useful multi-analyte blood test for cerebrotendinous xanthomatosis. Clinical biochemistry, 47(9), 860-863.

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Quantitative GC-MS Analysis of Formate

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Derivatized formate samples were analyzed with an Agilent 7890B GC system coupled to a 7000 triple quadrupole MS system. The column was a Phenomenex ZB-1701 column (30 m × 0.25 mm × 0.25 μm). Samples (2 μl) were injected into the GC-MS using split mode [0.5 bars; split flow (25 ml/min)]. Gas flow through the column was held constant at 1.0 ml of He per minute. The temperature of the inlet was 280°C, the interface temperature was 230°C, and the quadrupole temperature was 200°C. Reduced electron energy (60 eV) was used for analyte ionization. The column was equilibrated for 3 min before each analysis. The mass spectrometer was operated in selected ion monitoring (SIM) mode between 3.0 and 4.3 min with SIM masses of 136, 137, and 138 for M⁰, M⁺¹, and M⁺² (internal standard) formate, respectively. The utilized oven program is described in table S1.
The total run time of a sample was 5.97 min. Natural isotope abundance was taken into account and subtracted, as described in the Supplementary Materials.
Recorded data were processed using the MassHunter Software (Agilent). Integrated peak areas were extracted and used for further quantifications using in-house software packages.

Meiser J., Tumanov S., Maddocks O., Labuschagne C.F., Athineos D., Van Den Broek N., Mackay G.M., Gottlieb E., Blyth K., Vousden K., Kamphorst J.J, & Vazquez A. (2016). Serine one-carbon catabolism with formate overflow. Science Advances, 2(10), e1601273.

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Cholesterol and 7-dehydrocholesterol Quantification

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Both control and SLOS cells were cultured in LPDS medium to confluence. Cells were collected in 1X PBS, and pelleted by centrifugation at 500 × g for 10 min. Cell pellets were solubilized in 1.0 ml of 0.05% SDS. One aliquot was used for protein measurement (BCA ^™ Protein Assay Kit, Pierce, Rockford IL). The sterols were extracted from the remaining cell pellet using chloroform:methanol (2:1), after the internal standard (epicoprostanol, EPIC, Sigma C-2882) had been added. The solvent was removed by evaporation under nitrogen at 40° C. Sterols were saponified by the addition of ethanol/KOH and incubated at 37°C for 1 hour. Sterols were extracted 2 times with hexane. The combined hexane extracts were evaporated to dryness and derivatized with BSTFA at 80°C for 30 min. Concentrations of the trimethylsilyl ether derivatives of cholesterol and 7-dehydrocholesterol were measured using a ZB1701 column (30 m, 0.25 mm ID, 0.25μm film thickness: Phenomenex, Torrance, CA) and selected-ion mass spectrometry (Agilent GC 6890N and MS 5975). The mass spectra data were collected in selected ion mode m/z= 355 and 370 for EPIC; m/z= 329 and 458 for cholesterol, and m/z = 325 and 351 for 7-dehydrocholesterol. Internal standard (EPIC) and authentic standards of cholesterol and 7-dehydrocholesterol (Steraloids, Newport, RI C3000-000) were used for calibration

Chang S., Ren G., Steiner R.D., Merkens L., Roullet J.B., Korade Z., DiMuzio P.J, & Tulenko T.N. (2014). Elevated autophagy and mitochondrial dysfunction in the Smith–Lemli–Opitz Syndrome. Molecular Genetics and Metabolism Reports, 1, 431-442.

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Sterols quantification in mammalian cells

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Both control and SLOS cells were cultured in LPDS medium to confluence. Cells were collected in 1 × PBS and pelleted by centrifugation at 500 ×g for 10 min. Cell pellets were solubilized in 1.0 ml of 0.05% SDS. One aliquot was used for protein measurement (BCA™ Protein Assay Kit, Pierce, Rockford IL). The sterols were extracted from the remaining cell pellet using chloroform:methanol (2:1), after the internal standard (epicoprostanol, EPIC, Sigma C-2882) had been added. The solvent was removed by evaporation under nitrogen at 40 °C. Sterols were saponified by the addition of ethanol/KOH and incubated at 37 °C for 1 h. Sterols were extracted 2 times with hexane. The combined hexane extracts were evaporated to dryness and derivatized with BSTFA at 80 °C for 30 min. Concentrations of the trimethylsilyl ether derivatives of cholesterol and 7-dehydrocholesterol were measured using a ZB1701 column (30 m, 0.25 mm ID, 0.25 μm film thickness: Phenomenex, Torrance, CA) and selected-ion mass spectrometry (Agilent GC 6890N and MS 5975). The mass spectra data were collected in selected ion mode m/z = 355 and 370 for EPIC; m/z = 329 and 458 for cholesterol, and m/z = 325 and 351 for 7-dehydrocholesterol. Internal standard (EPIC) and authentic standards of cholesterol and 7-dehydrocholesterol (Steraloids, Newport, RI C3000-000) were used for calibration.

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