Simultaneous quantification of terpenelactones and flavonol aglycones in hydrolyzed ginkgo biloba extract by liquid chromatography with inline ultraviolet and evaporative light scattering detection

We report here a liquid chromatography (LC) method with inline ultraviolet/evaporative light scattering (UV/ELS) detection for the simultaneous quantification of the terpenelactones and flavonol aglycones in a single sample of hydrolyzed Ginkgo biloba extract (GBE). The sample is hydrolyzed by a rapid and convenient oven heating method for 1 h at 90 degrees C with 10% hydrochloric acid. The 1 h hydrolysis was found to be equivalent to the 2.25 h reflux treatment for dry powder extract, where total flavonol glycosides were 28.4 and 28.1%, respectively. Acceptable precision was achieved for total terpenelactones [relative standard deviation (RSD) = 4.8%] by ELS detection, and total flavonol aglycones (RSD = 2.3%) by UV detection. The analytical range was 1.5 to 7.3% (w/w) for the individual terpenelactones (ELS) and 2.5 to 15.0% (w/w) for the individual glycosides (UV) calculated from the aglycones quercetin, kaempferol, and isorhamnetin. This improved method allows for the first time high throughput sample preparation coupled with the quantification of the predominant compounds generally used for quality control of GBE in a single assay.     

Characterisation of proanthocyanidin aglycones and glycosides from rose hips by high-performance liquid chromatography-mass spectrometry, and their rapid quantification together with vitamin C

Fifteen individual proanthocyanidin aglycones and 19 glycosides, together with a complex mixture of chromatographically non-separated tetra- to octameric proanthocyanidin glycosides were detected--the non-separated glycosides being novel natural products--and characterised from dog rose hips using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS). Along with these phenolics, a 50% aqueous ethanol extract of rose hips was found to contain high levels of Vitamin C. A simple and rapid HPLC method assisted by diode array detection for the estimation of the total concentration of proanthocyanidin aglycones and glycosides, as well as Vitamin C, in rose hip extracts was developed.     

Qualitative and quantitative analysis of phenolic acid glycosides in Ginkgo biloba L. leaf,G. biloba leaf extract and its injection

Ginkgo biloba L. leaf (GBL) is one of the most commonly used medicinal plants in the world. Phenolic acids with biological activities have a relatively high content in G. biloba leaf extracts (GBE); therefore they are of great significance for the quality control of GBL, GBE and its preparations. However, there have been few studies focused on their analysis. In this work, 12 phenolic acids, including 11 phenolic acid glycosides, were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–Q-TOF/MS). Then, a method combining enzymolysis with HPLC was established for quantification of phenolic acid glycosides. It was found that the aglycones of phenolic acid glycosides mainly comprised five phenolic acids: 2,4,6-trihydroxybenzoic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid. The quantitative method was validated, and the correlation coefficient (0.9993–0.9999), recovery (≥88.4%), repeatability (≤0.8%), and inter-day precision (≤5.5%) were satisfactory. Finally, the contents of glycosides of five phenolic acids in GBL, GBE and GBE injection from different sources were determined by the developed method. The method was accurate, repeatable and practicable, which could be helpful for the quantification of phenolic acid glycosides in other products containing GBL or GBE.     

Evaluation of a method to determine flavonol aglycones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography: collaborative study

An interlaboratory study was conducted for evaluation of a method to determine the flavonol aglycones quercetin, kaempferol, and isorhamnetin in Ginkgo biloba products. The method calculates total glycosides based on these aglycones formed after acid hydrolysis. Twelve matrixes were chosen for study by 12 collaborating laboratories in 2 countries. Test materials included crude leaf material, standardized dry powder extract, single and multiple entity finished products, ethanol and glycerol tinctures, and National Institute of Standards and Technology (NIST) standard reference materials (SRMs). Results from 11 laboratories were used for the final calculations. Eight of the 12 matrixes evaluated produced acceptable results for total flavonol glycosides, with HorRat scores ranging from 1.31 to 2.05; repeatability relative standard deviations (RSDr) from 1.46 to 4.14; and reproducibility relative standard deviations (RSDR) from 4.67 to 9.69. These 8 matrixes consisted primarily of simple dosage forms (e.g., dry powder extracts, crude leaf samples, liquid extracts, and SRMs) and a single tablet product (Ginkgo Awareness). Four additional matrixes, consisting of 3 tablets and 1 soft gel product (Ginkgold, Ginkoba, Ginkogen, and Ginkgo Phytosome, respectively), showed greater total flavonol glycoside HorRat scores in comparison, ranging from 2.39 to 5.13, with RSDr values from 2.83 to 8.16, and RSDR values from 8.53 to 20.4. Based on the results presented here, the method is recommended for Official First Action for determination of total flavonol glycosides calculated from quercetin, kaempferol, and isorhamnetin in dry powder extracts, crude leaf material, liquid extracts, and a select finished product, Ginkgo Awareness.     

Simultaneous estimation of pantoprazole and domperidone in pure powder and a pharmaceutical formulation by high-perfomance liquid chromatography and high-performance thin-layer chromatography methods

This paper describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous estimation of pantoprazole (PANT) and domperidone (DOM) in pure powder and capsule formulations. The HPLC separation was achieved on a Phenomenex C18 column (250 mm id, 4.6 mm, 5 pm) using 0.01 M, 6.5 pH ammonium acetate buffer-methanol-acetonitrile (30 + 40 + 30, v/v/v, pH 7.20) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol (60 + 40, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 287 nm over the concentration range 400-4000 and 300-3000 ng/mL with mean recovery of 99.35+/-0.80 and 99.08+/-0.57% for PANT and DOM, respectively (HPLC method). Quantification was achieved with UV detection at 287 nm over the concentration range 80-240 and 60-180 ng/spot with mean recovery of 98.40+/-0.67 and 98.75+/-0.71% for PANT and DOM, respectively (HPTLC method). These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of PANT and DOM in pure powder and capsule formulations.     

Assay of tinidazole in human serum by high-performance thin-layer chromatography--comparison with high-performance liquid chromatography

Determination of tinidazole in human serum by high-performance thin-layer chromatography (HPTLC) is presented. It includes use of 10 x 10 cm plates coated with silica gel 60 and chloroform-acetonitrile-acetic acid (60 + 40 + 2) as mobile phase. Quantitation was performed by densitometry at 320 nm. The linearity (1-10 ng), precision (6%), reproducibility (5%), recovery (96%), and detection limit (1 mg/L) of tinidazole determination by HPTLC were comparable with corresponding method parameters by reversed-phase HPLC. A satisfactory correlation was found between the 2 analytical methods. The procedure was used to quantitate tinidazole in patient sera.     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

Rapid quantification of iridoid glycosides analogues in the formulated Chinese medicine Longdan Xiegan Decoction using high-performance liquid chromatography coupled with mass spectrometry [corrected

Longdan Xiegan Decoction (LXD) is a formulated preparation composed of 10 ingredient herbs, with iridoids as the main bioactive components. In this study, a rapid, simple and reliable method of simultaneous determination of four iridoid glycosides in LXD using high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (MS) was first developed and validated. The four iridoid glycosides references were isolated from LXD extract and purified using a preparative HPLC chromatography. The sample preparation for quantification comprised of a simple ultrasonic extraction and the satisfactory chromatographic separation of the four structurally similar iridoid glycosides was effected in less than three minutes on a CAPCELL PAK C(18) MGII column (3 microm, 100 mm x 2.0 mm), using an elution system of 10% methanol and their concentrations in different batches of LXD and ingredient herbs were simultaneously determined by HPLC-MS/MS using a multiple reaction monitoring (MRM) mode. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the four iridoid glycosides were 35, 20, 37 and 33 ng/mL, respectively.     

Rapid microwave-assisted hydrolysis for determination of ginkgo flavonol glycosides in extracts of Ginkgo biloba leaves

A rapid microwave-assisted hydrolysis (MAH) method is presented for the sample pretreatment of the determination of ginkgo flavonol glycosides in extracts of Ginkgo biloba L. (EGb). By this method, flavonol glycosides can be completely hydrolyzed within 2 min. After investigating the effects of solvents, acidity, microwave power, and microwave radiation time on hydrolysis, the optimal hydrolysis conditions are as follows: 300 W of microwave power, 2 min of hydrolysis time, 5.7% of hydrochloric acid in the hydrolysis solution, and n-propanol as the hydrolysis solvent. After MAH of the samples, three flavonol aglycones, such as quercetin, kaempferol, and isorhamnetin are analyzed by high-performance liquid chromatography. Compared with conventional reflux hydrolysis, this method owns offers several advantages: it saves time, costs less, and is environmentally friendly.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Determination of flavonol aglycones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography: single laboratory validation

A single laboratory validation (SLV) was completed for a method to determine the flavonol aglycones quercetin, kaempferol, and isorhamnetin in Ginkgo biloba products. The method calculates total glycosides based on these aglycones formed following acid hydrolysis. Nine matrixes were chosen for the study, including crude leaf material, standardized dry powder extract, single and multiple entity finished products, and ethanol and glycerol tinctures. For the 9 matrixes evaluated as part of this SLV, the method appeared to be selective and specific, with no observed interferences. The simplified 60 min oven heating hydrolysis procedure was effective for each of the matrixes studied, with no apparent or consistent differences between 60, 75, and 90 min at 90 degrees C. A Youden ruggedness trial testing 7 factors with the potential to affect quantitative results showed that 2 factors (volume hydrolyzed and test sample extraction/hydrolysis weight) were the most important parameters for control during sample preparation. The method performed well in terms of precision, with 4 matrixes tested in triplicate over a 3-day period showing an overall repeatability (relative standard deviation, RSD) of 2.3%. Analysis of variance testing at alpha = 0.05 showed no significant differences among the within- or between-group sources of variation, although comparisons of within-day (Sw), between-day (Sb), and total (St) precision showed that a majority of the standard deviation came from within-day determinations for all matrixes. Accuracy testing at 2 levels (approximately 30 and 90% of the determined concentrations in standardized dry powder extract) from 2 complex negative control matrixes showed an overall 96% recovery and RSD of 1.0% for the high spike, and 94% recovery and RSD of 2.5% for the low spike. HorRat scores were within the limits for performance acceptability, ranging from 0.4 to 1.3. Based on the performance results presented herein, it is recommended that this method progress to the collaborative laboratory trial.     

Analytical methodology for the determination of aflatoxins in peanut butter: comparison of high-performance thin-layer chromatographic, enzyme-linked immunosorbent assay and high-performance liquid chromatographic methods

High-performance thin-layer chromatography (HPTLC) was applied to the separation and quantification of aflatoxin in 300 jars of "crunchy" peanut butter. A critical evaluation of the proposed HPTLC method has been carried out by statistical comparisons with a commercially available enzyme-linked immunosorbent assay (ELISA) kit and a high-performance liquid chromatographic (HPLC) method. The statistical tests indicated that whilst the distributions of the data sets obtained with each method were similar, the HPLC method was found to be biased. Over-all results indicated that the HPTLC method gave more consistent data, relatively lower standard deviations and lower coefficients of variation. The ELISA kit was found to be less precise than the HPTLC and HPLC methods and prone to some loss of sensitivity caused by matrix interference.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.     

油菜蜂花粉黄酮含量的HPLC测定

以95％乙醇为溶剂,采用索氏提取器提取青海产油菜蜂花粉中的黄酮类化合物,将黄酮提取物中的黄酮甙水解为黄酮甙元后,利用HPLC法测定其中槲皮素、山萘酚、异鼠李素含量。结果表明,青海油菜蜂花粉中槲皮素、山萘酚、异鼠李素的平均含量分别为0．928％、0．295％、0．0834％,换算成总黄酮含量为3．28％。     

Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.     

Forced Degradation of Flavonol Glycosides Extraced from Ginkgo biloba

The degradation of flavonol glycosides extracted from Ginkgo biloba was performed under different conditions and the degraded products were determined by reversed-phase high performance liquid chromatography (RP-HPLC) method. Four stress conditions including acid(0.1 mol/L HCl), base(0.1 mol/L NaOH), temperature (70 ℃) and oxidation(0.03% H₂O₂, volume fraction) were used for the forced degradation studies. The pH stabilities of the flavonol glycosides were determined in phosphate buffers of varying pH values from 4.5 to 7.4. The degradation rate constants and half-life of three Ginkgo flavonol aglycones(quercetin, kaempferol and isorhamnetin) which represent Ginkgo flavonol glycosides were calculated in forced degradation and pH-stability studies of them. The results indicate that the three substances were more stable when incubated under acid condition and showed pH-dependent stability. The degradation was observed to follow first-order kinetics in all degradation studies. The stability results could provide important bases on development, preparation and storage of products of Ginkgo biloba extract and should be significantly considered during the further formulation development.     

Utilization of a scientifically operated charge-coupled device detector for high-performance thin-layer chromatographic analysis of tetracyclines

A high-performance thin-layer chromatography (HPTLC) method using a scientifically operated charge-coupled device detector is described for the assay of tetracycline pharmaceutical products. Quantitative information can be obtained for all samples on a TLC plate within a few seconds. The separation efficiency and detection limits were determined on both normal phase and reverse phase TLC plates. Fluorescence detection mode offers higher sensitivity than fluorescence quenching mode. The dynamic range, sensitivity, accuracy and precision of the system were evaluated. Detection limits of the impurities are in the range of 0.1 to 0.5 ng or 0.3 to 1% of tetracycline, depending on the compound, with a recovery percentage over 85%. The existing impurities in tetracycline capsules were determined using both HPLC and HPTLC techniques. All of the impurities were below the regulation level.     

Heart-cutting two-dimensional (size exclusion × reversed phase) liquid chromatography–mass spectrometry analysis of flavonol glycosides from leaves of Maytenus ilicifolia

Two-dimensional liquid chromatography–electrospray ionization mass spectrometry was employed to analyze flavonol glycosides present in leaves of Maytenus ilicifolia, frequently used in traditional Brazilian medicine. Since they contain many flavonol glycosides, including isomers, one-dimensional liquid chromatography did not give complete separation and identification, yielding overlapping of compounds with different molecular weights. Thus, employing size exclusion chromatography in the first and reversed phase in the second dimension, a great number of flavonol glycosides could be identified and its relative abundances determined. The majority of glycosides contained kaempferol or quercetin as aglycones, and glycosides with previously unreported structures were also present and characterized.