Guduchyadi medhya yoga-An HRLCMS-orbitrap data
Description
Background and rationale. Guduchyadi Yoga (GY) is a classical eight-drug Ayurvedic formulation traditionally indicated for cognition. Constituents relevant to cognitive impairment and stress include: Tinospora cordifolia (anti-stress; memory support), Achyranthes aspera (antidepressant-like effects), Embelia ribes/embelin (anticonvulsant/CNS-depressant), Convolvulus pluricaulis (memory/stress benefits), Acorus calamus (CNS activity), Terminalia chebula (in-vitro neuroprotection via ROS-scavenging phenolics/tannins), and Asparagus racemosus (neuroprotection; memory support). Classical Rasapañcaka and medhya (cognitive) indications, together with small clinical observations (e.g., MMSE and HAM-A/HAM-D improvements), motivate systematic chemical profiling and computational evaluation. Hydro-alcoholic extract (HAE). Solvent: ethanol:water (70:30, v/v; LC–MS grade). Weighed 25.00 g GY into a Type-I glass bottle; added 250 mL solvent (1:10 w/v). Extraction: magnetic stirring 300 rpm at 20–25 °C for 8 h (covered/amber-wrapped), then 16 h static maceration. Clarification: Whatman No. 42 filtration followed by 0.22 µm PTFE filtration. Solvent removal: rotary evaporation at 40 °C (reduced pressure) to near-dryness. The dried extract was transferred to sterile amber vials, yield (%) = (extract mass/25 g)×100, optionally N₂-flushed, and stored at −20 °C, protected from light. For LC–MS, aliquots were reconstituted/diluted and filtered per SOP. UHPLC–HRMS conditions. Thermo Scientific Vanquish UHPLC (autosampler 4 °C; column oven 40 °C) with RP-C18 column (2.1 × 100 mm, 1.9–2.6 µm). Injection: 5 µL. Total run: 30.0 min at 0.300 mL min⁻¹. Mobile phases: A = water (0.1% formic acid); D = programmed organic channel (B,C = 0%). Gradient (A complements D): 0–2 min, 5% D; 2–15 min, 5→95% D; 15–20 min, 95% D; 21 min, return to 5% D; 21–30 min, re-equilibrate. MS: Q Exactive™ Plus (HESI), Full MS with data-dependent MS² (Top-5), acquired in separate positive/negative runs. Full MS: 70,000 (m/z 200), AGC 1e6, max IT 100 ms; dd-MS²: 17,500, AGC 1e5, max IT 100 ms, isolation 1.5 m/z, stepped NCE 30, dynamic exclusion 10 s. Tune files: Cal_positive_29082024.mstune; Cal_negative_29082024.mstune. Divert Valve A: Start in 1–2 (True). Data acquisition and processing. Data were acquired in Xcalibur/SII (profile). Sequence: blank → system suitability → three QCs → study samples (QC every 8–10) → final blanks. Autosampler: puncture offset 0 µm; tray 4 °C. Raw files were processed in ⟨software/version⟩ with centroiding; mass error ≤ 5 ppm; minimum peak width 6–8 s; RT alignment 0.2–0.3 min; gap-filling enabled. MS² matches were queried against ⟨mzCloud/MassBank/in-house⟩ at predefined score thresholds. Features with QC RSD > 20% were flagged/excluded unless justified. Putative annotations were reported at MSI Level 2; Level 1 required |Δm| ≤ 5 ppm, RT within ±0.20 min of an authentic standard, and concordant MS² fragments.
Files
Steps to reproduce
Materials GY powder (IMPCL, Lot 19-ACH-LDA-539). LC–MS-grade ethanol, water; Whatman No. 42; 0.22 µm PTFE filters. Type-I glass bottles, magnetic stirrer, rotary evaporator (40 °C), amber vials, −20 °C freezer. Thermo Vanquish UHPLC + Q Exactive™ Plus (HESI); RP-C18 (2.1×100 mm, 1.9–2.6 µm). Tune files: Cal_positive_29082024.mstune; Cal_negative_29082024.mstune. Pre-analytical Sieve through #80, homogenize, aliquot; store light-protected, airtight with desiccant. Rinse tools with LC–MS solvent between lots. Extraction (HAE) Weigh 25.00 g GY; add 250 mL EtOH:H₂O (70:30) → 1:10 w/v. Stir 8 h at 300 rpm, 20–25 °C (covered/amber). Stand 16 h (RT). Filter: Whatman 42 → 0.22 µm PTFE. Evaporate (40 °C, reduced pressure) to near-dry. Record mass; Yield (%) = extract mass/25 g × 100. Store in amber vials at −20 °C (optional N₂). LC–MS Reconstitution Reconstitute per SOP; 0.22 µm PTFE filter to vials. UHPLC Autosampler 4 °C; oven 40 °C; flow 0.300 mL min⁻¹; inject 5 µL. Mobile phases: A = water + 0.1% FA; D = organic channel (B,C = 0). Gradient (A complements D): 0–2 min 5% D; 2–15 min 5→95% D; 15–20 min 95% D; 21 min 5% D; 21–30 min re-eq. HRMS (Q Exactive™ Plus, HESI) Load tune files; Divert Valve A “Start in 1–2 (True)”. Run positive and negative separately. Full MS: 70k @ m/z 200, AGC 1e6, max IT 100 ms. dd-MS² (Top-5): 17.5k, AGC 1e5, max IT 100 ms, isolation 1.5 m/z, stepped NCE 30, dynamic exclusion 10 s. Run sequence & QC Blank → system suitability → 3× QC → samples (QC every 8–10) → blanks. Autosampler: puncture offset 0 µm, wash both, wash 2 s, draw/dispense 5 µL s⁻¹. Processing Acquire in Xcalibur/SII (profile). Process in ⟨software/version⟩ with centroiding; mass error ≤5 ppm; peak width 6–8 s; RT align 0.2–0.3 min; gap-filling ON. Library search: mzCloud/MassBank/in-house (pre-set scores). QC filter: exclude RSD >20% unless justified. Annotations: MSI L2 (putative). MSI L1 requires |Δm| ≤5 ppm, RT ±0.20 min vs standard, concordant MS². Annotation levels: MSI Level 2 (putative): accurate mass + MS² library match. MSI Level 1 (confirmed): |Δm| ≤ 5 ppm, RT ± 0.20 min vs authentic standard, concordant MS².