Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection

A rapid liquid-chromatography (LC) method is presented which uses fluorescence detection (FLD) for palytoxin analogues analysis in benthic dinoflagellates of the genus Ostreopsis. The amino-acidic reagent 6-aminoquinolyl-N-hydroxisuccinimidyl carbamate (AccQ) was used for fluorescence labelling followed by LC-FLD.The efficacy of the method is exemplified by comparison of the results of the quantification obtained by LC-FLD and the hemolytic assay performed for palytoxins for which a highly significant linear correlation was achieved (r² = 0.9118). The derivatized palytoxin analogues were determined in the range of 0.75-25 ng.The proposed method was successfully applied to the determination and quantification of palytoxin analogues in 14 samples from different strains of Ostreopsis from different locations (Western Mediterranean Sea, Canary Islands, Madeira Islands and Southern coasts of Brazil). To confirm the chemical structure of the toxins, samples were also analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) with a system that has a poorer sensitivity when compared with LC-FLD detection and the hemolytic assay. The successful use of this method with dinoflagellates is a good indicator of suitability for other types of marine samples.     

Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.     

Rapid analysis of six trace trichlorophenols in seawater by magnetic micro‐solid‐phase extraction and liquid chromatography with tandem mass spectrometry

A new facile, rapid, inexpensive, and sensitive method for the analysis of six trace trichlorophenols in seawater samples was developed by magnetic micro‐solid‐phase extraction coupled to liquid chromatography with tandem mass spectrometry. Core–shell covalently functionalized ferroferric oxide coated with aminated silicon dioxide and decorated with multiwalled carbon nanotubes was applied as an adsorbent to perform the extraction process. The effect of factors including solution pH, contact time, adsorbent amount, and ionic strength were investigated in detail. The obtained results revealed that the proposed adsorbent was a highly effective and low‐cost magnetic micro‐solid‐phase extraction material for the enrichment of 2,3,4‐trichlorophenol, 2,3,5‐trichlorophenol, 2,3,6‐trichlorophenol, 2,4,5‐trichlorophenol, 2,4,6‐trichlorophenol, and 3,4,5‐trichlorophenol from seawater. Under the optimized conditions, the recoveries ranged from 88.0 to 99.5% at the three spiking levels, the limits of detection and the limits of quantification were 0.002 and 0.007 μg/L for the six trichlorophenols, respectively. The intra‐ and interday relative standard deviations were 2.0–6.7 and 4.5–8.9%, respectively. The calibration curves showed a good linearity in the range of 0.02–5.0 μg/L. The routine run analyses showed that the developed method was fast, simple, accurate, solvent‐saving and high resolution, and it was suitable for the determination of trace trichlorophenols in seawater.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

全氟辛酸的3,4-二氯苯胺衍生化及高效液相色谱分析

介绍了一种可用于环境污染物全氟辛酸（PFOA）检测的高效液相色谱/紫外检测（HPLC/UV）分析方法。首先选用3,4-二氯苯胺为衍生化试剂,利用碳二亚胺法合成PFOA的酰胺化衍生产物（其在255 nm处紫外吸收最大）。然后确定四氢呋喃或水相介质中mg/L水平PFOA的衍生化条件及薄层硅胶色谱净化步骤。建立柱前衍生-HPLC/UV方法,以合成的全氟辛酸-3,4-二氯苯酰胺为对照品,外标法定量,PFOA上机测定的定量限为0.5 mg/L。通过加标回收试验评价方法的准确性,其中有机相及水相衍生法的回收率分别为91.8%~108.7%及40.1%~53.7%。与已报道的柱前衍生-HPLC/UV方法比较,本方法具有反应条件温和、衍生产物稳定、原料廉价易得、操作简单、成本低等优点。将本方法应用于光催化降解研究中PFOA的降解动力学实验,结果与液相色谱-质谱联用方法（LC/MS）的结果一致,说明本方法具有较好的应用前景。     

A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites

Plasmodium falciparum is the causative agent of malaria, a deadly infectious disease for which treatments are scarce and drug-resistant parasites are now increasingly found. A comprehensive method of identifying and quantifying metabolites of this intracellular parasite could expand the arsenal of tools to understand its biology, and be used to develop new treatments against the disease. Here, we present two methods based on liquid chromatography tandem mass spectrometry for reliable measurement of water-soluble metabolites involved in phospholipid biosynthesis, as well as several other metabolites that reflect the metabolic status of the parasite including amino acids, carboxylic acids, energy-related carbohydrates, and nucleotides. A total of 35 compounds was quantified. In the first method, polar compounds were retained by hydrophilic interaction chromatography (amino column) and detected in negative mode using succinic acid-¹³C₄ and fluorovaline as internal standards. In the second method, separations were carried out using reverse phase (C18) ion-pair liquid chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent in positive detection mode, using d₉-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r² > 0.99). The intra- and inter-day accuracy and precision as well as the extraction recovery of each compound were determined. The lower limit of quantitation varied from 50 pmol to 100 fmol/3 × 10⁷ cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites.     

Simultaneous measurement of trace monoadenosine and diadenosine monophosphate in biomimicking prebiotic synthesis using high-performance liquid chromatography with ultraviolet detection and electrospray ionization mass spectrometry characterization

The method for simultaneous separation and determination of trace monoadenosine and diadenosine monophosphate (i.e. 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA) in biomimicking prebiotic synthesis was developed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) identification. The separation was performed on a Supelco C₁₈ column with a gradient elution (solvent A: 10 mM NH₄Ac aqueous solution; solvent B: MeOH). The flow rate was set at 1.0 ml/min. The quantitative determination was achieved by HPLC with UV detection at 260 nm. The linearity ranged from 0.5 to 100 μg/ml for each nucleotide. The limits of detection (LODs) for the four nucleotides were less than 0.30 μg/ml. The recovery ranged from 95.2 to 100.7%. The intra-day relative standard deviations (RSDs) of the retention times were between 0.7 and 1.1%. Both full-scan ESI-MS and -MS² for the four nucleotides under both positive and negative polarity were carried out and the possible cleavage pathways of them were depicted. The specific ions, [AMP + H]⁺ at m/z 348 and [ApA + H]⁺ at m/z 597, were chosen to characterize the four nucleotides in biomimicking prebiotic synthesis between N-(O,O-diisopropyl) phosphoryl amino acid (Dipp-aa) and adenosine. Using the proposed HPLC/UV/ESI-MS method, the concentration of 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA in the biomimicking prebiotic synthesis samples were determined.     

Identification and quantification of domoic acid by UHPLC/QTOF tandem mass spectrometry,with simultaneous identification of non-target photodegradation products

Amnesic shellfish poisoning is a potentially lethal human toxic syndrome which is caused by domoic acid (DA), a neurotoxin produced by marine phytoplankton, principally from Pseudonitzschia genus. In this report, a method to identify and quantify the DA toxin, with simultaneous identification of its photodegradation products, has been developed. It uses an ultra high performance liquid chromatography coupled to a quadrupole-time-of-flight tandem mass spectrometer (UHPLC–QTOF) after solid-phase extraction (SPE). An unambiguous identification of DA was carried out by considering both the retention time of DA in UHPLC and the exact mass of protonated DA molecule ([M + H]⁺ = 312.1447 m/z) and of the most intense fragment ion (m/z 266.1391). The quantification was conducted using protonated DA molecule with protonated Glafenin as internal standard, obtaining a limits of detection of 0.75 µg L^?1. Large screening with UHPLC–QTOF could also give structural information about degradation products of DA present in samples after UV-irradiation. This method was applied for the determination of DA in complex liquid samples after SPE and is applicable for environmental monitoring of this toxic substance in the aquatic environment.     

Isolation, preconcentration and determination of rhamnolipids in aqueous samples by dispersive liquid-liquid microextraction and liquid chromatography with tandem mass spectrometry

An analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for the determination of rhamnolipids. A dispersive liquid-liquid microextraction (DLLME) procedure was used to isolate and concentrate target compounds from aqueous samples collected from surface water, sewage treatment plant effluent and cultivation of microbial culture. Development of the DLLME procedure included optimization of several important parameters such as kind and volume of extracting and dispersing solvents as well as sample pH. Under optimized conditions a two-step extraction with sonication was used. Chloroform was applied as the extracting and acetone as the dispersing solvent. The recoveries of the analytes were 70-87%. Matrix effects investigated for the analytes revealed existence of ionization enhancement for both mono- and dirhamnolipids.     

Rapid and selective determination of UV filters in seawater by liquid chromatography-tandem mass spectrometry combined with stir bar sorptive extraction

Fast liquid chromatography coupled to triple-quadrupole tandem mass spectrometry was employed for the determination of six UV filters in seawater. The separation of the analytes was achieved in less than 5 min; polarity switching was used as four of the analytes were ionized in positive mode and the remaining two in negative mode. Two ionization sources were employed and compared: atmospheric pressure chemical ionization (APCI) gave better results than electrospray ionization (ESI) for all analytes, with higher reproducibility and lower detection limits. Therefore APCI was chosen for the determination of the analytes in seawater samples using stir bar sorptive extraction-liquid desorption (SBSE-LD).Quantitative analysis was performed in multiple reaction monitoring (MRM) mode; fragmentation pathways of the analytes with regard to the formation of the MRM ions were also proposed.For the analysis of seawater samples, calibration curves were drawn using SBSE in spiked seawater. All figures of merit of the method were satisfactory; limits of detection were particularly low for the four analytes ionized in positive mode, being in the range 8-31 ng/L. The method was applied to the determination of the six UV filters in seawater samples from Liguria, Italy. Only benzophenone-3 (BP-3) and ethylhexyl methoxycinnamate (EHMC) were measured in the analyzed samples; some of the remaining analytes were also detected but always below the limit of quantitation.     

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

Summary A reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 m reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 l sample volume of a biological fluid.     

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 μm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.     

Characterization ofC-glycosylflavones fromDissotis rotundifolia by liquid chromatography — UV diode array detection — tandem mass spectrometry

Summary By high-performance liquid chromatography (HPLC) coupled with UV diode-array detection (DAD) and thermospray mass spectrometry (TSP-MS), four main constituents of a polar, whole plant extract fromDissotis rotundifolia T. were characterized. The fourC-glycosylflavones, isoorientin, orientin, vitexin and isovitexin were detected in the methanolic and hydroalcoholic extract of the plant as well as in the commercial drug preparation ‘Sirop de Dissotis’. Although the UV data and TSP mass spectra allowed rapid characterisation of all fourC-glycosylflavones, exact attribution of the peaks to their structures could not be achieved as neither the UV spectra nor the TSP mass spectra enabled differentiation of one position isomer from the other. Therefore a successful attempt was made to distinguish the 6-C from the 8-C-glycosylflavones by thermospray tandem mass spectrometry (TSP-MS-MS). The collision induced dissociation (CID) spectra of the particular ion [M+H-120]⁺ gave fragments which permitted differentiation of position isomers. To confirm the accuracy of on-line results, reference compounds were included in the HPLC study.     

Determination of flurbiprofen in pharmaceutical formulations by UV spectrophotometry and liquid chromatography

In this study, a new and rapid UV spectrophotometric (UV) method and a reversed phase high performance liquid chromatographic (LC) method were developed for quantitative estimation of flurbiprofen, a non-selective, non-steroidal, anti-inflammatory drug (NSAID), in pure form and in pharmaceutical dosage form. The solvent system, wavelength of detection, chromatographic conditions were optimized in order to maximize the sensitivity of both the proposed methods. The linear regression equations obtained by least square regression method were Abs=7.5906×10⁻² concentration (μg/ml) + (−) 4.6210×10⁻² for the UV method, and peak area=1.2652×10² concentration (ng/ml) + 1.4830×10³ for the LC method. The detection limit as per the error propagation theory was found to be 0.34 μg/ml for UV method and 15 ng/ml for LC method. The developed methods were successfully employed with high degree of precision and accuracy for the estimation of total drug content in two commercial ophthalmic drops of flurbiprofen. The results of analysis were treated statistically, as per USP 2000 and International Conference on Harmonization (ICH) guidelines for validation of analytical procedures, and by recovery studies. The results obtained from UV method were comparable with those obtained by using LC. It was concluded that both the developed methods are equally accurate, sensitive, precise, reproducible, robust and rugged and could be applied directly and easily to the pharmaceutical preparations of flurbiprofen. However, LC method is useful at very low level (ng/ml), whereas UV method is suitable at μg/ml level.     

Determination of biogenic amines in wines by pre-column derivatization and high-performance liquid chromatography coupled to mass spectrometry

A new HPLC method for determining biogenic amines in wines is developed. This method is based on pre-column amine derivatization, further separation of derivatives and on-line hyphenation of HPLC to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Biogenic amines have been derivatized with 1,2-naphthoquinone-4-sulfonate at 65 °C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C₁₈ analytical column using an elution gradient based on increasing the percentage of methanol. Derivatives have been ionized in positive mode and detected by selected ion monitoring. The operating conditions of the APCI-MS system (voltages, temperatures and gases) have been thoroughly optimized to obtain the maximum sensitivity for all analytes. In the selected conditions, APCI-MS spectra display little fragmentation and good signal-to-noise ratio. Depending on the amine characteristics, the main spectral peaks are due to mono- and di-derivative products. Figures of merit of the method have been established under the selected conditions using red wine samples. Recoveries ranging from 94% to 106% have been obtained which prove excellent accuracy of the method in the determination of histamine, putrescine, cadaverine, tryptamine, phenylethylamine, tyramine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.     

Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

Summary A rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C₁₈ reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Sensitive determination of salicylate and benzophenone type UV filters in water samples using solid-phase microextraction, derivatization and gas chromatography tandem mass spectrometry

A sensitive procedure for the determination of three UV filters: ethylhexyl salicylate (EHS), 3,3,5-trimethylcyclohexyl salicylate (Homosalate, HMS), 2-hydroxy-4-methoxybenzophenone (BP-3) and two related hydroxylated benzophenones (2,4-dihydroxybenzophenone, BP-1 and 2,2′-dihydroxy-4-methoxybenzophenone, BP-8) in water samples is presented. Analytes were first concentrated on the coating of a solid-phase microextraction (SPME) fibre, on-fibre silylated and then determined using gas chromatography combined with tandem mass spectrometry (GC-MS/MS). Factors affecting the performance of extraction and derivatization steps are thoroughly evaluated and their effects on the yield of the sample preparation discussed. Under final working conditions, a PDMS-DVB coated SPME fibre was exposed directly to 10 mL of water, adjusted at pH 3, for 30 min. After that, the fibre was placed in the headspace (HS) of a 1.5 mL vial containing 20 μL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). On-fibre silylation of hydroxyl groups contained in the structure of target compounds was performed at 45 °C for 10 min. The whole sample preparation process was completed in 40 min, providing limits of quantification from 0.5 to 10 ng L⁻¹ and acceptable precision (RSDs under 13%) for samples spiked at different concentrations. All compounds could be accurately determined in river and treated wastewater (relative recoveries from 89 to 115%) using standards in ultrapure water, whereas standard addition is recommended to quantify their levels in untreated wastewater. Analysis of wastewater revealed the systematic presence of BP-3 and BP-1 in raw samples with maximum concentrations close to 500 and 250 ng L⁻¹, respectively.     

Simultaneous quantification of three isoflavonoid glycosides in rabbit plasma after oral administration of Astragalus mongholicus extract by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A selective and sensitive liquid chromatography coupled with triple stage quadruple tandem mass spectrometry (HPLC/TSQ-MS/MS) was developed and validated for simultaneous quantification of calycosin-7-O-β-d-glycoside (CCSG), formononetin-7-O-β-d-glycoside (Ononin) and (6R,10R)-9,10-dimethoxypterocarpan-3-O-β-d-glycoside (DPG) in rabbit plasma. Plasma samples were extracted with solid-phase extraction (SPE), separated on an Inertsil ODS-3 column and detected by tandem mass spectrometry with electrospray ionization (ESI) interface in positive selective reaction monitoring (SRM) mode. 3,7,8-Trimethoxy-xanthone-1-O-primaverose was used as internal standard (IS) for quantitative measurement. For each analyte, one major product ion was chosen and used for screening of it. Calibration curves were generated over the range of 2-1000 ng mL⁻¹ with the correlation coefficients greater than 0.99 by using a weighted (1/χ) least squares linear regression. The method had the lower limit quantification of 0.15, 0.21 and 0.19 for CCSG, Ononin and DPG, respectively, with precision less than 20%. The intra- and inter-day precisions ranged from 2.48 to 6.38% and 4.81 to 11.78% (R.S.D.%), respectively. This assay is suitable for determining the above three trace glycosides in rabbit plasma simultaneously and thus investigating the pharmacokinetics of glycosides from Astragalus mongholicus extract in rabbits.     

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.