Performance Liquid Chromatography-atmospheric Pressure Research Articles

Simultaneous determination of three components of sodium nitrophenolate in foodstuffs of animal origin by high performance liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization

Sodium nitrophenolate (SNP) is a widely used universal growth regulator consisting of 5-nitroguaiacol sodium (5NG), 4-nitrophenol sodium (PNP), and 2-nitrophenol sodium (ONP). SNP has a positive influence on plants and animals as a feed additive that accelerates growth but is potentially hazardous to humans. SNP has been reported to be cytotoxic and mutagenic, which may increase the risk of cancer and pose a great threat to food safety. There are neither mature detection nor standard methods for the trace analysis of SNP in animal food. Therefore, the development of an accurate and precise analytical method is imperative. This innovative method has theoretical and practical significance for the control of SNP residues, offering advantages such as cost-effectiveness and time efficiency. It will be beneficial for the establishment of detection standards and management measures in foodstuffs of animal origin.In this study, a reliable method for the simultaneous determination of SNP residues in animal food (porcine muscle, chicken tissue, fish, and liver) was developed. For realizing the perfect limit of quantification, the application of back extraction coupled with high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) was proposed to combine high sensitivity and high selectivity. The optimal method was as follows. First, 2.0 g samples were extracted with 10 mL 0.5 mol/L sodium hydroxide solution, followed by adjustment of the pH to acidity with 3 mol/L hydrochloric acid and the addition of sodium chloride (5.0 g) to saturate the inorganic phase. After back-extraction twice with 16 mL acetonitrile, the solution was merged and again saturated with 5 mL of sodium chloride solution. Second, the merged organic phase was cleaned up with 10 mL of n-hexane for defatting. The middle acetonitrile layer was then concentrated to nearly 1.5 mL at 40 ℃ in a N2 stream before dilution with the mobile phase to a volume of 3.0 mL and filtered. Finally, the analytes were separated on a C18 column (100 mm×4.6 mm, 3 μm) and subjected to gradient elution with a mixed solution of methanol and water. Mass spectrometric analysis, which was quantified using the external standard method, was carried out with an atmospheric pressure chemical ionization negative ion source and based on multiple reaction monitoring (MRM) mode. The key parameters, such as the extraction solvent, extraction steps, and purification method, were optimized.The calibration curves were linear in the ranges of 0.5-10 (5NG), 1.0-20 (PNP), and 2.5-50 μg/L (ONP) with correlation coefficients greater than 0.9999. The limit of quantification (LOQ) for 5NG was 1.0 μg/kg, double for PNP, and five times for ONP. The recoveries of the three different concentration levels in all the four matrices were in the range of 81.5%-98.4%, 81.5%-102%, and 81.4%-95.1%. The repeatability, expressed as the relative standard deviations (RSDs) of the three compounds, ranged from 1.51% to 5.98%, 1.10% to 8.85% and 0.91% to 8.61% (n=6). The developed method is characterized by an excellent purification effect, sensitivity, and accuracy. This method is suitable for the simultaneous and quantitative determination of SNP residues in foodstuffs of animal origin.

Simultaneous determination of 14 sexual hormones in health care products by ultra performance liquid chromatography-atmospheric pressure chemical ionization-triple quadrupole mass spectrometry

A comprehensive analytical method was developed for the simultaneous determination of 14 sexual hormones in health care products by ultra performance liquid chromatography-atmospheric pressure chemical ionization-triple quadrupole mass spectrometry (UPLC-APCI-MS/MS). The samples were extracted twice with acetonitrile and the extracts were purified using hydrophilic-lipophilic balance (HLB) cartridges. Chromatographic separation was achieved on a Hypersil Gold C18 column (100 mm×2.1 mm, 1.9 μm) by gradient elution with acetonitrile and 10 mmol/L ammonium acetate aqueous solution as the mobile phases. Compounds were detected by APCI-MS/MS with external standard calibration curve method. All the 14 sexual hormones showed a good linear trend in their respective concentration ranges and the correlation coefficients (r) were no less than 0.996. The limits of detection (LODs) ranged from 0.0990 μg/kg to 2.09 μg/kg. The limits of quantification (LOQs) were in the range of 0.495-5.23 μg/kg. The average recoveries of the 14 sexual hormones in the health care product samples ranged from 65.8% to 118.8% at the three spiked levels. The precisions (n=6) ranged from 0.6% to 8.7% (n=6). The method is simple, sensitive, accurate and precise, and is suitable for the determination of the illegal added sexual hormones in the health care products.

Determination of acrylamide in coffee by ultra performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry

A method using ultra high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (UHPLC-APCI-MS/MS) was developed for the determination of acrylamide in coffee. The coffee samples spiked with 13C3-acrylamide as the internal standard were extracted with methanol, and cleaned using HLB solid phase extraction (SPE) cartridges. The liquid chromatography separation was performed on a Brownlee validated AQ C18 column with isocratic elution. Methanol and 0.1% (volume percentage) formic acid aqueous solution were used as the mobile phase. Identification of acrylamide was achieved by APCI-MS/MS with multiple reaction monitoring (MRM) in the positive mode. The quantification analysis was performed by the internal standard method. The calibration curve showed good linearity with a correlation coefficient of 0.999 in the range of 0.5-100.0 μg/L. The limit of detection (LOD) was 5.0 μg/kg. The limit of quantification (LOQ) was 10.0 μg/kg. Recovery of acrylamide from coffee sample was evaluated at concentrations of 100.0, 200.0 and 1000.0 μg/kg. The average recoveries of acrylamide were between 94.6%-115.0% with relative standard derivations (RSDs) in the range of 2.8%-3.6% (n=6). This simple, accurate and sensitive method was proven to be suitable for the determination of acrylamide in coffee.

Determination of vitamin K composition of fermented food

A rapid ultra-high performance liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method was developed for the analysis of vitamin K compounds: phylloquinone (PK) and menaquinones (MK-n). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 min. Sample treatment with liquid and solid phase extractions and by the use of MK-4 as an internal standard enabled the quantitation of microgram level of vitamin K compounds in food. The method was used to screen and quantitate vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42 µg/100 g), whereas the highest MK-7 content was detected in natto (902 µg/100 g). Some MK-9 was present in kefir (5 µg/100 g). Two Chinese fermented soybean pastes contained significant amount of MK-6 (5–36 µg/100 g), MK-7 (12–86 µg/100 g), and MK-8 (22–44 µg/100 g).

UHPLC-APCI-MS Profiling of Triacylglycerols in Vegetable Oils—Application to the Analysis of Four North African Sesame Seed Varieties

Here, we describe an isocratic ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (UHPLC-APCI-MS) method for the characterization and the quantification of triacylglycerols (TAGs) in vegetable oils. The linearity and the precision of the method are validated, and the limits of detection are estimated. The APCI-MS detector shows a linear response from 1 to 1000 μg/mL, and the correlation coefficients (r2) obtained for standards TAGs exceed 0.995. As compared to previous methods, the proposed method is rapid, simpler, and more sensitive. The limits of detection estimated for several standard TAGs varies from 0.03 to 0.05 injected nanograms (6 to 10 μg/L). The application of the proposed method to the analysis of sesame oil allowed us to unambiguously identify 30 TAG species whereas previous methods have detected 19 TAGs, at the most. Among vegetable oils, sesame oil became an attractive target for adulteration due to its high commercial value. Most of the previous studies of TAG profiles of sesame oils had been devoted to the analysis of sesame oils originating from Asia and some Sub-Saharan countries. To the best of our knowledge, the TAG profiles of sesame oils from other origins are still unknown. The results we obtained for the four Tunisian sesame seed varieties clearly indicate that sesame oils can be classified according to their equivalent carbon number differences (ΔECN) with some rules similar to those required for olive oils.

An approach based on ultrahigh performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry allowing the quantification of both individual phytosteryl and phytostanyl fatty acid esters in complex mixtures

A method for the analysis of both individual phytosteryl and phytostanyl fatty acid esters in complex mixtures was established. The approach was based on a previously not described combination of three elements: (i) the formation of [M−FA+H]+ fragment ions via APCI (atmospheric pressure chemical ionization), (ii) a highly efficient UHPLC-based separation on a 1.7μ C8 column, previously established for phytostanyl fatty acid esters, allowing the distinction of individual fatty acid esters sharing the same sterol/stanol nucleus and of isotope peaks of phytosteryl fatty acid esters and corresponding phytostanyl fatty acid esters based on these [M−FA+H]+ fragment ions, and (iii) the adjustment of the APCI conditions allowing the differential APCI–MS–SIM (single ion monitoring) detection of phytostanyl esters of linoleic and linolenic acid based on their distinct formation of a [M+H]+ ion. The usefulness of the methodology was demonstrated by the analysis of a commercially available enriched margarine. Two runs per sample allowed the quantification of 35 target analytes; the total amounts of esters were between 124.7 and 125.3g/kg, being in good agreement with the labelled 125g/kg. Validation data were elaborated for 35 individual fatty acid esters of sitosterol, campesterol, brassicasterol, stigmasterol, sitostanol and campestanol. Recovery rates ranged from 95 to 106%; the coefficients of variation were consistently <5%, except for stigmasteryl-18:1. The approach describes for the first time a quantification of both individual phytosteryl and phytostanyl fatty acid esters and thus closes an analytical gap related to this class of health-relevant food constituents.

Fast analysis of 29 polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs with ultra-high performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous in the environment. Some of them are probable carcinogens and some are source markers. This work presents an ultra-high performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (UHPLC-APPI-MS/MS) method for simultaneous analysis of 20 PAHs and nine nitro-PAHs. These compounds are separated in 15 minutes in the positive mode and 11 minutes in the negative mode, one half of GC/MS analysis time. Two pairs of precursor/product ions are offered, which is essential for confirmation. This method separates and quantifies benzo[a]pyrene (the most toxic PAHs) and non-priority benzo[e]pyrene (isomers, little toxicity) to avoid overestimation of toxin levels, demonstrating its importance for health-related researches. With 0.5% 2,4-difluoroanisole in chlorobenzene as the dopant, limits of detection of PAHs except acenaphthylene and those of nitro-PAHs except 2-nitrofluoranthene are below 10 pg and 3 pg, respectively, mostly lower than or comparable to those reported using LC-related systems. The responses were linear over two orders of magnitude with fairly good accuracy and precision. Certified reference materials and real aerosol samples were analyzed to demonstrate its applicability. This fast, sensitive, and reliable method is the first UHPLC-APPI-MS/MS method capable of simultaneously analyzing 29 environmentally and toxicologically important PAHs and nitro-PAHs.

Ultra performance liquid chromatography atmospheric pressure photoionization high resolution mass spectrometric method for determination of multiclass pesticide residues in grape and mango juices

A novel analytical method was developed for determination of organochlorine, synthetic pyrethroid, organophosphate and carbamate pesticide residues in fruit juices using ultra performance liquid chromatography–atmospheric pressure photoionization–high resolution mass spectrometry (UPLC–APPI–HRMS). The analytes were extracted from fruit juices by dispersive solid-phase extraction using multi-walled carbon nanotubes (MWCNTs). The analysis was carried out in full scan mode using dual ionization mode of APPI in the mass range of 100–650 units. The limit of detection and limit of quantification values for the pesticides were in the range of 0.025–0.15ngmL−1 and 0.1–0.5ngmL−1 respectively. The matrix effect of the method was found to be low and extraction recoveries were in the range of 60–110%. Some of the real fruits juice samples showed the presence of some pesticides in the range of 6.5–24.8ngL−1.

Analysis of C60-fullerene derivatives and pristine fullerenes in environmental samples by ultrahigh performance liquid chromatography–atmospheric pressure photoionization-mass spectrometry

In this work, a method is proposed for the simultaneous analysis of several pristine fullerenes (C60, C70, C76, C78, and C84) and three C60-fullerene derivatives (N-methyl fulleropyrrolidine, [6,6]-phenyl C61 butyric acid methyl ester and [6,6]-phenyl C61 butyric acid butyl ester) in environmental samples. The method involves the use of ultrahigh performance liquid chromatography coupled to atmospheric pressure photoionization mass spectrometry (UHPLC–APPI-MS) and allowed the chromatographic separation in less than 4.5min. The product ions from tandem mass spectrometry studies of fullerene derivatives were characterized and the most abundant one (m/z 720), corresponding to [C60]−, was selected for quantitation. Selected reaction monitoring (SRM at 0.7m/z FWHM) by acquiring two transitions using both isotopic cluster ions [M]− and [M+1]− as precursor ions was proposed for quantitation and confirmation purposes. For pristine fullerenes, highly selective selected ion monitoring (H-SIM) acquisition mode by monitoring the isotopic cluster ions [M]− and [M+1]− was used. Pressurized solvent extraction conditions were optimized in order to improve recoveries of the studied fullerene compounds from sediment samples. Values up to 87–92% for C60-fullerene derivatives and lower but still acceptable, 70–80%, for pristine fullerenes were obtained. Method limits of quantitation (MLOQs) ranging from 1.5pgL−1 to 5.5ngL−1 in water samples and from 0.1ngkg−1 to 523ngkg−1 in sediments were obtained with good precision (relative standard deviations always lower than 13%). The applicability of the developed method was evaluated by analyzing several environmental samples such as sediments and pond water and the detected levels for C60-fullerene derivatives were of 0.1–2.7ngkg−1 and 1.5–8.5pgL−1, respectively. C60 and C70 were the only pristine fullerenes detected in the analyzed samples (0.1–7.2ngkg−1 in sediments and 9–330pgL−1 in water pond samples).

UHPLC-(+)APCI-MS/MS determination of oxygenated and nitrated polycyclic aromatic hydrocarbons in airborne particulate matter and tree barks collected in Buenos Aires city

A study was undertaken to evaluate the content of oxygenated and nitrated polycyclic aromatic hydrocarbons (OPAHs and NPAHs) in airborne particulate matter (PM2.5 and PM10), and tree barks. For PM2.5 and PM10 collection different approaches were followed using different types of filters and sample collectors. Samples of PM2.5 were collected on glass fiber filters during one month with a medium volume sampler. For PM10, glass and quartz fiber filters were used and samples were collected simultaneously for one week using a high volume sampler. In addition, bark samples were collected at the same sites. Ultra high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (UHPLC–APCI–MS/MS) was used for PAH derivative quantification. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring to provide a high degree of sensitivity and specificity. Chromatographic separation was performed on a reverse phase rapid resolution column using a gradient mode (total run time: 5.0min). The study evidenced that in airborne particle matter, four PAH derivatives were detected at concentrations from 0.01 to 240.62ngm−3 (or 0.3μgg−1 to 30mgg−1). Meanwhile, in tree barks, only OPAHs were detected at concentrations varying from 0.18 to 0.72μgg−1.

Identification of absorbed constituents in the rabbit plasma and cerebrospinal fluid after intranasal administration of Asari Radix et Rhizoma by HS-SPME-GC-MS and HPLC-APCI-IT-TOF-MSn.

Traditional Chinese Medicine (TCM) nasal therapy has been utilized to treat numerous diseases for over two millennia. It has many advantages compared with other routes. In this article, headspace-solid phase microextraction-gas chromatography-mass spectrometry and high performance liquid chromatography-atmospheric pressure chemical ionization-ion trap-time of flight-multistage mass spectrometry were applied for the first time to analyze the absorbed constituents in rabbit plasma and cerebrospinal fluid (CSF) after intranasal administration of Asari Radix et Rhizoma (AR). In total, 47 absorbed AR constituents including 14 monoterpenes, 10 phenylpropanoids, four benzene derivatives, two alkanes, nine N-alkylamides and eight lignans were tentatively identified in the rabbit plasma and CSF. Thirty-three absorbed constituents are found to have different bioactivities related to the pharmacological actions of AR through bibliography data retrieval. These indicated that many types of constituents of TCM can be absorbed at the nasal cavity into both rabbit blood and CSF. This is the first study to explore the absorption of AR, and comprehensively analyze the absorbed constituents after intranasal administration of TCM. These findings extend our understanding of the effective substances of AR, and inspire us to make a hypothesis on the mechanism of additive effect of multiple constituents of TCMs, which is very worthy of further investigation.

Ultra-high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry for the analysis of benzimidazole compounds in milk samples

In this work the atmospheric pressure ionization behavior and the tandem mass spectrometry fragmentation of 19 benzimidazoles was studied. Several atmospheric pressure ionization (API) sources (ESI, APCI and APPI) were evaluated showing that APCI was the best option, mainly for KETO-TCB that provided a 20 times improvement versus ESI. From the studies of tandem mass spectrometry, fragmentation pathways were proposed thus characterizing the most abundant and interesting product ions and selecting the most abundant and selective ones for the confirmatory quantitative method performed in SRM mode in a triple quadrupole mass analyzer. For quantitative analysis, the 19 benzimidazoles were separated in less than 7min using a C18 column packed with superficially porous particles providing high efficiency within the range of UHPLC. A QuEChERS-like sample treatment was applied to milk samples before the UHPLC–APCI–MS/MS determination providing method limits of quantitation that ranged from 0.6 to 3μgkg−1 and showing good repeatability (RSD: 2–18%) and accuracy (relative errors: 1–23%) suitable for using this method for routine analysis.

Determination and differentiation of triacylglycerol molecular species in Antarctic and non-Antarctic yeasts by atmospheric pressure-chemical ionization-mass spectrometry

Yeast, particularly Saccharomyces cerevisiae, has long served as a model eukaryotic system for studies on the regulation of lipid metabolism. We developed a high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry method for the detailed analysis of triacylglycerols (TAGs) in 14 species of yeast consisting of seven Antarctic yeasts (grown at 15°C and 5°C) and seven non-Antarctic yeasts (grown at 25°C and 15°C), the latter including 3 strains of S. cerevisiae. Analysis of TAG molecular species established that the sn-2 position was invariably occupied by an unsaturated fatty acyl moiety. In S. cerevisiae the preference was for oleic acid 18:1>palmitoleic acid 16:1, in Candida albicans, Cryptococcus humicolus and Rhodotorula mucilaginosa 18:1>linoleic acid 18:2 and in Zygosaccharomyces rouxii 18:2>18:1. In the Antarctic yeasts (Cryptococcus watticus, Cryptococcus victoriae, Cryptococcus nyarrowii, Leucosporidium antarcticum, Leucosporidium fellii, Candida psychrophila and Rhodotorula mucilaginosa) the general pattern was for the sn-2 position to be occupied by 18:1, 18:2 or linolenic acid 18:3. A trend towards synthesis of increased unsaturated fatty acid in TAGs was observed as the growth temperature was lowered. The application of principal component analysis demonstrated that the yeasts were differentiated into three distinct groups. One group consisted of the three S. cerevisiae strains, a second the other four non-Antarctic yeasts and the third the seven Antarctic yeasts. The data for the Antarctic yeasts, to the best of our knowledge, have not been previously reported.

Simultaneous quantification of cefpodoxime proxetil and clavulanic acid in human plasma by LC–MS using solid phase extraction with application to pharmacokinetic studies

A simple, rapid and selective high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method was developed and validated for the simultaneous estimation of cefpodoxime proxetil (CDPX) and clavulanic acid (CA) in human plasma. Extraction of samples was done by solid phase extraction technique (SPE) and chloramphenicol used as internal standard. Chromatographic separation was carried out on a reverse phase Princeton SPHER C18 (150mm×4mm i.d., 5μm) column using mixture of methanol: acetonitrile: 2mM ammonium acetate (25:25:50, v/v, pH 3.5) at 0.8mL/min flow rate. Detection was performed on a single quadrupole MS by selected ion monitoring (SIM) mode via APCI source. The calibration curve was linear within the concentration range, 0.04-4.4μg/mL and 0.1-10.0μg/mL for CDPX and CA respectively. Pharmacokinetic parameters of tablet (CDPX 200mg, CA 125mg) were evaluated. Cmax, Tmax, T1/2, elimination rate constant (Kel), AUC0-t, and AUC0-∞ of tablet were 2.13±0.06μg/mL, 2h, 3.05±0.15h, 0.24±0.37h(-1), 6.81±0.14μg h/mL and 7.72±0.23μg h/mL respectively for cefpodoxime (CP), 5.34±0.28μg/mL, 2h, 2.73±0.25h, 0.26±0.31h(-1), 15.37±0.16μg h/mL and 16.59±0.53μg h/mL respectively for CA.

Triacylglyceride profiling in serum by silver ion high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and effective method was developed to investigate triacylglyceride (TAG) compounds in biological matrices using silver ion high performance liquid chromatography-mass spectrometry (Ag+ -HPLC-MS). The TAG compounds from mouse serum were extracted by a modified Folch method using the classical CHC13-MeOH solvent system. The extract was separated on a Varian ChromSpher 5 Lipids column with isocratic elution using acetonitrile-hexane (1: 99, v/v) as mobile phase at a flow rate of 0.75 mL/min, and detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in positive ion mode to acquire sufficient MS information of TAG compounds by enhanced mass spectrometry (EMS), enhanced product ion (EPI) and neutral loss (NL) scans. The identification of TAG compounds was based on their chromatographic behaviors and MS data. Besides, the NL scans of thirty fatty acids were performed to further validate the results. Finally, 66 TAG compounds as well as 5 cholesteryl ether (CE) compounds were obtained from the mouse serum extract. This method is simple, reproducible and also suitable for the analysis of TAG compounds in other samples.

Ultra high performance liquid chromatography–atmospheric pressure photoionization-mass spectrometry for high-sensitivity analysis of US Environmental Protection Agency sixteen priority pollutant polynuclear aromatic hydrocarbons in oysters

In response to Gulf of Mexico deepwater horizon oil spill, we have developed an atmospheric pressure photoionization (APPI) based ultra high performance liquid chromatography–mass spectrometry (UHPLC–MS) method for high-sensitivity analysis of United States Environmental Protection Agency (US EPA) 16 priority pollutant polynuclear aromatic hydrocarbons (PAHs) in oysters. Analyses were performed on an Agilent's Infinity 1290 UHPLC system coupled with a G6140A single quadrupole MS detector with Syagen's PhotoMate® APPI® source. Column separation was achieved using Zorbax Eclipse PAH column. Chlorobenzene was used as an APPI dopant for maximum overall sensitivity. Dynamic linear ranges were evaluated and found to cover 3.6–5.1 (Ave. 4.4) orders of magnitude with R2 of at least 0.995. A quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and cleanup procedure was used. The spike recoveries ranged from 77% to 110% with %RSD of 0.6–6.7 at spike concentrations below or substantially below the US Food and Drug Administration (FDA) level of concern in oysters. The on-column instrument detection limits (IDLs, 6σ S/N=3) ranged from 8 to 106pg with an average of 23pg for 16 PAHs. The method detection limits (MDLs, 6σ S/N=3) ranged from 0.013 to 0.129ppm with an average of 0.040ppm for all analytes. These MDLs were about 5 times to over 4 orders of magnitude lower than US FDA levels of concern in oysters.

A highly specific and sensitive quantification analysis of the sterols in silkworm larvae by high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols—7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and β-sitosterol—using a high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4- 13C 2 as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.

Determination of Pilocarpine in Human Plasma by LC–APCI–MS–MS and Application to a Pharmacokinetic Study

A sensitive, specific and rapid high performance liquid chromatography–atmospheric pressure chemical ionization source-tandem mass spectrometry (LC-APCI-MS-MS) method for the determination of pilocarpine in human plasma was developed and validated. The method is based on liquid–liquid extraction, followed by a reversed-phase liquid chromatographic separation, and detected by means of tandem mass spectrometry. The linear calibration curve covered a concentration range of 2–500 μg L−1. The intra- and inter-day precisions for pilocarpine were <10% and the accuracies were between 90 and 110%. The method was applied successfully to a pharmacokinetic study involving 20 healthy Chinese male volunteers after oral administration of 6 mg pilocarpine.

High performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry for sensitive determination of bioactive amines in donkey milk

In the present study we report on the optimization and validation of a sensitive high performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI–MS) method for the determination of 8 bioactive amines (histamine, tyramine, tryptamine, 2-phenylethylamine, cadaverine, putrescine, spermidine and spermine) in donkey milk samples. The method involves donkey milk pre-treatment to remove proteins and pre-column dansylation of the amines. HPLC in reversed phase mode has been used for bioactive amines separation and the operating condition of the APCI–MS system proved to be powerful and very efficient for peak assignment. The separation was accomplished in a short time with an excellent resolution for all the amine peaks. Quantification was carried out by monitoring the characteristic [M+H] + ion of each amine derivative. The method sensitivity, linearity and repeatability were assayed with satisfactory results. The detection limits of the analysed amines ranged from 0.5 μg L −1 to 15 μg L −1; the highest LOD was for spermine. Also remarkably good recovery values were obtained; at the lowest spiking level (1 μg L −1) the percent mean recoveries ranged from 77.7 to 109.7. Furthermore, as the investigations relate to a complex matrix as donkey milk, suitable studies on matrix effect were performed. Finally, the developed and validated method was applied to analyse 13 donkey milk samples. Among the identified bioactive amines, putrescine, spermine and spermidine proved to be the main amines in donkey milk. Their concentration levels in the present study were lower than the values determined in mature human, cow and sow milk.

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.