The Bacopa monnieri (L.) Wettst. (BM) used in the present study was that used in previous studies [8 (link),15 (link)]. It was collected in Ho Chi Minh city, Vietnam and identified by Dr. Pham Thanh Huyen, Department of Resource Medicinal Material, National Institute of Medicinal Material, Hanoi, Vietnam (NIMM) and submitted as voucher specimen 9967 in NIMM. BME was prepared as previously described [8 (link),15 (link)]. Briefly, BM aerial parts were dried at 50 °C, cut into small pieces, crushed and extracted with 50% ethanol (1:8 w/v) at 85 °C for 2 h, twice filtered through filter paper and concentrated under reduced pressure at 50 °C. After further extraction three times with n-butanol, the extract was subjected to chemical analysis using a high-performance liquid chromatographic system (HPLC; Shimazu, Japan) [8 (link)] and determined to contain 21.8% bacoside A and 11.0% bacopaside I. Bacopaside I, a triterpene saponin of BM, was isolated by Dr. Nguyen Van Tai, Department of Phytochemistry, National Institute of Medicinal Materials, Vietnam, as previously described [8 (link)] and determined to be ≥ 95% by HPLC-MS analysis. (Mass spectrometry data is available at http://wakandb.u-toyama.ac.jp/wiki/ LCMS: Bacopa_INM-484 ).
High performance liquid chromatography (hplc)
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HPLC (High-Performance Liquid Chromatography) is an analytical technique used to separate, identify, and quantify components in a liquid sample. It utilizes a high-pressure pump to pass the sample mixture through a column packed with a stationary phase, enabling the separation of the components based on their interactions with the stationary and mobile phases.
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Spd m20a, by Shimadzu (15 mentions)
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Lc 20ad, by Shimadzu (8 mentions)
Class vp 6, by Shimadzu (8 mentions)
Spd 20a, by Shimadzu (7 mentions)
Hpx 87h column, by Bio-Rad (7 mentions)
C18 column, by Shimadzu (7 mentions)
Lc 20ad pump, by Shimadzu (7 mentions)
Centrivap concentrator, by Labconco (6 mentions)
Cto 20ac, by Shimadzu (5 mentions)
Sil 20ac autosampler, by Shimadzu (5 mentions)
Millipore filter, by Merck Group (5 mentions)
C18 column, by Phenomenex (5 mentions)
Sil 20ac, by Shimadzu (5 mentions)
Optilab t rex, by Wyatt Technology (4 mentions)
Sil 20ac ht, by Shimadzu (4 mentions)
Refractive index detector, by Shimadzu (4 mentions)
Rid 10a refractive index detector, by Shimadzu (4 mentions)
Sil 20a, by Shimadzu (4 mentions)
Dgu 20a3, by Shimadzu (4 mentions)
813 protocols using high performance liquid chromatography (hplc)
1
Bacopa monnieri Extract Preparation
2
Quantitative Analysis of Fermentation Metabolites
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Cell density was measured by using microplate reader at 600 nm with 200 μL fermentation broth added in a cuvette. The metabolites 3-HP, lactic acid and acetic acid were determined by high performance liquid chromatography (HPLC) system (Shimazu, Kyoto, Japan) equipped with a C18 column and a SPD-20 A UV detector at 210 nm. The column temperature was 25 °C, and mobile phase was 0.05% phosphoric acid at a flow rate of 0.8 mL min−1. 1,3-PD and 2,3-BD were quantitatively analyzed by HPLC (Shimadzu, Japan) equipped with a column of Aminex HPX-87 H Ion Exclusion particles (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) using a differential refractive index detector. The column was maintained at 65 °C and mobile phase was 5 mM sulfuric acid (in Milli-Q water) at 0.6 mL min−1. Residual glycerol concentration was measured every 3 h by a titration method with NaIO4 (for control of glycerol). All samples were filtered through 0.22-μm membrane filter.
3
Quantitative HPLC-MS/MS Analysis Protocol
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For all measurements, a Shimadzu high performance liquid chromatography (HPLC) apparatus, including binary pumps, degasser, autosampler, column oven and control unit (LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Duisburg, Germany) was used. The HPLC was coupled to an API4000 triple quadrupole mass spectrometer (MS) from Sciex (Darmstadt, Germany). MS ion source parameters were set as follows: ionization voltage, 2.500 V; nebulizer gas, 50 psi; heating gas, 50 psi, curtain gas, 30 psi, temperature, 600 °C; collision gas level, 7. For data acquisition and processing, Analyst (Version 1.6.2, Sciex, Framingham, MA, USA) and MultiQuant software (Version 3.0.1, Sciex, Framingham, MA, USA) were used. Figures were drawn using OriginPro software (Version 2019, OriginLab, Northampton, MA, USA).
4
HPLC-MS/MS Analysis of Hydroxychloroquine
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Chromatographic conditions for the 1st dimension were as follows: HPLC (Shimadzu Corporation, Kyoto, Japan) equipped with ACE2/CMC column, ultrapure water as the mobile phase with flow rate at 0.2 mL/min, column was kept at 37 °C in a CTO-20A column oven, the detector wavelength for HCQ was set at 254 nm, injection volume was 5 μL.
The second dimension included an HPLC system coupled with an ion trap-time of flight mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with an electrospray ionization source. Chromatographic conditions for the 2nd dimension: Inertsil ODS-3 (150 × 4.6 mm, 3 μm, Shimadzu) was used as the analytical column for separation; 0.1% formic acid water solution (A)-acetonitrile (B) mixed mobile phase was used with gradient elution set as follows: 0–40 min, 5%–100% B; 40-50 min, 100% B; 50–50.01 min, 100%–5% B; 50.01–70 min, 5% B. Flow rate was 1 mL/min; column temperature was 30 °C; the detector wavelength was set at 254 nm. The optimal parameters for mass spectrometry were as follows: ESI source, nebulizer gas (N2, purity >99.999%) flow rate, 3 L/min; drying gas (N2, purity >99.999%) pressure, 109 kPa. Both curve desolvation line and heat block temperatures were 200 °C. Interface voltage was 4.5 kV; detector voltage, 1.57 kV; positive ionization scan mode with scan range at m/z 100–1000.
The second dimension included an HPLC system coupled with an ion trap-time of flight mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with an electrospray ionization source. Chromatographic conditions for the 2nd dimension: Inertsil ODS-3 (150 × 4.6 mm, 3 μm, Shimadzu) was used as the analytical column for separation; 0.1% formic acid water solution (A)-acetonitrile (B) mixed mobile phase was used with gradient elution set as follows: 0–40 min, 5%–100% B; 40-50 min, 100% B; 50–50.01 min, 100%–5% B; 50.01–70 min, 5% B. Flow rate was 1 mL/min; column temperature was 30 °C; the detector wavelength was set at 254 nm. The optimal parameters for mass spectrometry were as follows: ESI source, nebulizer gas (N2, purity >99.999%) flow rate, 3 L/min; drying gas (N2, purity >99.999%) pressure, 109 kPa. Both curve desolvation line and heat block temperatures were 200 °C. Interface voltage was 4.5 kV; detector voltage, 1.57 kV; positive ionization scan mode with scan range at m/z 100–1000.
5
Serum Biotin and Arginine Analysis
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The serum levels of biotin were analyzed using the high-performance liquid chromatography (HPLC-Shimadzu, Kyoto, Japan) method [26 (link), 27 (link)]. As C18-ODS-3 column (250 × 4.6 mm, 5 m), chromatography fractions containing reversed-phase column and biotin were used. The serum concentrations of arginine were determined using HPLC (Shimadzu, Japan) [28 (link)]. Serum samples were extracted 1:1 in 35% (wt/vol) sulfosalicylic acid dihydrate. After mixing and centrifugation, the supernatant was mixed 1:1 with a lithium-D buffer prior to analysis. The amino acid standard was obtained from Sigma-Aldrich Chemicals (St. Louis, USA).
6
HPLC Analysis of Fermentation Broth
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The products were analyzed using HPLC (Shimadzu Corporation, Kyoto, Japan). After fermentation in shake flasks, 500 μL of the fermentation broth was mixed with 500 μL of methanol and the mixture was centrifuged at 13,000 rpm for 3 min. The supernatant was filtered using a 0.22-μm filter membrane. The HPLC (Shimadzu Corporation) was equipped with a reverse-phase C18 column (4.6 × 150 mm, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 40 °C. The detection wavelength was set at 290 nm. A flow rate of 1.0 mL/min was used with a gradient elution method: (1) 0.1% (v/v) trifluoroacetic acid in water and (2) 0.1% (v/v) trifluoroacetic acid in acetonitrile, 0−10 min, 10−40% B; 10−20 min, 40−60% B; 20−22 min, 60%−10% B; and 22−25 min, 10% B.
7
In Vivo Pharmacokinetic Evaluation of Diltiazem Formulations
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In vivo study was performed on healthy albino rabbits (2.5 to 3 kg). Animals were kept in cages made of polypropylene having paddy husk bedding material under sterile conditions. Standard food pellets and water ad libitum were given as feed to them. CPCSEA rules were followed for handling and taking care of animals. Prior approval from the Institutional Animal Ethics Committee (IAEC) proposal No. 028, JSS College of Pharmacy, Mysuru, India was received for the animal study. For in vivo study, optimized formulation (product A) and marketed sustained-release tablets (product B), both containing 90 mg of DTZ, were orally administered. Blood samples (500 μL) were withdrawn at different time intervals and plasma concentrations of DTZ quantified by HPLC analysis as described previously.29 (link) Briefly, chromatographic separation was conducted using HPLC (Shimadzu, Kyoto, Japan) equipped with a C18 column (250 mm 3 4.6 mm ID; particle size 5 mm). The mobile phase consisted of a mixture of acetonitrile and water (containing 0.35% trimethylamine, 37:63, v/v). The column temperature was kept at 40°C, and the flow rate was set at 0.95 mL/min. The detection was conducted using a UV-VIS detector at a wavelength of 240 nm. Pharmacokinetic parameters (Cmax, tmax, Kel, t1/2 and AUC0-24h) were estimated from the individual plasma concentrations versus time profiles both the products.
8
Affinity Ultrafiltration for COX-2 Binding
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To evaluate the specificity of the established approach, a solution of celecoxib (specific COX-2 inhibitor) was prepared as the positive control, in contrast to adenosine which was used as the negative control, both at a concentration of 20 μM. The control samples were prepared with inactivated COX-2. Besides, comparison of the peak area of the ultrafiltrate was performed between the active and inactivated enzyme groups to determine the specific binding between ligands and COX-2.
HPLC condition: HPLC (Shimadzu Corporation, Japan) furnished with a binary pump, vacuum degasser, diode array detector (DAD), and automatic sampler, was employed in combination with the usage of an Ultimatetm XB-C18 (250 × 4.6 mm, 5 μm) to verify the affinity ultrafiltration approach. With reference to the modified approach recorded in the previous study (Jadhav and Shingare, 2005 (link)), the conditions for HPLC were set as follows: mobile phases, water (containing 0.1% formic acid; A) and acetonitrile (B); elution program, 0–30 min, 5%–95% B; column temperature, 35°C; sample injection volume, 20 μL; detection wavelength, 254 nm; and the flow rate, 1.0 mL/min.
HPLC condition: HPLC (Shimadzu Corporation, Japan) furnished with a binary pump, vacuum degasser, diode array detector (DAD), and automatic sampler, was employed in combination with the usage of an Ultimatetm XB-C18 (250 × 4.6 mm, 5 μm) to verify the affinity ultrafiltration approach. With reference to the modified approach recorded in the previous study (Jadhav and Shingare, 2005 (link)), the conditions for HPLC were set as follows: mobile phases, water (containing 0.1% formic acid; A) and acetonitrile (B); elution program, 0–30 min, 5%–95% B; column temperature, 35°C; sample injection volume, 20 μL; detection wavelength, 254 nm; and the flow rate, 1.0 mL/min.
9
Synthesis and Characterization of WCopW Analogs
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All l -form WCopW analogs (except YCopW, SCopW, HCopW, WCopW, WhCopW, WtCopW, and WCopW1, 2) were prepared using solid-phase peptide synthesis and validated with HPLC (Shimadzu, Kyoto, Japan) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Shimadzu, Kyoto, Japan). Other l -form and d -form WCopW analogs as well as cecropin P1, melittin, and protegrin-1 (linear) were purchased from Anygen Inc. (Gwangju, Republic of Korea). Protegrin-1 was refolded with 2 mM glutathione (Bioworld, Tokyo, Japan) and 0.2 mM glutathione disulfide (Bioworld, Tokyo, Japan) for 48 h. The purity and molecular weight of each peptide were measured using HPLC (Shimadzu, Kyoto, Japan) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Shimadzu, Kyoto, Japan). All WCopW analogs and cecropin P1, melittin and protegrin-1 were prepared as TFA salts except the WCopW43 used in the in vivo test, which was an acetate salt.
10
Characterizing Protein Behavior via FSEC
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Fluorescence size exclusion chromatography (FSEC) is an efficient method to efficiently characterize protein behavior without purification (67 (link)). FSEC is typically performed on proteins tagged with GFP. We demonstrated using FSEC to characterize PCNA without tagging it with GFP. Cell pellets were harvested from genome-edited cells and resuspended in 180 μL ice-cold TBS buffer and then combined with 20 μL of 100 mM DDM and 20 mM CHS to lyse cells. The mixtures were rotated at 4 °C for 45 min followed by spin down at 21,000g for 20 mins at 4 °C. The supernatant was incubated with 1 μL of ALFANB-eGFP, 0.1 mg/mL stock, for 2 h at 4 °C, followed by injecting onto a Superdex 200 increase 10/300 GL column (Cytiva), pre-equilibrated with TBS buffer, at a flow rate of 0.5 mL/min. The GFP signal was collected by HPLC (Shimazu) equipped with an RF-20A fluorescence detector (excitation 488 nm and emission 508 nm, Shimazu).
To visualize cellular location of PCNA-GFP-11, 30 to 50 ng of Superfolder GFP1-10 in pEG plasmid was transfected into the PCNA knock-in cells without mNeonGreen. The cells were grown in 37 °C, 8% CO2 for 20 h before imaging. Fluorescence imaging was performed as described above.
To visualize cellular location of PCNA-GFP-11, 30 to 50 ng of Superfolder GFP1-10 in pEG plasmid was transfected into the PCNA knock-in cells without mNeonGreen. The cells were grown in 37 °C, 8% CO2 for 20 h before imaging. Fluorescence imaging was performed as described above.
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