Lovenox® and Clexane®, the innovator versions of enoxaparin marketed in the U.S. and Europe were purchased from Sanofi-Aventis (Bridgewater, NJ). Generic versions of Lovenox® were provided by three different manufactures (three current lots of each). Online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was performed as previous described [4 (link)]. Briefly, enoxaparin injections were diluted into 1 μg/μL and directly injected into a HILIC column (2.0 mm × 150 mm, 200 Å, Phenomenex, Torrance, CA) by an Agilent 1200 autosampler. The LC column was directly connected online to the standard ESI source of LTQ-Orbitrap XL FT-MS (Thermo Fisher Scientific, San-Jose, CA). The enoxaparin intact chain compositions were analyzed under the negative mode. Following raw data acquisition, charge deconvolution was auto-processed using DeconTools [14 –15 (link)] software. Enoxaparin structural assignment was performed by automatic processing with GlycReSoft 1.0 software, developed at Boston University (http://code.google.com/p/glycresoft/downloads/list ) [4 (link),12 (link)]. The output on enoxaparin composition from GlycReSoft was then processed using GlycCompSoft to provide automated relative quantification of intact chains present in enoxaparin.
Ltq orbitrap xl ft ms
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LTQ-Orbitrap XL FT MS is a high-performance hybrid mass spectrometer that combines a linear ion trap with an Orbitrap mass analyzer. It provides high-resolution, accurate-mass measurements and tandem mass spectrometry capabilities for a wide range of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Luna hilic column, by Phenomenex (4 mentions)
Lovenox, by Sanofi (1 mentions)
Hilic column, by Phenomenex (1 mentions)
Clexane, by Sanofi (1 mentions)
Agilent 1200 autosampler, by Agilent Technologies (1 mentions)
Agilent poroshell 120 ec c18 column, by Agilent Technologies (1 mentions)
Arixtra, by Sanofi (1 mentions)
6 protocols using ltq orbitrap xl ft ms
1
Enoxaparin Structural Characterization by HILIC-FTMS
2
LMWH Separation and Characterization
3
Glycan and Fatty Acid Analysis via FTMS
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Glycan samples were dissolved in HPLC grade water as 0.2–0.5 μg/μL, and each sample (5 μL) was run in direct infusion mode by the standard ESI source of LTQ-Orbitrap XL FTMS (Thermo Fisher Scientific, San-Jose, CA, USA). LC parameters: Agilent Poroshell 120 ECC18 column (2.7 μm, 3.0 × 50 mm), mobile phase A was 5-mM ammonium acetate prepared with HPLC grade water, and mobile phase B was 5-mM ammonium acetate prepared in 98% HPLC grade acetonitrile with 2% of HPLC grade water. The flow was used 50% A and 50% B at a rate of 250 μL/min. The source parameters for FTMS were in the negative-ion mode, a spray voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens voltage of −50 V, a capillary temperature of 275 °C, a sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6 L/min. All FT mass spectra were acquired at a resolution 60,000 with 200–2000 Da mass range. Fatty acids sample was dissolved in HPLC acetonitrile and used 100% B as flow; all other conditions were the same.
4
LMWH Separation and Characterization
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A Luna HILIC column (2.0 ×
50 mm, 200 Å, Phenomenex, Torrance, CA) was used to separate
the LMWHs. Mobile phase A was 5 mM ammonium acetate prepared with
HPLC grade water. Mobile B was 5 mM ammonium acetate prepared in 98%
HPLC grade acetonitrile with 2% of HPLC grade water. The gradient
was used from 5% A to 70% A in 7 min then reset to 5% A at a flow
rate of 250 μL/min. The LC column was directly connected online
to the standard ESI source of LTQ-Orbitrap XL FT MS (Thermo Fisher
Scientific, San-Jose, CA). The source parameters for FTMS detection
were optimized using Arixtra (a synthetic ultra LMWH from Sanofi-Aventis,
Paris, France) to minimize the insource fragmentation and sulfate
loss and maximize the signal/noise in the negative-ion mode. The optimized
parameters, used to prevent in-source fragmentation, included a spray
voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens
voltage of −50 V, a capillary temperature of 275 °C, a
sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6
L/min. External calibration of mass spectra routinely produced a mass
accuracy of better than 3 ppm. All FT mass spectra were acquired at
a resolution 60 000 with 200–2000 Da mass range.
50 mm, 200 Å, Phenomenex, Torrance, CA) was used to separate
the LMWHs. Mobile phase A was 5 mM ammonium acetate prepared with
HPLC grade water. Mobile B was 5 mM ammonium acetate prepared in 98%
HPLC grade acetonitrile with 2% of HPLC grade water. The gradient
was used from 5% A to 70% A in 7 min then reset to 5% A at a flow
rate of 250 μL/min. The LC column was directly connected online
to the standard ESI source of LTQ-Orbitrap XL FT MS (Thermo Fisher
Scientific, San-Jose, CA). The source parameters for FTMS detection
were optimized using Arixtra (a synthetic ultra LMWH from Sanofi-Aventis,
Paris, France) to minimize the insource fragmentation and sulfate
loss and maximize the signal/noise in the negative-ion mode. The optimized
parameters, used to prevent in-source fragmentation, included a spray
voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens
voltage of −50 V, a capillary temperature of 275 °C, a
sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6
L/min. External calibration of mass spectra routinely produced a mass
accuracy of better than 3 ppm. All FT mass spectra were acquired at
a resolution 60 000 with 200–2000 Da mass range.
5
Heparin Oligosaccharide Profiling by HILIC-FTMS
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Each sample (100 μg) was added to 100 μL digestion buffer (50 mmol/L pH 7.0). Heparin
lyase II (10 mU in Tris–HCl buffer, pH 7.0) was added and the sample was digested in 37°C
water bath for 12 hours to produce fragments. Enzymatic digestion was terminated by
removing the enzymes using an YM-3 centrifugal filter unit. The filtrates were lyophilized
and redissolved in 100 µL of distilled water at a concentration of 1 µg/mL. Online
hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS)
was used to analyze the resulting oligosaccharides.12 (link) A Luna HILIC column (2.0 mm2 × 50 mm2, 200 Å; Phenomenex,
Torrance, California) was connected online to the standard ESI source of LTQ-Orbitrap XL
FTMS (Thermo Fisher Scientific, San Jose, California). Mass spectra were acquired at a
resolution 60 000 with 200 to 1800 m/z range.
lyase II (10 mU in Tris–HCl buffer, pH 7.0) was added and the sample was digested in 37°C
water bath for 12 hours to produce fragments. Enzymatic digestion was terminated by
removing the enzymes using an YM-3 centrifugal filter unit. The filtrates were lyophilized
and redissolved in 100 µL of distilled water at a concentration of 1 µg/mL. Online
hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS)
was used to analyze the resulting oligosaccharides.12 (link) A Luna HILIC column (2.0 mm2 × 50 mm2, 200 Å; Phenomenex,
Torrance, California) was connected online to the standard ESI source of LTQ-Orbitrap XL
FTMS (Thermo Fisher Scientific, San Jose, California). Mass spectra were acquired at a
resolution 60 000 with 200 to 1800 m/z range.
6
Heparin Lyase II Digestion and HILIC-FTMS Analysis
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Samples (100 mg) were added to 100 mL digestion buffer (50 mmol/L NH 4 OAc, 2 mmol/L CaCl 2 , pH 7.0). Heparin lyase II (10 mU in Tris-HCl buffer, pH 7.0) was added and mixed well. Samples were digested in 37 C water bath for 12 hours to produce fragments. Enzymatic digestion was terminated by removing the enzymes using a YM-3 centrifugal filter unit. The filtrates were lyophilized and redissolved in 100 mL of distilled water at a concentration of 1 mg/mL. Online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments. 14 A Luna HILIC column (2.0 Â 50 mm 2 , 200 A ˚; Phenomenex, Torrance, California) was directly connected online to the standard ESI source of LTQ-Orbitrap XL FTMS (Thermo Fisher Scientific, San Jose, California). Mass spectra were acquired at a resolution 60 000 with 200 to 1800 m/z range.
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