Oasis MCX Research Articles

Analysis of Intact Glycosidic Aroma Precursors in Grapes by High-Performance Liquid Chromatography with a Diode Array Detector.

Nowadays, the techniques for the analysis of glycosidic precursors in grapes involve changes in the glycoside structure or it is necessary the use of very expensive analytical techniques. In this study, we describe for the first time an approach to analyse intact glycosidic aroma precursors in grapes by high-performance liquid chromatography with a diode array detector (HPLC-DAD), a simple and cheap analytical technique that could be used in wineries. Briefly, the skin of Muscat of Alexandria grapes was extracted using a microwave and purified using solid-phase extraction combining Oasis MCX and LiChrolut EN cartridges. In total, 20 compounds were selected by HPLC-DAD at 195 nm and taking as a reference the spectrum of phenyl β-D-glucopyranoside, whose DAD spectrum showed a first shoulder from 190 to 230 nm and a second around 200–360 nm. After that, these glycosidic compounds were identified by High-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-qTOF-MS). Disaccharides hexose pentose were the most abundant group observed with respect to the sugars and monoterpendiols the main aglycones found.

Development and validation of an automated solid-phase extraction-LC-MS/MS method for the bioanalysis of fluoxetine in human plasma.

A wide-range, specific, and precise liquid chromatography tandem mass spectrometric (LC-MS/MS)technique for quantifying fluoxetine (FLX) in human plasma was developed using the RapidTrace® automated solid-phase extraction (SPE) method; the analyte and internal standard (IS) were extricated on Oasis MCX SPE cartridges. Acetonitrile and 5 mM ammonium formate buffer (90:10 v/v) were used as mobile phase to achieve chromatographic separation on the reverse phase (C18 column). The analyte and IS were ionized using +ve electrospray ionization approach which was further traced by multiple-reaction monitoring on a tandem mass spectrometer. To quantify the FLX and FLX-d5, the parent-to-daughter ion transition of m/z of 310.0/44.1 and 315.0/44.0 was used, respectively. The method demonstrated a linear active limit of 0.20–30 ng/ml with recoveries ranging from 63.04% to 79.39% for quality control samples and 61.25% for IS samples. The concentrations over the calibration range demonstrated acceptable precision and accuracy. Due to the high inconsistency of the FLX concentration data, the minimum threshold of the assay was kept at 0.20 ng/ml. The flow rate was maintained at 500 μL/min, and the time for sample analysis for each injection was 3.5 min. The method was found to be specific, sensitive, and faster with minimum utilization of organic solvents and was utilized further for metabolic and pharmacokinetic studies.

Simultaneous determination of 11 illicit drugs and metabolites in wastewater by UPLC-MS/MS.

Assessing collective drug consumption based on the concentrations of illicit drugs and their metabolites in wastewater is a new technology. Currently, this technology is receiving attention in China, and methods for multiple illicit drug detection in wastewater are urgently needed. In our study, a method with a short runtime (7 min), a small solid-phase extraction (SPE) loading volume (50 mL) and high sensitivity (lower limits of quantitation (LLOQs) ranged from 0.2 to 5 ng/L) was developed for the simultaneous determination of amphetamines, ketamine, opiates, cocaine and their metabolites in wastewater. Samples were enriched by SPE on a mixed-mode sorbent (Oasis MCX) and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LODs) ranged from 0.1 to 2 ng/L, and the LLOQs varied between 0.2 and 5 ng/L. Moreover, the method developed was applied to real wastewater samples collected from 15 different wastewater treatment plants (WWTPs). In the results, the most abundant compounds were morphine (1.8-46.6 ng/L) and codeine (3.7-24.9 ng/L), which were detected in 13 WWTPs. After successful optimization of the UPLC-MS/MS conditions and sample loading pH, the method developed is able to meet the needs of common illicit drug monitoring and high-throughput analysis requirements.

Role of urinary cations in the etiology of interstitial cystitis: A multisite study.

To determine whether patients with interstitial cystitis have elevated levels of toxic urinary cations, to identify and quantify these cationic metabolites, and to assess their cytotoxicity. Isolation of cationic fraction was achieved by solid phase extraction using an Oasis MCX cartridge on urine specimens from interstitial cystitis patients and controls. C18 reverse phase high-performance liquid chromatography was used to profile cationic metabolites, and they were quantified by the area under the peaks and normalized to creatinine. Major cationic fraction peaks were identified by reverse phase high-performance liquid chromatography and liquid chromatography-mass spectrometry. HTB-4 urothelial cells were used to determine the cytotoxicity of cationic fraction and of individual metabolites. The reverse phase high-performance liquid chromatography analysis was carried out on cationic fraction metabolites isolated from urine samples of 70 interstitial cystitis patients and 34 controls. The mean for controls versus patients was 3.84 (standard error of the mean 0.20) versus 6.71 (0.37) mAU*min/µg creatinine, respectively (P=0.0001). The cationic fraction cytotoxicity normalized to creatinine for controls versus patients in mean percentage was -7.79% (standard error of the mean 3.32%) versus 20.03% (standard error of the mean 2.75%; P<0.0005). The major toxic cations were 1-methyladenosine, 1-methylguanine, N2 ,N2 -dimethylguanosine and L-tryptophan. These data confirm significant elevation of toxic cations in the urine of interstitial cystitis patients. These toxic cations likely represent a primary cause of interstitial cystitis, as they can injure the bladder mucus and initiate an epithelial leak.

Determination of antipsychotic drugs in nails and hair by liquid chromatography tandem mass spectrometry and evaluation of their incorporation into keratinized matrices

Nail samples are an alternative to hair for long-term monitoring of drug use, although there are a limited number of studies about its applicability. This study presents the development and validation of a LC–MS/MS method for the determination of five antipsychotic drugs (clozapine, haloperidol, levomepromazine, olanzapine and quetiapine) in nail and in hair samples. Samples were washed with dichloromethane, pulverized with a ball mill, and incubated in water:acetonitrile (50:50 v/v) with horizontal agitation for 90 min. Then, samples were purified by solid phase extraction with OASIS MCX cartridges and analysed by LC–MS/MS. The analytical method was fully validated in nails and in hair, including: limits of detection (2.5 pg/mg and 2.5−10 pg/mg, respectively), limits of quantification (LOQ) (10 pg/mg and 10−20 pg/mg, respectively), linearity (LOQ to 10,000 pg/mg), selectivity (no endogenous or exogenous interferences), accuracy (97.4–106.9% and 97.3–108.2%, respectively), imprecision (<7.9% and <8.6%, respectively), extraction efficiency (62.3%–109.8% and 45.1%–83.6%, respectively), matrix effect (-35.6% to 654.4% and -71.0% to -10.8%, respectively) and autosampler stability after 72 h (%loss <12.4%). Moreover, paired fingernail, toenail and hair samples from 13 patients under chronic treatment were analysed, and concentrations in the different matrices were compared. Concentrations in real samples ranged from 11.3–8306 pg/mg in fingernails, from 12.7–1755.4 pg/mg in toenails and from 17.6–24045.4 pg/mg in hair. Hair concentrations were generally higher than nail concentrations; however, a variable pattern was found in fingernails and toenails depending on the case. In addition, concentrations in paired hair and nail samples from a patient under chronic treatment with quetiapine at different doses were studied for 1-year.

Enantiomeric determination of cathinones in environmental water samples by liquid chromatography-high resolution mass spectrometry

The enantiomeric determination of chiral drugs in the environment is of emerging concern since their enantiomers often exhibit stereoselectivity in environmental occurrence, fate and toxicity. In this study a method based on solid-phase extraction followed by chiral liquid chromatography and high-resolution mass spectrometry has been developed for the enantiomeric determination of a group of cathinones in river water and effluent wastewater. The enantioseparation was carried out using a Chiralpak CBH column in reversed-phase mode, and optimised by evaluating the effects of flow rate, buffer concentration and organic modifier. Under optimal conditions, good enantioseparations (Rs ≥1.2) were achieved for all the analytes. Two mixed-mode cation-exchange sorbents (Oasis WCX and Oasis MCX) in solid-phase extraction were evaluated in river water. Oasis MCX sorbent showed better performance with apparent recoveries ranging from 57 to 91% and matrix effect ranging from -10 to 15%. It is worth noting that a shifting of retention times and loss of enantioresolutions in environmental water samples was observed for all the analytes when the Oasis WCX sorbent was used. The method was validated with river water and effluent wastewater samples and its overall performance was satisfactory. The method quantification limits for all the analyte enantiomers ranged from 1.0 to 2.9ng/L in river water, and from 2.3 to 6.0ng/L in effluent wastewater. The repeatability and reproducibility values, expressed as% relative standard deviation (n=5) were less than 15%. The method was then applied to the analysis of river water and effluent wastewater. The racemic methylone and methedrone (EF=0.49 and 0.46, respectively) were detected at low ng/L in some of the river water samples.

Analysis of valsartan, clindamycin and mesalamine in human plasma by LC–MS/MS using different extraction methodologies to overcome matrix effect

Interference of endogenous components present in biological fluids has a profound effect on the performance of LC–MS/MS-based assays. The present work evaluates the effect of plasma matrix components during sample preparation for accurate and precise determination of valsartan, clindamycin and mesalamine by LC–ESI–MS/MS. The methods were thoroughly assessed using different approaches for sample preparation such as solid-phase extraction (SPE) with different cartridges for valsartan, conventional protein precipitation and Phree phospholipid removal cartridge for clindamycin and liquid–liquid extraction (LLE) with and without derivatization for mesalamine to minimize matrix interference. The extent of matrix effect was determined by post-column analyte infusion, post-extraction spike method and by calculation of slope of calibration lines for different plasma sources. The influence of matrix ion suppression/enhancement on the extraction recovery and sensitivity of the methods was identified and quantified. The mean recovery obtained was 96.8% for valsartan using SPE on Oasis MCX, 92.2% for clindamycin using Phree phospholipid cartridge and 96.5% for mesalamine using LLE following derivatization. The intra-batch and inter-day precision (% CV) was ˂4.0 for the methods and was validated as per the current regulatory guidance.

Detection of azithromycin residue in broiler feathers by liquid chromatography-tandem mass spectrometry

In the study, a sensitive and reproducible method for the quantitative analysis of azithromycin in broiler feather samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Feather samples were rinsed after being wrapped in medical gauze, then chopped and then added to 5% (v/v) ammonia in methanol solution for ultrasonic extraction. The extract was purified by the combination of commercial polymeric microparticles (Oasis MCX) and Fe3O4 nanoparticles. The LC separation was performed on an Agilent Eclipse plus C18 column. Multiple reaction monitoring was used for the selective detection of azithromycin. The good linearity curve of azithromycin in feather sample was in the range from 1.0 μg kg−1 to 100.0 μg kg−1 with 0.9935 of correlation coefficient. And the limit detection and limit of quantification was 0.5 μg kg−1 and 2.0 μg kg−1 in spiked feather samples. The recoveries of azithromycin were 85.2–94.7% with the relative standard deviation less than 10%. The established method is simple, rapid, sensitive and specific, and could meet the need of government and enterprises to monitor the illegal use of azithromycin in livestock and poultry breeding.

A simpler potentiometric method for histamine assessment in blood sera.

Histamine intolerance results from a disequilibrium of accumulated histamine and the capacity for histamine degradation. An impaired histamine degradation based on reduced DAO activity and the resulting histamine excess may cause numerous symptoms mimicking an allergic reaction. For that, the determination of histamine in blood or in food products has great importance to identify risk factors. A new histamine-selective electrode is proposed using cucurbit[6]uril (CB[6]), as ionophore, in the analysis of biological samples. The selection of this smart supramolecular organic framework was based on its apparent stability constant of histamine-CB[6] (log β) of 4.33. The optimized electrode based on a polymeric membrane (PVC) combines the histamine-selective ionophore with 2-nitrophenyl octyl ether as solvent mediator and potassium tetrakis(4-chlorophenyl)borate as anionic additive. Furthermore, multi-walled carbon nanotubes particles were included in the membrane composition to partly lower the detection limit of the method, while improving stability and lowering the response drift (± 4mV). The electrodes showed a rapid response (≃ 13s) in the pH operational range of 2.7-5.4, with a Nernstian slope of 30.9 ± 1.2mV/dec, a detection limit of (3.00 ± 0.61) × 10-7mol/L, and a lower limit of the linear range of (3.00 ± 0.00) × 10-7mol/L. After miniaturization, the electrode was used as a detector in a sequential-injection lab-on-valve flow setup. The optimized flow conditions were achieved for sample injection volumes of 197μL propelled towards the cell under detection, at a flow rate of 30μL/s during 100s, making the analysis of 30 samples per hour possible. The developed system was used to analyze spiked blood serum samples previously cleaned by using solid-phase extraction. The sample pretreatment of the serum samples using Oasis MCX cartridges showed outstanding efficiency for histamine determination. The recovery values for three different levels of histamine concentration (1 × 10-4mol/L, 1 × 10-5mol/L, and 1 × 10-6mol/L) were (97 ± 6)%, (103 ± 1)%, and (118 ± 9)%, respectively, showing that this method was suitable for biological samples.

Solid-phase extraction based on cation-exchange sorbents followed by liquid chromatography high-resolution mass spectrometry to determine synthetic cathinones in urine

Cathinone derivates have become very popular as legal highs and are easily obtained on the market as “bath salts”, getting around the legislation due to their modified structure based on banned substances. The aim of this project is to evaluate two mixed-mode cation-exchange sorbents in solid-phase extraction (SPE) and their efficacy in pretreating urine samples for the determination of synthetic cathinones by liquid chromatography high-resolution mass spectrometry (LC-HRMS). A method for determining a group of eleven trending synthetic cathinones in urine using SPE followed by LC-HRMS using Orbitrap as analyser was developed and validated. In the extraction step, two different cation-exchange sorbents were evaluated and compared, a weak one (Oasis WCX) and a strong one (Oasis MCX). Better results were obtained for Oasis MCX in terms of recoveries and matrix effects (lower, if a clean-up step was applied). Method quantification limits were set at 0.2 ng mL−1 and method detection limits were between 0.04 and 0.16 ng mL−1. The results obtained enabled the studied compounds to be quantified at the usual levels at which they are present in urine samples. Moreover, these limits were lower than the ones found in the literature using similar extraction strategies, which shows that the reported strategy can be a useful tool in forensic and toxicological analyses.

A LC-MS/MS method for the determination of common synthetic cathinones in meconium

New psychoactive substances have been introduced into the market in the last years due to their unregulated status. Synthetic cathinones are one of their main representatives, and they have shown to produce neonatal complications. It is important to have objective tools to identify in utero exposure to drugs that have shown to produce neonatal complications. An analytical method was developed and fully validated for the determination of common synthetic cathinones, including methylone, methedrone, mephedrone, 3,4-methylenedioxypyrovalerone (MDPV), (±)-4-fluoromethamphetamine and 4-fluoromethcathinone in meconium. Meconium (0.25 ± 0.02 g) was homogenized with methanol by sonication for 30 min. After centrifugation, the sample was extracted with Oasis MCX columns. The analysis was performed by LC-MS/MS using an Atlantis T3 column (3 μm, 2.1 × 50 mm) and a gradient with acetonitrile and 0.1% formic acid in water. Method validation included the following parameters: selectivity (no endogenous or exogenous interferences), limits of detection (n = 3, 0.5–1 ng/g) and quantification (n = 3, 1–2 ng/g), linearity (n = 5, LOQ-200 ng/g), imprecision (n = 15, 0% to 10%), accuracy (n = 15, 87.3% to 97.8%), matrix effect (n = 10, −76% to −28.1%), extraction efficiency (n = 6, 63.7% to 91.3%), total process efficiency (n = 6, 16% to 60.2%) and stability for 72 h in the autosampler (n = 3, %loss = −6.7% to 5.1%). The method was applied to 28 meconium specimens.

Development and application of the analytical method for illicit drugs and metabolites in fish tissues

In this study, we developed a screening method for highly accurate and sensitive analysis of 12 illicit drugs and metabolites in fish tissues. The approach was based on ultrasonic-assisted extraction and solid phase extraction, followed by ultra-performance liquid chromatography-tandem mass spectrometry analysis. The homogenized samples were ultrasonic-assisted extracted with 1% acetic acid in methanol followed by SPE cleanup with Oasis MCX cartridge. The method was validated in accordance with the European Medicines Agency guidelines by evaluating the following required parameters: the limits of detection and limits of quantification, linearity, accuracy, repeatability, recovery of extraction, and matrix effect. For the tissues involved in this study, the recoveries ranged from 60% to 127%, and the matrix effect ranged from −19% to 83% with the inter- and intra-day variability below 12%. The method has been successfully applied to wild fish caught from six sampling sites of four urban rivers in Beijing, China. The analysis showed that the target compounds, including amphetamine, 3,4-methylenedioxyamphetamine, codeine and heroin were detected with high detection frequencies. Codeine exhibited the highest bioaccumulation factor (up to 73,986) in the muscle of Crucian carp, while ketamine tended to accumulate in the skin, 3,4-methylenedioxyamphetamine and heroin tended to accumulate in gastrointestinal tract, respectively.

Skin exposure: requirements to methods determining active ingredient of pesticides in washings

The article presents results of studies exemplified by diquat on analysis concerning influence of lower limit value of quantitative assessment in washing sample for safety coefficient in exposure and in absorbed dose, if acting substance is absent in workplace ambient air samples and in dermal washings of workers. To control diquat in dermal washings, there is a method based on ion-pair liquid chromatography with ultraviolet detection (working wavelength 310 nm). To concentrate sample, cartridges for solid-phase extraction, containing ion exchange sorbent (Oasis MCX 6cc/500 mg), are used. Lower limit of assessment in washing sample — 0,15 micrograms. Experimentally set washing completeness is within range of 80–92%, standard deviation of repetition is 7,0% at most. The method created was tested in nature studies determining dermal exposure in workers subjected to 5 various preparations based on diquat dibromide when used for surface spraying from tractor and from aircraft. For lower limit of detection in washing sample (0,15 micrograms/washing), calculated risk value of exposure varied within 0,26–0,36; risk of absorbed dose was low — 0,23 (the allowable one ≤1). Findings are that present measuring methods which provide lower limit of detection 1 and 5 micrograms in washing sample could result in unallowable risk establishment even with absence of the substance in all samples of workplace air and dermal washings. The calculation formula suggested enables to give theoretic basis for requirements to lower limit of detecting active substances in dermal washing samples for evaluating risk of pesticides use in agriculture.

Design of selective molecularly imprinted sorbent for the optimized solid-phase extraction of S-pramipexole from the model multicomponent sample of human urine.

The objective of this article was to design the selective molecularly imprinted sorbent dedicated to the solid-phase extraction of S-pramipexole from the complex matrix such as human urine. For that purpose, S-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole was used as the template acting as the structural analog of S-pramipexole and five various monomers were employed in the presence of ethylene glycol dimethacrylate to produce molecularly imprinted polymers. The binding capabilities of resulted polymers revealed that the highest imprinting effect was noted for polymer prepared from the itaconic acid. The comprehensive analysis of morphology and the characterization of binding sites showed not only negligible differences in the extension of surfaces of imprinted and nonimprinted polymers but also higher heterogeneity of binding sites in the imprinted material. Comprehensive optimization of the molecularly imprinted solid-phase extraction allowed to select the most appropriate solvents for loading, washing, and elution steps. Subsequent optimization of mass of sorbent and volumes of solvents allowed to achieve satisfactory total recoveries of S-pramipexole from the model multicomponent real sample of human urine that equals to 91.8 ± 3.2% for imprinted sorbent with comparison to only 37.1 ± 1.1% for Oasis MCX.

Total determination of triclabendazole and its metabolites in bovine tissues using liquid chromatography-tandem mass spectrometry

A reliable LC-MS/MS analytical method for the determination of residual triclabendazole and its principal metabolites (triclabendazole sulfoxide, triclabendazole sulfone and keto-triclabendazole) in bovine tissues was developed, in which triclabendazole and its metabolites are oxidized to keto-triclabendazole as a marker residue. The method involves sample digestion with hot sodium hydroxide, thus releasing the bound residues of various triclabendazole metabolites in bovine tissues. The target compounds are extracted from the digest mixture with ethyl acetate, defatted by liquid-liquid partitioning using n-hexane and acetonitrile, then oxidized with hydrogen peroxide in a mixture of ethanol and acetic acid. The reaction mixture is cleaned up using a strong cation exchange cartridge (Oasis MCX) and the analytes are quantified using LC-MS/MS. The optimal conditions for the complete oxidation of triclabendazole and its metabolites to keto-triclabendazole are an incubation time of 16 h and a temperature of 90 °C. The developed method was evaluated using three bovine samples: muscle, fat, and liver. Samples were spiked with triclabendazole and its principal metabolites at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The validation results show excellent recoveries (81–102%) and precision (<10%) for all target compounds. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. These results suggest the developed method is applicable to quantifying residual triclabendazole in bovine tissues in compliance with the MRLs established by the Codex Alimentarius and EU and Japanese regulations, and thus the proposed method will be a useful tool for the regulatory monitoring of residual triclabendazole and its metabolites.

Determination of 11 sulfonamides in pork by two-step liquid-liquid extraction-solid phase extraction purification coupled with high performance liquid chromatography-tandem mass spectrometry

A method based on two-step liquid-liquid extraction-solid phase extraction purification coupled with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of 11 common sulfonamides in pork. The samples were extracted first with ethyl acetate containing 2%(v/v) formic acid and then with acetone for the second step. The supernatant was degreased with n-hexane and cleaned using an Oasis MCX SPE column. Finally, nitrogen was blown over the obtained mixture, and the mixture was filtered for conducting HPLC-MS/MS analysis. The sulfonamides were detected in the multiple reaction monitoring positive ion mode, and quantification was performed by employing the external standard method with matrix calibration curves. All the 11 sulfonamides demonstrated good linearity in the range of 20-400 μg/L, with the correlation coefficient (r2) higher than 0.99. The limits of detection (LODs; S/N=3) and limits of quantification (LOQs; S/N=10) were found to be in the ranges of 0.1-1.0 μg/kg and 0.2-3.0 μg/kg, respectively. Furthermore, the results indicated that the average recoveries of the 11 sulfonamides ranged from 79.3% to 105.5% at three spiked levels of 50, 100, and 200 μg/kg, with the relative standard deviations (RSDs) in the range of 1.3%-11.6% (n=6). This method showed higher extraction efficiency than the one-step liquid-liquid extraction, and the detection sensitivity was highly improved.

Determination of Flunixin in Swine Plasma, Urine and Feces by UPLCMS/ MS and its Application in the Real Samples

Background: Flunixin is a Non-Steroidal Anti-Inflammatory Drug (NSAID), because it can effectively alleviate the organism of pyrexia, inflammation and pain, it has been widely used in veterinary practice. In order to better study flunixin in the body's absorbed, distributed, metabolized and excreted, our team developed a UPLC-MS/MS method for determination of flunixin in swine plasma, urine and feces. Methods: Flunixin was extracted from the sample by liquid-liquid extraction and cleaned-up using a mixed-mode Oasis MCX solid-phase extraction column. Analysis was performed on UPLC-MS/MS operating in Multiple Reaction Monitoring (MRM) mode. Internal standard was used for quantitation of target drug. Results: Recoveries of fortified samples ranged from 90.2% to 101.4%, with Relative Standard Deviations (RSD) lower than 17.0%. The Limits Of Detection (LODs) and Quantification (LOQs) in plasma were 0.25 and 0.5 µg L-1, in urine were 0.25 and 0.5 µg L-1, and in feces were 0.5 and 1 µg kg-1, respectively. This validated method was successfully applied to the determination of flunixin in real samples. The half-life of flunixin after the last dose in pigs was 7.37±1.60 h after intramuscular administration of 2.2 mg/kg of flunixin, and approximately 6.8% and 1.9% of the administered dose was excreted as parent compound in urine and feces respectively. Conclusion: The developed UPLC-MS/MS method for determination of flunixin in swine plasma, urine and feces was validated and successfully applied to monitor flunixin from real samples.

Mixed-mode solid phase extraction combined with LC-MS/MS for determination of empagliflozin and linagliptin in human plasma

A selective, sensitive, precise and accurate liquid chromatography-tandem mass spectrometry method was developed and validated for the concurrent determination of antidiabetic drugs, empagliflozin and linagliptin in human plasma. Sample preparation was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries like hydrophilic-lipophilic balance (Oasis HLB), mixed-mode cation exchange (Oasis MCX) and weak-cation exchange (Oasis WCX). SPE conditions like sample pH, washing and elution solvents were suitably optimized. Best results were obtained using Oasis MCX cartridges in terms of extraction recovery (78–88%) and matrix effects (~3.0%) for both the analytes. Chromatographic conditions for the separation of analytes and their labeled internal standards (ISs) were established on XSelect HSS Cyano (50 × 2.1 mm, 3.5 μm) column using 2 mM ammonium acetate buffer and acetonitrile as the mobile phase. Detection of analytes was achieved using electrospray ionization in the positive mode. For quantitative analysis, multiple reaction monitoring transitions were m/z 451.3 → 71.1 for empagliflozin and m/z 473.2 → 420.2 for linagliptin. Standard curve concentrations were validated in the range of 1.50–1500 ng/mL for empagliflozin and 0.015–15.0 ng/mL for linagliptin. The intra-batch and inter-batch precision (% CV) was <3.7 for both the drugs. The stability of the analytes established under different storage conditions was found to be appropriate for routine laboratory practices. The validated method was used to study the pharmacokinetics of the drugs using a single oral dose of fixed-dose combination tablet consisting of 25 mg empagliflozin and 5 mg linagliptin in healthy subjects.

Optimization and Validation of the Analytical Method to Detect New Psychoactive Substances in Wastewater

New psychoactive substances (NPS) are emerging continuously, as the crackdown against traditional drugs becomes stricter. Metabolites of NPSs and the unchanged drugs enter wastewater through urine and are collected and treated by wastewater treatment plants before being discharged into the aquatic environment. Based on previous research, solid phase extraction (SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) were used and the detection method of 11 NPSs was optimized. Influences of wastewater pretreatment procedures, including SPE columns; pH values of water samples; and processes of flushing and redissolving on NPS recoveries were compared. It was shown that the best recovery was achieved when Oasis MCX columns were used and the pH value of wastewater samples was adjusted to 2. A flushing process with 2 mL water at pH=2 followed by 2 mL methanol was needed. Redissolution of the residue after evaporation was best achieved with 400 μL of a 20% methanol water solution. It was indicated by retention times, recoveries, matrix effects, limits of detection, limits of quantification, as well as precision that C18-UPLC-MS/MS and the optimized method are efficient and valid. Influent and effluent samples from 11 municipal wastewater treatment plants in Beijing were analyzed using the optimized method for validation. The validated method can be used as an important reference for monitoring NPSs via wastewater-based epidemiology and for assessing the risk of NPS abuse in China.

MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS

Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture.Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4-D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC).Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%±1.9, 80.60%±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealed its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C. albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis.Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens.