UPLC-DAD Method Research Articles

Chemical profiling and quantification of potential active constituents responsible for the antiplasmodial activity of Cissampelos pareira

Ethnopharmacological relevanceCissampelos pareira is used traditionally in India as a remedy for the treatment of various diseases including malaria but the active ingredients responsible for antiplasmodial activity have not yet been investigated.Aim of the study: The identification and quantification of compounds responsible for antiplasmodial activity in different parts (leaf, stem and root) of C. pareira is the target of current study. Material and methodsThe hydro ethanolic parent extracts of different parts of C. pareira and fractions prepared from these extracts were evaluated against Pf3D7 (chloroquine sensitive) and PfINDO (chloroquine resistance) strains in culture to quantify the IC50 for extracts and fractions. Promising fractions of root part of plant were subjected to silica gel column chromatography to obtain pure compounds and their structures were elucidated by detailed spectroscopic analysis. Pure compounds were also tested against Pf3D7 and PfINDO strains. A rapid and simple UPLC-DAD method was developed for the identification and quantification of pharmaceutically important metabolites of C. pareira. ResultsAmong different extracts, the hydro ethanolic extract of root part of C. pareira was found most active with IC50 values (μg/ml) of 1.42 and 1.15 against Pf 3D7 and Pf INDO, respectively. Tested against Pf 3D7 the most potent fractions were root ethyl acetate fraction (IC50 4.0 μg/ml), stem water fraction (IC50 4.4 μg/ml), and root water fraction (IC50 8.5 μg/ml). Further, phytochemical investigation of active fractions of root part led to the isolation and characterization of a new isoquinoline alkaloid, namely pareirarine (8), along with five known compounds magnoflorine (5), magnocurarine (10), salutaridine (11), cissamine (13) and hayatinine (15). Hayatinine (15), a bisbenzylisoquinoline alkaloid, isolated from root ethyl acetate fraction was most promising compound with IC50 of 0.41 μM (Pf INDO) and 0.509 μM (Pf 3D7). Magnocurarine (10) and cissamine (13) were also found active with IC50 values of 12.51 and 47.34 μM against Pf INDO and 12.54 and 8.76 μM against Pf 3D7, respectively. A total of thirty compounds were detected in studied extracts and fractions, structures were assigned to 15 of these and five of these biologically important compounds were quantified. Isolation of saluteridine (11) from C. pareira and the evaluation of antiplasmodial activity of pure compound from C. pariera is disclosed for the first time. ConclusionThis study concludes that the antimalarial potential of C. pareira may be attributed to isoquinoline type alkaloids present in this plant and also provides the scientific evidence for the traditional use of this plant in treatment of malaria.

Simultaneous Determination of the Flavonoids and Limonoids in Citrus junos Seed Shells Using a UPLC-DAD-ESI/MS

Citrus junos seeds (CS) have been traditionally used for the treatment of cancer and neuralgia. They are also used to manufacture edible oil and cosmetic perfume. A large amount of CS shells without oil (CSS) are discarded after the oil in CS is used as foods or herbal remedy. To efficiently utilize CSS as a by-products, it needs to be studied through chemical analysis. Therefore, we developed an ultra-performance liquid chromatography (UPLC)-diode array detection (DAD) method for simultaneous determination and quantitative analysis of five components (two flavonoids and threes limonoids) in CSS. A Waters Acquity UPLC HSS T3 column C18 (2.1 × 100 mm, 1.8 μm) was used for this separation. It was maintained at 40℃. The mobile phase used for the analysis was distilled water and acetonitrile with gradient elution. To identify the quantity of the five components, a mass spectrometer (MS) with an electrospray ionization (ESI) source was used. The regression equation showed great linearity, with correlation coefficient ≥ 0.9912. Limits of detection (LOD) and limits of quantification (LOQ) of the five compounds were 0.09 - 0.13 and 0.26 - 0.38 μg/mL, respectively. Recoveries of extraction ranged from 97.45% to 101.91%. Relative standard deviation (RSD) values of intra- and inter-day precision were 0.06 - 1.15% and 0.19 - 0.25%, respectively. This UPLC-DAD method can be validated to simultaneously analyze quantities of marker flavonoids and limonoids in CSS.

Impact of pumpkin seed oil and coffee treatment on the characteristics of semi-hard cheese

Semi-hard cheese was made from fresh domestic cow milk and treated with pumpkin seed oil and coffee during its ageing. The energy value and composition of the cheese were examined by determining the content of dry matter, minerals, proteins, fat, caffeine and fatty acids. During the ageing period, the cheese was dipped into a sweet, freshly-prepared, high-quality coffee drink and coated with a mixture of milled coffee grains and cold pressed pumpkin seed oil to improve the sensory properties of the cheese, but also to improve its nutritional value. It is considered that treated cheese could have greater nutritional and pro-health properties then untreated cheese, due to nutritional and healing properties of pumpkin seed oil and coffee used during the maturation period. Pumpkin seed oil contains carbohydrates, minerals, proteins, and important unsaturated fatty acids, while coffee is rich in antioxidants and helps in the prevention of type 2 diabetes, Parkinson's and Alzheimer's disease, and high cholesterol. To determine the impact of the treatment, the fatty acid and caffeine contents were determined by the GC-FID and the UPLC-DAD method. The cheese treated with pumpkin seed oil and coffee had a higher amount of unsaturated fatty acids (UFA) and a lower amount of saturated fatty acids (SFA) than the untreated cheese. The proportions of long-chain UFA, such as the C18:2n6c (Omega 6) and the C18:3n3 (Omega 3), were higher in the treated cheese than those in the untreated cheese, as well as the C20:1, the C22:2 and the C24:1, which were not detected in the untreated cheese. Caffeine concentration in the treated cheese was 33.08 mg/L.

Development of a Robust UPLC Method for Simultaneous Determination of a Novel Combination of Sofosbuvir and Daclatasvir in Human Plasma: Clinical Application to Therapeutic Drug Monitoring.

A rapid and selective UPLC-DAD method was developed and validated for simultaneous analysis of the novel two-drug combination Darvoni® for the treatment of HCV: Sofosbuvir (SF)/Daclatasvir (DC) in human plasma using Ledipasvir as internal standard (IS) where the extraction process was conducted using automated SPE. Although the analysis of the combination after concomitant oral intake of two tablets of SF and DC individually was reported in literature, yet simultaneous analysis of this new combination in human plasma after a single oral dose was not previously reported. The adopted chromatographic separation was achieved on Waters® Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) as a stationary phase using isocratic elution using a mobile phase system of ammonium formate (pH 3.5; 5 mM) and acetonitrile (60:40 v/v) pumped at a flow rate of 0.2 mL.min−1. The UV detection was carried out at 261 nm for SF and 318 nm for DC and IS. SF was eluted at 1.123 min while DC was eluted at 3.179 min. The proposed chromatographic method was validated in accordance with guidelines of FDA for bioanalytical method validation. A linear range was achieved in the range of 25-6400 and 50-12800 ng.mL−1 for SF and DC, respectively. The proposed UPLC-DAD method was found to be accurate with % bias ranging between -10.0-7.2 for SF and -6.9-8.0 for DC. Also it was proved to be precise with % CV for intraday precision ranging between 3.8-9.6 for SF and 2.8-9.2 for DC whereas interday precision ranged between 5.1-9.3 for SF and 3.7-9.1 for DC. Moreover, % extraction recovery ranged between 90.0-107.2 for SF and 93.1-108.0 for DC using the suggested method. The adopted chromatographic method was successfully applied to the therapeutic drug monitoring of SF and DC in healthy volunteers after the oral intake of one Darvoni® tablet.

Stability of sufentanil and baclofen mixtures for intrathecal analgesia at different concentrations in polypropylene syringes

Intrathecal analgesia is a method using various molecules alone or in combination. Among these, the association sufentanil/ baclofen is widely used. Instead of moving patients to the few expert centers taking charge of these specific preparations, it could be better to transport syringes to peripheral centers managing pump refilling. That is why, it is interesting to demonstrate the stability of the mixture, and so to be able to ensure the best transport conditions of syringes. A stability indicating UPLC-DAD method was developed and validated according to the ICH guidelines. Four mixtures of sufentanil baclofen stored in 5±3°C and 25±2°C were evaluated for seven days and compared to the initial observed concentrations. The stability is demonstrated only for preparations stored at 5±3°C for seven days thanks to relative concentrations (95% confidence intervals of the mean of 3 samples) systematically positioned between 90% and 110%. On the other hand, after few days, degradation products of sufentanil appeared for all mixtures stored at 25°C±2°C. This study shows the stability of a weakly and a highly concentrated mixture of sufentanil and baclofen solutions in polypropylene syringes stored at 5±3°C for seven days. This result will allow the transport of the preparation under optimal conditions. Advance preparations for intrathecal pump refills could also be feasible.

Stability Study of Morphine and Baclofen Solution in Polypropylene Syringes

AbstractBackgroundThe association of morphine and baclofen is used for the treatment of spasticity related pain. Moving these patients is sometimes difficult. In order to transport these syringes for pump refilling, it could be interesting to demonstrate the stability of the mixture, and so to be able to ensure the best transport conditions of syringes.MethodsA stability indicating UPLC-DAD method was developed and validated according to the ICH guidelines. Two mixtures of morphine and baclofen – a low concentration mixture (morphine 1 mg/mL – baclofen 0.08 mg/mL) and a high concentration mixture (morphine 10 mg/mL – baclofen 1.6 mg/mL) stored in 5±3 °C and 25±2 °C were evaluated for seven days and compared to the initial observed concentrations.ResultsThe stability of the low and high mixture is demonstrated for both storage conditions for seven days thanks to relative concentrations (95 % confidence intervals of the mean of 3 samples) systematically positioned between 95 % and 105 %. No degradation product was observed during the stability study.ConclusionThis study shows the stability of a weakly concentrated mixture and a highly concentrated mixture of morphine and baclofen. Extrapolation of these data to an intermediate mixture may be considered. Further studies will support this hypothesis. This result will allow the transport of the preparation under optimal conditions. Advance preparations for intrathecal pump refills could also be feasible.

A new strategy for choosing “Q-markers” via network pharmacology, application to the quality control of a Chinese medical preparation

Due to its chemical complexity, proper quality control for a Chinese medical preparation (CMP) has been a great challenge. Choosing the appropriate quality markers (Q-markers) for quality control of CMP is an important work. Best of all, the chosen Q-markers are the main chemical compounds from the herbals as well as the active constituents of this CMP. Only in this way the established quality control system can really achieve the purpose of controlling the quality of CMP and ensuring the safely and effectively use of CMP. To achieve the purpose, network pharmacology combined with the contents of chemical compounds in the CMP has been used in this research. We took an anti-arrhythmic CMP, Shenxian-Shengmai oral liquid (SSOL), as an example. Firstly, UPLC-QTOF-MS/MS method was used to analyze the main components of SSOL. A total of 64 compounds were unambiguously or tentatively identified and 32 of them were further validated by reference compounds. Secondly, the network was constructed based on the identified compounds to predict the effective compounds related to cardiac arrhythmias. Based on the existing database and the operation method of topology, a method of double network analysis (DNAA) was proposed, from which 10 important targets in the pathway of arrhythmia were screened out, and 26 compounds had good antiarrhythmic activity. Based on the prediction results of network pharmacology along with the contents of the compounds in this CMP, ten representative compounds were chosen as the Q-markers for the quality control of SSOL. We find that five of these ten compounds, including danshensu, rosmarinic acid, salvianolic acid A, epimedin A and icariin, have antiarrhythmic activity. Then, the UPLC-DAD method was established as the control method for SSOL.

Stability Indicating HPLC-UV vs. UPLC-DAD for Estimation of Atorvastatin Simultaneously with Aspirin, Clopidogrel and their Related Impurities in Bulk and Capsules

Aspirin (ASP) (analgesic; anti-inflammatory; anti-platelet), clopidogrel bisulphate (CLP) (anti-platelet) and atorvastatin calcium (ATV) (anti-hyperlipidemia) are co-formulated in several pharmaceutical formulations. Salicylic acid (SAL), clopidogrel carboxylic acid metabolite related impurity A (CLP-RA) and clopidogrel inactive R-enantiomer related impurity C (CLP-RC) are considered to be the main synthesis impurities and degradation products of ASP and clopidogrel. On the other hand, high efficiency and less elution are the basic requirements of high-speed chromatographic separation. So a comparative study of two simple, precise, and accurate reversed-phase HPLC-UV and UPLC-DAD methods were developed and validated for simultaneous estimation of ASP, CLP and ATV in bulk and capsules and in the presence of SAL, CLP-RA and CLP-RC. An Agilent Eclipse plus C18 (250 mm × 4.6 mm, 5 μm) for HPLC method and an Agilent Eclipse plus C18 (100 × 2.1 mm, 1.7 μm) for UPLC method were used. A gradient mobile phase for HPLC method and an isocratic mobile phase for UPLC, were used. Detection was performed for both methods at 220 nm for ASP, CLP, SAL, CLP-RA and CLP-RC, and at 240 nm for ATV. The propsed UPLC-DAD method had advantages, of being economic on the large scale with relatively much satisfactory results for sensitivity and specificity.

HPLC-UV vs. UPLC-DAD for Estimation of Tinidazole, Benzyl Alcohol and Hydrocortisone Acetate Simultaneously with Tioconazole and Its Related Impurities in Bulk and Pharmaceutical Formulations

Tioconazole (TCN) (anti fungal) is commonly co-formulated with tinidazole (TND) (antiprotozoal), hydrocortisone acetate (HYD) (anti infalmmatory) and benzyl alcohol (BZL) (preservative) in different pharmaceutical preparations. Tioconazole has three related impurities namely; tioconazole related impurities A, B and C (TCN-RA), (TCN-RB) and (TCN-RC), respectively. As the main purpose of a test for related substances is to control degradation impurities and to limit impurities arising during the synthesis of active ingredients and on the other hand, high efficiency and less elution are the basic requirements of high-speed chromatographic separation. So, two simple, precise, and accurate reversed-phase HPLC-UV and UPLC-DAD methods have been developed and compared for simultaneous estimation of TND, BZL, HYD and TCN in bulk and pharmaceutical formulations in the presence of TCN related impurities. A Eurospher C18 (250 mm × 4.6 mm, 5 μm) for HPLC method and an Eclipse plus C18 (100 × 2.1 mm, 1.7 μm) for UPLC method were used. An isocratic mobile phase consisted of 20 mM anhydrous KH2PO4 (containing 0.2% TEA v/v), pH adjusted to 6 with orthophosphoric acid: acetonitrile, (25:75, v/v) for HPLC method and a gradient mobile phase of 20 mM anhydrous KH2PO4 (containing 0.2% TEA v/v), pH adjusted to 6 with orthophosphoric acid and acetonitrile mixed in different proportions for UPLC method, were prepared. Flow rate was set at 1.0 mL min-1 and 0.3 mL min-1 for HPLC and UPLC methods, respectively and the detection was performed for both methods at 295 nm, 210 nm, 245 nm and 225 nm for TND, BZL, HYD and TCN and its related impurities, respectively. The propsed UPLC-DAD method had advantages, of being economic on the large scale with relatively much satisfactory results for linearity, sensitivity and specificity. The two proposed methods are applicable for the purity assessment of TCN in bulk, vaginal tablets and creams.

Cytotoxic New Nortriterpene from Roots of Potentilla atrosanguinea var. argyrophylla and its UPLC Quantification

Potentilla atrosanguinea is well known for its ethnomedicinal uses since ancient times. The present study includes the isolation of a new nortriterpene, 28-methyl-acanthochlamate (1), from the roots of P. atrosanguinea. The structure of 1 was established from HR-ESI-MS and NMR spectroscopic analysis. Its relative stereochemistry was set with the help of NOESY correlation experiment. Compound (1) has been quantified by a new developed UPLC-DAD method (1.1 mg/g). The in vitro cytotoxicity of the new compound (1) was evaluated by sulforhodamine B assay against three cancer cells; human cervical cancer (SiHa), epidermal carcinoma (KB), and human adenocarcinoma (Colo-205). The new isolated compound (1) showed a significantly higher cytotoxicity against all the cells (SiHa, IC50 30.5 µg/mL; KB, IC50 22.6 µg/mL and Colo-205, IC50 18.8 µg/mL).

A new ultra-high pressure liquid chromatography method for the determination of antioxidant flavonol aglycones in six Lysimachia species

UPLC-DAD method was developed and validated for the quantitative determination of free flavonol aglycones (kaempferol, quercetin and myricetin) after acidic hydrolysis in six Lysimachia species. Quantitative analyses showed that the amounts of various flavonol aglycones were significantly different in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, Lysimachia ciliata and Lysimachia clethroides. The L. clethroides sample was found to be the richest in kaempferol (25.77 ± 1.29 μg/mg extract) and quercetin (97.67 ± 4.61 μg/mg extract), while the L. nummularia sample contained the highest amount of myricetin (20.79 ± 1.00 μg/mg extract). The antioxidant capacity of hydrolysed extracts was evaluated using in vitro DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS•+ [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] decolourisation tests. The observed radical scavenging capacities of the extracts showed a relationship with the measured flavonol aglycone content and composition. The acidic treatment resulted in an increased free radical scavenging activity compared to the untreated methanol extract.

Synthesis and Pharmacological Evaluation of Novel Adenine–Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H2S slow-releasing moiety, coupled via a stable ether bond. The H2S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H2S donor, were selected for further in vivo evaluation. Their ability to release H2S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.

Simultaneous determination of baicalin, oroxylin A-7-O-glucuronide and wogonoside in rat plasma by UPLC-DAD and its application in pharmacokinetics of pure baicalin, Radix Scutellariae and Yinhuang granule.

A novel UPLC-DAD method was developed and validated for the simultaneous determination of baicalin (baicalein-7-glucuronide, BG), oroxylin A-7-O-glucuronide (OAG) and wogonoside (WG) in rat plasma using rutin as the internal standard. Plasma samples were precipitated using acetonitrile containing 0.1% formic acid. Separation was performed on an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) using gradient acetonitrile and 0.2% formic acid water solution as mobile phase. The flow-rate was set at 0.4 mL/min and the eluate was detected at 275 nm. The method was linear over the ranges of 0.075-17.50, 0.050-12.60 and 0.056-14.10 µg/mL for BG, OAG and WG, respectively. The intra- and inter-day precisions were respectively <4.8% and 6.4%. All of the limits of detection of three analytes in rat plasma were 0.01 µg/mL, whereas the limits of quantification were, respectively, 0.035, 0.025 and, 0.025 µg/mL. This assay has been successfully applied to pharmacokinetics of BG, OAG and WG in rats after oral administration of Yinhuang granule (YHG) and comparative pharmacokinetics of BG in rats following oral administration of the pure BG, Radix Scutellariae (RS) or YHG. We speculate that some co-existing ingredients in RS or YHG may increase the absorption and elimination of BG in rat. This work may be helpful for the quality control of Yinhuang granule.

Simultaneous quantitation of chloroquine and primaquine by UPLC-DAD and comparison with a HPLC-DAD method.

BackgroundChloroquine and primaquine are the first-line treatment recommended by World Health Organization for malaria caused by Plasmodium vivax. Since the problem of counterfeit or substandard anti-malarials is well established all over the world, the development of rapid and reliable methods for quality control analysis of these drugs is essential. Thus, the aim of this study was to develop and validate a novel UPLC-DAD method for simultaneously quantifying chloroquine and primaquine in tablet formulations.MethodsThe UPLC separation was carried out using a Hypersil C18 column (50 × 2.1 mm id; 1.9 μm particle size) and a mobile phase composed of acetonitrile (A) and 0.1% aqueous triethylamine, pH 3.0 adjusted with phosphoric acid (B), at a flow rate 0.6 mL/min. Gradient elution was employed. UV detection was performed at 260 nm. UPLC method was fully validated and the results were compared to a conventional HPLC-DAD method for the analysis of chloroquine and primaquine in tablet formulations.ResultsUPLC method was shown to be linear (r2 > 0.99), precise (CV < 2.0%), accurate (recovery rates from 98.11 to 99.83%), specific, and robust. No significant differences were observed between the chloroquine and primaquine contents obtained by UPLC and HPLC methods. However, UPLC method promoted faster analyses, better chromatographic performance and lower solvent consumption.ConclusionsThe developed UPLC method was shown to be a rapid and suitable technique to quantify chloroquine and primaquine in pharmaceutical preparations and may be successfully employed for quality control analysis.

Qualitative and quantitative analysis of the major constituents in Chinese medical preparation Lianhua-Qingwen capsule by UPLC-DAD-QTOF-MS.

Lianhua-Qingwen capsule (LQC) is a commonly used Chinese medical preparation to treat viral influenza and especially played a very important role in the fight against severe acute respiratory syndrome (SARS) in 2002-2003 in China. In this paper, a rapid ultraperformance liquid chromatography coupled with diode-array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS) method was established for qualitative and quantitative analysis of the major constituents of LQC. A total of 61 compounds including flavonoids, phenylpropanoids, anthraquinones, triterpenoids, iridoids, and other types of compounds were unambiguously or tentatively identified by comparing the retention times and accurate mass measurement with reference compounds or literature data. Among them, twelve representative compounds were further quantified as chemical markers in quantitative analysis, including salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid. The UPLC-DAD method was evaluated with linearity, limit of detection (LOD), limit of quantification (LOQ), precision, stability, repeatability, and recovery tests. The results showed that the developed quantitative method was linear, sensitive, and precise for the quality control of LQC.