Authors:András Makó, Hilda Hernádi, Gyöngyi Barna, Réka Balázs, Sándor Molnár, Viktória Labancz, Brigitta Tóth and Zsófia Bakacsi
The particle size distribution (PSD) values obtained for a soil database representing the main Hungarian soil types using the Hungarian standard (MSZ-08-0205-78) and the international standard (ISO/DIS 11277:1994) were compared with the pipette method. The relationship between these PSDs and other physical soil characteristics (upper limit of plasticity according to Arany, water vapour adsorption according to Sík) was also analysed, and a suggestion was made of how these results could be converted into each other.
Experience showed that the pre-treatments applied as part of the ISO/DIS method may change the ratio of particle size fractions: there was a significant increase in the clay content, while the silt content decreased to a lesser and the sand content to a greater extent, possibly because some of the particles remain in microaggregate form when the MSZ method is used. The results confirmed the greater accuracy of the ISO/DIS method: the clay contents measured with the ISO/DIS method exhibited stronger correlations with the upper limit of plasticity according to Arany and with hygroscopicity values than those measured with the MSZ method.
The estimated ISO/DIS fractions became much closer to the measured ones when the suggested pedotransfer functions were applied. The conversion method proved to be more reliable for the prediction of clay and sand content than for silt content. In its present form the estimation method is not suitable for replacing the ISO/DIS method, but it could be of good service in research and comparative analysis in cases where only the MSZ method can be used or where only old MSZ PSD data exist.
Authors:Jong-Woo Jeong, Yun-Hwan Seol, Hun-Chan Hyun, Hye-Rim Kim, Jong-Hwa Lee, Young-Dae Gong, Nam Sook Kang and Tae-Sung Koo
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of an anticancer drug, supinoxin (RX-5902), in rat plasma. Following precipitation pretreatment using 0.1% formic acid in acetonitrile, separation was performed using a reverse phase liquid chromatography column packed with C18 (3.5 μm, 2.1 × 50 mm) along with a mobile phase of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL min−1. Detection was achieved using MS/MS by multiple reaction monitoring via an electrospray ionization source at mass/charge transitions of m/z 442.30 → 223.30 for supinoxin and m/z 430.08 → 223.20 for the internal standard DGG-200064. This method demonstrated a linear standard curve (r = 0.9980) over a supinoxin concentration range of 0.0005–1 μg mL−1, as well as intra- and inter-assay precisions below 7.08% and 13.74%, respectively, and an accuracy of 1.15–4.50%. The matrix effect, recovery, and process efficiency were 93.63%, 99.70%, and 93.33%, respectively. Thus, a sensitive and reliable LC–MS/MS method was developed and validated for the quantification of supinoxin in rat plasma. This method was successfully applied to the evaluation of pharmacokinetic studies after single intravenous and oral administration of 1 mg kg−1 supinoxin in rats.
Authors:N. H. Hoang, N. L. Huong, S.-Y. Hong and Je Won Park
A highly sensitive analytical tool for the fast quantification of irsogladine in human plasma was developed. Cleanup using a solid-phase extraction technique is a simple method for extracting both irsogladine and lamotrigine (internal standard) spiked into human plasma. The resolvable separation of both analytes through reversed-phase high-performance liquid chromatography (HPLC) was carried out within 5 min. The HPLC–electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) method, which was operated in a selected reaction monitoring mode specific to the target analytes, was verified for use in the quantification of irsogladine. The inter- and intra-day precision (relative standard deviation, RSD) of irsogladine spiked into quality control samples were <7%, and their accuracies were between 96.6% and 102.1%. The calibration curve for irsogladine spiked into human plasma was linear over the range from 1.8 to 100 ng mL−1 with lower limit of quantification at 1.8 ng mL−1. The established method was successfully applied for a bioequivalence study of irsogladine.
Authors:Wioleta Jesionek, Barbara Majer-Dziedzic, Györgyi Horváth, Ágnes M. Móricz and Irena M. Choma
Thin-layer chromatography—direct bioautography (TLC—DB) followed by liquid chromatography—tandem mass spectrometry (LC—MS/MS) was used for screening and tentative identification of the antibacterial constituents of Salvia officinalis L. ethanol extract. Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, that is, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, luminescence gene-tagged Pseudomonas syringae pv. maculicola, and naturally luminescent marine bacterium Aliivibrio fischeri. Eight fractions with the widest antimicrobial spectrum were detected using TLC—DB, isolated by semi-preparative TLC, and subjected to LC—MS/MS analyses. Finally, five bioactive components were tentatively identified, based on their fragmentation pattern, such as salvigenin, cirsimaritin, rosmanol, carnosic acid, and 12-O-methyl carnosic acid.
Authors:Qiang Ren, Tianrui Xia, Xian-Gao Quan, Lin Ding and Hui-Yun Wang
Scutellaria barbata D. Don has been used as a traditional Chinese medicine for antitumor and anti-inflammatory. However, there were just a few investigations about S. barbata D. Don according to bioactivity-directed isolation and online identification for the chemical constituents. In this work, eight compounds were isolated from S. barbata D. Don. The three flavonoids indicated the cytotoxic activity against human leukemic Reh cell lines. In addition, the constituents of S. barbata D. Don were further characterized and identified by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The UHPLC- Q-TOF-MS method was in negative ion mode. HPLC separation was performed on a Tosoh TSK gel ODS-100V (4.6 × 150 mm, 3.0 μm) column by gradient elution using water containing 0.3% formic acid and acetonitrile as mobile phase at a flow rate of 0.8 mL min−1. A total of 18 compounds, including 4 phenolic acids and 14 flavonoids were tentatively characterized and identified by means of the retention time, accurate mass, and characteristic fragment ions.
Authors:Zhongming Han, Miaomiao Xu, Yunhe Wang, Limin Yang and Mei Han
Ionic liquid-based ultrasonic-assisted extraction (ILUAE) was successfully applied to the extraction of the four chromones (prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, and sec-O-glucosylhamaudol) from Saposhnikovia divaricata (Radix Saposhnikoviae) for the first time. A series of l-alkyl-3-methylimidazolium ILs differing in anion and cation compositions was evaluated for extraction efficiency, and [C3MIM]Br was selected as the optimal solvent. In addition, ultrasound extraction parameters were optimized, and the chromones were directly quantified and analyzed by rapid resolution liquid chromatography-electrospray ionization/mass spectrometry (RRLC-ESI/MS). The optimal conditions were as follows: 0.4 M concentration of [C3MIM]Br, 20:1 solvent to solid ratio, and ultrasonic time, temperature, and frequency of 5 min, 40 °C, and 50 kHz, respectively. This approach obtained the highest extraction yield of 10.188 ± 0.473 mg g−1 for total chromones. Compared with regular UAE, the proposed approach exhibited a higher efficiency (61.56% increase) and shorter extraction time (nine times shorter). Also, ILUAE was an efficient, rapid, and simple sample preparation technique for extraction of chromones, and the established RRLC-DAD method could serve as a rapid and effective technique for extracting chromones from Radix Saposhnikoviae.
Continuous-flow processing in the manufacturing of modern biotherapeutics represents a great potential and could significantly improve productivity and product quality as well as reduce operating costs. Microfluidic perfusion systems are not only capable for producing therapeutic proteins but also suitable for organ-on-a-chip based drug testing and toxicology studies. Integrating modular unit operations for protein purification in the microfluidic cell culture device can lead to point-of-care therapeutic protein production. The multi-organ microfluidic platforms that integrate several organ-on-a-chip microfluidic units will help in preclinical testing of drug substances and toxicological studies by producing highly reliable preclinical pharmacokinetic data. In this perspective, the current state of the art and future trends of continuous flow systems are summarized for biopharmaceutical production and organ-on-a-chip drug testing.
Authors:Daniel Blanco-Ania and Floris P. J. T. Rutjes
While continuous-flow chemistry is steadily increasing its footprint in academic research and in the manufacturing of pharmaceutical intermediates and fine chemicals, the attention for flow chemistry in educational programs is on average rather limited. This account is meant to provide a personal overview of the possibilities to address the involvement of flow chemistry in the various stages of chemical education.
Authors:K. Wróblewski, A. Petruczynik, B. Buszewski, M. Szultka-Młyńska, H. Karakuła-Juchnowicz and M. Waksmundzka-Hajnos
Vortioxetine is a new drug against major depressive disorder with high affinity for a range of different serotonergic targets in the central nervous system. Therapeutic drug monitoring is an important tool for the clinical management of patients receiving a pharmacotherapy, particularly in psychiatry. For this reason, determination of drug concentration in biological fluids is important for a rational dosage of drugs. Rapid and reliable analytical assays are also required to detect and identify drugs of toxicological importance. For analysis of vortioxetine by high-performance liquid chromatography (HPLC), no procedures for its determination in saliva have been reported and there are only a few ones for its determination in serum. A sensitive and selective highperformance liquid chromatography with diode array detector (HPLC-DAD) or mass spectrometer (HPLC-MS) method was developed for the fast quantification of vortioxetine in human saliva and serum. The determination was performed on a Synergi Polar RP column in isocratic mode under the optimal mobile phase containing 70% methanol, 20% acetate buffer at pH 3.5, 10% double distilled water, and 0.025 M L−1 diethylamine.
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of doxazosin mesylate (DOX) and finasteride (FIN) in bulk powders and pharmaceutical formulations. The compounds were separated on a Pinnacle II C18 column (250 × 4.6 mm i.d.; particle size, 5 μm) with an isocratic mobile phase at a flow rate of 1.0 mL min−1. The mobile phase was a mixture of 25 mM ammonium acetate and acetonitrile in the ratio of 50:50 %v/v. The pH of the buffer was adjusted to 4.0 ± 0.05 with glacial acetic acid. The detection was performed at 230 nm. The total chromatographic analysis time per sample was 15 min with DOX and FIN eluting at 3.9 and 7.2 min, respectively. The accuracy, precision, specificity, linearity, and sensitivity of the method were validated according to the International Conference on Harmonization (ICH) guidelines. The calibration plots were linear (r2 > 0.999) over the concentration range 24.25–291.0 μg mL−1 and 122.5–1470.0 μg mL−1 for DOX and FIN, respectively. The method was used for the simultaneous determination of DOX and FIN in capsules.
Rhizome extracts of Hedychium coronarium are widely used as phytotherapeutics. As of date, there is no documented study on the standardization of H. coronarium extract, and the following research is an effort in this direction. Coronarin D is an important bioactive compound present in H. coronarium which shows chemopreventive activity against cancer. H. coronarium extracts were assessed for coronarin D content for the first time. The extraction was checked using different solvents: n-hexane, acetone, and methanol. Coronarin D was separated on silica gel 60F254 high-performance thin-layer chromatography (HPTLC) plates by isocratic gradient method using n-hexane-ethyl acetate (80:20 v/v) as mobile phase. Densitometric quantification was performed at 231 nm in absorption mode. This method gave a well-defined peak at Rf 0.20 corresponding to coronarin D. The method was validated using International Conference on Harmonization (ICH) guidelines in terms of precision, repeatability, and accuracy. Linearity range of coronarin D was 200–1000 ng spot−1 with a correlation coefficient of R2 ± SD = 0.9987 ± 2.62% in the concentration range of 200–1000 ng spot−1. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 35 and 115 ng, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels, and the average percentage recovery was found to be 98.22 % for coronarin D. Among the different solvents, acetone produced maximum extraction efficiency of coronarin D. The proposed HPTLC method can be applied for robust identification and quantitative determination of coronarin D in H. coronarium extracts.
Authors:Biljana Petanovska-Ilievska, Lenche Velkoska-Markovska and Mirjana S. Jankulovska
Reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of sodium benzoate and potassium sorbate in beverages was developed using high speed column. The simple and rapid reverse-phase method for quantitative determination of both preservatives was established on LiChroCART® Purospher STAR RP-18e (30 mm × 4 mm; 3 μm) column, mobile phase consisted of acetonitrile-phosphate buffer (pH = 3.5) in volume ratio of 8:92 (v/v), flow rate of 1 mL min−1, ultraviolet (UV) detection at 195 nm for sodium benzoate and 260 nm for potassium sorbate, and constant column temperature at 25 °C. Linearity, precision, accuracy, limit of quantification (LOQ), and limit of detection (LOD) were tested for method validation. Linearity range for sodium benzoate was 6.04–200.27 mg L−1 (R2 = 0.999) while, for potassium benzoate (R2 = 0.999), 12.19–406.36 mg L−1. The RSD values ≤1.03% demonstrate excellent intra-day precision. LOD for sodium benzoate and potassium sorbate was 0.004 and 0.003 mg L−1, while LOQ was 0.012 and 0.009 mg L−1, respectively. This method was applied for quantitative determination of investigated preservatives in beverages which were taken from Macedonian markets.
Authors:Dong-Hyeon Ko, Ki-Won Gyak and Dong-Pyo Kim
The past three decades have seen increasing progress in the integration and process diversification of microfluidic systems for use in chemistry, biochemistry, and analysis. Here we summarize recent achievements in microreaction modules and microseparation units. We look into recent developments of microreaction systems fabricated by various 3D printing techniques for chemical synthetic applications. Moreover, we take a look at the recent achievements of newly developed microseparation technologies with enhanced separation efficiency realized by adopting single or hybrid principles as well as novel device concepts. Emerging technologies of 3D printing have potential to realize a vertically stacking the microchannels and miniaturization of bulky microreaction accessories. When the advanced microreaction systems are integrated with newly developed microseparation technologies, automated synthesis of industrial compounds, such as pharmaceuticals which need multiple types of salification chemistry, will be almost completed. Many opportunities are open to developing innovative microreaction systems with these techniques that can also be highly durable under harsh conditions.
Flow chemistry has become a vibrant area for research over the past decade. This perspective is intended to capture insights on how these advances have and will continue to impact the development and commercialization of active pharmaceutical ingredients. A series of chemistry examples from a number of pharmaceutical companies will highlight the influence of flow chemistry on this industry.
Authors:Gellért Sipos, Tamás Bihari, Dorottya Milánkovich and Ferenc Darvas
For successful deep space exploration, a vast amount of chemistry-related challenges has to be overcome. In the last two decades, flow chemistry has matured enough to take the lead in performing chemical research in space. This perspective article summarizes the state of the art of space chemistry, analyzes the suitability of flow chemistry in extraterrestrial environment, and discusses some of the challenges and opportunities in space chemistry ranging from establishing an end-to-end microfactory to asteroid mining.
In this perspective article, the use of continuous flow synthesis to prepare advanced pharmaceutical intermediates in developing economies is highlighted. Case studies are presented to suggest that cost effective local manufacture of life saving drugs, may potentially be implemented to facilitate better access to drugs to the underprivileged.
Optimizing current chemical processes alone does not yield the improvements required in the fine chemical and pharmaceutical industries. At least partially, a switch from batch to continuous manufacturing is needed. Cost-, time-, and atom-efficient routes frequently demand the application of high temperatures, pressures, and concentrations, and/or the use of highly reactive reagents. These chemistries often cannot be employed in conventional reactors. Costly and long alternative synthetic routes are chosen instead. The application of continuous-flow microreactors allows to access “harsh” or “hazardous” reaction conditions and, furthermore, enables entirely new transformations.
Authors:Long Wang, Yuan-Yuan Jiang, Li Zhang, Tao Wang, Rui-Wu Yang, Chun-Bang Ding, Xiao-Li Wang and Yong-Hong Zhou
A high-performance liquid chromatography (HPLC) method has been developed for the simultaneous identification and quantification of active compounds (cryptotanshinone, dihydrotanshinone I, tanshinone IIA, tanshinone I, salvianolic acid A, salvianolic acid B, protocatechuic aldehyde, and rosmarinic acid) contained in traditional Chinese folk medicine Salvia przewalskii Maxim. The herb samples (including wild, cultivated, and yin pian) from fourteen main regions were investigated. Chromatographic separation was performed on an Agilent Eclipse XDB-C18 reserved-phase column (250 mm × 4.6 mm i.d., 5 μm) using gradient elution with water-formic acid (99.9: 0.1, v/v) and acetonitrile at a flow rate of 0.8 mL min−1, an operating temperature of 30 °C, and a wavelength of 275 nm. Similarity analysis (SA), principal component analysis (PCA), and hierarchical cluster analysis (HCA) were used to analyze the data based on fingerprints. For fingerprint analysis, 27 peaks were selected as the common peaks to evaluate the similarities among different samples. The results of SA showed that the method permits to obtain desired linearity, precision, accuracy, and recovery. All samples were divided into three categories by PCA and HCA, and the concentration of the eight bioactive compounds varied significantly from different regions. It was demonstrated that chromatographic fingerprinting by HPLC combined with the simultaneous determination of eight bioactive compounds was a helpful method for the quality control of S. przewalskii.
Authors:Huba Kalász, Attila Hunyadi, Kornélia Tekes, Rafael Dolesal and Gellért Karvaly
Blood-brain penetration of 20-hydroxyecdysone 2,3;20,22-diacetonide (20DA) has been scouted using chromatographic methods. In vivo experiments were performed by treating male Wistar rats intraperitoneally (i.p.) with a dose of 50 mg kg−1 20DA. Control experiment was done by using 20-hydroxyecdysone (20E). Definite brain penetration of 20DA was found by using high-performance liquid chromatography (HPLC), while 20E does not show this type of distribution.
Authors:Jun-ichi Yoshida, Heejin Kim and Aiichiro Nagaki
This perspective article discusses the basic concept of time control by space based on flow and micro, some examples that realized extremely fast reactions which were difficult to achieve by conventional flask chemistry, and the future of this fascinating chemistry.
There are great opportunities for innovation in the drug discovery process, particularly in the lead development phase. The traditional “design–synthesize–screen” cycle has seen little innovation as a whole despite major advances at each stage, including automated purification and synthesis as well as high throughput biological screening. It could be argued that the hit-to-lead and lead optimization processes remain slow and modular with inefficient flow of information, resulting in a loss of time and money. New flow technologies may provide a promising foundation for developing a continuous integrated small molecule optimization platform that would greatly enhance hit-to-lead and lead optimization programs. Herein, we discuss major developments in integrating synthesis, purification, screening, and machine learning into a single continuous-flow platform and provide some insight into future directions of this field.
Authors:Bartholomäus Pieber, Kerry Gilmore and Peter H. Seeberger
The way organic multistep synthesis is performed is changing due to the adoption of flow chemical techniques, which has enabled the development of improved methods to make complex molecules. The modular nature of the technique provides not only access to target molecules via linear flow approaches but also for the targeting of structural cores with single systems. This perspective article summarizes the state of the art of continuous multistep synthesis and discusses the main challenges and opportunities in this area.
Photochemistry and photoredox catalysis have witnessed a remarkable comeback in the last decade. Flow chemistry has been of pivotal importance to alleviate some of the classical obstacles associated with photochemistry. Herein, we analyze some of the most exciting features provided by photo flow chemistry as well as future challenges for the field.
Authors:Victor Sebastian, Saif A. Khan and Amol A. Kulkarni
Continuous-flow synthesis of specific functional materials is now seen as a reliable synthesis approach that gives consistent product properties. This perspective article aims to survey recent work in some of the relevant areas and to identify new domains where flow synthesis of functional materials can be better than the conventional synthesis methods. It also emphasizes the need for developing high-throughput integrated synthesis and screening systems for almost all functional materials so that laboratory-scale recipes can be transformed into reliable manufacturing processes. New areas relevant to functional materials which have remained unexplored in flow synthesis are also highlighted.
Electrosynthesis is an old method currently moving again in the focus of organic synthesis. Some limitations of conventional electrosynthesis can be overcome by the use of electrochemical flow devices. This perspective indicates where the pitfalls, where the advantages and where the challenges are in implementing flow electrosynthesis as an alternative tool for the synthetic chemist.
The current state of the art of polymer synthesis in (microstructured) continuous-flow reactors is given, focusing on controlled/living polymerization methods that allow for precision polymer design. Emerging trends and the most notable developments are discussed. Especially, the field of multistep reactions and online monitoring are highlighted, which in combination may give access to fully automated high-throughput polymer synthesis reactors in the future.
We present a new simple thin-layer chromatographic method designed for determination of the main alkaloids of Chelidonium majus L. In this study, we used roots and herb of the plant collected in spring and autumn. The alkaloid fractions were prepared according to modified pharmacopeial procedure .
In our method, we performed two-step elution onto silica gel plates. The first eluent consisted of chloroform, methanol, and water mixed with 70:30:4 proportion. The second eluent comprised of toluene, ethyl acetate, and methanol with 83:15:2 proportion. The described thin-layer chromatography (TLC) system allows qualitative and quantitative determination of the following alkaloids: sanguinarine, chelerythrine, chelidonine, coptisine, and berberine. For determination of protopine, eluent with n-buthanol, acetic acid, and water in 15:1.5:4 proportion was investigated.
The dominant alkaloids observed in studied fractions were coptisine (1027.096 ± 13.367–287.474 ± 3.069 mg/100 g dry matter ± sdv) and chelidonine (1780.667 ± 263.522– 115.929 ± 14.694 mg/100 g dry matter ± sdv). The alkaloid detected in the least amount was chelerythrine (30.74 ± 7.526–1.143 ± 0.0651 mg/100 g dry matter ± sdv). The highest total amount of all alkaloids was determined in the fractions obtained from herbs in spring, and the lowest amount was detected in herbs autumn.
Additionally, we compared amounts of studied alkaloids in different parts of plants (aerial parts and roots). The plants were collected in spring and autumn.
Authors concluded that the presented method can be used as a valuable tool for screening studies on C. majus L.
Authors:Dariusz Szeremeta, Magdalena Knaś, Ewa Długosz and Mieczysław Sajewicz
The present research is focused on identification of volatile components of different commercial products containing raw herbs of Cistus incanus L. The dried herbal material was hydrodistilled, and the obtained essential oils were analyzed by means of gas chromatography with mass spectrometric detection. Alternatively, the headspace analysis of the volatile sample components was also performed. It was found out that the investigated samples of the C. incanus L. species show a wide variation in terms of quality and quantity of the respective essential oils, which might result in their variable biological activity also. In conclusion, a postulate for standardization of chemical composition of the raw plant material used in therapeutic preparations is formulated.
Ultra high-performance liquid chromatography (UHPLC) coupled with high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometry was used for the preliminary photodegradation study of nine new generation psychotropic drugs. Based on the above method, two ionization sample modes — electrospray and atmospheric pressure chemical ionization were used for the registration of photodegradation profiles of the selected drugs. Multivariate chemometric analysis showed that electrospray ionization (ESI) method is more specific than atmospheric pressure chemical ionization (APCI) in high-resolution mass spectrometry (HR-MS) analysis of the analyzed psychotropic drugs. It was noticed that, with the use of ESI method, more potential photodegradation products can be identified and lower limits of its detection can be obtained.
Authors:Bernhard Barwinski, Pedro Migowski, Fabrice Gallou, Giancarlo Franciò and Walter Leitner
A process comprising a continuous-flow hydrogenation reaction integrated with selective water-organic solvent biphasic extraction using CO2 as molecular switch to control partitioning was devised for the synthesis of arylpiperidines from arylpyridines. The selective hydrogenation of 4-phenylpyridine using heterogeneous carbon-supported metal catalysts was chosen as model reaction. A design-of-experiment approach was used for the identification of suitable reaction conditions under continuous-flow operation. A maximum selectivity for 4-phenylpiperidine of 96% was achieved at 87% conversion suppressing the deep hydrogenation to 4-cyclohexylpiperidine almost completely (≤5%). The higher basicity of piperidines over pyridines was exploited for selective and reversible protonation of the product upon pressurization with CO2 separating it quantitatively from the remaining starting material in a water—EtOAc biphasic system. This concept enabled a fully integrated and a salt-free synthetic process using a standard Pd/C catalyst for the hydrogenation coupled with the CO2-triggered isolation of the desired product 4-phenylpiperidine in 81% yield and 98% purity.
Authors:K. Wróblewski, A. Petruczynik, I. Radzik, S. J. Czuczwar and M. Waksmundzka-Hajnos
Three independent reversed phase high-performance liquid chromatography (HPLC) procedures with diode array detection (DAD) for the analysis of carbamazepine (CBZ), topiramate (TPM), and valproic acid (VPA) have been developed in order to determine drug penetration of the blood—brain barrier. Determination of CBZ was performed on C18 column with mobile phases containing methanol (55%, v/v), acetate buffer at pH 3.5 (20%, v/v), double distilled water (25%, v/v), and 0.025 M L−1 diethylamine (DEA). The mobile phase containing acetonitrile and water (8:2, v/v) or acetonitrile and phosphate—citrate buffer at pH 2.6 (1:1), respectively, for analysis of VPA and TPM was applied. Quantification of carbamazepine was performed at 285 nm without extraction procedure before the analysis. Determination of topiramate and valproic acid was performed using precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). FMOC-Cl is a suitable agent, which reacts with both primary and secondary amines and also with acidic groups. Topiramate was determined at 263 nm and valproic acid at 300 nm. The proposed procedures are simple, not time-consuming, and suitable for the determination of investigated compounds in mouse brain homogenates.
This study aims to develop and validate a high-performance size-exclusion chromatography (HPSEC) method to determine the amount of polymer in cefmetazole sodium for injection and to compare this method with gel chromatography. A Zenix SEC-150 column was used with the mobile phase of phosphate buffer solution (pH 7.0; 0.01 M)—acetonitrile (90:10 v/v) at a flow rate of 0.8 mL min−1 and a detection wavelength of 240 nm. The polymer was quantified by an external standard method with self-control, and the amount was expressed by the percentage of cefmetazole. The HPSEC method was validated for specificity, linearity, and precision. The chromatographic conditions, chromatographic performances, sensitivity, linearity, and precision of the developed HPSEC method and gel chromatography were compared, and both methods were subsequently used to determine the amount of polymer from seven batches of samples. The HPSEC method was fully validated. The time of isocratic elution for sample assay was less than 14 min. The results of comparison indicate that the developed HPSEC method was superior to gel chromatography. The Student t test results also showed significant difference in the amount of polymer from the samples obtained by the two methods. Thus, the HPSEC method with two obvious advantages, the superior sensitivity and a shorter analysis time, is more suitable for determination of polymer amount in cefmetazole sodium for injection to control the quality of the product.
Authors:Sinan Zazoğlu, Beril Anilanmert, Muhammed Aydin and Salih Cengiz
A fast, reliable, inexpensive, and practical method with a low determination limit and high recovery has been developed for the determination of the marijuana metabolite in routine analysis. THC-COOH in urine was validated using liquid chromatography—tandem mass spectrometry (LC—MS/MS). Before an easy single-step extraction with Toxi-Tubes, basic hydrolysis was performed at 60 °C for 30 min. LC—MS/MS analysis takes 2.5 min for each sample, and the retention time of the analyte is 1.75 min. Specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, repeatability, and intermediate precision (inter-day) system suitability parameters were determined in the validation study. The recovery of the extraction method was 88.67 (±5.91). LOD and LOQ values were 1.41 and 5.00 ng mL−1, respectively. The method showed linear response between the values 5.00 and 500.00 ng mL−1. The repeatability was 9.64% (relative standard deviation, RSD%), and the intermediate precisions (RSDR%) were 10.73%, 13.74%, and 8.11% at 10.00, 100.00, and 200.00 ng mL−1 concentration levels, respectively. No statistically significant difference was found in ANOVA analysis, between three consecutive days in intermediate precision study, for 90% confidence level. HorRat values were between 0.34 and 0.61. The method was applied to CEDIA positive samples, obtained from the Trabzon Group Presidency of Turkish Council of Forensic Medicine, successfully.
Authors:Biljana K. Tubić, Bojan D. Marković, Sandra S. Vladimirov, Slavica M. Ristić, Branka M. Ivković, Miroslav M. Savić, Jelena M. Poljarević and Tibor J. Sabo
A series of new (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate esters has shown cytotoxic activity towards human leukemic cell lines. The aim of this study was to develop and validate a bioanalytical method for quantification of (S,S)-O,O-diethyl-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate dihydrochlorides (DE-EDCP) and its metabolite, substituted propanoic acid (EDCP), in mouse serum by ultra high-performance liquid chromatography—tandem mass spectrometry (UHPLC—MS/MS). Structural analog, derivative of 1,3-propanediamine, was used as an internal standard (IS). Sample preparation employed protein precipitation by acetonitrile and subsequent centrifugation. Optimal UHPLC separation conditions were set to achieve simultaneous determination of both compounds in a short run time of 6 min. Additionally, the selected reaction monitoring (SRM) mode developed in this method allowed a highly sensitive, accurate, and precise identification of compounds of interest. The lower limit of quantitation (LOQ) was 1.3 ng mL−1 for DE-EDCP and 0.3 μg mL−1 for EDCP. The calibration curves were linear over the concentration range of 1.3–26.7 ng mL−1 and 0.3–6.7 μg mL−1 for DE-EDCP and EDCP, respectively. Precision (%CV) and accuracy (%RE) for DE-EDCP and EDCP ranged from 3.5% to 16.0% and from 1.8% to 14.4%, respectively.
The validation process was performed in accordance with the regulatory guidance/guideline, and all of the obtained results met the established acceptance criteria. The newly developed and validated UHPLC—MS/MS method is rapid, sensitive, and selective, and it can be successfully applied to drug monitoring in nonclinical studies.
Authors:János Kalmár, László Kuti, János Kátai, Renáta Figler and György Füleky
A Nyírség egyik jellegzetes talajtípusa a kovárványos barna erdőtalaj. Mind hazánkban, mind külföldön sokan vizsgálták a kovárványképződést és állítottak fel természettudományosan megalapozott elméleteket a képződés mikéntjére vonatkozóan. Leggyakrabban a vas-vegyületek lefelé irányuló mozgásávbal és adott mélységben történő kicsapódásával próbálták meg leírni a jelenséget. Munkánk célja ásványtani, talajtani és mikrobiológiai vizsgálatok segítségével megválaszolni a kovárványképződésének kérdését.
A Nyírség tamáspusztai homokdomb alapvetően homogénnek tekinthet ásványi összetételét tekintve. A kovárványszintek képződésének korábban leírt kémiai és szemcseösszetételbeli kritériumai teljesülnek (pH 4.5–6.5 közé esik, a szemcseösszetétel pedig a kovárványrétegben meghaladja a leiszapolható rész 10 %-ot). Részletesen vizsgálva a kovárványrétegeket benne a homokszemcsék korrodáltak, töredezettebbek, ami mind egykori gyökérnedvek korróziójának eredménye lehet. A homokszemcsék kötőanyaga a kovárványrétegben elsősorban vas-oxihidroxidból áll, és a kovárványréteg eredetileg képlékeny, gyúrt, szakadozott szerkezetet mutat. A kovárványréteg felső része erodált, ami egykori talajfelszínen történő elhelyezkedését jelzi. A réteg alsó része tagolt és gyakran benyúlik az alatta elhelyezkedő homoktestbe. Véleményünk szerint a vas-oxihidroxid kiválások a kovárványrétegben alapvetően biológiai (növényi vas felvétel, majd elhalás után mikrobiális bontás segítségével létrejött vas-oxihidroxid akkumuláció) akkumulációs és kiválási folyamatokra utalnak. Mindezek alapján úgy gondoljuk, hogy a kovárványrétegek az egykori homokdomb felszínén képződtek és nem később bekövetkezett vasmozgás során jöttek létre.
Authors:Yan-Mei Zhong, Xun-Long Zhong, Ji-Hua Wang and Liang Han
Patrinia scabiosaefolia Fisch. (PSF), a well-known traditional Chinese medicine, has been demonstrated to show therapeutic effects on inflammatory bowel disease. In this study, a rapid and sensitive method using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was developed for identification of the major constituents in PSF. The separation analysis was performed on Waters Acquity UPLC system, and the accurate mass of molecules and their fragment ions were determined by Q-TOF-MS. Thirty-one constituents, including triterpenoids, iridoids, flavonoids, and organic acids were detected and tentatively deduced on the basis of their element compositions, tandem mass spectrometry (MS/MS) data, and relevant literatures. Twelve constituents were discovered for the first time in PSF. The results demonstrated that hederagenin-type and oleanolic acid-type saponins were the main constituents of PSF. Our work provides a certain foundation for further quantitation of major chemical constituents and in vivo pharmacokinetic studies of PSF. Moreover, the analytical approach developed herein has proven to be generally applicable for profiling the chemical constituents in traditional Chinese medicines (TCMs) and other complicated mixtures.
New polyacrylate-based monosized-porous polymer beads were proposed as a stationary phase for the separation of polar compounds by microbore reversed-phase chromatography. For this purpose, monosized-porous poly(glycerol dimethacrylate-co-glycerol-1,3-diglycerolate diacrylate), poly(GDMA-co-GDGDA), beads with hydroxyl functionality were synthesized by a modified seeded polymerization. The selected octadecylating agent, stearoyl chloride (SC), was covalently attached onto the hydrophilic beads via a direct, single stage reaction with a simple synthetic route. SC attached-poly( GDMA-co-GDGDA) beads were slurry-packed into the microbore columns and used as separation medium microbore reversed-phase chromatography. The stationary phase was used for separation of alkylbenzenes and polar analytes by micro reversed-phase chromatography, using mobile phases with low acetonitrile content. Theoretical plate number (TPN) values up to 12,000 plates m−1 and 10,000 plates m−1 for alkylbenzenes and polar analytes, respectively, were achieved. The results also showed that poly(GDMA-co-GDGDA) hydrogel beads are a promising starting material for a number of chromatographic applications like reversed-phase (RP) chromatography, hydrophilic interaction chromatography (HILIC), ion-exchange chromatography (IEC), and affinity chromatography with a single-stage surface modification.
The use of polypropylene materials in industry for food packaging is increasing. The presence of additives in the polymer matrix enables the modification or improvement of the properties and performance of the polymer, but these additives are potential risk for human health. In this context, an efficient analytical method for the quantitative determination of three antioxidants (2,6-di-tert-butyl-4-methylphenol (BHT), dibutylhydroxyphenylpropionic acid stearyl ester (Irganox 1076), and tns-(2.4-di-tert-butyl)-phosphite (Irgafos 168)) and five ultraviolet stabilizers (2-(2′-hydroxy-5′-methylphenyl) (UV-P), (2′-hydroxy-3′-tert-5′-methylphenyl)-5-chloroben zotriazole (UV-326), 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)-5-chlorobenzotriazole (UV-327), 2-(2H-benzotriazol- 2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol(UV-329), and 2-hydroxy-4(octyloxy) benzophenone (UV-531)) in polypropylene food packaging and food simulants by high-performance liquid chromatography (HPLC) has been developed. Parameters affecting the efficiency in the process such as extraction and chromatographic condition were studied in order to determine operating conditions. The analytical method showed good linearity, presenting correlation coefficients (R ≥ 0.9977) for all additives. The limits of detection and quantification were between 0.03 and 0.30 μg mL−1 and between 0.10 and 1.00 μg mL−1 for eight analytes, respectively. Average spiked recoveries in blank polypropylene packaging and food simulants were in the range of 80.4–99.5% and 75.2–106.7%, with relative standard deviations in the range of 0.9–9.1% and 0.2–9.8%. Dissolving the polypropylene food packaging with toluene and precipitating by methanol was demonstrated more effective than ultrasonic extract with acetonitrile or dichloromethane for extracting the additives. The method was successfully applied to commercial polypropylene packaging determination, Irgafos 168 and UV-P were frequently found in six commercial polypropylene films, and the content ranged from 166.47 ± 5.11 to 845.27 ± 29.31 μg g−1 and 2.10 ± 0.29 to 19.23 ± 1.26 μg g−1, respectively.
Authors:Mei-Xia Zhu, Sheng-Nan Li, Hai-Dan You, Bin Han, Zhi-Ping Wang, Yan-Xi Hu, Jin Li and Yu-Feng Liu
High-performance liquid chromatography coupled with photodiode array detection and evaporative light scattering detection (HPLC—DAD—ELSD) was established to determine paeoniflorin and albiflorin simultaneously in Radix Paeoniae Rubra. The assay was performed on a Diamonsil C18 (4.6 mm × 250 mm, 5 μm) column by a gradient elution program with acetonitrile and aqueous formic acid (0.05% v/v) as mobile phase at a flow rate of 1.0 mL min−1. The detection wavelength of DAD was 230 nm, and the evaporator tube temperature of ELSD was set at 110 °C with the nebulizing gas flow rate of 3 L min−1. The temperature of column was kept at 30 °C. The linear ranges of paeoniflorin and albiflorin were within 0.050–1.510 mg mL−1 and 1.007–5.035 mg mL−1. The recoveries of paeoniflorin and albiflorin were 96.2–102.9% and 95.0–102.4%, respectively, while the relative standard deviation (RSD) of them was 0.2–2.5%. This method was quick, simple, accurate, and specific. It could be used for the quality control of Radix Paeoniae Rubra. The proposed approach was expected as a powerful tool for the quality control of Radix Paeoniae Rubra.
Authors:Sree Rama Murthy Pyla, Prafulla Kumar Sahu and K. Srinivas
Linaclotide, a first-in-class guanylate cyclase-C agonist, was recently approved by US Food and Drug Administration (FDA) as a promising pharmacotherapy for the management of constipation-predominant irritable bowel syndrome (IBS). In this communication, we present a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of linaclotide along with its degradation products. During the International Conference on Harmonization (ICH) prescribed stress study, linaclotide was found susceptible to degrade under hydrolytic (acid and base) and oxidative (peroxide) conditions. The separation of the degradants from the analyte was achieved on a Zorbax Eclipse XDB C8 Column (250 mm × 4.6 mm, 5 μm) using 0.01 N potassium dihydrogen orthophosphate buffer and acetonitrile (80:20 v/v) as mobile phase at a flow rate of 1.00 mL min−1 at column temperature of 40 °C. The detection of the column effluents was realized on a photodiode array detector set at 220 nm. Under the above optimal condition, the method was validated with respect to specificity, linearity, range, precision, robustness, and sensitivity in compliance to the regulatory requirements.
Authors:Wioleta Jesionek, Barbara Majer-Dziedzic, Györgyi Horváth, Ágnes M. Móricz and Irena M. Choma
In this study, thin-layer chromatography—direct bioautography (TLC—DB) was used for guiding the isolation and identification of antibacterial constituents of Thymus vulgaris L. ethanol extract. This TLC—bioassay method enables the separation and detection of active components directly on the surface of chromatographic plates. They can be identified by comparison with reference substances or using physicochemical methods, preferably spectroscopic ones (liquid chromatography—tandem mass spectrometry [LC—MS/MS], in the presented paper). The described method belongs to the effect-directed analyses (EDA). Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, including methicillin-resistant Staphylococcus aureus as well as luminescent bacteria like Aliivibrio fischeri. Five fractions with the widest antimicrobial spectra were detected using TLC—DB, isolated by semi-preparative TLC and subjected to LC—MS/MS analyses. Finally, two bioactive components were tentatively identified, basing on their fragmentation pattern, as eriodictyol and 4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-bifenylodion.
Authors:Rui Jiang, Tao Liu, Shu Yang, Liquan Sun and Aiqin Luo
Four geminal ionic liquids (GILs), namely, 1,4-bis(1,1′-butyl-3,3′- methylene- imidazolium)-benzene bis[(trifluoromethyl)sulfonyl]imide (BBMIB-NTf2), 1,4- bis(1,1′-butyl-3,3′-methylene-imidazolium)-benzene tetrafluoroborate (BBMIB-BF4), 1,4- bis(1,1′-butyl-3,3′-methylene-imidazolium)-benzene hexafluophosphate (BBMIB-PF6), and 1,4-bis(1,1′-methyl-3,3′-methylene-imidazolium)-benzene bis[(trifluoromethyl) sulfonyl] imide (BMMIB-NTf2), were synthesized. They were statically coated onto the inner walls of fused-silica capillary columns and used as stationary phases for gas chromatography. The evaluation of BBMIB-NTf2, BBMIB-BF4, BBMIB-PF6, and BMMIB-NTf2 as stationary phases is reported here for the first time. These new stationary phases exhibit efficiencies of at least 2.3 × 103 plates per meter. Abraham solvation parameter model was used to evaluate the solvation characteristics. The system constants indicated that the dipolarity/polarizability and the hydrogen-bond basicity play a major role among five molecular interactions between stationary phases and solute molecules. A fundamental understanding into the solvation characteristics of these GILs can be used as a guide to choosing the appropriate geminal ionic liquids for specific applications in various fields. The chromatographic separation performance was evaluated by a Grob test mixture, n-alkanes, alcohols, and aromatic isomers. Furthermore, the thermal stability was tested. The present results demonstrate that these geminal ionic liquids stationary phases possess excellent chromatographic separation performance and good thermal stability (at least up to 270 °C) and may be applicable as gas chromatography stationary phases for more application.
Authors:A. Maciejowska, A. Godziek, M. Sajewicz and T. Kowalska
In this short communication, we report on three striking phenomena of the circadian rhythm. One was observed with the non-linear concentration changes of the monomeric L-Cys and the non-linear yields of the L-Cys derived peptides, when undergoing spontaneous non-linear peptidization. The other one was observed with the binary L-Phe-L-Pro system, and the third one with L-Ser, D-Ser, and DL-Ser. So far, no analogous reports have been released on the circadian rhythm of the spontaneous non-linear peptidization of proteinogenic amino acids in a sterile abiotic environment (70% aqueous acetonitrile, or 70% aqueous methanol solutions). At the moment, we cannot find any rational explanation of this phenomenon, yet it seems highly probable that its origin is analogous to or even of a primordial nature for the circadian rhythm phenomena abundantly found in biological samples by other researchers. An experimentally established lack of the circadian rhythm with peptidization of the non-proteinogenic amino acid (D-Ser) can encourage us to revisit a still unsolved question of homochirality preconditions.
Authors:J.F.F. Anderson, M.C.G. Gerlin, R.A. Sversut, L.C.S. Oliveira, A.K. Singh, M.S. Amaral and N.M. Kassab
The objective of this study was to develop and validate an assay method for simultaneous determination of atenolol, furosemide, losartan, and spironolactone in pharmaceutical formulations. A reverse-phase high-performance liquid chromatography procedure was developed, using a Kinetex® C-18 column (100 mm × 4.6 mm, 2.6 μm). The mobile phase was composed of methanol—water (75:25 v/v, pH 3.0, adjusted with phosphoric acid), with a flow rate of 0.4 mL min−1. All drugs were separated in less than 5 min. The method was validated according to International Conference on Harmonization (ICH) and Association of Official Analytical Chemists (AOAC) guidelines. The method showed linearity in a concentration range of 0.75–12.0 μg mL−1 for atenolol (r = 0.9995), 0.30–12.00 μg mL−1 for furosemide (r = 0.9997), 0.45–12.00 μg mL−1 for losartan (r = 0.9995), and 0.45–12.0 μg mL−1 for spironolactone (r = 0.9999). The method also showed repeatability and precision. The three-day average intra-day precisions were 101.35 ± 0.74% for atenolol, 95.84 ± 1.44% for furosemide, 98.90 ± 1.16% for losartan, and 97.19 ± 0.18% for spironolactone. Similarly, the inter-day precisions were 101.34 ± 0.72% for atenolol, 95.84 ± 0.1.50% for furosemide, 98.90 ± 1.17% for losartan, and 97.19 ± 0.83% for spironolactone. The method accuracy was also tested and validated — in this case, the average recovery values were 100.18 ± 1.20% for atenolol, 99.83 ± 1.54% for furosemide, 100.07 ± 0.95% for losartan, and 99.94 ± 0.93% for spironolactone. Finally, the method was successfully applied in the simultaneous determination of atenolol, furosemide, losartan, and spironolactone in magisterial formulas, as well as in commercial pharmaceutical formulations.
The hydrodistilled essential oil from flowering aerial parts of Ocimum gratissimum L. (Lamiaceae) growing desolately in South India was examined to determine its composition. The oil was analyzed by gas chromatography equipped with flame ionization detector (GC–FID) and gas chromatography coupled with mass spectrometry (GC– MS). Forty-one constituents were identified, representing 99.4% of the total oil. The main components were identified as eugenol (57.1%), α-bulnesene (15.6%), and β-caryophyllene (14.2%). Phenylpropanoids (57.3%) and sesquiterpene hydrocarbons (31.6%) were the prominent groups of compounds, followed by oxygenated sesquiterpenes (6.5%), oxygenated monoterpenes (3.0%), and monoterpene hydrocarbons (1.0%). The compound α-bulnesene was identified for the first time in this report. The essential oil was found to be eugenol–α-bulnesene–β-caryophyllene chemotypes.
Authors:S.M. Nurulain, S. Ojha, S. Dhanasekaran, K. Kuča, N. Nalin, C. Sharma, A. Adem and H. Kalász
Distribution of K027, a hydrophilic, positively charged compound is monitored in the body of pregnant mice using high-performance liquid chromatography (HPLC). Intraperitoneal injection was done on the 18th day of pregnancy; the plasma and brains of the mother mice, placentae and the fetuses’ brains were dissected following 5, 15, 30, 60, and 120 min of treatment. Significant incorporation of K027 was found in the placentae and in fetuses’ brains relative to its levels in the mothers’ plasma and brains. This incorporation warns of a possible adjustment of dose of pyridinium aldoxime antidotes in case of pregnancy. Further studies with different gestational periods and animal models are warranted.
Authors:S.E. Kepekci Tekkeli, I. Gazioglu and M.V. Kiziltas
A new, sensitive, and selective high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of moxifloxacin (MOX) in human breast milk. MOX was precolumn derivatized with fluorescamine; the fluorescent derivative was separated on an RP C18 column using a mobile phase composed of acetonitrile–10 mM orthophosphoric acid by isocratic elution with flow rate of 0.5 mL min−1. The method was based on the measurement of the derivative using fluorescence detection at 481 nm with excitation at 351 nm. The calibration curve was linear over the range of 1–40 μg mL−1. Limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.3 and 1 μg mL−1, respectively. Intra-day and interday repeatabilities were less than 3.15%.
Authors:Gül Fidan Yenel Avci, Beril Anilanmert and Salih Cengiz
The analysis of trace levels of explosives in post-blast debris is critical in homeland security, environmental analysis, and crime scene forensic investigations. A fast and a selective determination method with high recovery was developed for the common explosives 2,4,6-trinitrotoluene (TNT), 3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil, using liquid chromatography—tandem mass spectrometry (LC—MS/MS). An easy and practical sample preparation method was developed using 4.00 mL acidified acetone with 0.25% HCl. After the easy evaporation of acetone extract, 10 min LC—MS/MS analysis provided a clear separation in column. Short duration of the whole procedure allows the use of this method in routine analysis. As a result of the analysis performed in spiked soils in 50.0, 100.0, and 250.0 ng g−1 concentrations, high recoveries such as 100.4 (±8.8)% for RDX, 96.9 (±10.5)% for HMX, and 97.6 (±13.9)% for TNT were obtained. Limit of detection (LOD) and limit of quantification (LOQ) values obtained from the analysis of the spiked soils were 4.3 ng g−1 and 7.00 ng g−1 for RDX, 6.8 ng g−1 and 10.0 ng g−1 for HMX, and 18.9 ng g−1 and 38.0 ng g−1 for TNT, respectively. The Horwitz Ratio (HorRat) calculation was used to evaluate if the inter-day and inter-analyst precisions were in the acceptable limits. The method was successfully applied to three artificial explosion samples for detection of explosives.
Selection of adsorbent for the development of purification process for biomolecules is crucial due to the requirement of large number of binding sites and adsorption area. Considering this, porous structure with high charge density is selected as an adsorbent for macromolecule purification. Such selection may provide high static binding capacity but causes loss of separation performance due to improper porosity of adsorbent in comparison to solute sizes involved. To address this problem for the screening of adsorbent, this work reports adsorbent selection procedure on the basis of adsorbent pore diameter (dp), solute hydrodynamic dimensions (RH), and flow velocity in support of binding capacity. Towards that end, this study evaluated the pore accessibility performance of varying characteristics adsorbents using tracers like acetone, lysozyme, and bovine serum albumin (BSA) by designing nonbinding conditions. All screened adsorbents showed certain loss of total surface area depending on the solute dimensions and pore size. Sepharose type adsorbents showed accessible area loss up to 25% for lysozyme and 50% for BSA. Sepabeads type showed 30% loss, while macroporous UNO type showed only 7% loss of surface area for lysozyme. The study correlates accessibility with size ratio β (dp/RH). The value of β > 38 is found to be required for the accessibility of total pore area and optimum separation performance of ion exchangers investigated. Accessibility and β provide useful information for the selection of suitable adsorbent for the purification of macromolecules.
Authors:Aida Begic, Ana Djuric, Borko Gobeljic, Ivana Stevanovic, Vera Lukic, Ivan Stanojevic, Milica Ninkovic, Luciano Saso, Danilo Vojvodic and Mirjana Djukic
The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC—UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG—GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01—200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision, accuracy (bias < 2%), and high reproducibility.
Through suitable sample handling, an overestimation of GSSG was prevented. High recovery (>99%) was achieved. The method was successfully applied for the analysis of GSH and GSSG in liver homogenates of Wistar rats intraperitoneally exposed to cadmium (Cd) (1 mg kg−1 CdCl2/21 days). Regardless of other Cd-mediated hepatotoxicity mechanisms, herein, we have exclusively interpreted/emphasized oxidative GSH depletion.
The presented method is acceptable for a routine analysis of GSH and GSSG in biological matrix, while the calculated ratio GSSG—GSH is considered as a valuable OS marker.
Authors:Gui-Hua Gu, Da-Jian Yang, Shi-Yun Wang, Wei Zeng, Wei Wang and Jing-Song Yuan
A high-performance liquid chromatography—diode-array detection method was developed and validated to determine simultaneously eleven major alkaloids in Corydalis decumbens (Thunb.) Pers. The alkaloids detected were corlumidine, protopine, coptisine, tetrahydrojatrorrhizine, palmatine, berberine, sanguinarine, papaverine hydrochloride, tetrahydropalmatine, bicuculline, and corydaline. Chromatographic separation was achieved using a C-18 column with a mobile phase composed of A (0.2% acetic acid solution, adjusted with triethylamine to pH 5.0) and B (acetonitrile), with stepwise gradient elution. Ultraviolet diode-array detection was used; chromatograms were examined at the wavelength of 280 nm. The regression equations showed a good linear relationship between the peak area of each marker and concentration (r = 0.9994–0.9999). The recovery values ranged between 93.66% and 100.54%. The method was fully validated with respect to detection and quantification limits, precision, reproducibility, and accuracy. The described high-performance liquid chromatography (HPLC) method was successfully used for the differentiation and quantification of the eleven major alkaloids in C. decumbens (Thunb.) Pers. and can be considered an effective procedure for the analyses of this important class of natural compounds.
Authors:Y. Zhou, Y. Yang, X.L. Li, Z.Y. Chen, Q.B. Liu, X.L. Zhu and J. Yang
An efficient and sensitive analytical method based on precolumn derivatization and gas chromatography—mass spectrometry—selected ion monitoring (GC—MS—SIM) was proposed and validated for analysis of two cembrenediols (CBDs) which are α-cembrenediol and β-cembrenediol in tobacco samples. CBDs in tobacco samples were extracted by sonication with 50 mL dichloromethane for 10 min before derivatized with 2:3 (v/v) bis(trimethylsilyl)trifluoroacetamide (BSTFA)—pyridine at 20 °C for 100 min. CBDs’ level in tobacco samples was analyzed by GC—MS—SIM and quantified by the internal standard method. The linear range for α-CBD and β-CBD was 13.6–554.6 μg mL−1 and 4.11–162.6 μg mL−1, and the correlation coefficients of both were 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) of α-cembrenediol and β-cembrenediol were 0.40 μg g−1 and 1.34 μg g−1, and 0.27 μg g−1 and 0.90 μg g−1, respectively. Average recoveries of α-CBD and β-CBD were 94.4–99.9% and 91.9–98.2% while the relative standard deviations (RSDs, n = 5) were ranged from 2.67 to 5.6% and 2.04 to 4.22%, respectively. This proposed analytical method has been successfully applied to analyze CBDs in tobacco samples.
Authors:Camila C. Pires, Moacir Kaiser, Lauren D. Grünspan, Fabiano Barreto, Adrine Innocente, Simone Gnoatto, João V. Laureano, Bibiana V. Araujo, Teresa Dalla Costa and Leandro Tasso
An accurate and reliable LC—MS/MS assay was firstly developed and validated for quantitative determination of a new antimalarial prototype drug, 3β-hydroxyurs-12-en-28-oic acid (LAFIS 01), in rat plasma. Dexamethasone was employed as internal standard. Simple protein precipitation by acetonitrile for the sample preparation was used. Effective separation was achieved with Phenomenex Luna C18 (50 × 2 mm, 5 μm) column. The mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.1% acetic acid, delivered by gradient elution. The column temperature was maintained at 40 °C. The LAFIS 01 was monitored by electrospray ionization interface, operating in the negative mode (ESI−) in multiple reactions monitoring (MRM), checking the transitions 455 > 455 for LAFIS 01 and 451 > 361 for the IS. Once LAFIS 01 demonstrated low fragmentation by collision-induced dissociation (CID) nonpresenting abundant high-intensity fragments to meet the desired concentration levels quantification, only pseudomolecular ion was monitored. The flow rate was 500 μL min−1. The lower limit of quantitation achieved was 10 ng mL−1 and linearity was observed from 10 to 500 ng mL−1. The relative standard deviation (RSD) values of the intra- and inter-assay precisions of the method were below 8.42 and 7.94%, respectively. The accuracy ranged from 92.05 to 102.94%. The extraction recovery of LAFIS 01 and IS was up to 90%. The method showed linearity, precision, accuracy, sensitivity, and stability required to quantify LAFIS 01 in preclinical pharmacokinetic study.
Authors:Narendra A. Gajbhiye, Jayanti Makasana, Tushar Dhanani and Raju Saravanan
Aegle marmelos Correa (Bael tree) is a medicinal fruit tree, widely used for healing purposes in various systems of medicines. Coumarins and alkaloids present in various parts of bael tree including roots and fruit pulp are the primary active constituents implicated for its biological activities. An efficient liquid chromatography–electrospray ionization—tandem mass spectrometry (LC—ESI—MS/MS) method was developed for identification and simultaneous determination of four coumarin derivatives, namely, umbelliferone, psoralene, marmin, and imperatorin, and an alkaloid, skimmianine, in root and stem bark of A. marmelos. The chromatographic separation of analytes was performed on Altima C18 (50 × 4.6 mm, 3 μm) column using methanol and 0.1% acetic acid in water (54:46, v/v) as the mobile phase under isocratic conditions. The LC–MS/MS parameters were optimized in the positive ionization mode using electrospray ionization source. The quantification of the analytes was performed using multiple reaction monitoring (MRM) transitions, umbelliferone (m/z 163.1 → 107.1), psoralene (m/z 187.2 → 131.1), marmin (m/z 333.5 → 163.2), imperatorin (m/z 271.1 → 203.1), and skimmianine (m/z 260.1 → 227.0). The extraction method was standardized for optimum yield of coumarin derivatives and the alkaloid in different extraction solvents. Higher yield of the analytes was found in methanolic extracts in comparisons to petroleum ether, chloroform, ethyl acetate, ethanol, and water. The method was validated for linear range, intra- and inter-batch precision and accuracy. The distribution of coumarin derivatives and an alkaloid was found to vary significantly in different plant samples, and their concentration was much higher in roots as compared to stem bark.
Authors:C.M.M. Silveira, C.M. Della Lucia, M.R. Pirozi, T.A. Montini and H.M. Pinheiro-Sant’Ana
This study aimed to optimize and validate methods for the analysis of thiamin and folic acid in fortified rice, pure and mixed to the milled rice (raw and cooked). The analysis was performed by high-performance liquid chromatography coupled to a diode array detector (HPLC—DAD). Different mobile phases were tested. Different ratios of organic modifier, pH ranges, triethylamine concentrations, and flow rates were used. For the validation, tests of recovery, repeatability, linearity, limit of detection (LOD), and limit of quantification (LOQ) were performed. The optimized methods showed good resolution of vitamins’ peaks, excellent recovery (82.6 to 104%), repeatability with relative standard deviation of peak areas, and retention times less than 10% and high coefficients of determination (0.9998 for thiamin and 0.9997 for folic acid). The LOD and LOQ were 0.00193 μg and 0.0193 μg for thiamin and 0.000934 μg and 0.00934 μg for folic acid. The optimized methods demonstrated reliability and sensitivity in the detection and quantification of these vitamins in fortified rice, pure and mixed to milled rice (raw and cooked). Furthermore, the methods were performed in isocratic mode, with short run time (<13 min), reflecting positively on the economy of reagents and analysis times.
Authors:P. Zhu, S. Wang, J. Wang, L. Zhou and P. Shi
In order to assess the contribution of adenosine triphosphate and its metabolites to the cellular metabolism process in Saccharomyces cerevisiae, it is very important to simultaneously determine the relative concentrations of ATP and its metabolites. In this study, a fast, simple reversed-phase high-performance liquid chromatography with high selectivity was developed to simultaneously measure adenosine triphosphate and its metabolites (adenosine diphosphate, adenosine monophosphate, and cyclic adenosine monophosphate) in yeast. The method was performed under the gradient grogram, and the detection was monitored at 254 nm. Analysis was achieved within 25 min. The four components can be detected with linear response over the concentration range from 1 to 100 mg L−1 with excellent correlation coefficients (r2) > 0.999. The recovery of the four analytes was 92.9%, 90.4%, 99.1%, and 105.1%, respectively. To demonstrate the good analysis of yeast samples, changes in the four adenine nucleotides levels caused by caloric restriction in yeast were determined. It is expected that the current method may contribute to further metabolomics and system biology investigations of yeast.
Authors:Y.T. Kamal, Mhaveer Singh, Shahana Salam and Sayeed Ahmad
The alternative system of medicines like Unani and Ayurveda is preferred worldwide nowadays due to its therapeutic efficacy, lower side effects, holistic approach, psychological dimensions, and qualitative action of weather and seasonal requirement. A simple procedure is described for the simultaneous extraction and estimation of piperlongumine and piperine in a well-known Unani polyherbal formulation using reversed-phase high-performance liquid chromatography (HPLC). The chromatography was carried out on reversed-phase C18 (250 × 4.6 mm) column with a mobile phase containing acetonitrile—water (50:50 v/v). Detection was accomplished with ultraviolet (UV) detection at λ = 325 nm. The flow rate was kept as 1.0 mL−1. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines for accuracy (94.4–105.0%), precision (0.37–2.17% RSD), and robustness (0.14–2.11% RSD). The limit of detection (LOD) values were found as 30 and 10 ng mL−1, while limit of quantification (LOQ) was 100 and 30 ng mL−1 for piperlongumine and piperine, respectively, which proved the sensitivity of the method satisfactory enough for accurate analysis of the both piperlongumine and piperine.
Authors:Ankita Misra, Amit Srivastava, Sharad Srivastava and A.K.S. Rawat
Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correlation coefficient of more than 0.9949. Relative standard deviation (RSD) (%), LOD, LOQ, and recovery (%) are within the acceptable limit. Besides that, methanolic extract shows the inhibition (%) range from 24.45 to 68.75% at 0.02–0.12 mg mL−1. IC50 of extract was observed at 46.75 μg mL−1, suggesting the promising activity in methanol extract. Hence, the proposed method for simultaneous quantification of five bioactive phenolics in the tuber of C. benghalensis using HPLC–PDA detection under the specified conditions is specific and accurate, and validation proves its selectivity and reproducibility.