Spd m20a detector

Chromatographic determinations were performed using a HPLC system equipped with a LC-20AT quaternary gradient pump, DGU-20A5 degasser, SIL-20A autosampler, CTD-10ASvp thermostatted column compartment, CBM-20A communication bus module, and SPD-M20A detector (Shimadzu, Kyoto, Japan). Chromatographic separation was performed using an Agilent 5 TC-C18(2) column (250 mm × 4.6 mm, 5.0 μm, Santa Clara, CA, USA). The mobile phase components were 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 1.0 mL/min. The following gradient elution program was used: 0~13 min, 89~85% A; 13~25 min, 85~75% A; 25~27 min, 75~73% A; 27~45 min, 73% A. The column temperature was set at 32 °C and the injection volume was 10 μL. Additionally, the chromatographic data at a wavelength of 325 nm were applied.

Quantification of organic acids by Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) was performed as described previously [34 ] with some modifications. The mobile phase was 3 mM sulfuric acid aqueous solution. Sample (20 µL) was separated on a column set at 42 °C with a flow rate of 0.6 mL/min; and the absorbance at 215 nm was monitored by an SPD-M20A detector (Shimadzu, Japan). The HPLC system consisted of two pumps (LC-20 AT, Shimadzu, Kyoto, Japan), a degasser (DGU-20A, Shimadzu), an autosampler (SIL-20 A, Shimadzu) and a column oven (CTO-20A, Shimadzu). Chromatograms were evaluated with the Clarity software package (LabSolutions, Shimadzu). This method was termed as HPLC method 1.

The extracts obtained from plant sample were prepared in HPLC-grade methanol for quantitative analysis. Standard scopolamine (0.6 mg/5 mL) and atropine (1.0 mg/5 mL) were prepared in HPLC-grade methanol. Analysis was performed with a Shimadzu (Nexera) HPLC system equipped with LC-30AD Liquid Chromatograph, SIL-30AC Autosampler, SPD-M20A Detector, CBM-20A Communication Bus Module, CTO-20AC Column Oven and Lab solutions software. All samples were filtered through 0.45 μM (Millipore, Bedford, MA) filter. Extracts were separated on a RP-18 (4 × 250 mm, 5 μm; Merck, Bangalore, India) column. The mobile phase consisted of 0.05 M KH₂PO₄ in water (A) and acetonitrile (B) delivered at a flow rate of 1.0 mL/min. The gradient system was as follows: at 0 minutes 10% B, at 20 minutes 60% B, at 23 minutes 60% B, and at 25 minutes 10% B. The samples were analyzed at 30°C to provide efficiency to the peaks and the UV chromatograms were recorded at 210 nm.

The analysis was carried out on a Shimadzu CBM-20A liquid chromatograph (Shimadzu Europa GmbH, Duisburg, Germany) equipped with an SIL-20AC auto sampler and a CTO-20AC column oven. Detection was carried out using a Shimadzu SPD-M20A detector. The system was interfaced by Shimadzu LC solution software. Chromatography was carried out on a Phenomenex Luna C18(2) column (100 Å, 5 μm, 4.6 × 250 mm) (Phenomenex, Inc., Torrance, CA, USA). Columns were maintained at a temperature of 40 °C. Eluents were (A) 0.5% aqueous formic acid and (B) 0.5% formic acid in MeCN/water (6:4), and the flow rate was 1 mL min⁻¹. A 20 μL sample was injected into the high-performance liquid chromatography (HPLC). Following is the elution program used: 100% A to 60% A in 40 min, 60% A to 50% A in 10 min, 50% A to 30% A in 10 min, and then isocratic elution for another 10 min. The column was washed with 100% MeCN and re-equilibrated with 100% eluent A before the next injection. Quantification was performed with calibration curves (0–50 μg mL⁻¹) constructed with kaempferol 3-O-glucoside (R² = 0.9999), rutin (R² = 0.9990), and pelargonin (R² = 0.9999).

Ten capsules were weighted, and the average capsule mass was calculated. A quantity of powder equivalent to one capsule containing 1.5 mg of cytisine was transferred into a 50 mL volumetric flask. To this flask, 50 mL of methanol were added, and the solution was shaken for 10 min. From this solution, aliquots of appropriate volume were transferred to 10 mL volumetric flasks and diluted with methanol to obtain a final concentration of 20 µg/mL of cytisine. Twenty microliters of solution were injected into the column. The measurements were performed in triplicate.
Standard curve was prepared using seven concentration: 0.1, 0.5, 1, 10, 20, 30, 40 and 50 μg/mL in triplicate LOD and LOQ were calculated according to the formula: LOD = 3.3 (SD/S), and LOQ = 10 (SD/S), where SD is the standard deviation of response (peak area) and S is the slope of the calibration curve.
Linearity was determined by injections of above solutions in triplicate. The average peak areas were plotted against concentrations. The linearity was evaluated by calculating coefficient of correlation, slope and intercept.
The specificity was evaluated to ensure that there was no interference from the excipients present in the capsules. Peak purity was confirmed by comparison of investigated drug UV spectra (Shimadzu SPD-M20A detector (Shimadzu Corporation, Canby, OR, USA)) with the spectra of standard.

IR spectra were recorded as ATR-FTIR spectra using a Perkin-Elmer UART TWO FT-IR-spectrometer. The UV/VIS spectra were recorded in high-purity solvents (UVASOl^®) using the JASCO double-beam photometer (V-630). NMR spectra were recorded in MeOD using a Bruker NEO-500 instrument operating at 500 and 125 MHz for ¹H and ¹³C{¹H} NMR, respectively. Spectra referencing was accomplished using the CD₂HOD solvent peak for ¹H and the CD₃OD solvent peak for ¹³C NMR spectra (δ = 3.31 and 49.0 for ¹H and ¹³C{¹H} signals, respectively). Multiplicities in ¹H NMR spectra were described using the common descriptors s (singlet), d (doublet), t (triplet), or m (multiplet). High-resolution mass spectra were obtained by EI-TOF (70 eV) using Waters Micromass instruments. Reversed-phase semi-preparative HPLC was performed on Shimadzu LC-20AP pump equipped with DGU-20A5R degassing unit, a Shimadzu SPD-M20A detector, a Shimadzu SIL-20ACHT auto-sampler and a Phenomenex Gemini C₁₈ column (10 × 250 mm, 10 μm). Data were recorded and analyzed using LabSolutions software. For column chromatography, Silica gel 60 (0.063–0.2 mm, Macherey-Nagel) was used as solid matrix. TLC was carried out on precoated Silica gel 60 plates (0.20 mm). Compounds were visualized under UV light and further by spraying with H₂SO₄–EtOH (1:9, v/v). All solvents used were of analytical grade.