Liquid Chromatography Detection Research Articles

Improved method for morphine determination in biological fluids and tissues: rapid, sensitive and selective

Morphine was assayed using a simple two step solvent extraction--acid back extraction sample preparation method, coupled with normal phase high-performance liquid chromatography (HPLC) and dual electrode coulometric detection. HPLC is performed with a 1.0 M Tris-methanol (5:95) mobile phase with subtle pH adjustments to separate morphine and internal standard from any interfering compounds. The use of normal phase HPLC (silica column) substantially reduces problems from interfering lipophilic substances sometimes encountered with reverse phase HPLC following solvent extraction and which would otherwise require more time-consuming sample preparation. Dual electrode detection further improves the selectivity for morphine and gives excellent sensitivity (0.5 ng mL-1), reproducibility and stability for automated sample injection. This method has proven suitable for pharmacokinetic studies of morphine.

Alcohol and metal determination in alcoholic beverages through high-temperature liquid-chromatography coupled to an inductively coupled plasma atomic emission spectrometer

In the present work, an inductively coupled plasma atomic emission spectrometry (ICP-AES) system was used as a high temperature liquid chromatography (HTLC) detector for the determination of alcohols and metals in beverages. For the sake of comparison, a refractive index (RI) detector was also employed for the first time to detect alcohols with HTLC. The organic compounds studied were methanol, ethanol, propan-1-ol and butan-1-ol (in the 10–125 mg/L concentration range) and the elements tested were magnesium, aluminum, copper, manganese and barium at concentrations included between roughly 0.01 and 80 mg/L. Column heating temperatures ranged from 80 to 175 °C and the optimum ones in terms of peak resolution, sensitivity and column lifetime were 125 and 100 °C for the HTLC-RI and HTLC-ICP-AES couplings, respectively. The HTLC-ICP-AES interface design ( i.e., spray chamber design and nebulizer type used) was studied and it was found that a single pass spray chamber provided about 2 times higher sensitivities than a cyclonic conventional design. Comparatively speaking, limits of detection for alcohols were of the same order for the two evaluated detection systems (from 5 to 25 mg/L). In contrast, unlike RI, ICP-AES provided information about the content of both organic and inorganic species. Furthermore, temperature programming was applied to shorten the analysis time and it was verified that ICP-AES was less sensitive to temperature changes and modifications in the analyte chemical nature than the RI detector. Both detectors were successfully applied to the determination of short chain alcohols in several beverages such as muscatel, pacharan, punch, vermouth and two different brands of whiskeys (from 10 to 40 g of ethanol/100 g of sample). The results of the inorganic elements studied by HTLC-ICP-AES were compared with those obtained using inductively coupled plasma mass spectrometry (ICP-MS) obtaining good agreement between them. Recoveries found for spiked samples were close to 100% for both, inorganic elements (with both HLTC-ICP-AES and ICP-MS) and alcohols (with both HTLC-ICP-AES and HTLC-RI hyphenations).

SEC‐MR‐NMR: Online Coupling of Size Exclusion Chromatography and Medium Resolution NMR Spectroscopy

Online coupling of size exclusion chromatography together with medium resolution nuclear magnetic resonance (SEC-MR-NMR) might be one solution to the problem of chemically sensitive detection in liquid polymer chromatography. By use of a combination of SEC with a table-top, specially designed 20 MHz NMR spectrometer, based on a permanent magnet, online (1) H NMR spectra of SEC fractions can be obtained. The integration of digital filters, mechanical shims and electronic shims led to substantially improved sensitivity and chemical selectivity compared to former TD (time domain) 20 MHz instruments. (1) H NMR spectra of PMMA and PS homopolymers as well as PS-PMMA block copolymers were of sufficient quality to enable detection and de-formulation of unknown polymer compounds. (1) H NMR spectra of acceptable resolution and S/N ratio were collected online during the chromatography. The SEC separation online with the NMR measurements performed well and resulted in the proof of principle of the SEC-MR-NMR combination.

Chemiluminescence detectors for liquid chromatography

In this tutorial we describe the construction of chemiluminescence detectors for high performance liquid chromatography (HPLC), comprising the components required to deliver the chemiluminescence reagent, a coiled-tubing flow cell, photomultiplier tube and detector housing, and various options for data acquisition. We also discuss two state-of-the-art commercially available chemiluminescence detectors for HPLC and other flow analysis methodology.

Liquid Chromatography Automatic System with Optical Activity Laser Detector and Its Applications

A new liquid chromatographic system with polarimetric detection and a computer program allowing the output of chromatograms to a display on line and electronic data storing was designed, built and put to work. The chromatographic system includes the laser polarimetric detector, having a measuring interval of one second, the chromatographic columns, the continuous flux polarimeter tubes of 50 and 100 mm, the programs for data acquisition, processing and storing, and the technical know-how for its most efficiently application. 30 min is all the time needed to obtain a chromatogram by this method which is reasonably shorter than the time required for any other known comparable technique, and offering, besides, lower operation cost. The combination of molecular exclusion liquid chromatography and laser polarimetric detection has turned into a carbohydrate separation and quantification system of basic importance for the evaluation of plants fluids of industrial interest (sugarcane, agave, vegetable extracts, etc.). It is described here the application of this system as an early or complementary indicator of leaf scald —disease that affects sugarcane plants. Another application on algae extracts gave good results in the separation and identification of biologically active components. The introduction of this system in several research centers in Cuba and abroad has resulted in practical information for the industry.

Preconcentration of emerging contaminants in environmental water samples by using silica supported Fe 3O 4 magnetic nanoparticles for improving mass detection in capillary liquid chromatography

A magnetic material based on Fe 3O 4 magnetic nanoparticles incorporated in a silica matrix by using a sol–gel procedure has been used to extract and preconcentrate emerging contaminants such as acetylsalicylic acid, acetaminophen, diclofenac and ibuprofen from environmental water samples prior to the analysis with Capillary LC–MS. The use of the proposed silica supported Fe 3O 4 magnetic nanoparticles enables surfactant free extracts for the analysis with MS detection without interferences in the ionisation step. Under the optimum conditions, we demonstrated the reusability of the magnetic sorbent material during 20 uses without loss in the extraction efficiency. In addition, no cleanup was necessary. The preconcentration factor was 100 and the detection limits were between 50 and 150 ng/L. The proposed procedure has been applied to the analysis of water samples obtaining recoveries between 80 and 110% and RSD values lower than 12%. Concentrations of the target analytes over the range 1.7 and 0.1 μg/L have been found in different water samples.

Optimization and Validation of a Confirmatory Method for Determination of Ten Sulfonamides in Feeds by LC and UV-Diode Array Detection

A simple, rapid, and reliable multiresidue method, based on liquid chromatography (LC) and ultraviolet (UV)-diode array detection (DAD), is described for assaying ten sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, sulfamethoxazole, sulfaquinoxaline, and sulfadimethoxine) in feeds. The chromatographic separation is accomplished using a C18 column, eluted with a mobile phase consisting of acetate buffer, acetonitrile, and methanol. The sample preparation requires a simple extraction with chloroform/acetone and a purification step by solid-phase extraction. The analytical parameters of precision, detection and quantification limits, recovery, and ruggedness have been evaluated by a validation procedure following the European guidelines of Regulation 882/2004/EC and Decision 657/2002/EC.

Simple, stable and sensitive electrogenerated chemiluminescence detector for high-performance liquid chromatography and its application in direct determination of multiple fluoroquinolone residues in milk

A simple, stable and sensitive electrogenerated chemiluminescence (ECL) detector was developed. It was based on tris(2,2-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+) immobilized on the surface of a Pt wire with Nepem-105D ion exchange solution. The detector was prepared by inserting a Pt wire with immobilized Ru(bpy) 3 2+ (working electrode) into a capillary tube, followed by inserting another Pt wire (counter electrode) in this tube and sealing. ECL behavior was investigated using ofloxacin as an analyte. Under optimal conditions, stable ECL intensity was obtained. This detector has been used in HPLC-ECL for the determination of multiple target fluoroquinolone residues in milk. There is no post column reagent addition, which would dilute the analytes, potentially leading to chromatographic band-broadening. The system is very simple with low dead volume, low baseline and background noise, together with high sensitivity and stability. The as-prepared ECL detector, when was used for the determination of ofloxacin, pefloxacin, enrofloxacin and difloxacin in milk, demonstrated adequate sensitivity to allow quantification of trace FQ levels in commercial milk samples. One or more of the target FQ analytes were present at levels above the LOD of the new ECL detector in each and every one of the 22 milk samples analysed.

Evaluation of the dietary protein using an optimized HPLC method

Protein quality is determined by the type and concentration of the essential amino acids and by their bioavailability. Hence, the content of dietary essential amino acids from a protein or mixture of proteins is a factor which determines the feeding quality of the protein. The purpose of the study was to evaluate the quality of the protein from a high protein raw ingredient (corn gluten) using a chromatographic method (HPLC) under optimized experimental conditions. In this study, we used pre-column derivatization, separation by high-performance liquid chromatography (HPLC) and UV detection. The optimized method was used to determine the amino acids from high-protein raw ingredients commonly used in animal feeding (corn gluten, 61%CP). A set of 6 amino acids analyses has been performed in 6 different days, each sample being prepared in double. For characterization of data strings we used quality parameters: average, standard deviation, standard error, confidence level, precision, accuracy. For verifying the Gaussian shape of the strings we used the Kernel Density. For identifying and rejecting the outliers from the data strings we used the Q test. The very low values of the accuracy for several amino acids (glutamic acid, threonine, alanine, valine, phenylalanine, isoleucine, leucine) determined us to introduce the dilution stage (1:2) for the hydrolysed samples. The method can be considered as repeatable (precision in different days) and accurate (evaluation by tracing yields) for all the determined amino acids.

Chiral Separation, Determination of Absolute Configuration, and High-performance Liquid Chromatography Detection of Enantiomeric 3-hydroxyhexadecanoyl-CoA

The enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of 2-enoyl-CoA to yield 3-hydroxyacyl-CoA in mitochondrial and peroxisomal β-oxidation. However, the stereospecificities of these hydratases differ from each other. To provide clear evidence of the stereospecificities of hydratases, the absolute configuration of 3-hydroxyhexadecanoyl-CoAs was determined, and they were subjected to a high-performance liquid chromatography (HPLC) using a chiral separation column. The retention time of 3(R)-hydroxyhexadecanoyl-CoA was shorter than that of 3(S)-hydroxyhexadecanoyl-CoA. The HPLC analysis carried out using a chiral separation column is considered to be useful for the study of enoyl-CoA hydratase.

Development and Possibilities of Multichannel Voltammetric Detection in Liquid Chromatography

AbstractVoltammetric detection in HPLC by using linear potential sweeps applied to dual electrode cells is evaluated. The application of an offset between both potential sweeps, applied to either serial or parallel setup of both working electrodes, and the further subtraction of the corresponding datasets are discussed. The use of multivariate curve resolution for the treatment of overlapping signals is also investigated. Despite some noise problems, mostly derived from the differences between the working electrodes, the proposed multichannel method appears to be promising. The different approaches are tested by analyzing some thiols mixtures.

Increased flow rate compatibility for universal acoustic flame detection in liquid chromatography

The liquid chromatography (LC) flow rate tolerance of the universal Acoustic Flame Detector (AFD) is characterized and significantly expanded through using larger bore burners. For example, increasing the burner i.d. from 1.00 to 4.00 mm increases the AFD upper flow rate limit from 20 to 100 μL/min. While signal and noise each reduce as the burner i.d. widens, the best current performance is obtained with a 2.30 mm i.d. burner. This approach also allows AFD operation over a broader range of mobile phase temperatures. As a result, the overall increased flow rate compatibility of the detector can facilitate improved chromatography and further development of LC-AFD applications.

Ultrasensitive detection of malondialdehyde with surface-enhanced Raman spectroscopy

Malondialdehyde (MDA) is a biomarker of lipid peroxidation that has been widely associated with food rancidity as well as many human diseases. Most current MDA detection methods involve MDA reaction with thiobarbituric acid (TBA), followed by UV-visible and/or fluorescence detection of high-performance liquid chromatography (HPLC)-separated TBA-MDA. Herein, we report the first proof-of-concept study of surface-enhanced Raman detection of a TBA-MDA adduct using silver nanoparticles as the SERS substrate and the 632.8 nm HeNe laser as a Raman excitation source. Current SERS detection limit of TBA-MDA is 0.45 nM, ~100 times higher than the 36 nM fluorescence sensitivity recently reported with the HPLC-purified TBA-MDA. Molecular specificity of the SERS technique was studied by comparing the SERS spectrum of TBA-MDA with those acquired with TBA adducts of other TBA-reactive compounds (TBARCs) that includes formaldehyde, acetaldehyde, butyraldehyde, trans-2-hexenal, and pyrimidine. Compared to TBA and TBA adducts with those TBARCs, the SERS activity of TBA-MDA adduct is significantly higher. The possibility of direct SERS detection of TBA-MDA in a reaction mixture (without HPLC separation) has also been investigated.

On the isomerisation of cefozopran in solution

In this study, we first described the two configurations of cephalosporins by cefozopran, then determined their molecular structure and finally evaluated the stability and biological activity of the two configurations. Our results showed that cefozopran existed as two different configurations in the aqueous solution with acetonitrile. Using mass spectrometry with liquid chromatography, nuclear magnetic resonance with liquid chromatography and optical rotation detection technology, we determined the spatial structures of both configurations and the detailed mechanism for change. By molecular docking and determining their antimicrobial activities, we showed that the biological activity of cis-isomer was stronger than that of trans-isomer.

Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: A review

Literature published in the last decade concerning the use of mid-infrared spectrometry as a detection system in separation techniques employing a liquid mobile phase is reviewed. In addition to the continued use of isocratic liquid chromatographic (LC) techniques, advances in chemometric data evaluation techniques now allow the use of gradient techniques on a routine basis, thus significantly broadening the range of possible applications of LC–IR. The general trend towards miniaturized separation systems was also followed for mid-IR detection where two key developments are of special importance. Firstly, concerning on-line detection the advent of micro-fabricated flow-cells with inner volumes of only a few nL for transmission as well as attenuated total reflection measurements enabled on-line mid-IR detection in capillary LC and opened the path for the first successful realization of on-line mid-IR detection in capillary zone electrophoresis as well as micellar electrokinetic chromatography. Secondly, concerning off-line detection the use of micro-flow through dispensers now enables to concentrate eluting analytes on dried spots sized a few tens of micrometers, thus matching the dimensions for sensitive detection by mid-IR microscopy. Finally in an attempt to increase detection sensitivity of on-line mid-IR detection, mid-IR quantum cascade lasers have been used. Applications cover the field of food analysis, environmental analysis and the characterization of explosives among others. Best detection sensitivities for on-line and off-line detection have been achieved in miniaturized systems and are in the order of 50 ng and 2 ng on column, respectively.

Comprehensive comparison of liquid chromatography selectivity as provided by two types of liquid chromatography detectors (high resolution mass spectrometry and tandem mass spectrometry): “Where is the crossover point?”

The selectivity of mass traces obtained by monitoring liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was compared. A number of blank extracts (fish, pork kidney, pork liver and honey) were separated by ultra performance liquid chromatography (UPLC). Detected were some 100 dummy transitions respectively dummy exact masses (traces). These dummy masses were the product of a random generator. The range of the permitted masses corresponded to those which are typical for analytes (e.g. veterinary drugs). The large number of monitored dummy traces ensured that endogenous compounds present in the matrix extract, produced a significant number of detectable chromatographic peaks. All obtained chromatographic peaks were integrated and standardized. Standardisation was done by dividing these absolute peak areas by the average response of a set of 7 different veterinary drugs. This permitted a direct comparison between the LC-HRMS and LC-MS/MS data. The data indicated that the selectivity of LC-HRMS exceeds LC-MS/MS, if high resolution mass spectrometry (HRMS) data is recorded with a resolution of 50,000 full width at half maximum (FWHM) and a corresponding mass window. This conclusion was further supported by experimental data (MS/MS based trace analysis), where a false positive finding was observed. An endogenous matrix compound present in honey matrix behaved like a banned nitroimidazole drug. This included identical retention time and two MRM traces, producing an MRM ratio between them, which perfectly matched the ratio observed in the external standard. HRMS measurement clearly resolved the interfering matrix compound and unmasked the false positive MS/MS finding.

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC). Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis. We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/β-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of β-actin was 26.12 ± 13.39 ng/mL for healthy, and 49.61 ± 46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC. This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results.

High performance liquid chromatography with on-line dual quantum cascade laser detection for the determination of carbohydrates, alcohols and organic acids in wine and grape juice

In the present study the simultaneous use of two quantum cascade lasers (QC-lasers) was investigated for the on-line detection in high performance liquid chromatography (HPLC). An optical set-up based on three gold mirrors and a ZnSe beam splitter was used to direct the emitted laser light trough a liquid flow cell with an optical path length of 52 μm onto a mercury-cadmium-telluride (MCT) detector. Using the separation of eight components of wine and grape juice as an example, on-line dual QC-laser detection in HPLC could be shown successfully for the first time.

Development and single laboratory validation of a method for citrinin

Citrinin (CIT) is a nephrotoxic secondary metabolite produced by several fungal species. CIT contamination has been reported in many cereals. Recently CIT contamination of red mold rice, used as an artificial colour, received attention because a CIT producing Monascus species was used in its production. Therefore, we developed a relatively simple method, immunoaffinity column clean-up followed by high performance liquid chromatographic (IAC-HPLC) detection, to determine CIT in red mold rice and several other commodities, and performed a single laboratory validation of the method. CIT was extracted by shaking for 30 min with 70% methanol, after which the extract was purified using an immunoaffinity column. The isolated CIT was quantitated by HPLC with fluorescence detection. Recoveries of CIT from red mold rice and koji-rice at levels of 50-2,500 µg/kg were 81-88%. RSDs, estimated with naturally contaminated red mold rice at 50, 200 and 400 µg/kg were 5.1%, 2.8% and 4.1%, respectively. Recoveries of CIT from cereals and mushroom samples at 50, 200 and 400 µg/kg were 60-123%, 69-88% and 73-100% with RSDs of 2.1-7.8%, 1.7-9.2% and 0.6-5.3%, respectively.

Intracerebral microdialysis during deep brain stimulation surgery

BackgroundThis report describes the use of microdialysis in conjunction with deep brain stimulation (DBS) surgery to assess extracellular levels of neurotransmitters within the human basal ganglia (BG). Electrical stimulation of the subthalamic nucleus (STN) is an efficacious treatment for advanced Parkinson's disease, yet the mechanisms of STN DBS remain poorly understood. Measurement of neurotransmitter levels within the BG may provide insight into mechanisms of DBS, but such an approach presents technical challenges. MethodsAfter microelectrode recordings confirmed location of STN, a custom microdialysis guide cannula was inserted. A CMA (Stockholm, Sweden) microdialysis probe was then positioned to the same depth as the microrecording electrode in STN or 2mm inferiorly to record in the substantia nigra. The catheter was perfused at a rate of 2.0μL/min with a sterile mock CSF solution and samples of extracellular fluid were collected at regular intervals. Dialysate samples were analyzed using high-pressure liquid chromatography (HPLC) detection procedures for quantitation of glutamate, gamma-aminobutyric acid (GABA), and dopamine. ResultsLevels of neurotransmitters were reliably identified in dialysate samples using HPLC. By monitoring concentrations of glutamate, GABA and dopamine, we were able to demonstrate what seemed to be a steady state baseline within approximately 30min. ConclusionMicrodialysis during DBS surgery is a feasible method for assessing levels of glutamate, GABA and dopamine within the human BG. Obtaining a steady state baseline of neurotransmitter levels appears feasible, thus making future studies of intraoperative microdialysis during DBS meaningful.