Validation of a simple HPLC method to quantify methotrexate concentrations in human plasma

Methotrexate (MTX) is a chemotherapy and immunosuppressive agent widely used to treat cancer, autoimmune diseases in children and adult patients, and ectopic pregnancy. However, MTX is highly toxic to the liver, kidney, and nervous system. This study aimed to quantify the concentration of MTX in human plasma using high-performance liquid chromatography (HPLC). MTX and its internal standard (para aminoacetophenone-PAPA) in plasma samples were extracted simultaneously with methanol. Sample purity was performed using the 1 cc OASIS HLB cartridges. Sample injection volume of 10 µL was analyzed on a Lichrocart Supersil 125-4 column C18 maintained at 40 °C on a Waters 2695 XE equipped with a PDA detector set at 303 nm. The mobile phase contained phosphate buffer (pH 6.0) and methanol at a ratio of 80:20 (v/v) and was maintained at a flow rate of 1 ml/min. The results showed that the total time of chromatographic analysis was 15 min. MTX and PAAP were found in the chromatograms at retention times of 2.3 and 5.2 min, respectively. The linear range of the MTX from 0.5 to 25 µg/mL. Intra-day and inter-day imprecision for MTX ranged from 3.42 to 8.128%. LLOQ of MTX was 0.5 µg/mL and the extraction effects were above 77%. In conclusion, we developed and validated a simple HPLC method to determine the MTX concentrations in human plasma.

Lab-In-Syringe with Bead Injection Coupled Online to High-Performance Liquid Chromatography as Versatile Tool for Determination of Nonsteroidal Anti-Inflammatory Drugs in Surface Waters

We report on the hyphenation of the modern flow techniques Lab-In-Syringe and Lab-On-Valve for automated sample preparation coupled online with high-performance liquid chromatography. Adopting the bead injection concept on the Lab-On-Valve platform, the on-demand, renewable, solid-phase extraction of five nonsteroidal anti-inflammatory drugs, namely ketoprofen, naproxen, flurbiprofen, diclofenac, and ibuprofen, was carried out as a proof-of-concept. In-syringe mixing of the sample with buffer and standards allowed straightforward pre-load sample modification for the preconcentration of large sample volumes. Packing of ca. 4.4 mg microSPE columns from Oasis HLB® sorbent slurry was performed for each sample analysis using a simple microcolumn adapted to the Lab-On-Valve manifold to achieve low backpressure during loading. Eluted analytes were injected into online coupled HPLC with subsequent separation on a Symmetry C18 column in isocratic mode. The optimized method was highly reproducible, with RSD values of 3.2% to 7.6% on 20 µg L−1 level. Linearity was confirmed up to 200 µg L−1 and LOD values were between 0.06 and 1.98 µg L−1. Recovery factors between 91 and 109% were obtained in the analysis of spiked surface water samples.

Characterization of the Permeation Properties of Membrane Filters and Sorption Properties of Sorbents Used for Polar Organic Chemical Integrative Samplers

<p>Polar organic chemical integrative samplers (POCIS) are promising devices for measuring time-weighted average concentrations of hydrophilic compounds in aquatic environments. However, the mechanism of POCIS uptake remains unclear. This study characterizes the permeation properties of polyethersulfone and polytetrafluoroethylene and the sorption properties of Oasis HLB (Waters), Envi-Carb (Supelco), and Oasis WAX (Waters) under identical conditions via calibration experiments of the POCIS. Plant protection products, neonicotinoid herbicides, and linear alkyl benzene sulfonates (LAS) were tested. The permeation experimental results suggested that the penetration rate constants (<i>k</i>_m) with high <i>K</i>_ow values (<i>K</i>_ow: octanol–water partition coefficient) were low, which indicated that the sorption of chemicals on the membrane may constitute a limiting factor for the permeation. The sorption experiments indicated that the sorption rate constants (<i>k</i>_s) depended on the type of sorbent instead of the <i>K</i>_ow values. Low <i>k</i>_s/<i>k</i>_m ratios were obtained for chemicals with low <i>K</i>_ow values, which implied that the POCIS uptake for highly hydrophilic compounds was controlled by both membrane and sorbent kinetics. The <i>k</i>_m and <i>k</i>_s values corresponded to the model and predicted the values of the sampling rates<i> </i>(<i>R</i>_s) for the LAS. These findings revealed the possibility of using <i>k</i>_s and <i>k</i>_m values to predict <i>R</i>_s values.</p>

Characterization of the Permeation Properties of Membrane Filters and Sorption Properties of Sorbents Used for Polar Organic Chemical Integrative Samplers

<p>Polar organic chemical integrative samplers (POCIS) are promising devices for measuring time-weighted average concentrations of hydrophilic compounds in aquatic environments. However, the mechanism of POCIS uptake remains unclear. This study characterizes the permeation properties of polyethersulfone and polytetrafluoroethylene and the sorption properties of Oasis HLB (Waters), Envi-Carb (Supelco), and Oasis WAX (Waters) under identical conditions via calibration experiments of the POCIS. Plant protection products, neonicotinoid herbicides, and linear alkyl benzene sulfonates (LAS) were tested. The permeation experimental results suggested that the penetration rate constants (<i>k</i>_m) with high <i>K</i>_ow values (<i>K</i>_ow: octanol–water partition coefficient) were low, which indicated that the sorption of chemicals on the membrane may constitute a limiting factor for the permeation. The sorption experiments indicated that the sorption rate constants (<i>k</i>_s) depended on the type of sorbent instead of the <i>K</i>_ow values. Low <i>k</i>_s/<i>k</i>_m ratios were obtained for chemicals with low <i>K</i>_ow values, which implied that the POCIS uptake for highly hydrophilic compounds was controlled by both membrane and sorbent kinetics. The <i>k</i>_m and <i>k</i>_s values corresponded to the model and predicted the values of the sampling rates<i> </i>(<i>R</i>_s) for the LAS. These findings revealed the possibility of using <i>k</i>_s and <i>k</i>_m values to predict <i>R</i>_s values.</p>

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF–MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Determination of Pharmaceuticals Discharged in Wastewater from a Public Hospital Using LC-MS/MS Technique

The presence of pharmaceuticals classified as emerging contaminants (EC) in surface water, groundwater, and drinking water generates uncertainty concerning the interactions that could be occurred with aquatic organisms and living beings. Thus, the monitoring of hospital wastewater is of great importance to identify the main classes of pharmaceuticals that could be discharged to the municipal sewage system and wastewater treatment plants (WWTPs). This work described the implementation and validation of a highly selective and sensitive analytical method using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the detection and quantification of these emerging compounds. The solid-phase extraction (SPE) method was employed using Oasis HLB cartridges. For LC-MS/MS analysis, the chromatographic separation was conducted in a C-18 Kinetex column (Phenomenex), and detection was achieved in an AB SCIEX QTrap 3200 tandem mass spectrometer (MS/MS) in the multiple reaction monitoring (MRM) mode. The quantitative analysis was performed by using the internal standard (IS) method with isotopically labeled analogs. The implemented method presented good linearity within the concentration range of 0.1–400 µg L-1 showing correlation coefficients (R2) ranged from 0.991 to 0.999. The limits of detection (LODs) were from 0.02 to 0.59 µg L-1, while the limits of quantification (LOQs) ranged from 0.07 to 1.80 µg L-1. The analytical method was successfully applied to the analysis of wastewater samples discharged by a public hospital in San Nicolas de los Garza, Nuevo Leon, Mexico, in two sampling periods: May 2017 and March 2018. Resumen. La presencia de fármacos, clasificados como contaminantes emergentes, en agua superficial, subterránea y potable, genera incertidumbre sobre las interacciones que podrían ocurrir con organismos acuáticos y los seres vivos. Por lo tanto, el monitoreo del agua residual hospitalaria es de gran importancia para identificar los principales productos farmacéuticos que podrían descargarse al sistema de alcantarillado municipal y por lo tanto, estar presentes en el agua a tratar en las plantas de tratamiento de agua residual (PTAR). Este trabajo describe la implementación y validación de un método analítico altamente selectivo y sensible utilizando extracción en fase sólida (SPE) y cromatografía líquida acoplada a espectrometría de masas tándem (LC-MS/MS) para la detección y cuantificación de estos compuestos emergentes. Se empleó el método de extracción en fase sólida utilizando cartuchos Oasis HLB. Para el análisis LC-MS/MS, la separación cromatográfica se realizó en una columna Kinetex C-18 (Phenomenex), y la detección se realizó en un espectrómetro de masas en tándem AB SCIEX QTrap 3200 (MS/MS) en el modo de monitoreo de reacciones múltiples (MRM). El análisis cuantitativo se llevó a cabo utilizando el método de estándar interno (IS) con análogos marcados isotópicamente. El método presentó una buena linealidad dentro del rango de concentración de 0.1 a 400 µg L-1, con coeficientes de correlación (R2) que oscilaron entre 0.991 y 0.999. Los límites de detección (LOD) fueron de 0.02 a 0.59 µg L-1, mientras que los límites de cuantificación (LOQ) variaron de 0.07 a 1.80 µg L-1. El método analítico se aplicó con éxito al análisis de muestras de agua residual vertidas por un hospital público de San Nicolás de los Garza, Nuevo León, México, en dos períodos de muestreo: mayo de 2017 y marzo de 2018.

Идентификация и определение биологически активных веществ шалфея лекарственного Salvia officinalis L., полученных в условиях субкритической экстракции

Объектами исследования были листья шалфея лекарственного (Salvia officinalis L.) из серии«Зеленая аптека», г. Горячий Ключ, Травы Кавказа. Работа посвящена изучению возможности использования альтернативного способа извлечения, идентификации и определения биологически активных веществ из лекарственных трав под воздействием субкритической экстракции. Целью исследований является компонентный состав биологически активных веществ шалфея лекарственного, образующийся в условиях субкритической экстракции. Субкритическая экстракция биологически активных веществ из шалфея лекарственного (Salvia officinalis L.) проводилась с использованием деионизированной воды и 70% раствора этилового спирта в температурном диапазоне от 100 до 250°С. Хроматографическое определение аналитов в экстракте шалфея лекарственного проводили методом газовой и жидкостной хромато-масс-спектрометрии в сочетании с техникой твердофазного концентрирования. Идентификацию аналитов осуществляли с использованием стандартных образцов и индивидуальных соединений. Проведена оптимизация условий субкритической и твердофазной экстракции компонентов эфирных масел и фенольных соединений из экстракта шалфея лекарственного. Оптимальный выход эфирномасличных компонентов из шалфея лекарственного достигается при 200оС, дальнейшее повышение температуры приводит к деградации исследуемых соединений и снижению эффективности их извлечения. Аналиты переводили в органическую фазу твердофазной экстракцией с использованием сорбентов «Strata C18-E», «Strata X», «Oasis HLB». Эфирномасличные и фенольные компоненты в органической фазе получены при субкритической экстракции в условиях повышенных температуры и давлении. Идентифицированный компонентный состав экстракта, образующийся в условиях субкритической экстракции, существенно отличается от веществ, полученных традиционным гидродистилляционным способом. В составе шалфея лекарственного идентифицированы 52 эфирномасличных соединения. Субкритическая экстракция позволила в отличие от гидродистилляционного способа одновременно извлекать компоненты фенольной природы: коричные кислоты, флавоноиды, антоцианы, а также дитерпеновые химические соединения. Достоинством исследуемого способа является возможность одновременного концентрирования и определения эфирномасличных и фенольных компонентов. Результаты данного исследования представляют несомненный интерес для специалистов в области химии лекарственного растительного сырья.

Optimization of SPE method for the extraction of 12 neurotransmitters from sheep brain

The present paper presents our attempts concerning the development of an extraction method for catecholamines. In order to achieve the extraction of all the selected solutes using a single SPE cartridge, several types of support were tested, among them: cation exchange supports, hydrophilic-lipophilic supports, C18 supports and PGC supports. As unfortunately none of the supports tested offered us the possibility of carrying out the extraction of 12 catecholamines from our standard mixture, we chose to use a coupling of two different cartridges: Oasis HLB and PGC which together ensure the extraction of all the compounds of the mixture with good extraction yields and with simple protocols. The selected cartridges were successfully tested for the extraction of a sample spiked from sheep brain with the 12 catecholamines in our mixture. The SPE method that we have developed allows the purification of the samples (a significant part of the components of the matrix is eliminated during this step) and also a preconcentration of the samples.

Simultaneous determination of steviol glycosides in stevia by high performance liquid chromatography

A method for determination of steviol glucosides in stevia using high performance liquid chromatography was developed and validated. The compounds were extracted from the matrices with methanol for 60 minutes, cleaned by Oasis HLB column and then determined by HPLC using C18 column (250 mm x 4.6 mm x 5 &micro;m) and PDA detector. The calibration curves were linear in range of 1 to 100 &mu;g/mL; the RSD was of 1.39 &shy; 2.85%; and the recovery was of 93 &shy; 105%. The method was applied to analyze 09 stevia samples collected from markets in Hanoi (including dry stevia rebaudiana, stevia powder). The results showed that composition of steviol glycosides differs from sample to sample. Besides, stevioside and rebaudioside A are the most abundant steviol glycosides in the samples.