High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts

The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC(50) values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 microM, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown".     

On-Line HPLC with Biochemical Detection for Screening Bioactive Compounds in Complex Matrixes

On-line high-performance liquid chromatography (HPLC) coupled with biochemical detection (BCD) has been developed to screen compounds showing antioxidant action, enzyme inhibition and receptor affinity in complex matrixes. This review summarizes HPLC methods combining different post-column detection methods, such as diode-array detection (DAD), mass spectrometry (MS), chemiluminescence (CL) and nuclear magnetic resonance, for antioxidant screening. The methods based on a single relatively stable reagent such as DPPH^• and ABTS^•+ were the most popular. Oxygen free radical scavengers mainly depended on post-column CL detection. The on-line hyphenated HPLC–BCD systems based on post-column UV/DAD fluorescence and MS detection were also widely applied to screen enzyme- and receptor-active compounds. These strategies provide a convenient tool for quick identification and quantification of active compounds in complex matrixes.

     

Multiple on‐line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection

On‐line high performance liquid chromatography (HPLC) coupled with three biochemical detection (BCD) methods was applied to evaluate bioactive components in Danshen injection. On‐line HPLC‐photo‐diode array–fluorescence detection based on the fluorogenic substrate 7‐acetoxy‐1‐methyl quinolinium iodide, was built to search acetylcholinesterase (AChE) inhibitors in Danshen injection. On‐line HPLC coupled with the scavenging assay of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) free radicals was developed to screen antioxidants. The three active profiles were obviously different. Radical scavenging profiles revealed seven strong peaks in the chromatographic fingerprint possessing obvious free radical inhibition effects, while some minor peaks exhibited stronger AChE inhibition activities. The main radical scavengers and AChE inhibitors were identified by HPLC‐MS. Several unknown ingredients showing strong AChE inhibition activities needed further identification except protocatechuic aldehydrate, salvianolic acid H or I and lithospermic acid. The on‐line multiple on‐line HPLC‐BCD methods will provide powerful tools in the field of pharmacognosy for fast‐track identification of interesting and/or novel bioactive compounds.     

Size-exclusion chromatography with on-line ultraviolet, proton nuclear magnetic resonance and mass spectrometric detection and on-line collection for off-line Fourier transform infrared spectroscopy.

The coupling of HPLC with UV detection and on-line NMR spectroscopy and mass spectrometry combined with a dedicated interface for the collection of the chromatographic eluent for subsequent Fourier transform (FT) IR has been investigated using a number of polymer additives as model compounds. Size-exclusion chromatography was performed using deuterated chloroform as eluent with the separation monitored on-line by UV detection at 254 nm and on-flow 1H-NMR and MS. The effluent from the NMR probe was directed to a dedicated HPLC interface where it was deposited on a germanium plate for subsequent FT-IR. NMR and MS spectra were successfully obtained for 2,6-di-tert.-butyl-4-methylphenol, octadecyl-3-(3,5-di-tert.-butyl-4-hydroxyphenyl) propionate (Irganox 1076) and diisooctyl phthalate on-line and FT-IR spectra for all three compounds were obtained off-line. Practical problems encountered with this multiple hyphenation are described.     

Development of an on-line automated sample clean-up method and liquid chromatography–tandem mass spectrometry analysis: application in an in vitro proteolytic assay

Fluorescence detection has been a method of choice in industry for screening assays, including identification of enzyme inhibitors, owing to its high-throughput capabilities, excellent reproducibility, and sensitivity. Occasionally, inhibitors are identified that challenge the fluorescence assay limit, necessitating the development of more sensitive detection methods to assess these compounds. For data mining purposes, however, original assay conditions may be required. A direct method transfer to highly sensitive and specific LC-MS-based methods has not always been possible due to the presence of MS-incompatible neutral detergents and non-volatile salts in the assay matrix. Utilizing an in vitro proteolytic screening assay for the serine protease hepatitis C virus (HCV) nonstructural (NS) 3 protease as a test case, we report the development of an automated sample clean-up procedure implemented on-line with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis to complement fluorescence detection. Ion exchange and peptide microtraps were employed to remove MS-incompatible assay matrix components. Three protease inhibitors were used to validate the MS/MS method. Comparable potencies were achieved for these compounds when assessed by fluorescence and MS/MS detection. Furthermore, four-fold less enzyme could be utilized when employing the MS/MS method compared to fluorescence detection. The longer analysis time, however, resulted in reduced sample capacity. The potency of our designed HCV NS3 protease inhibitors are thus routinely evaluated using a continuous fluorescence-based assay. Only pertinent inhibitors approaching the fluorescence assay sensitivity limit are subsequently analyzed further by LC-MS/MS. This methodology allows us to maintain a database and to compare results independent of the detection method. Despite the relatively slow sample turnaround time of this LC-MS approach, the versatility of the automated on-line clean-up procedure and sample analysis can be applied to assays containing reagents which were historically considered to be MS incompatible.     

Determination of methotrexate in environmental samples by solid phase extraction and high performance liquid chromatography: ultraviolet or tandem mass spectrometry detection?

In order to assess exposure levels of hospital personnel involved in the preparation and administration of antineoplastic drugs, environmental monitoring should be carried out. Wipe samples, pads, gloves and air samples should be collected at the end of each work shift, properly treated and then analysed using instrumental techniques which are sufficiently sensitive and specific to detect even trace amounts of drug. In this study, a method using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), incorporating solid phase extraction (SPE), was validated for determination of methotrexate (MTX) in wipe and air samples. Each step of the method was first developed and optimised using ultraviolet detection (UV), and afterwards tandem mass spectrometry was used to obtain a lower limit of quantitation when the expected drug level was less than the analytical UV detection limit. SPE enabled a 20-fold preconcentration of the analyte when using HPLC/UV and a further 30-fold preconcentration was obtained when analysing samples by HPLC/MS/MS. For example, the limit of quantitation (LLQ) was lowered from 3000 ng on wipe (direct injection onto an HPLC/UV system) to 5 ng on wipe (SPE plus HPLC/MS/MS). 7-hydroxymethotrexate was used as internal standard to assess precision and accuracy.     

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures

In this study, an end-point-based fluorescence assay for soluble epoxide hydrolase (sEH) was transformed into an on-line continuous-flow format. The on-line biochemical detection system (BCD) was coupled on-line to liquid chromatography (LC) to allow mixture analysis. The on-line BCD was based on a flow system wherein sEH activity was detected by competition of analytes with the substrate hydrolysis. The reaction product was measured by fluorescence detection. In parallel to the BCD data, UV and MS data were obtained through post-column splitting of the LC effluent. The buffer system and reagent concentrations were optimized resulting in a stable on-line BCD with a good assay window and good sensitivity (S/N > 60). The potency of known sEH inhibitors (sEHis) obtained by LC–BCD correlates well with published values. The LC–BCD system was applied to test how oxidative microsomal metabolism affects the potency of three sEHis. After incubation with pig liver microsomes, several metabolites of sEHis were characterized by MS, while their individual potencies were measured by BCD. For all compounds tested, active metabolites were observed. The developed method allows for the first time the detection of sEHis in mixtures providing new opportunities in the development of drug candidates.     

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

Analyses of non-steroidal anti-inflammatory drugs by on-line concentration capillary electrochromatography using poly(stearyl methacrylate-divinylbenzene) monolithic columns

This study describes the ability of on-line concentration capillary electrochromatography (CEC) coupled with UV or mass spectrometry (MS) for the determination of nine common non-steroidal anti-inflammatory drugs (NSAIDs) in water samples. A series of poly(stearyl methacrylate-divinylbenzene) (poly(SMA-DVB)) monolithic columns, which were prepared by single step in situ polymerization of divinylbenzene (DVB), stearyl methacrylate (SMA) and vinylbenzenesulfonic acid (VBSA, charged monomer), were developed as separation columns for the first time. The effects of polymerization condition of monolithic columns on analyte separations were examined, and the results indicated that separation performances were markedly improved in monolithic columns prepared with short reaction time (3 h) and low SMA:DVB ratio (40/60 ratio of SMA:DVB). Subsequently, an on-line concentration step of step-gradient elution was combined to this CEC system, and by optimizing the difference in eluent strength between the sample matrix and mobile phase, all NSAIDs detection sensitivity were improved (limit of detection (LOD) was 3.4-10 μg/L for UV, and 0.01-0.19 μg/L for MS). When compared to the best CE and LC reports on NSAIDs analyses so far, this on-line concentration CEC method provided better detection ability within shorter separation time (12 min) when either UV or MS detector was employed. This is the first report for on-line concentration CEC with MS detection applied in trace solute analyses of real samples.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.     

Determination of abamectin in soil samples using high-performance liquid chromatography with tandem mass spectrometry

Abamectin, which is comprised of a mixture of avermectins B1a and B1b, is a natural pesticide used as an anti-parasitic agent in livestock, ornamental, and agricultural crops, which can potentially be transported to aquatic systems. These compounds are highly toxic to both aquatic vertebrates and invertebrates at low concentrations in water. This investigation developed high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) techniques to support automated extraction by an accelerated solvent extraction (ASE) system and chromatographic techniques to measure residues of avermectins in complex soil samples. HPLC along with atmospheric pressure chemical ionization (APCI) MS/MS was used for separation and determination of avermectin isomers in soil samples. Average method recovery for abamectin by UV was 91%, while detection by MS/MS resulted in a 68% recovery for abamectin. Individual method recoveries by MS/MS were 53.6% for avermectin B1a and 36.8% for avermectin B1b. The use of tandem technology eliminated matrix interferences and resulted in an approximately eight-fold increase in sensitivity.     

On-line determination of carboxylic acids, aldehydes and ketones by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionisation mass spectrometry after derivatization with 2-nitrophenylhydrazine

2-Nitrophenylhydrazine (2-NPH) is widely used for the derivatization of carboxylic acids, aldehydes and ketones, in industrial and biological samples. These compounds react with 2-NPH to form derivatives, which are separated by high-performance liquid chromatography (HPLC) and detected with diode array detection (DAD). The UV spectra give information about the functionality of the compounds: carboxylic acid or ketone/aldehyde. Most of the eluting compounds in "known" samples are well characterised by the retention time (comparison with those of standards) of the 2-NPH derivative and their UV spectrum. The identification of different unknown 2-NPH derivatives of carboxylic acids, ketones and/or aldehydes, in industrial or biological samples, based on retention time and/or UV spectrum is not sufficient. These unknown 2-NPH compounds can be identified with on-line atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) based on the molecular mass or/and the fragmentation of the derivative. A novel and specific on-line HPLC-DAD-APCI(-)-MS method is described for the determination of carboxylic acids, ketones and aldehydes, after on-line pre-column derivatization with 2-NHP. The fragmentation of different 2-NPH derivatives were investigated and the possibilities of APCI(-)-MS detection were demonstrated by the on-line identification of an unknown derivative, which turned out to be a side product between 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-NPH in the presence of high concentrations of a cyclic amide in the sample solution.     

On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium

An on-line analysis method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining HPLC with hydrogen dioxide radical scavenging and HPLC-DAD-MS-CL system. The structural identification and activity characteristics of various constituents could be rapidly achieved by the on-line assay of UV, MS and CL in one run. In 4 species of Epimedium studied 32 compounds, including phenolic acids, 8-isopentenyl-flavonoid glycosides and flavonoid glycosides containing a ortho-hydroxyl group, were identified by comparison with authentic standards and published mass data. Among these compounds, phenolic acids and flavonoid glycosides containing an ortho-hydroxyl group could obviously inhibit CL, which suggested their strong radical scavenging activity. These four species each exhibited different active properties, which might correlate to their respective quality. The results indicated that the on-line HPLC-DAD-MS-CL system would be a potential method to rapidly and sensitively screen radical scavengers in herbal medicines, and could display an integrated fingerprint based on different detectors.     

On-line capillary electrophoresis-electrospray mass spectrometry for the stereoselective analysis of drugs and metabolites

The on-line combination of partial-filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the enantioseparation of pharmaceutical drugs and metabolites, namely amphetamines, methadone, venlafaxine and selected tropane alkaloids. The partial-filling technique proved to be a suitable and efficient approach to avoid mass spectrometry (MS) source contamination, as well as signal suppression due to nonvolatile additives. To achieve chiral separation, various chiral selectors were applied, including neutral and particularly negatively charged cyclodextrins. Because of the countercurrent contribution, charged cyclodextrins were found more suitable for the on-line MS detection of separated enantiomers. Hyphenation of capillary electrophoresis (CE) with mass spectrometry was found appropriate for the stereoselective analysis of methadone in real serum samples. Moreover, the use of MS in the selected ion monitoring mode resulted in a very high selectivity, as well as improved sensitivity compared to UV detection. Finally, with atropine as a model compound, the quantitative performances of the method were evaluated and showed high sensitivity, as well as good repeatability in terms of migration time and peak area ratio.     

On-line dual-precolumn-based trace enrichment for the determination of polar and acidic microcontaminants in river water by liquid chromatography with diode-array UV and tandem mass spectrometric detection

Dual-pre-column-based trace enrichment combined on-line with liquid chromatography-diode-array UV and tandem mass spectrometric detection was used to determine a wide polarity range of organic microcontaminants in river water. Various sorbents were studied for their extraction efficiency of (highly) polar and acidic compounds and their ability to selectively remove humic substances, which are normally co-extracted and interfere in the UV detection of polar microcontaminants. An optimised on-line dual-pre-column set-up with PLRP-S in the first pre-column and Hysphere-1 in the second pre-column was used to study the analytical performance of the procedure. Tandem MS was used for confirmation purposes and to quantify the organic microcontaminants in river water at the low-ng/l level. In addition, the influence of the type of sample (drinking and river water) on suppression of analyte responses in electrospray ionization MS was studied.     

Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged,derivatized glycans

The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.     

Ultrapure water for liquid chromatography-mass spectrometry studies

Improvements in trace enrichment techniques combined with the sensitivity of mass spectrometry offer enhanced opportunities to analyze ever lower concentrations of drugs, metabolites, pesticides or environmental pollutants. To perform HPLC and liquid chromatography-mass spectrometry (LC-MS) analyses under optimum conditions, the water used for mobile phase preparation needs to be highly purified and delivered on demand. Indeed, both UV photodiode array detection and MS detection methods are sensitive to organic contaminants (total organic carbon, TOC), and the water quality has a direct impact on the achievable detection limits. The benefits of UV photooxidation on TOC reduction for LC-MS studies were highlighted using electrospray ionization MS detection by comparing HPLC-grade bottled water, freshly produced UV185/254-treated water, and freshly produced non-UV-treated water.