On-line Solid-phase Extraction Column Research Articles

Extension of the Temporal Window for the Determination of Alpha-Methylthiofentanyl and Thiofentanyl in Rat Urine by Monitoring the Metabolite Norfentanyl Using Online Solid-Phase Extraction (SPE) Coupled with Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS)

Alpha-methylthiofentanyl and thiofentanyl are psychoactive substances that have been abused in recent years, strongly threatening social and public security. Because they are rapidly metabolized in vivo, their determination presents a great challenge for forensic science. An automated method of online solid-phase extraction coupled with ultra-high performance liquid chromatography—tandem mass spectrometry was developed for the determination of alpha-methylthiofentanyl, thiofentanyl, and the metabolite norfentanyl in urine. The analytes were first enriched and purified by an Oasis HLB online SPE column and eluted on a Hypersil GOLD C18 analytical column. Chromatographic separation was achieved with mobile phase components of 2 mM ammonium formate and 0.1% formic acid in water and same composition in methanol in gradient elution mode. The limit of detection and lower limit of quantitation of the method for the three analytes were 0.2 and 2 pg/ml, respectively. Acceptable linearity was obtained from 2 to 800 pg/ml. The accuracy was in the range of 87.98–108.95%; and precision, calculated as the relative standard deviation, was less than 14.63%. The method recoveries were from 65.56 to 113.72%. This highly sensitive method was applied to study the detection window of the precursor compounds and metabolites in rat urine. Both alpha-methylthiofentanyl and thiofentanyl are metabolized to norfentanyl, which was first revealed in this study. With the aid of norfentanyl, the detection windows of alpha-methylthiofentanyl and thiofentanyl were extended to 18–26 days and 20–27 days, respectively. This study provides important auxiliary tools for the forensic identification of fentanyl analogs.

Simultaneous Determination of Five Hormones in Milk by Automated Online Solid-Phase Extraction Coupled to High-Performance Liquid Chromatography.

Many hormones show the effects of protein assimilation and growth promotion, and they are frequently used as veterinary drugs in livestock, which has harmful effects on human health. It is necessary to determine their contamination level in animal-derived food, especially in milk. In this study, a detailed procedure is described for an automated online solid-phase extraction (SPE)-HPLC method capable of detecting five hormones (i.e., estriol, prednisone acetate, hydrocortisone, diethylstilbestrol, and estrone) in cow milk. The corresponding milk samples were precipitated by addition of acetonitrile and then purified as well as enriched by a polar-enhanced polymer (PEP) online SPE column. The supernatants were directly injected into the online SPE-HPLC system using methanol-water as the mobile phase mixture. The linearity range of the method was 0.1-25 μg/mL for prednisone acetate, hydrocortisone, and diethylstilbestrol, 0.2-25 μg/mL for estriol, and 0.5-25 μg/mL for estrone, with correlation coefficients (r) ranging from 0.9994 to 0.9996. The recovery rates determined at three concentration levels for the five compounds were in the range of 70.82-112.90%. LODs of estriol, prednisone acetate, hydrocortisone, diethylstilbestrol, and estrone were 0.023, 0.005, 0.006, 0.004, and 0.054 μg/mL, respectively. This automated online SPE-HPLC method was both effective and reliable in the simultaneous measurement of five hormones, and the method was successfully applied to the detection of five hormone species in milk. An automated online SPE-HPLC method has been developed for the analysis of five hormones in cow milk. Online SPE proved to be a powerful technique for determining five hormones simultaneously. This method ensured simple sample pretreatment and less operation time. The established method was successfully applied to the analysis of five hormone species in milk.

Development of an SPE Method Using Ionic Liquid-Based Monolithic Column for On-Line Cleanup of Human Plasma and Simultaneous Determination of Five Steroid Drugs

An 1-vinyl-3-hexyl imidazolium bromide ionic liquid-based monolithic column was prepared via in situ free radical polymerization, which was used as the sorbent of on-line solid phase extraction. The prepared material was characterized by scanning electron microscopy, nitrogen adsorption–desorption instrument and mercury intrusion porosimetry, respectively, and the results showed that the homemade monolith occupied relatively uniform porous structure and high-specific surface area of 212.53 m2 g−1. On-line solid phase extraction–high-performance liquid chromatography was performed to quantitatively analyze five steroid drugs, including betamethasone, norgestrel, halcinonide, beclomethasone dipropionate and testosterone propionate in human plasma. The determination procedure was carried out following the optimization of the mobile phase composition. Methodological validation showed that correlation coefficients of the linear regressions were in the range of 0.9989–0.9992; the values of relative standard deviation for precision were in the range of 1.39–6.15% for intra-day and 0.32–6.34% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 99.02–101.98, 93.27–102.89, 102.79–104.01, 100.04–102.61 and 100.77–102.44% for the five drugs in order, respectively; the values of relative standard deviation for repeatability calculated according to retention times and peak areas of the five drugs were in the ranges of 0.28–0.76% (n = 5) and 0.84–2.63% (n = 5), respectively. The results showed that the polymer monolithic column was feasible for an on-line solid phase extraction column, exhibiting good selectivity and high permeability.

Simultaneous determination of metolazone and valsartan in plasma by on-line SPE coupled with liquid chromatography/tandem mass spectrometry.

Combination of metolazone (0.5 mg) and valsartan (80 mg) has been verified as a promising therapy treatment for hypertension. In order to facilitate to pharmacokinetic research, it needs a method for the simultaneously determination of metolazone and valsartan in biological samples. However, there are no relative reports so far. In order to facilitate to pharmacokinetic research, an on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry method for the simultaneous determination of metolazone and valsartan in beagle dog plasma was developed and validated in this study. An on-line solid phase extraction column Retain PEP Javelin (10 mm × 2.1 mm) was used to remove impurities in plasma samples. The metolazone, valsartan and internal standard (losartan) were separated on a Poroshell 120 SB-C18 column (4.6 mm × 50 mm × 2.7 µm) with a gradient elution procedure. Acidified acetonitrile/water mixture was used as a mobile phase. The selected multiple-reaction monitoring mode in positive ion was performed and the parent to the product transitions m/z 366/259, m/z 436.2/291 and m/z 423.4/207 were used to measure the metolazone, valsartan and losartan. The method was linear over the range of 0.1-100 ng/mL and 1-1000 ng/mL for metolazone and valsartan, respectively. This method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability and then successfully applied to pharmacokinetic studies of the metolazone and valsartan combination tablets in beagle dogs.

On-line SPE Using Ionic Liquid-Based Monolithic Column for the Determination of Antihypertensives in Human Plasma

A poly(ionic liquid-glycidylmethacrylate-co-ethyleneglycol dimethacrylate) monolithic column was prepared via in situ free-radical polymerisation and used as an on-line solid-phase extraction column. The obtained monolithic column was characterized by scanning electron microscopy, Fourier transform infrared spectrometer, nitrogen adsorption–desorption measurement, and mercury intrusion porosimetry, which showed a relatively uniform and porous structure. During the on-line solid-phase extraction–high performance liquid chromatography protocol, nifedipine, nitrendipine, and felodipine were separated efficiently from human plasma using a methanol–water linear gradient at a flow rate of 1.0 mL min−1. The extraction efficiency for the targets was satisfactory with no matrix interference. Under the optimized extraction conditions, the linear regression coefficient was >0.9973; precisions for inter- and intra-day assays presented as relative standard deviations were less than 7.29 %, and the accuracy and recovery was in the range of 96–105 and 90–106 %, respectively. As a result, the polymerization monolithic column exhibited high selectivity and good permeability, and it was feasible to be used as an on-line SPE column. Meanwhile, validation assays also demonstrated acceptable sensitivity and precision for simultaneous quantitative screening of nifedipine, nitrendipine, and felodipine in human plasma.

An on-line solid phase extraction–liquid chromatography tandem mass spectrometry method for the determination of perfluoroalkyl substances in the Antarctic ice core samples

An on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry method for the analysis of perfluoroalkyl substances (PFASs) in water samples was developed. The optimal analytical conditions were obtained through the optimization of the extraction efficiency of on-line solid phase extraction column, sample loading rate and loading volume, and the concentration of ammonium acetate in mobile phase. Under the optimal condition, the analytical method displayed good linearity (r2 > 0.99) for 12 PFASs (C5–C14 perfluoroalkyl carboxylic acids and C6/C8 perfluoroalkyl sulfonic acids) over a concentration range of 0.5–100 ng/L. The limits of quantitation for samples were between 0.025 ng/L and 0.5 ng/L and the relative standard deviations (RSD) of five consecutive analyses were less than 10% for 1 ng/L standard solution. Satisfactory results were obtained using this analytical method for the analysis of perfluoroalkyl substances in Antarctic ice core samples. The recoveries of all perfluoroalkyl substances were in a range of 73%–117% when the samples were spiked with standards at the concentrations of 2.5 ng/L and 25 ng/L.

Analysis of Acidic Endogenous Phytohormones in Grapes by Using Online Solid-Phase Extraction Coupled with LC-MS/MS

Phytohormones play important roles in regulating numerous plant physiological and developmental processes, even during the postharvest storage period. In order to determine the functions and changes of gibberellins acid (GA3), indoleacetic acid (IAA), abscisic acid (ABA), indolebutyric acid (IBA) and jasmonic acid (JA) in grape berries during storage, an ultrasensitive method based on direct injection online solid-phase extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. Grape berries were extracted with cold methanol. After centrifugation, the supernatants were concentrated with a vacuum centrifugal concentrator and injected into an online solid-phase extraction column. After the cleanup procedure, the analytes were determined by LC-MS/MS. The results showed that the linearity of the proposed method was 10-210 µg kg(-1) for ABA, 20-200 µg kg(-1) for IBA, 15-320 µg kg(-1) for IAA, 20-320 µg kg(-1) for GA3 and 3.0-90.0 µg kg(-1) for JA. The limits of detection of the method were 0.71, 2.79, 0.94, 0.39 and 0.57 µg kg(-1), respectively. The proposed method was successfully applied to the analysis of endogenous phytohormones in grape berries during the postharvest storage period.

Simultaneous determination of five estrogens and four androgens in water samples by online solid-phase extraction coupled with high-performance liquid chromatography–tandem mass spectrometry

A novel method for simultaneous determination of five estrogens and four androgens by online solid-phase extraction (SPE) coupled with high-performance liquid chromatography–tandem mass spectrometry (HPLC–ESI-MS/MS) in water samples was developed. An aliquot of 50mL water sample after filtration was injected directly into autosampler and the analytes were preconcentrated on a NG1 online SPE column. After cleanup step the analytes were eluted in back flush mode and then separated on a liquid chromatography column. The experimental parameters, such as sample loading flow rate, cleanup condition and elution time, were optimized in detail. Estrogens and androgens were detected in negative and positive mode, respectively. High ionization efficiency of all the analytes was achieved by adding of 1‰ ammonia in the mobile phase. The recoveries ranged from 31.8% to 119.0% and the inter-day RSDs ranged from 2.7% to 19.6%. The limits of detections (LODs) were between 0.1 and 2.5ng/L. The proposed method was successfully applied to the analysis of three types of water samples, including river water, influent and effluent water from a wastewater treatment plant (WWTP). The recoveries of androgens were not that good and a further study is being planned to improve the sensitivity for them. The proposed method is simple, sensitive and suitable for simultaneous analysis and monitoring of estrogens and androgens in water samples.

Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6mm×37mm, 25μm), and the biological matrix was washed out with the solvent (20mM KH2PO4 adjusted pH 3.0) at flow rate of 2mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6mm×50mm, 5μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4min. The UV detection was performed at 286nm. The calibration curves showed excellent linear relationship (R2=0.9995) over the concentration range of 0.05–50μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.

Automated on-line solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry for determination of lipophilic marine toxins in shellfish

Automated on-line solid-phase extraction (SPE) coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for fast determination of lipophilic marine toxins in shellfish samples. The direct coupling of an on-line SPE column to LC–MS/MS was accomplished using column switching techniques. Suitable chromatographic separation was performed on a reversed-phase C18 column under alkaline conditions (pH 11).The selected reversed-phase C18 SPE column allowed rapid and efficient on-line desalting of hydrolysed shellfish samples, avoiding signal suppression during mass spectrometry detection. Furthermore, the on-line SPE procedure allowed reducing matrix effects observed in raw and hydrolysed shellfish extracts.The proposed method was evaluated in terms of linearity, precision, accuracy and limits of detection (LODs). Quantitative recovery (97–102%) and satisfactory inter-day precision (RSD<8%) were achieved for all target compounds. LODs in the sub-μgkg−1 level (0.37–0.68μgkg−1) were obtained for all toxins except for okadaic acid, which showed a value of 2.75μgkg−1.Several mussel samples from North-western Spain were finally analysed in order to demonstrate the applicability of the method. Okadaic acid was the predominant toxin in all samples, although other lipophilic toxins were also detected.

An improved on-line solid phase extraction coupled HPLC–MS/MS system for quantification of Sifuvirtide in human plasma

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/ z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1–6250 ng/mL, and the correlation coefficients ( r 2) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from −7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC–MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).

On-line solid-phase extraction of large-volume injections coupled to liquid chromatography-tandem mass spectrometry for the quantitation and confirmation of 14 selected trace organic contaminants in drinking and surface water

We describe the development and validation of an on-line solid-phase extraction of large-volume injections coupled to liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation and confirmation of 14 selected trace organic contaminants in drinking and surface water. Selected compounds were: anti-infectives (clarithromycin, sulfamethoxazole and trimethoprim), an anticonvulsant (carbamazepine) and its transformation product 10,11-dihydrocarbamazepine, an antihypertensive (enalapril), antineoplastics (cyclophosphamide and methotrexate), herbicides (atrazine, cyanazine, and simazine) and two of their transformation products (deethylatrazine and deisopropylatrazine) and an antiseptic (triclocarban). The breakthrough volume determinations showed that out of all the investigated sorbents, the Strata-X on-line solid-phase extraction column showed the best performance. The method used a load volume of 10.0 mL and was validated using the corresponding matrices, yielding for most compounds, R 2 > 0.99. Extraction recoveries ranged from 60 to 109%. The intra- and inter-day precision were <14 and <16%, respectively. The method detection limits ranged from 0.6 to 6 ng L −1. Matrix effects were in general low. The performance of the on-line method was demonstrated with the analysis of real water samples. The application of alternative techniques of confirmation was also explored using accurate mass measurements on a time-of-flight mass spectrometer and the data-dependent reverse energy ramp scan on a triple quadrupole.

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography–tandem mass spectrometry and its applications to a pharmacokinetic study

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2 mL min −1. Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39–400.00 ng mL −1, the correlation coefficients ( r 2) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39 ng mL −1. The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC–MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.

On-line solid-phase extraction with on-support derivatization for high-sensitivity liquid chromatography tandem mass spectrometry of estrogens in influent/effluent of wastewater treatment plants

A solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry for the determination of estrogens in sewage influent and effluent has been performed. The possibility of preparing estrogen derivatives directly on a solid-phase extraction bed in which the targeted analytes have been previously isolated and pre-concentrated was explored. This method uses an Oasis HLB column (2.1 mm × 20 mm i.d.) for on-line sample cleanup and derivatization support, and a Sunfire C 18 column (150 mm × 2.1 mm i.d.) with a mobile phase consisting of acetonitrile–water–formic acid for separation. After sample introduction, some matrix interferences are removed by washing up SPE column with methanol–water. Phenolic hydroxyl group of estrogens is subsequently derivatized with dansyl chloride. Reaction takes place in the on-line solid-phase extraction column. The excess of reagent and other matrix interferences are then removed by a second washing. Dansylated estrogens are further back eluted and analyzed with HPLC–MS–MS. The optimized on-line protocol is emphasized owing to a low limit of quantification (1 ng L −1) is achieved with only around 1 mL of sample and a low sensitive MS instrument. Developed strategy has been demonstrated to be an improvement over existing methods due to its greater sensitivity and the low volume of matrix used and the total analysis time (extraction, derivatization, analysis) is less than 17 min.

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.