Simultaneous determination of five active compounds in compound Salvia Miltiorrhiza gel via multi-channel HPLC detection

As an empirical medicine of traditional Chinese medicine, Fuzhengjiedu Granules have shown an effect against COVID-19 in clinical and inflammatory animal models. It is formulated with eight herbs, including Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma, Glycyrrhizae Radix Et Rhizoma, Lonicerae Japonicae Flos, Gleditsiae Spina, Fici Radix, Pogostemonis Herba, and Citri Reticulatae Pericarpium. This study established a high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method by simultaneously determining 29 active compounds in the granules with significant content differences. Separation by gradient elution using acetonitrile and water (0.1% formic acid) as mobile phases was performed on a Waters Acquilty UPLC T3 column (2.1 mm × 100 mm, 1.7 μm). A triple quadrupole mass spectrometer, operating in positive and negative ionization modes, was used for multiple reaction monitoring to detect the 29 compounds. All calibration curves showed good linear regression (r2 > 0.998). RSDs of precision, reproducibility, and stability of active compounds were all lower than 5.0%. The recovery rates were 95.4–104.9%, with RSDs< 5.0%. This method was successfully used to analyze the samples, and the results showed that 26 representative active components from 8 herbs were detected in the granules. While aconitine, mesaconitine, and hypaconitine were not detected, indicating that the existing samples were safe. The granules had the maximum and minimum content of hesperidin (27.3 ± 0.375 mg/g) and benzoylaconine (38.2 ± 0.759 ng/g). To conclude, a fast, accurate, sensitive, and reliable HPLC-QQQ-MS/MS method was established, which can simultaneously detect 29 active compounds that have a considerable difference in the content of Fuzhengjiedu Granules. This study can be used to control the quality and safety of Fuzhengjiedu Granules and provide a basis and guarantee for further experimental research and clinical application.

Kleeb Bua Daeng formula is a popular traditional remedy sold by the Chao Phya Abhaibhubejhr Hospital, Thailand. This formula contains Piper nigrum L., Nelumbo nucifera Gaertn. and Centella asiatica L. components. A reliable and accurate analytical method for the determination of five major active compounds in this formula was developed and validated. High performance liquid chromatography with a diode-array detector was used to quantify piperine, quercitin, kaempferol, asiaticoside and madecassoside in Kleeb Bua Daeng formula. The separation was carried out using a hypersil C18 column with detection at wavelengths 210, 280 and 370 nm. Gradient chromatographic conditions using a mixture of 0.05% phosphoric acid and acetonitrile allowed for complete resolution of the 5 active compounds. The validation outcomes for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness demonstrated that this HPLC method was accurate and reliable for the simultaneous determination of active compounds in this formula. Hence, this developed and validated HPLC fingerprint method is appropriate for quality control of Kleeb Bua Daeng formula and could be applied to modified Kleeb Bua Daeng formulations.

Four different techniques to quantify unresolved chromatographic peaks with known spectral features combined with photodiode array detection, are investigated as regards their efficiency for the accurate and precise determination of drugs in the low μg-range. The comparison includes peak suppression utilising difference chromatograms, first-order derivative chromatograms, selective chromatograms, generated by the calculation of orthogonal polynomial shares, and the powerful least-squares multicomponent analysis approach. Each of these methods uses UV-spectra taken throughout, the peak. The results presented and conclusions reached should enable the chromatographer to come to a decision about the reasonable use of these options now provided by multichannel detection in HPLC.

A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector

Poplar bark and leaves can be an attractive source of salicylates and other biologically active compounds used in medicine. However, the biochemical variability of poplar material requires a standardization prior to processing. The official analytical protocols used in the pharmaceutical industry rely on the extraction of active compounds, which makes their determination long and costly. An analysis of plant materials in their native state can be performed using vibrational spectroscopy. This paper presents for the first time a comparison of diffuse reflectance in the near- and mid-infrared regions, attenuated total reflection, and Raman spectroscopy used for the simultaneous determination of salicylates and flavonoids in poplar bark and leaves. Based on 185 spectra of various poplar species and hybrid powdered samples, partial least squares regression models, characterized by the relative standard errors of prediction in the 4.5–9.9% range for both calibration and validation sets, were developed. These models allow for fast and precise quantification of the studied active compounds in poplar bark and leaves without any chemical sample treatment.

HPLC stability-indicating method has been developed for the simultaneous determination of Butamirate citrate and Benzoic acid in pharmaceutical dosage forms. Active compounds were separated on Zorbax SB-C8 column (250 mm × 4.6 mm, 5 μm Agilent) at an ambient temperature. Elution was performed with a mobile phase consisting of acetonitrile (solvent A) and a mixture of 10 gm of sodium lauryl sulphate and 5ml of 1 N sulphuric acid in 1000 ml distilled water (solvent B) at a ratio of 70:30 v/v and a flow-rate of 1.7 ml/min. Detection was performed with UV detector at 205 nm. Cough syrup preparation was subjected to long-term stability in order to demonstrate that storage conditions and degradants from both active compounds did not interfere with the quantification of Butamirate citrate and Benzoic acid. Typical validation characteristics such as linearity, range, precision, accuracy, and selectivity were evaluated for Butamirate citrate and Benzoic acid. Using the novel method, both Butamirate citrate and Benzoic acid were separated successfully. Validation studies demonstrated that the novel method possessed a linear UV response, good system precision and accuracy, high sensitivity and specificity for Butamirate citrate and Benzoic acid.

Distribution of the active components from Xianglian Pill in tissues of healthy and antibiotic-associated diarrhea model mice and the mechanism study

Rhodiola L. has a long history as traditional Chinese medicine (TCM) with a medicinal efficacy similar to Ginseng and Manyprickle Acathopanax roots. There exist three classes of important active constituents, i.e., phenylethanoids (salidroside, p-tyrosol), phenylpropanoides (rosarin, rosavin, rosin), and monoterpene (rosiridin) in this TCM. In this study, by optimizing the extraction, separation and analytical conditions, a sensitive and accurate high performance liquid chromatographic method has been developed for the simultaneous determination of the six active compounds in the different species of Rhodioa L. for the first time. The analysis was performed on a Purospher STAR C18 column at 30 °C using 20 mmol L−1 aqueous ammonia acetate/methanol gradient system at a flow rate of 1.0 mL min−1 and photodiode array detection (DAD ) at wavelengths 276, 250 and 205 nm, respectively. The optimized method provided good linear relation (r2 > 0.9993 for all the target compounds), satisfactory precision (RSD values less than 1.53%) and good recovery (from 96.3–104.8%). The limits of detection ranged between 0.012 and 0.047 μg mL−1 for the different analytes. The developed method has been successfully applied to analysis and quality control of Rhodiola L.

In this study, a novel infrared-assisted extraction (IRAE) method, in which infrared radiation was employed to extract the active compounds from plant, was developed and coupled with HPLC for simultaneous determination of four phenolic acids and four diterpenoids in Radix Salviae miltiorrhizae (Chinese name Danshen). The extraction conditions of IRAE were optimized and the optimal conditions were as follows: extraction time of 15 min; extraction solvent of 70% v/v methanol in water solution, and solid/liquid ratio of 0.1:15 (g/mL). Chromatography was performed on a 200 mm×4.6 mm id, 5-μm particle size, C18 column. Good linearity (r>0.9994) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. Recoveries of the eight compounds were from 96.90 to 104.30% and RSD was below 2.5%. By using this novel IRAE method, with a shorter extraction time, the determined amounts of the eight active components in Danshen were comparable with or even higher than those extracted with conventional heat-reflux extraction and ultrasound-assisted extraction methods and microwave-assisted extraction. The developed IRAE method is simple, low-cost and efficient, offering a great promise for quick determination of active compounds in natural plants.

To develop a RP-HPLC method for simultaneous determination of three active compounds, dictamnine, obacunone and fraxinellone in root bark of Dictamnus dasycarpus and supply a reference for the establishment of the quality standard of D. dasycarpus. A Kromasil C18 column was used with methanol-water (60:40) as the mobile phase, at the flow rate of 1 mL x min(-1). 236 nm was selected as the detected wavelength. The determined three compounds were well separated with a linear range of 0.0021-0.1060, 0.0201-0.9200 and 0.0102-1.020 g x L(-1), respectively. The recoveries of them were 100.5%, 99.2% and 100.2%, respectively. This method is simple, rapid and accurate, particularly suitable for the quality control of D. dasycarpus.

Inhibition of porcine liver carboxylesterase by a new flavone glucoside isolated from Deverra scoparia

Microcomponents which contain cysteine, cystine and cysteic acid are supposed to be in gelatin. The most active compound seems to be cysteine.Eight lAG-gelatins-bone and hide, lime and aCId treated, oxidized and not oxidized-were investigated by means of HPLC and fluorescence detection. The resulting data provide the sum of cysteine and cystine and by a second determination the sum of cysteine/cystine and cysteic acid content.In spite of the fact that gelatins originate from dIfferent manufacturers it turned out that limed bone gelatins always contain less cysteine/cystine than limed hide gelatins. The same holds for the aCId treated gelatins.In an independent experiment we determined the cysteine content by its specific colour reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) whereby We also found the difference between oxidized and not oxidized gelatins described above.

Na-K-ATPase inhibitor dissociated from hypertension-associated plasma protein

Many psychotropic substances are illegally available on the streets and/or via the Internet. This wide distribution has become a serious social problem. To control this problem, many substances have been controlled as 'designated substances' (Shitei-Yakubutsu) in Japan since April 2007 by the Pharmaceutical Affairs Law, including tryptamines, phenethylamines and piperazines. In the present study, simultaneous determination methods using HPLC with multi-channel electrochemical detection (MECD) were developed for the designated substances. The proposed methods utilizing online electrochemical oxidation are the first report on the simultaneous determination of various designated substances. The methods involve direct determination and require no complicated pretreatments such as fluorescence labeling. The designated substances were separated by reversed-phase chromatography using a TSK-gel ODS-100V (4.6 × 250 mm, i.d., 3 µm) and gradient elution by a mixture of potassium phosphate buffer, methanol and acetonitrile. The total separation of 31 designated substances was successfully performed but required long chromatographic run times. Thus, the designated substances were divided into three groups: (1) tryptamines, (2) phenethylamines and (3) piperazines and others. They were then analyzed by HPLC-MECD as another separation method. The suitable applied voltages for each designated substance were determined based upon the hydrodynamic voltammogram. The limits of detection (signal-to-noise ratio of 3) of the designated substances for the most suitable voltages were in the range of 17.1 pg (5-MeO-MIPT) to 117 ng (indan-2-amine). The calibration curves based on the peak heights were linearly related to the amounts of the designated substances (R(2) > 0.999). Good accuracy and precision by intra-day assay and inter-day assay were also obtained using the present procedures. The proposed methods were applied to the analyses of the designated substance in several real samples.

A simple, precise and rapid stability-indicating ultra-performance liquid chromatography (UPLC) method is developed for the simultaneous quantitative determination of Telmisartan, Amlodipine besylate and Hydrochlorothiazide from their innovative poly pill combination drug product in the presence of degradation products. It involves a 100 mm x 2.1 mm, 1.7 μm C-18 column. The separation is achieved on a simple gradient method. The mobile phase A contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 90:10, v/v, and mobile B contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 20:80, v/v. The flow rate is 0.6 mL min−1 and the column temperature is maintained at 55°C.The gradient program (T/%B) is set as 0/5, 1.2/5, 1.6/40, 4/40, 4.1/5 and 4.5/5. The detector wavelength is 271 nm for Hydrochlorothiazide and Telmisartan and 237 nm for Amlodipine. The retention times of Telmisartan, Amlodipine, and Hydrochlorothiazide are 3.6 minutes, 3.2 minutes and 0.9 minutes; respectively. The total runtime for the separation of the three active compounds and their degradation products is 4.5 minutes. The described method is validated with respect to system suitability, specificity, linearity, precision and accuracy. The precision of the assay method is evaluated by carrying out six independent assays of T, A and H (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H). The accuracy of the method is evaluated in triplicate at three concentration levels, i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL−1 of T, 0.08 mg mL−1 of A, 0.2 mg mL−1 of H). The described method is linear over the range, 16 to 48 μg mL−1 for T, 2 to 6 μg mL−1A and 5 to 15 μg mL−1 for H. The method is fast and suitable for high-throughput analysis allowing the analysis of about 250 samples per working day.