Lcq advantage ion trap mass spectrometer

LC-ESI-MS was performed using an LC Surveyor system hyphenated to an LCQ Advantage ion trap mass spectrometer (both instruments ThermoFinnigan, Hemel Hempsted, UK). A Gemini 3 µm C18, 100 × 2.00 mm column fitted with a Gemini C18, 4.0 × 2.0 mm guard column (Phenomenex, Cheshire, UK) was used and was thermostatically controlled at 30°C. The LC method employed two separate mobile phases: acetonitrile + 1% acetic acid and water + 5% acetonitrile + 1% acetic acid. LC-grade water was obtained from a Millipore purification system. The mass spectrometer was operated in positive electrospray ionization mode. Selection and tuning of the mass spectrometer settings for each of the alkaloid analytes, including thebaine, were performed using direct infusion, involving the direct introduction of each of the analytes dissolved in methanol. LC-MS data analysis was carried out using XCaliber 2.0 software package supplied with the Thermo Finnigan LC system and LCQ Advantage ion trap mass spectrometer. The LC conditions for the analysis are shown in table 1.
Table 1.

Liquid chromatography conditions for the analysis of thebaine and other opium alkaloids.

mobile phase composition solvent A: acetonitrile + 1% acetic acid solvent B: water + 5% acetonitrile + 1% acetic acid
time (minutes)	%A	%B	flow rate (μl min−1)
0.00	0	100	200
10.00	65	35	200
12.00	65	35	200
14.00	0	100	300
20.00	0	100	300

In z preliminary study, various kinds of solid reagents such as sodium borate, diatomaceous earth, basic aluminum oxide, Ca₂CO₃, Na₂CO₃ and NaHCO₃ were screened at an excess dosage of 25% (wt %, mass ratio of solid reagents to S. flavescens particles). The extraction procedure was as follows: S. flavescens roots (10.0 g), solid reagents, and 72 g of stainless steel balls with 12 mm diameter were added into a 50 mL vial (PM-200 planetary mill, Retsch, Haan, Germany). After co-grinding at 400 rpm for 10 min, the powders were extracted with water for 20 min and then centrifuged at 3077 g for 10 min. The solution pH was adjusted to 4–5 with citric acid. The solution was condensed, centrifuged at 9391 g, the supernatant was discarded and the precipitants were analyzed by ultraviolet spectrophotometry (UV-2550 PC, Shimadzu, Kyoto, Japan) and HPLC/MS (Agilent 1100 HPLC system, Santa Clara, CA, USA) consisting of a Surveyor autosampler, pumps and photodiode array detector connected to an LCQ-Advantage ion trap mass spectrometer (Thermo Finnigan, San Francisco, CA, USA) fitted with an ESI source. Acidification pH value was optimized by acidifying the extracted solution to the pH values of 10 to 4, and then analyzing the extracts.

Each sample (20 μL) was injected twice into a reverse‐phase HPLC system using a Phenomenex Kinetex C18 column (5μ, 100 mm × 4.60 mm) with a gradient of 5–99% ACN in water with 0.1% formic acid over 12 min, held at 99% for 5 min, and then returned to 5% at 22 min for another 3 min. The solvents were LC–MS grade (JT Baker, Center Valley, PA). The flow rate was 0.7 mL min^‐1.
The HPLC eluate was electrospray ionized (capillary temperature at 325°C, source voltage at 5 kV, and a sheath gas flow rate of 69 L min^‐1) and analyzed in the positive mode in the mass range of m/z from 300 to 2000 using a Thermo‐Finnigan LCQ Advantage ion trap mass spectrometer (Thermo‐Finnigan, San Jose, CA). MS/MS spectra were obtained in a data‐dependent manner using collision‐induced dissociation at 35 eV.

The eluting peaks of cell-free culture filtrates corresponding to standard L-DOPA and HGA peaks by HPLC were collected and further analyzed by mass spectrometry (MS). MS analysis was performed using a Thermo-Finnigan LCQ advantage ion trap mass spectrometer (San Jose, CA, USA) using an ESI interface in negative-ion mode.

Qualitative HPLC-PDA/UV-ESI-MS/MS analyses were performed using a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan) equipped with Xcalibur 3.1 software. Analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Fusion-RP column (Phenomenex). The eluent was a mixture of methanol (solvent A) and a 0.1% v/v aqueous solution of formic acid (solvent B). A linear gradient of increasing 55% to 85% A was developed within 45 min. The column was successively washed for 15 min with methanol and equilibrated with 55% A for 10 min. Elution was performed at a flow rate of 0.8 ml/min with a splitting system of 2:8 to MS detector (160 ml/min) and PDA detector (640 ml/min), respectively. The volume of the injected methanol solutions was 20 μl. Analyses were performed with an ESI interface in the positive mode. The ionization conditions were optimized and the parameters used were as follows: capillary temperature, 270 °C; capillary voltage, 29.0 V; tube lens offset, 50.0 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 3.00 arbitrary units; spray voltage, 4.50 kV; scan range of m/z 150–1200. N₂ was used as the sheath and auxiliary gas. PDA data were recorded with 200–600 nm range with preferential channel as the detection wavelength 260 nm.

Melting points were determined on a Kofler apparatus and are uncorrected. Chemical shifts (δ) are reported in parts per million and are calibrated using residual undeuterated solvent as an internal reference. 1H NMR,³¹P NMR and¹³C NMR spectra of all compounds were recorded with a Varian Gemini 200 spectrometer operating at 200 MHz or Bruker TopSpin 3.2 spectrometer operating at 400 MHz, in a ~2% solution of deuterated water (D₂O), unless otherwise stated. Data for¹H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, or combinations thereof. The 95% purity of tested compounds was confirmed by combustion analysis. ESI-MS/MS experiment was performed in negative ion mode, using a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with Xcalibur 3.1 software. Merck gel plates (60 F254) were used for analytical TLC. UV light was used to examine the spots. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was used as the drying agent. Commercially available chemicals were purchased from Sigma-Aldrich and TCI Chemicals. The synthesis of DM-22 was performed in 3 steps characterized by the preparation of the following intermediate products.