Analysis was carried out on an Agilent 1100 series HPLC system (Agilent Corporation, Germany) consisting of a G1315B Diode Array Detector (DAD), a G1311A low-pressure quatpump, a G1379A online degasser, a G1316A thermostat column compartment and a G1313A automatic sample injector. The YF-20 Donghua automatic decocting machine, YBS250E liquid packing machine and YBS liquid packing machine were all obtained from Beijing Donghuayuan Medical Equipment Co., Ltd.
G1315b diode array detector
Manufactured by Agilent Technologies
Sourced in Germany, United States
The G1315B diode array detector is a laboratory instrument manufactured by Agilent Technologies. It is designed to detect and measure the absorption of light by samples in various analytical applications. The core function of the G1315B is to provide high-sensitivity, full-spectrum detection capabilities for liquid chromatography (LC) systems.
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Lab products found in correlation
G1312a binary pump, by Agilent Technologies (5 mentions)
G1311a quaternary pump, by Agilent Technologies (5 mentions)
G1316a thermostat, by Agilent Technologies (4 mentions)
G1379b degasser, by Agilent Technologies (4 mentions)
Agilent 1100 series hplc system, by Agilent Technologies (2 mentions)
G1316a thermostat column compartment, by Agilent Technologies (2 mentions)
G1311a pump, by Agilent Technologies (2 mentions)
G1322a degasser, by Agilent Technologies (2 mentions)
G1329a autosampler, by Agilent Technologies (2 mentions)
1200 hplc system, by Agilent Technologies (2 mentions)
Esquire 6000, by Bruker (2 mentions)
Luna omega polar c18 analytical column, by Phenomenex (2 mentions)
Polar c18 security guard cartridge, by Phenomenex (2 mentions)
Agilent 1100 series, by Agilent Technologies (2 mentions)
G1392a autosampler, by Agilent Technologies (2 mentions)
Agilent chemstation software rev a 10.02, by Agilent Technologies (2 mentions)
G1379a degasser, by Agilent Technologies (2 mentions)
Zorbax eclipse xdb c18 80, by Agilent Technologies (2 mentions)
Hip sampler g1367a, by Agilent Technologies (2 mentions)
Synergy fusion c18, by Phenomenex (1 mentions)
21 protocols using g1315b diode array detector
1
HPLC-based Analytical Protocol
2
HPLC-DAD-MS Analysis of Triacylglycerols
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Prior to HPLC analysis, samples (50 mg mL À1 ) were prepared in acetonitrile, isopropanol and n-hexane (2:2:1, v/v/v) and filtered through a 0.2 lm PTFE-filter (Ziemer Chromatographie, Langerwehe, Germany). TAGs were analysed using an Agilent 1100 Series HPLC system equipped with a G1315B diode array detector (DAD) (all from Agilent Technologies, Waldbronn, Germany). Sample tray temperature was set to 30 °C. Injection volume was 10 µL. The column set used consisted of two C18 columns (both Kinetex TM , 250 9 4.6 mm i.d., 5 lm particle size; Phenomenex, Aschaffenburg, Germany) connected in series and operated at 20 °C. Acetonitrile (eluent A) and isopropanol (eluent B) represented the mobile phase. The gradient was programmed as follows: from 30 to 70% B in 60 min, from 70 to 30% B in 3 min and isocratic 30% B for 10 min. Total run time was 73 min at a flow rate of 0.9 mL min À1 . TAGs were monitored at 210 nm.
TAG identification was performed by coupling the above HPLC system to a 3000 + ion trap mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionisation (ESI) source. ESI-MS n parameters were detailed previously (Lieb et al., 2019) .
Relative TAG composition was expressed as percentage of the total peak area.
TAG identification was performed by coupling the above HPLC system to a 3000 + ion trap mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionisation (ESI) source. ESI-MS n parameters were detailed previously (Lieb et al., 2019) .
Relative TAG composition was expressed as percentage of the total peak area.
3
NMR and Mass Spectrometry Characterization
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A Bruker DRX-600 NMR spectrometer, operating at 599.19 MHz for 1H and 150.858 MHz for 13C, using the TopSpin 3.2 software package, was used for the NMR experiments in CD3OD. The chemical shifts are expressed in δ (parts per million), referring to the solvent peaks δH 3.31 and δC 49.05 for CD3OD, with coupling constants, J, in Hertz. Conventional pulse sequences have been used for 1H-1H DQF-COSY, 1H-13C HSQC, and HMBC experiments [50 (link)]. ESI-MS experiments [51 (link)] were conducted using a Finnigan LC-Q Deca spectrometer (Thermoquest, San Jose, CA, USA), which was equipped with Xcalibur 3.1 software (Thermoquest, San Jose, CA, USA). Molecular exclusion chromatography was performed on Sephadex LH-20. HPLC separations were performed with a Waters 515 series pump system, equipped with a Waters R401 refractive index detector and a Rheodyne injector (100 μL loop), using Synergy Fusion C18 (250 × 10 mm i.d., 4 μm, Phenomenex, Torrance, CA, USA), at a flow rate of 2 mL/min. Thin-layer chromatography (TLC) was performed with silica gel plates 60F-254 (Delchimica, Naples, Italy) and cerium sulphate spray reagent, and a UV lamp (254 and 366 nm) was used to visualize the spots. Quantitative analysis of the extract was performed with Agilent 1100 HPLC with G1312A Binary Pump, G1315B Diode Array Detector (DAD), and G1328B Injector (Agilent Technologies, Santa Clara, CA, ISA).
4
HPLC Analysis of Medicinal Plant Extracts
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The whole dried roots were cut into small pieces and ground into fine powder. Dried samples (0.20 g) were extracted with 70% EtOH (50 mL) for 30 min under ultrasonication (250 W, 40 °C). Then, the solution was cooled to room temperature, adjusted with 70% EtOH to original volume, mixed, and passed through a membrane filter having 0.45 μm porosity. A volume of 10 μL was used for HPLC analysis.
HPLC analysis was performed with Agilent series 1200 equipment consisting of a G1311A pump, G1315B-Diode array detector, and G1316A column compartment. Samples were separated on an Agilent TC-C18 column (4.6 mm i.d. × 250 mm, 5 μm particle). The mobile phase was a gradient prepared from CH3CN (component A) and water containing 0.05% H3PO4 (component B), and the conditions used for gradient elution were: 0–8 min, 20% A; 8–30 min, 20–38% A; 30–42 min, 38–50% A; 42–50 min, 50–95% A. The flow rate was 1.0 mL/min. The wavelength conversion based on the maximum absorption peak was changed as follows: 0–15 min, 237 nm; 15–23 min, 365 nm; 23–30 min, 237 nm; 30–37 min, 370 nm; 37–45 min, 237 nm. The injection volume for all samples was 10 μL. The column was maintained at 30 °C during the analysis.
HPLC chromatograms of the standards of five main active components can be seen inFigure 5 A, and the samples are shown in Figure 5 B.
HPLC analysis was performed with Agilent series 1200 equipment consisting of a G1311A pump, G1315B-Diode array detector, and G1316A column compartment. Samples were separated on an Agilent TC-C18 column (4.6 mm i.d. × 250 mm, 5 μm particle). The mobile phase was a gradient prepared from CH3CN (component A) and water containing 0.05% H3PO4 (component B), and the conditions used for gradient elution were: 0–8 min, 20% A; 8–30 min, 20–38% A; 30–42 min, 38–50% A; 42–50 min, 50–95% A. The flow rate was 1.0 mL/min. The wavelength conversion based on the maximum absorption peak was changed as follows: 0–15 min, 237 nm; 15–23 min, 365 nm; 23–30 min, 237 nm; 30–37 min, 370 nm; 37–45 min, 237 nm. The injection volume for all samples was 10 μL. The column was maintained at 30 °C during the analysis.
HPLC chromatograms of the standards of five main active components can be seen in
5
Levonorgestrel Tablet Analysis via HPLC-DAD
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HPLC-DAD was used to determine the limit of dextronorgestrel, related substances, to perform the content uniformity assay, and analyze standards and samples from dissolution tests, following the International Pharmacopeia specifications for levonorgestrel tablets [20] . HPLC-DAD analysis was conducted with an Agilent HP1200 consisting of a G1329A autosampler, a G1322A degasser, a G1311A quaternary pump, a G1316A column thermostat, and a G1315B diode array detector (Agilent Technologies, Santa Clara, CA, USA). Data was acquired using ChemStation software (Agilent Technologies). Specific details for each assay are provided below.
6
Chromatographic Analysis of API Samples
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The chromatographic conditions used for the analysis were based on compendial methods for the API [8 ]. HPLC-DAD coupled with tandem mass spectrometry (MS/MS) was conducted with Agilent instrumentation using G1311A pump, G1329A autosampler, G1330A autosampler thermostat, G1316A column compartment, G1315B diode array detector, G6340 mass spectrometer modules (Agilent Technologies, Santa Clara, California, USA). Instrument software comprised Agilent ChemStation B.01.03 with 6300 Series Ion Trap LC/MS software Version 6.1. The following chromatographic conditions were used: 0.9 mL/min flow rate; Agilent column ZORBAX SB-C18 3.0 mm x 250 mm, 5 μm; 60 °C column temperature; 10 μL injection volume (MPA powder samples); 20 μL injection volume (finished product samples). The diode array detector (DAD) was set at 254 nm, where all spectra were stored from 190 nm to 400 nm. The mass spectrometer used the following parameters: capillary - 3500 V; Nebulizer 50.0 psi; Dry Gas 12.0 L/min; Dry Temp 350 °C; alternating polarity; Scan: 50–500 m/z; ESI Source.
7
HPLC and Mass Spectrometry Analysis of Steroids
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HPLC analyses for product separation were performed using an Agilent 1100 series system (G1311A Quaternary pump, G1379A Solvent degasser, G1315B Diode array detector, and G1313A Standard autosampler; Agilent Technologies, USA). This device was connected to a reversed-phase C18 GP column (4.6 × 250 mm, 5 μm; Mightysil; Kanto Chemical, Japan), and the analysis temperature was maintained at 40°C. The mobile phase was mixed with two solvents, water (A) and acetonitrile (B), at a rate of 1 ml·min−1. The HPLC system started with acetonitrile and water at a ratio of 15:85, increased to 50:50 for 8 min, and then to 90:10 for 18 min. The ratio was maintained for 19 min, reduced to 15:85 for 21 min, and finally, ran for 25 min. To detect the substrate and product, the UV detector was set to 242 or 245 nm. Mass analysis was performed using quadrupole time-of-flight/electrospray ionization mass spectrometry in the positive ion (+) mode using ultra-performance liquid chromatography (SYNAPT G2‐S/ACUITY; Waters Corp., USA). The products isolated from the steroids were analyzed using a 700 MHz NMR spectrometer (Korea Basic Science Institute, Korea). For 1H, 13C NMR, HMBC, HSQC, COSY, and ROESY, 7.3 and 15 mg progesterone and androstenedione products, respectively, were dissolved in 1 ml CDCl3.
8
HPLC Analysis of Gel Formulation MET
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MET content was determined after extraction of gel formulation samples in ethanol (99.9%) and analyzed by HPLC method using an Agilent Technologies 1200 HPLC system equipped with a G1312A binary pump, a G1316A thermostat, a G1379B degasser and a G1315B diode array detector (Agilent, Waldbronn, Germany) in the following conditions: Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 µm column (Agilent, Waldbronn, Germany); mobile phase: acetonitrile—0.01M phosphate buffer pH 4.7 (15:85, v/v); flow rate 1.0 mL/min; detection at 319 nm; retention time 3.0 min [22 (link)]; the standard calibration curve was linear over the range of 2.5–150 µg/mL (R2 = 0.999).
9
Quantification of Ketoconazole in Gel Formulations
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KTZ content was determined after extraction of the gel formulation samples using ethanol 99.9% and analysis by the HPLC method with an Agilent Technologies 1200 HPLC system equipped with a G1312A binary pump, a G1316A thermostat, a G1379B degasser, and a G1315B diode array detector (Agilent, Waldbronn, Germany) in the following conditions: a Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 µm column (Agilent, Waldbronn, Germany); mobile phase: an acetonitrile–methanol–phosphate buffer, pH 6.8 (40:35:25, v/v); a flow rate of 1.0 mL/min; detection at 231 nm; and a retention time of 4.0 min [61 (link)]. The standard calibration curve was linear over the range of 2.5–65 µg/mL (R2 = 0.998; y = 29.676x − 24.455).
10
HPLC Analysis of Pharmaceutical Compounds
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The HPLC system used in this study consisted of an Agilent G1322A degasser, G1311A quaternary pump, G1313A auto sampler, G1316A thermostat column compartment and G1315B diode array detector (Agilent Technologies, Palo Alto, CA, USA.). The software used to acquire and analyze the data was ChemStation® for LC 3D, Rev. A. 09.03 (Agilent Technologies). Analysis was performed with a Luna® Omega 5 µm PS C18 150 × 4.60 mm column (Phenomenex, Macclesfield, UK), equipped with a universal HPLC guard column packed with a SecurityGuard™ C18 cartridge. The mobile phase consisted of 20 mM sodium phosphate buffer at pH 3 ± 0.2 and acetonitrile (60:40, v/v) and the flow rate was 1 mL/min. The column temperature and injection volume were set to 30 °C and 10 μL, respectively. Ultraviolet (UV) detection at 213 nm was employed. The HPLC method was validated in terms of specificity, linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) according to the International Conference of Harmonization guidelines [16 ]. Calibration curves in the concentration range of 0.5–100 μg/mL were constructed (r2 ≥ 0.99) and the LOD and LOQ values were 0.23 and 0.69 µg/mL, respectively.
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