Api 2000

Mass spectra were acquired using an Applied Biosystems API2000 liquid chromatography/tandem mass spectrometry triple quadrupole mass spectrometer (Applied Biosystems, Carlsbad, CA, USA) equipped with an electrospray ionization source in positive ion mode (m/z 400–1800, with a declustering potential of 10–20 V and 0.1 Da steps). The molecular weight of the peptide was deduced from the multiply charged species using Analyst v1.4 with Bioanalyst extensions (Applied Biosystems).

Mass spectra were recorded on an API 2000 liquid chromatography-tandem mass spectrometry (LC-MS)/MS spectrometer (electron spray ion source, Applied Biosystems) coupled with an Agilent 1100 HPLC system using a Phenomenex Luna HPLC C18 column (50 × 2.00 mm, particle size 3 μm). Compound purity was determined by HPLC-ultraviolet using the following procedure. A amount of 10 μl of compound (1 mg ml⁻¹ in methanol) was injected and eluted with a gradient of water/methanol containing 2 mM ammonium acetate from 60:40 to 0:100 for 10 min, and subsequently with 0:100 for 10 min at a flow rate of 300 μl min⁻¹, starting the gradient after 10 min. Ultraviolet absorption was detected from 220 to 400 nm using a diode array detector.

To optimize MS conditions, a solution containing 100 ng/mL of matrine was infused into the tandem mass spectrometer (API 2000; Applied Biosystems, Foster City, CA, USA) at a flow rate of 10 μL/min. The turbo ion spray interface was operated in positive ion mode at 5500 V for spray voltage and ion spray temperature was set to 350°C. The precursor ions of the analytes were optimized as protonated molecular ions, [M + H]⁺. Matrine was quantified using the multiple reaction monitoring mode and the peak-area ratio method with an IS.

Glycerol concentration was monitored by HPLC analysis. Coculture samples were centrifuged at 12,000g for 2 min to collect cell-free broth, which was then filtered through 0.2-μm-pore-size polytetrafluoroethylene membrane syringe filters (VWR International) before subjected to HPLC analysis. The HPLC system consisted of a Bio-rad HPX-87H column, a Waters 2695 separation module and a Waters 410 differential refractometer. Isocratic elution was conducted using 14 mM sulfuric acid as mobile phase at a flow rate of 0.7 mL/min.
The concentration of MA product and the pathway intermediates were determined by LC/MS/MS analysis. Cell-free broth was collected by 12,000g centrifugation for 5 min followed by filtration through 0.2-μm-pore-size polytetrafluoroethylene membrane syringe filters (VWR International). 10 μL 1 g/L p-coumaric acid internal standard was added into 1 mL of the filtered broth. 10 μL of the mixed solution was then injected into an Applied Biosystems API2000 LC/MS/MS running on a previously established method [20 (link)].

Chemicals were purchased from Merck (Darmstadt, Germany), ABCR (Karlsruhe, Germany) or TCI (Eschborn, Germany). Analytical thin layer chromatography (TLC) was performed on TLC plates F₂₅₄ (Merck) and analyzed using UV light. High resolution mass spectra (HR-MS) were recorded on a micrOTOF-Q mass spectrometer (Bruker), low resolution mass spectra (LR-MS) on an API 2000 (Applied Biosystems) mass spectrometer. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ or (CD₃)₂SO on a Bruker Ascend 600 MHz NMR-spectrometer operating at 600.18 MHz (¹H), and 150.93 MHz (¹³C). Chemical shifts (δ) are reported in ppm and are referenced to the chemical shifts of the residual solvent proton(s) present in chloroform δ [(CHCl₃) = 7.26 ppm for the ¹H NMR spectra and δ (CDCl₃) = 77.16 ppm for the ¹³C NMR spectra] and in dimethylsulfoxide δ ((CH₃)₂SO) = 2.50 ppm for the ¹H NMR spectra and δ ((CD₃)₂SO) = 39.52 ppm for the ¹³C NMR spectra. Multiplicity: s, singlet; d, doublet; q, quartet; m, multiplet. Coupling constants (J) are shown in Hertz (Hz). The infrared spectra were recorded as solid samples on an ALPHA-T (Bruker) with a Platinum ATR Module using the Opus software. The IR spectra were measured in the attenuated total reflection (ATR) mode in the region of 4,000–385 cm⁻¹ (s, strong; m, medium; w, weak) and are reported in cm⁻¹.

The amino acids in dried blood pots on filter paper were determined by MS/MS (Applied Biosystems, API 2000). All the chemicals used in MS/MS (methanol, and acetonitrile) were liquid chromatographic/mass purity grade and were purchased from Fisher Scientific. The preparation and detection methods were described in previous studies.19, 20 In brief, dried blood spots were extracted using methanol, which contained amino acids as internal standards. After derivatization with n‐butyl alcohol hydrochloric acid, the samples were tested using MS/MS. A total of 17 amino acids were determined, including Phe, Tyr, alanine (Ala), Asp, Glu, Met, Val, Arg, Gly, Gln, His, Ser, Thr, Leu, Trp, Cit, and Orn.